US20170335295A9 - Production of fatty acid derivatives - Google Patents
Production of fatty acid derivatives Download PDFInfo
- Publication number
- US20170335295A9 US20170335295A9 US14/709,232 US201514709232A US2017335295A9 US 20170335295 A9 US20170335295 A9 US 20170335295A9 US 201514709232 A US201514709232 A US 201514709232A US 2017335295 A9 US2017335295 A9 US 2017335295A9
- Authority
- US
- United States
- Prior art keywords
- fatty acid
- host cell
- fatty
- ester
- acyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000004519 manufacturing process Methods 0.000 title claims description 74
- 229940053200 antiepileptics fatty acid derivative Drugs 0.000 title abstract description 45
- 150000002194 fatty esters Chemical class 0.000 claims abstract description 102
- 239000000203 mixture Substances 0.000 claims abstract description 22
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 344
- 229930195729 fatty acid Natural products 0.000 claims description 344
- 239000000194 fatty acid Substances 0.000 claims description 344
- 150000002148 esters Chemical class 0.000 claims description 247
- 108090000623 proteins and genes Proteins 0.000 claims description 235
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 174
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 171
- 229920001184 polypeptide Polymers 0.000 claims description 169
- 108020002982 thioesterase Proteins 0.000 claims description 145
- 102000005488 Thioesterase Human genes 0.000 claims description 140
- 102000004190 Enzymes Human genes 0.000 claims description 137
- 108090000790 Enzymes Proteins 0.000 claims description 137
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 133
- 244000005700 microbiome Species 0.000 claims description 73
- 230000004133 fatty acid degradation Effects 0.000 claims description 72
- 101710116435 Outer membrane protein Proteins 0.000 claims description 38
- 238000006467 substitution reaction Methods 0.000 claims description 30
- 108010001058 Acyl-CoA Dehydrogenase Proteins 0.000 claims description 28
- 102000002735 Acyl-CoA Dehydrogenase Human genes 0.000 claims description 28
- 241000894006 Bacteria Species 0.000 claims description 23
- 150000001413 amino acids Chemical group 0.000 claims description 23
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 22
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 22
- 230000003247 decreasing effect Effects 0.000 claims description 15
- 230000001965 increasing effect Effects 0.000 claims description 15
- 241000588724 Escherichia coli Species 0.000 claims description 13
- 241000908790 Alcanivorax jadensis Species 0.000 claims description 11
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 10
- 235000019387 fatty acid methyl ester Nutrition 0.000 claims description 10
- 241000219195 Arabidopsis thaliana Species 0.000 claims description 9
- 241000206589 Marinobacter Species 0.000 claims description 9
- 241000516659 Roseiflexus Species 0.000 claims description 9
- 241000588625 Acinetobacter sp. Species 0.000 claims description 8
- 241000588986 Alcaligenes Species 0.000 claims description 8
- 241000337031 Anaeromyxobacter Species 0.000 claims description 8
- 241000968725 Gammaproteobacteria bacterium Species 0.000 claims description 8
- 241000699660 Mus musculus Species 0.000 claims description 8
- 241001440631 Rhodoferax ferrireducens Species 0.000 claims description 8
- 241000045411 Hahella chejuensis Species 0.000 claims description 7
- 108010062385 long-chain-alcohol O-fatty-acyltransferase Proteins 0.000 claims description 7
- 241000611270 Alcanivorax borkumensis Species 0.000 claims description 6
- 241000282414 Homo sapiens Species 0.000 claims description 6
- 241000839458 Limnobacter sp. Species 0.000 claims description 6
- 241000186367 Mycobacterium avium Species 0.000 claims description 6
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims description 6
- 241000588626 Acinetobacter baumannii Species 0.000 claims description 5
- 239000004471 Glycine Substances 0.000 claims description 5
- 241001508003 Mycobacterium abscessus Species 0.000 claims description 5
- 241000186366 Mycobacterium bovis Species 0.000 claims description 5
- 241000186362 Mycobacterium leprae Species 0.000 claims description 5
- 241000187492 Mycobacterium marinum Species 0.000 claims description 5
- 241000187480 Mycobacterium smegmatis Species 0.000 claims description 5
- 241000187479 Mycobacterium tuberculosis Species 0.000 claims description 5
- 241000187917 Mycobacterium ulcerans Species 0.000 claims description 5
- 241001503673 Nocardia farcinica Species 0.000 claims description 5
- 241001524101 Rhodococcus opacus Species 0.000 claims description 5
- 241000187392 Streptomyces griseus Species 0.000 claims description 5
- 241001519901 Acidobacteria bacterium Species 0.000 claims description 4
- 241001134630 Acidothermus cellulolyticus Species 0.000 claims description 4
- 241001165345 Acinetobacter baylyi Species 0.000 claims description 4
- 241000607528 Aeromonas hydrophila Species 0.000 claims description 4
- 241000607525 Aeromonas salmonicida Species 0.000 claims description 4
- 241001135315 Alteromonas macleodii Species 0.000 claims description 4
- 241000337032 Anaeromyxobacter dehalogenans Species 0.000 claims description 4
- 241000328888 Candidatus Solibacter usitatus Species 0.000 claims description 4
- 241000580885 Cutaneotrichosporon curvatus Species 0.000 claims description 4
- 241001534811 Erythrobacter litoralis Species 0.000 claims description 4
- 241000190842 Erythrobacter sp. Species 0.000 claims description 4
- 241000187808 Frankia sp. Species 0.000 claims description 4
- 241000064284 Janibacter sp. Species 0.000 claims description 4
- 241000662215 Marinobacter algicola Species 0.000 claims description 4
- 241000206597 Marinobacter hydrocarbonoclasticus Species 0.000 claims description 4
- 241000501784 Marinobacter sp. Species 0.000 claims description 4
- 241000190928 Microscilla marina Species 0.000 claims description 4
- 241001313618 Moritella sp. Species 0.000 claims description 4
- 241000907999 Mortierella alpina Species 0.000 claims description 4
- 241000187910 Mycobacterium gilvum Species 0.000 claims description 4
- 241000187488 Mycobacterium sp. Species 0.000 claims description 4
- 241000142559 Mycobacterium vanbaalenii Species 0.000 claims description 4
- 241000863422 Myxococcus xanthus Species 0.000 claims description 4
- 241000204971 Natronomonas pharaonis Species 0.000 claims description 4
- 241001495390 Nocardioides sp. Species 0.000 claims description 4
- 241001260361 Photobacterium profundum Species 0.000 claims description 4
- 241001423781 Plesiocystis pacifica Species 0.000 claims description 4
- 241001599925 Polaromonas naphthalenivorans Species 0.000 claims description 4
- 241001472610 Polaromonas sp. Species 0.000 claims description 4
- 241000355705 Psychrobacter arcticus Species 0.000 claims description 4
- 241000309106 Psychrobacter cryohalolentis Species 0.000 claims description 4
- 241000557299 Psychrobacter sp. Species 0.000 claims description 4
- 241001443285 Reinekea sp. Species 0.000 claims description 4
- 241000187562 Rhodococcus sp. Species 0.000 claims description 4
- 241000516658 Roseiflexus castenholzii Species 0.000 claims description 4
- 241000187559 Saccharopolyspora erythraea Species 0.000 claims description 4
- 241001312746 Salinibacter ruber Species 0.000 claims description 4
- 241000237098 Sphingopyxis alaskensis Species 0.000 claims description 4
- 241000863001 Stigmatella aurantiaca Species 0.000 claims description 4
- 241001468227 Streptomyces avermitilis Species 0.000 claims description 4
- 241000187432 Streptomyces coelicolor Species 0.000 claims description 4
- 241000618895 Tenacibaculum sp. Species 0.000 claims description 4
- 244000301083 Ustilago maydis Species 0.000 claims description 4
- 235000015919 Ustilago maydis Nutrition 0.000 claims description 4
- 229940011158 alteromonas macleodii Drugs 0.000 claims description 4
- 241001464430 Cyanobacterium Species 0.000 claims description 3
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 3
- 238000011144 upstream manufacturing Methods 0.000 claims description 3
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 claims description 2
- 230000002255 enzymatic effect Effects 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 160
- 239000000446 fuel Substances 0.000 abstract description 22
- 210000004027 cell Anatomy 0.000 description 444
- 150000004665 fatty acids Chemical class 0.000 description 319
- 229940088598 enzyme Drugs 0.000 description 136
- 239000000758 substrate Substances 0.000 description 108
- 230000000694 effects Effects 0.000 description 103
- 230000014509 gene expression Effects 0.000 description 72
- 125000000539 amino acid group Chemical group 0.000 description 62
- -1 gaseous Substances 0.000 description 61
- 125000003275 alpha amino acid group Chemical group 0.000 description 60
- 102000040430 polynucleotide Human genes 0.000 description 60
- 108091033319 polynucleotide Proteins 0.000 description 60
- 239000002157 polynucleotide Substances 0.000 description 60
- 230000002238 attenuated effect Effects 0.000 description 58
- 235000021588 free fatty acids Nutrition 0.000 description 58
- 239000003208 petroleum Substances 0.000 description 53
- 239000000047 product Substances 0.000 description 53
- 125000003729 nucleotide group Chemical group 0.000 description 49
- 239000002773 nucleotide Substances 0.000 description 48
- 235000001014 amino acid Nutrition 0.000 description 47
- 102000005962 receptors Human genes 0.000 description 46
- 108020003175 receptors Proteins 0.000 description 46
- 150000007523 nucleic acids Chemical group 0.000 description 45
- 150000007970 thio esters Chemical class 0.000 description 43
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 42
- 238000012258 culturing Methods 0.000 description 42
- 238000006243 chemical reaction Methods 0.000 description 39
- 102000039446 nucleic acids Human genes 0.000 description 38
- 108020004707 nucleic acids Proteins 0.000 description 38
- 108700016155 Acyl transferases Proteins 0.000 description 36
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 36
- 230000015572 biosynthetic process Effects 0.000 description 35
- 102000057234 Acyl transferases Human genes 0.000 description 34
- 150000001298 alcohols Chemical class 0.000 description 29
- 230000002103 transcriptional effect Effects 0.000 description 27
- 239000013598 vector Substances 0.000 description 24
- 229940024606 amino acid Drugs 0.000 description 23
- 150000002191 fatty alcohols Chemical class 0.000 description 23
- 108020004414 DNA Proteins 0.000 description 20
- 239000003225 biodiesel Substances 0.000 description 20
- 102000004169 proteins and genes Human genes 0.000 description 20
- 101150087812 tesA gene Proteins 0.000 description 20
- 102000053602 DNA Human genes 0.000 description 19
- 229910052799 carbon Inorganic materials 0.000 description 19
- 101100390711 Escherichia coli (strain K12) fhuA gene Proteins 0.000 description 18
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 18
- 239000012634 fragment Substances 0.000 description 18
- 229930195733 hydrocarbon Natural products 0.000 description 18
- 150000002430 hydrocarbons Chemical class 0.000 description 18
- 235000018102 proteins Nutrition 0.000 description 18
- 239000002028 Biomass Substances 0.000 description 17
- 230000001976 improved effect Effects 0.000 description 17
- BBMCTIGTTCKYKF-UHFFFAOYSA-N 1-heptanol Chemical compound CCCCCCCO BBMCTIGTTCKYKF-UHFFFAOYSA-N 0.000 description 16
- AMQJEAYHLZJPGS-UHFFFAOYSA-N N-Pentanol Chemical compound CCCCCO AMQJEAYHLZJPGS-UHFFFAOYSA-N 0.000 description 16
- ZSIAUFGUXNUGDI-UHFFFAOYSA-N hexan-1-ol Chemical compound CCCCCCO ZSIAUFGUXNUGDI-UHFFFAOYSA-N 0.000 description 16
- 241000196324 Embryophyta Species 0.000 description 15
- 239000002551 biofuel Substances 0.000 description 15
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 15
- 238000012217 deletion Methods 0.000 description 15
- 230000037430 deletion Effects 0.000 description 15
- KBPLFHHGFOOTCA-UHFFFAOYSA-N 1-Octanol Chemical compound CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 description 14
- 101150071111 FADD gene Proteins 0.000 description 14
- 241000235015 Yarrowia lipolytica Species 0.000 description 14
- 101150016526 fadE gene Proteins 0.000 description 14
- 150000002185 fatty acyl-CoAs Chemical class 0.000 description 14
- 238000002703 mutagenesis Methods 0.000 description 14
- 231100000350 mutagenesis Toxicity 0.000 description 14
- 230000002018 overexpression Effects 0.000 description 14
- 230000037361 pathway Effects 0.000 description 14
- HLZKNKRTKFSKGZ-UHFFFAOYSA-N tetradecan-1-ol Chemical compound CCCCCCCCCCCCCCO HLZKNKRTKFSKGZ-UHFFFAOYSA-N 0.000 description 14
- 238000000855 fermentation Methods 0.000 description 13
- 230000004151 fermentation Effects 0.000 description 13
- 239000000463 material Substances 0.000 description 13
- 239000003921 oil Substances 0.000 description 13
- 235000019198 oils Nutrition 0.000 description 13
- 238000007792 addition Methods 0.000 description 12
- 150000001299 aldehydes Chemical class 0.000 description 12
- MWKFXSUHUHTGQN-UHFFFAOYSA-N decan-1-ol Chemical compound CCCCCCCCCCO MWKFXSUHUHTGQN-UHFFFAOYSA-N 0.000 description 12
- LQZZUXJYWNFBMV-UHFFFAOYSA-N dodecan-1-ol Chemical compound CCCCCCCCCCCCO LQZZUXJYWNFBMV-UHFFFAOYSA-N 0.000 description 12
- 239000000126 substance Substances 0.000 description 12
- 241000233866 Fungi Species 0.000 description 11
- 108091028043 Nucleic acid sequence Proteins 0.000 description 11
- 241000223252 Rhodotorula Species 0.000 description 11
- 238000000338 in vitro Methods 0.000 description 11
- 238000003780 insertion Methods 0.000 description 11
- 230000037431 insertion Effects 0.000 description 11
- 239000002609 medium Substances 0.000 description 11
- 229910052717 sulfur Inorganic materials 0.000 description 11
- 239000011593 sulfur Substances 0.000 description 11
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 10
- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 10
- 230000000295 complement effect Effects 0.000 description 10
- 239000001993 wax Substances 0.000 description 10
- 101100127701 Bacillus subtilis (strain 168) lcfB gene Proteins 0.000 description 9
- 108010073254 Colicins Proteins 0.000 description 9
- 108700033865 EC 2.3.1.86 Proteins 0.000 description 9
- 241000701968 Enterobacteria phage phi80 Species 0.000 description 9
- 101100502354 Escherichia coli (strain K12) fadK gene Proteins 0.000 description 9
- 241000701988 Escherichia virus T5 Species 0.000 description 9
- 108010067157 Ferrichrome Proteins 0.000 description 9
- 241000238631 Hexapoda Species 0.000 description 9
- 108010011449 Long-chain-fatty-acid-CoA ligase Proteins 0.000 description 9
- 102100034337 Long-chain-fatty-acid-CoA ligase 6 Human genes 0.000 description 9
- 101100119785 Vibrio anguillarum (strain ATCC 68554 / 775) fatB gene Proteins 0.000 description 9
- 230000001851 biosynthetic effect Effects 0.000 description 9
- 101150061398 fabR gene Proteins 0.000 description 9
- GGUNGDGGXMHBMJ-UHFFFAOYSA-N ferrichrome Chemical compound [Fe+3].CC(=O)N([O-])CCCC1NC(=O)CNC(=O)CNC(=O)CNC(=O)C(CCCN([O-])C(C)=O)NC(=O)C(CCCN([O-])C(C)=O)NC1=O GGUNGDGGXMHBMJ-UHFFFAOYSA-N 0.000 description 9
- 230000035772 mutation Effects 0.000 description 9
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 9
- 239000002994 raw material Substances 0.000 description 9
- 101150026728 tesB gene Proteins 0.000 description 9
- 101150077217 yhfL gene Proteins 0.000 description 9
- 241000611272 Alcanivorax Species 0.000 description 8
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 8
- 150000001875 compounds Chemical class 0.000 description 8
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 8
- 238000009396 hybridization Methods 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 230000001105 regulatory effect Effects 0.000 description 8
- 102000000452 Acetyl-CoA carboxylase Human genes 0.000 description 7
- 108010016219 Acetyl-CoA carboxylase Proteins 0.000 description 7
- 108010018763 Biotin carboxylase Proteins 0.000 description 7
- 102000014914 Carrier Proteins Human genes 0.000 description 7
- 108010078791 Carrier Proteins Proteins 0.000 description 7
- 108010039731 Fatty Acid Synthases Proteins 0.000 description 7
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 7
- 150000001335 aliphatic alkanes Chemical class 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 7
- 230000001413 cellular effect Effects 0.000 description 7
- BXWNKGSJHAJOGX-UHFFFAOYSA-N hexadecan-1-ol Chemical compound CCCCCCCCCCCCCCCCO BXWNKGSJHAJOGX-UHFFFAOYSA-N 0.000 description 7
- 239000012074 organic phase Substances 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- 229920002477 rna polymer Polymers 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- 241000589291 Acinetobacter Species 0.000 description 6
- 102100022089 Acyl-[acyl-carrier-protein] hydrolase Human genes 0.000 description 6
- 239000004475 Arginine Substances 0.000 description 6
- 241000195493 Cryptophyta Species 0.000 description 6
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 6
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 6
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 6
- 239000004473 Threonine Substances 0.000 description 6
- 239000000654 additive Substances 0.000 description 6
- 150000001336 alkenes Chemical class 0.000 description 6
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 6
- 125000004122 cyclic group Chemical group 0.000 description 6
- 230000007613 environmental effect Effects 0.000 description 6
- 239000000284 extract Substances 0.000 description 6
- 210000005254 filamentous fungi cell Anatomy 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 230000000670 limiting effect Effects 0.000 description 6
- 108020004999 messenger RNA Proteins 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 230000002829 reductive effect Effects 0.000 description 6
- 229920006395 saturated elastomer Polymers 0.000 description 6
- 235000000346 sugar Nutrition 0.000 description 6
- 238000013518 transcription Methods 0.000 description 6
- 230000035897 transcription Effects 0.000 description 6
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 5
- 241000219194 Arabidopsis Species 0.000 description 5
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 5
- 241001149698 Lipomyces Species 0.000 description 5
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 5
- 239000004472 Lysine Substances 0.000 description 5
- 241000589516 Pseudomonas Species 0.000 description 5
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 5
- 241000223230 Trichosporon Species 0.000 description 5
- 230000002378 acidificating effect Effects 0.000 description 5
- 125000003118 aryl group Chemical group 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 230000004071 biological effect Effects 0.000 description 5
- 235000010633 broth Nutrition 0.000 description 5
- 229910002092 carbon dioxide Inorganic materials 0.000 description 5
- 239000005516 coenzyme A Substances 0.000 description 5
- 229940093530 coenzyme a Drugs 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 239000012530 fluid Substances 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 150000002632 lipids Chemical class 0.000 description 5
- 239000000314 lubricant Substances 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- 241000894007 species Species 0.000 description 5
- 235000015112 vegetable and seed oil Nutrition 0.000 description 5
- 210000005253 yeast cell Anatomy 0.000 description 5
- 102100027841 Acyl-CoA wax alcohol acyltransferase 2 Human genes 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 241001536303 Botryococcus braunii Species 0.000 description 4
- 240000002791 Brassica napus Species 0.000 description 4
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 4
- 241000192733 Chloroflexus Species 0.000 description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 4
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 4
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 4
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 4
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
- 241000186359 Mycobacterium Species 0.000 description 4
- 108091034117 Oligonucleotide Proteins 0.000 description 4
- 241001520808 Panicum virgatum Species 0.000 description 4
- 241000588671 Psychrobacter Species 0.000 description 4
- 241000316848 Rhodococcus <scale insect> Species 0.000 description 4
- 241000190932 Rhodopseudomonas Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 241000187747 Streptomyces Species 0.000 description 4
- 241000192560 Synechococcus sp. Species 0.000 description 4
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 4
- 241000235013 Yarrowia Species 0.000 description 4
- 240000008042 Zea mays Species 0.000 description 4
- 101150070497 accC gene Proteins 0.000 description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 4
- 238000004422 calculation algorithm Methods 0.000 description 4
- 150000007942 carboxylates Chemical group 0.000 description 4
- 239000001913 cellulose Substances 0.000 description 4
- 229920002678 cellulose Polymers 0.000 description 4
- 230000004186 co-expression Effects 0.000 description 4
- 101150048956 coaA gene Proteins 0.000 description 4
- RGJOEKWQDUBAIZ-UHFFFAOYSA-N coenzime A Natural products OC1C(OP(O)(O)=O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-UHFFFAOYSA-N 0.000 description 4
- KDTSHFARGAKYJN-UHFFFAOYSA-N dephosphocoenzyme A Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 KDTSHFARGAKYJN-UHFFFAOYSA-N 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 108010083294 ethanol acyltransferase Proteins 0.000 description 4
- 239000003502 gasoline Substances 0.000 description 4
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 4
- 229960000310 isoleucine Drugs 0.000 description 4
- 150000002576 ketones Chemical class 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- 150000004702 methyl esters Chemical class 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 238000005192 partition Methods 0.000 description 4
- 239000003348 petrochemical agent Substances 0.000 description 4
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 4
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 4
- 239000004474 valine Substances 0.000 description 4
- 239000008158 vegetable oil Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000580482 Acidobacteria Species 0.000 description 3
- 241001134629 Acidothermus Species 0.000 description 3
- 241000607534 Aeromonas Species 0.000 description 3
- 241000590031 Alteromonas Species 0.000 description 3
- 241000186063 Arthrobacter Species 0.000 description 3
- 101100012355 Bacillus anthracis fabH1 gene Proteins 0.000 description 3
- 244000063299 Bacillus subtilis Species 0.000 description 3
- 235000014469 Bacillus subtilis Nutrition 0.000 description 3
- 101001074429 Bacillus subtilis (strain 168) Polyketide biosynthesis acyltransferase homolog PksD Proteins 0.000 description 3
- 101100012357 Bacillus subtilis (strain 168) fabHA gene Proteins 0.000 description 3
- 101000936617 Bacillus velezensis (strain DSM 23117 / BGSC 10A6 / FZB42) Polyketide biosynthesis acyltransferase homolog BaeD Proteins 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical class C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 241000589173 Bradyrhizobium Species 0.000 description 3
- 241000589174 Bradyrhizobium japonicum Species 0.000 description 3
- 241000328900 Candidatus Solibacter Species 0.000 description 3
- 239000004215 Carbon black (E152) Substances 0.000 description 3
- 241001337994 Cryptococcus <scale insect> Species 0.000 description 3
- 241000192700 Cyanobacteria Species 0.000 description 3
- 241000190844 Erythrobacter Species 0.000 description 3
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 3
- 239000005977 Ethylene Substances 0.000 description 3
- 241000187809 Frankia Species 0.000 description 3
- 229930091371 Fructose Natural products 0.000 description 3
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 3
- 239000005715 Fructose Substances 0.000 description 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 3
- 101000937642 Homo sapiens Malonyl-CoA-acyl carrier protein transacylase, mitochondrial Proteins 0.000 description 3
- 241000520764 Janibacter Species 0.000 description 3
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 3
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 3
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 3
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 3
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 3
- 241000890160 Limnobacter Species 0.000 description 3
- 102100027329 Malonyl-CoA-acyl carrier protein transacylase, mitochondrial Human genes 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 241000085284 Marinobacter hydrocarbonoclasticus ATCC 49840 Species 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 241001303121 Methylibium Species 0.000 description 3
- 241001512042 Methylibium petroleiphilum Species 0.000 description 3
- 241000190905 Microscilla Species 0.000 description 3
- 241001074116 Miscanthus x giganteus Species 0.000 description 3
- 241000592260 Moritella Species 0.000 description 3
- 241000863420 Myxococcus Species 0.000 description 3
- 241000894753 Natronomonas Species 0.000 description 3
- 241000187654 Nocardia Species 0.000 description 3
- 241000187580 Nocardioides Species 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 241000607568 Photobacterium Species 0.000 description 3
- 241001423784 Plesiocystis Species 0.000 description 3
- 241000512220 Polaromonas Species 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- 241000582398 Reinekea Species 0.000 description 3
- 241001134718 Rhodoferax Species 0.000 description 3
- 241000187560 Saccharopolyspora Species 0.000 description 3
- 241001466077 Salina Species 0.000 description 3
- 241001312748 Salinibacter Species 0.000 description 3
- 241000383873 Sphingopyxis Species 0.000 description 3
- 241000863002 Stigmatella Species 0.000 description 3
- 241001496920 Tenacibaculum Species 0.000 description 3
- 241000221566 Ustilago Species 0.000 description 3
- 235000007244 Zea mays Nutrition 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 235000004279 alanine Nutrition 0.000 description 3
- 230000000692 anti-sense effect Effects 0.000 description 3
- 235000003704 aspartic acid Nutrition 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000007845 assembly PCR Methods 0.000 description 3
- 230000001651 autotrophic effect Effects 0.000 description 3
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 235000014633 carbohydrates Nutrition 0.000 description 3
- 238000012219 cassette mutagenesis Methods 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 238000002485 combustion reaction Methods 0.000 description 3
- 239000000356 contaminant Substances 0.000 description 3
- 239000002537 cosmetic Substances 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 239000003599 detergent Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 101150090981 fabG gene Proteins 0.000 description 3
- 101150035981 fabH gene Proteins 0.000 description 3
- 150000002192 fatty aldehydes Chemical class 0.000 description 3
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 230000037442 genomic alteration Effects 0.000 description 3
- 235000013922 glutamic acid Nutrition 0.000 description 3
- 239000004220 glutamic acid Substances 0.000 description 3
- 239000005431 greenhouse gas Substances 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 239000003317 industrial substance Substances 0.000 description 3
- 101150068528 mabA gene Proteins 0.000 description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 210000003463 organelle Anatomy 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 235000021317 phosphate Nutrition 0.000 description 3
- 239000002987 primer (paints) Substances 0.000 description 3
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 3
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 230000001568 sexual effect Effects 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000005809 transesterification reaction Methods 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- 108700037654 Acyl carrier protein (ACP) Proteins 0.000 description 2
- 102000048456 Acyl carrier protein (ACP) Human genes 0.000 description 2
- 241000864489 Alcanivorax borkumensis SK2 Species 0.000 description 2
- 241001655243 Allochromatium Species 0.000 description 2
- 241000190857 Allochromatium vinosum Species 0.000 description 2
- 101100388296 Arabidopsis thaliana DTX51 gene Proteins 0.000 description 2
- 240000002900 Arthrospira platensis Species 0.000 description 2
- 235000016425 Arthrospira platensis Nutrition 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 101100000756 Bacillus subtilis (strain 168) acpA gene Proteins 0.000 description 2
- KAKZBPTYRLMSJV-UHFFFAOYSA-N Butadiene Chemical compound C=CC=C KAKZBPTYRLMSJV-UHFFFAOYSA-N 0.000 description 2
- 241000588565 Caenorhabditis tropicalis Species 0.000 description 2
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 description 2
- 241000195597 Chlamydomonas reinhardtii Species 0.000 description 2
- 241000191382 Chlorobaculum tepidum Species 0.000 description 2
- 241000191366 Chlorobium Species 0.000 description 2
- 241000398616 Chloronema Species 0.000 description 2
- 241000190831 Chromatium Species 0.000 description 2
- 241000192721 Clathrochloris Species 0.000 description 2
- 241000186566 Clostridium ljungdahlii Species 0.000 description 2
- 101100490145 Clostridium perfringens (strain 13 / Type A) ackA2 gene Proteins 0.000 description 2
- RGJOEKWQDUBAIZ-IBOSZNHHSA-N CoASH Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCS)O[C@H]1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-IBOSZNHHSA-N 0.000 description 2
- 108091033380 Coding strand Proteins 0.000 description 2
- 241000223233 Cutaneotrichosporon cutaneum Species 0.000 description 2
- 241000206584 Cyanidium caldarium Species 0.000 description 2
- 241000235646 Cyberlindnera jadinii Species 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 101150042222 DGAT1 gene Proteins 0.000 description 2
- 101710088194 Dehydrogenase Proteins 0.000 description 2
- 101710088335 Diacylglycerol acyltransferase/mycolyltransferase Ag85A Proteins 0.000 description 2
- 101710088334 Diacylglycerol acyltransferase/mycolyltransferase Ag85B Proteins 0.000 description 2
- 101710088427 Diacylglycerol acyltransferase/mycolyltransferase Ag85C Proteins 0.000 description 2
- 102000015303 Fatty Acid Synthases Human genes 0.000 description 2
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 2
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 241001655241 Halochromatium Species 0.000 description 2
- 241000192729 Heliothrix Species 0.000 description 2
- 241000863029 Herpetosiphon Species 0.000 description 2
- 241000223198 Humicola Species 0.000 description 2
- 241001655238 Isochromatium Species 0.000 description 2
- 241000235058 Komagataella pastoris Species 0.000 description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 2
- 101100433987 Latilactobacillus sakei subsp. sakei (strain 23K) ackA1 gene Proteins 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- LTYOQGRJFJAKNA-KKIMTKSISA-N Malonyl CoA Natural products S(C(=O)CC(=O)O)CCNC(=O)CCNC(=O)[C@@H](O)C(CO[P@](=O)(O[P@](=O)(OC[C@H]1[C@@H](OP(=O)(O)O)[C@@H](O)[C@@H](n2c3ncnc(N)c3nc2)O1)O)O)(C)C LTYOQGRJFJAKNA-KKIMTKSISA-N 0.000 description 2
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 2
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 2
- 241001655239 Marichromatium Species 0.000 description 2
- 241000192041 Micrococcus Species 0.000 description 2
- 241000235395 Mucor Species 0.000 description 2
- 241001497385 Oscillochloris Species 0.000 description 2
- 241000228150 Penicillium chrysogenum Species 0.000 description 2
- 241001671240 Phaeospirillum Species 0.000 description 2
- 101100462488 Phlebiopsis gigantea p2ox gene Proteins 0.000 description 2
- 244000298647 Poinciana pulcherrima Species 0.000 description 2
- 241000192725 Prosthecochloris Species 0.000 description 2
- 241000589540 Pseudomonas fluorescens Species 0.000 description 2
- 108050006183 Pyruvate dehydrogenase E1 component Proteins 0.000 description 2
- 241000191025 Rhodobacter Species 0.000 description 2
- 241000191023 Rhodobacter capsulatus Species 0.000 description 2
- 241000191035 Rhodomicrobium Species 0.000 description 2
- 241000190937 Rhodopila Species 0.000 description 2
- 241000190984 Rhodospirillum rubrum Species 0.000 description 2
- 241001671222 Rhodothalassium Species 0.000 description 2
- 241000221523 Rhodotorula toruloides Species 0.000 description 2
- 241001671221 Rhodovibrio Species 0.000 description 2
- 241001478305 Rhodovulum Species 0.000 description 2
- 102000002278 Ribosomal Proteins Human genes 0.000 description 2
- 108010000605 Ribosomal Proteins Proteins 0.000 description 2
- 241000006388 Roseospira Species 0.000 description 2
- 101100215626 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) ADP1 gene Proteins 0.000 description 2
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 2
- 244000044822 Simmondsia californica Species 0.000 description 2
- 235000004433 Simmondsia californica Nutrition 0.000 description 2
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 241000122973 Stenotrophomonas maltophilia Species 0.000 description 2
- 101100398785 Streptococcus agalactiae serotype V (strain ATCC BAA-611 / 2603 V/R) ldhD gene Proteins 0.000 description 2
- RAHZWNYVWXNFOC-UHFFFAOYSA-N Sulphur dioxide Chemical compound O=S=O RAHZWNYVWXNFOC-UHFFFAOYSA-N 0.000 description 2
- 241000192707 Synechococcus Species 0.000 description 2
- 241000192581 Synechocystis sp. Species 0.000 description 2
- 108010006785 Taq Polymerase Proteins 0.000 description 2
- 241001655242 Thermochromatium Species 0.000 description 2
- 241000190988 Thermochromatium tepidum Species 0.000 description 2
- 241001504076 Thermosynechococcus elongatus BP-1 Species 0.000 description 2
- 241000191001 Thiocapsa Species 0.000 description 2
- 241000736901 Thiocystis Species 0.000 description 2
- 241000579678 Thiorhodococcus Species 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- 241000607598 Vibrio Species 0.000 description 2
- 241000607618 Vibrio harveyi Species 0.000 description 2
- 241000588902 Zymomonas mobilis Species 0.000 description 2
- 101100386830 Zymomonas mobilis subsp. mobilis (strain ATCC 31821 / ZM4 / CP4) ddh gene Proteins 0.000 description 2
- 101150008263 accD gene Proteins 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 101150006213 ackA gene Proteins 0.000 description 2
- 101150023061 acpP gene Proteins 0.000 description 2
- 101150051130 acpP1 gene Proteins 0.000 description 2
- 125000002252 acyl group Chemical group 0.000 description 2
- 102000045404 acyltransferase activity proteins Human genes 0.000 description 2
- 108700014220 acyltransferase activity proteins Proteins 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 101150014383 adhE gene Proteins 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 238000003915 air pollution Methods 0.000 description 2
- 125000001931 aliphatic group Chemical group 0.000 description 2
- 150000001345 alkine derivatives Chemical class 0.000 description 2
- 125000000217 alkyl group Chemical group 0.000 description 2
- 125000003368 amide group Chemical group 0.000 description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 2
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 230000001588 bifunctional effect Effects 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 229910052793 cadmium Inorganic materials 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002091 carbon monoxide Inorganic materials 0.000 description 2
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 150000002016 disaccharides Chemical class 0.000 description 2
- 229920001971 elastomer Polymers 0.000 description 2
- 239000003995 emulsifying agent Substances 0.000 description 2
- 125000004494 ethyl ester group Chemical group 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 101150026389 fabF gene Proteins 0.000 description 2
- 239000003925 fat Substances 0.000 description 2
- 235000019197 fats Nutrition 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 238000000769 gas chromatography-flame ionisation detection Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 108090001018 hexadecanal dehydrogenase (acylating) Proteins 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 239000000543 intermediate Substances 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000004922 lacquer Substances 0.000 description 2
- 101150026107 ldh1 gene Proteins 0.000 description 2
- 101150041530 ldha gene Proteins 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 239000010687 lubricating oil Substances 0.000 description 2
- 238000005461 lubrication Methods 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- LTYOQGRJFJAKNA-DVVLENMVSA-N malonyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)CC(O)=O)O[C@H]1N1C2=NC=NC(N)=C2N=C1 LTYOQGRJFJAKNA-DVVLENMVSA-N 0.000 description 2
- 239000011565 manganese chloride Substances 0.000 description 2
- 239000013028 medium composition Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 150000002772 monosaccharides Chemical class 0.000 description 2
- 239000003471 mutagenic agent Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000012038 nucleophile Substances 0.000 description 2
- 150000002894 organic compounds Chemical class 0.000 description 2
- 238000012261 overproduction Methods 0.000 description 2
- 239000003973 paint Substances 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 239000004014 plasticizer Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 229920000098 polyolefin Polymers 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 101150060030 poxB gene Proteins 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000001902 propagating effect Effects 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 239000011535 reaction buffer Substances 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 230000003362 replicative effect Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000002741 site-directed mutagenesis Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 229940082787 spirulina Drugs 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 239000004753 textile Substances 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 150000003626 triacylglycerols Chemical class 0.000 description 2
- 229910052725 zinc Inorganic materials 0.000 description 2
- 239000011701 zinc Substances 0.000 description 2
- 239000004711 α-olefin Substances 0.000 description 2
- GWSURTDMLUFMJH-FOCLMDBBSA-N (e)-hexadec-1-en-1-ol Chemical compound CCCCCCCCCCCCCC\C=C\O GWSURTDMLUFMJH-FOCLMDBBSA-N 0.000 description 1
- JEGNXMUWVCVSSQ-ISLYRVAYSA-N (e)-octadec-1-en-1-ol Chemical compound CCCCCCCCCCCCCCCC\C=C\O JEGNXMUWVCVSSQ-ISLYRVAYSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 description 1
- YZMJITMBAOBDKN-UHFFFAOYSA-N 1,3,3,4,4,5,6,6,6-nonachlorohex-1-ene Chemical compound ClC(C(Cl)(Cl)Cl)C(C(C=CCl)(Cl)Cl)(Cl)Cl YZMJITMBAOBDKN-UHFFFAOYSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- MXHRCPNRJAMMIM-SHYZEUOFSA-N 2'-deoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 MXHRCPNRJAMMIM-SHYZEUOFSA-N 0.000 description 1
- CKTSBUTUHBMZGZ-SHYZEUOFSA-N 2'‐deoxycytidine Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 CKTSBUTUHBMZGZ-SHYZEUOFSA-N 0.000 description 1
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 1
- LUCHPKXVUGJYGU-XLPZGREQSA-N 5-methyl-2'-deoxycytidine Chemical compound O=C1N=C(N)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 LUCHPKXVUGJYGU-XLPZGREQSA-N 0.000 description 1
- 241001532786 Acanthoceras Species 0.000 description 1
- 241000178217 Acanthococcus Species 0.000 description 1
- 241000023308 Acca Species 0.000 description 1
- 241001607836 Achnanthes Species 0.000 description 1
- NLHHRLWOUZZQLW-UHFFFAOYSA-N Acrylonitrile Chemical compound C=CC#N NLHHRLWOUZZQLW-UHFFFAOYSA-N 0.000 description 1
- 241000542937 Actinastrum Species 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 241000971563 Actinochloris Species 0.000 description 1
- 241001085823 Actinotaenium Species 0.000 description 1
- 101710146995 Acyl carrier protein Proteins 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- OSDWBNJEKMUWAV-UHFFFAOYSA-N Allyl chloride Chemical compound ClCC=C OSDWBNJEKMUWAV-UHFFFAOYSA-N 0.000 description 1
- 241000513952 Amphikrikos Species 0.000 description 1
- 241000091673 Amphiprora Species 0.000 description 1
- 241000611184 Amphora Species 0.000 description 1
- 241000192542 Anabaena Species 0.000 description 1
- 241000149144 Anabaenopsis Species 0.000 description 1
- 241000511264 Ankyra Species 0.000 description 1
- 241001607821 Anomoeoneis Species 0.000 description 1
- 241000832614 Apatococcus Species 0.000 description 1
- 241000192660 Aphanizomenon Species 0.000 description 1
- 241000192698 Aphanocapsa Species 0.000 description 1
- 241001491231 Aphanochaete Species 0.000 description 1
- 241000192705 Aphanothece Species 0.000 description 1
- 241000203069 Archaea Species 0.000 description 1
- 241000688154 Arthrodesmus Species 0.000 description 1
- 239000000592 Artificial Cell Substances 0.000 description 1
- 241000488546 Ascochloris Species 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241001513093 Aspergillus awamori Species 0.000 description 1
- 241000892910 Aspergillus foetidus Species 0.000 description 1
- 241001225321 Aspergillus fumigatus Species 0.000 description 1
- 241000351920 Aspergillus nidulans Species 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- 241001491696 Asterionella Species 0.000 description 1
- 241001450642 Asterococcus <scale insect> Species 0.000 description 1
- 241001442168 Audouinella Species 0.000 description 1
- 241000195520 Bacillaria Species 0.000 description 1
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 1
- 241000193752 Bacillus circulans Species 0.000 description 1
- 241001328122 Bacillus clausii Species 0.000 description 1
- 241000193749 Bacillus coagulans Species 0.000 description 1
- 241000193422 Bacillus lentus Species 0.000 description 1
- 241000194108 Bacillus licheniformis Species 0.000 description 1
- 241000194107 Bacillus megaterium Species 0.000 description 1
- 241001328127 Bacillus pseudofirmus Species 0.000 description 1
- 241000194103 Bacillus pumilus Species 0.000 description 1
- 101100378010 Bacillus subtilis (strain 168) accC1 gene Proteins 0.000 description 1
- 101100322122 Bacillus subtilis (strain 168) accC2 gene Proteins 0.000 description 1
- 241000193388 Bacillus thuringiensis Species 0.000 description 1
- 241000156986 Balbiania Species 0.000 description 1
- 241001442195 Bangia Species 0.000 description 1
- 241000218490 Basichlamys Species 0.000 description 1
- 241000893961 Blidingia Species 0.000 description 1
- 241000180102 Botrydium Species 0.000 description 1
- 241001536324 Botryococcus Species 0.000 description 1
- 241000192550 Botryosphaerella Species 0.000 description 1
- 241000809324 Brachiomonas Species 0.000 description 1
- 241000357879 Brachysira Species 0.000 description 1
- 241000937743 Brebissonia Species 0.000 description 1
- 241000193764 Brevibacillus brevis Species 0.000 description 1
- 241000546114 Bulbochaete Species 0.000 description 1
- 241000023782 Caloneis Species 0.000 description 1
- 241000192685 Calothrix Species 0.000 description 1
- 241001607768 Campylodiscus Species 0.000 description 1
- 241001200840 Capsosiphon Species 0.000 description 1
- 108090000489 Carboxy-Lyases Proteins 0.000 description 1
- 102000004031 Carboxy-Lyases Human genes 0.000 description 1
- 241000218459 Carteria Species 0.000 description 1
- 241001167556 Catena Species 0.000 description 1
- 241000227752 Chaetoceros Species 0.000 description 1
- 241000196298 Chaetomorpha Species 0.000 description 1
- 241000565393 Chaetopeltis Species 0.000 description 1
- 241000499536 Chaetophora <green alga> Species 0.000 description 1
- 241000382419 Chaetosphaeridium Species 0.000 description 1
- 241000736839 Chara Species 0.000 description 1
- 241000412430 Characiochloris Species 0.000 description 1
- 241001454319 Characiopsis Species 0.000 description 1
- 241000195641 Characium Species 0.000 description 1
- 241001195790 Charales Species 0.000 description 1
- 241000199906 Chilomonas Species 0.000 description 1
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 description 1
- 241000977954 Chlainomonas Species 0.000 description 1
- 241001134016 Chlamydocapsa Species 0.000 description 1
- 241000195585 Chlamydomonas Species 0.000 description 1
- 241000195649 Chlorella <Chlorellales> Species 0.000 description 1
- 241001454308 Chlorobotrys Species 0.000 description 1
- 241000086006 Chlorochytrium Species 0.000 description 1
- 241001353638 Chlorogloea Species 0.000 description 1
- 241000192703 Chlorogloeopsis Species 0.000 description 1
- 241000508318 Chlorogonium Species 0.000 description 1
- 241000908325 Chlorolobion Species 0.000 description 1
- 241000500710 Chloromonas Species 0.000 description 1
- 241000131978 Choricystis Species 0.000 description 1
- 241000391100 Chromophyton Species 0.000 description 1
- 241000199485 Chromulina Species 0.000 description 1
- 241000531074 Chroococcidiopsis Species 0.000 description 1
- 241001460448 Chroodactylon Species 0.000 description 1
- 241000195492 Chroomonas Species 0.000 description 1
- 241001414754 Chroothece Species 0.000 description 1
- 241000391085 Chrysamoeba Species 0.000 description 1
- 241000384556 Chrysocapsa Species 0.000 description 1
- 241000391098 Chrysochaete Species 0.000 description 1
- 241001306464 Chrysonebula Species 0.000 description 1
- 241000206751 Chrysophyceae Species 0.000 description 1
- 241001247786 Chrysosaccus Species 0.000 description 1
- 241000123346 Chrysosporium Species 0.000 description 1
- 241000186650 Clavibacter Species 0.000 description 1
- 241001633062 Closteriopsis Species 0.000 description 1
- 241001478806 Closterium Species 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 241000134090 Coccomyxa <Trebouxiophyceae> Species 0.000 description 1
- 241001608005 Cocconeis Species 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 241001293172 Coelastrella Species 0.000 description 1
- 241000542911 Coelastrum Species 0.000 description 1
- 241001470365 Coelosphaerium Species 0.000 description 1
- 241000680729 Coenochloris Species 0.000 description 1
- 241001633026 Coenocystis Species 0.000 description 1
- 241001472322 Colacium Species 0.000 description 1
- 241000196257 Coleochaete Species 0.000 description 1
- 241001408563 Collodictyon Species 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 241001442162 Compsopogon Species 0.000 description 1
- 241001430862 Coronastrum Species 0.000 description 1
- 241001465364 Cosmarium Species 0.000 description 1
- 241001495677 Cosmocladium Species 0.000 description 1
- 241000023818 Craticula Species 0.000 description 1
- 241000973888 Crinalium Species 0.000 description 1
- 241000163983 Crucigenia Species 0.000 description 1
- 241001167735 Crucigeniella Species 0.000 description 1
- 241000195618 Cryptomonas Species 0.000 description 1
- 241000700108 Ctenophora <comb jellyfish phylum> Species 0.000 description 1
- 241000410565 Cyanodictyon Species 0.000 description 1
- 241000206574 Cyanophora Species 0.000 description 1
- 241000159506 Cyanothece Species 0.000 description 1
- 241000391115 Cyclonexis Species 0.000 description 1
- 241000491670 Cyclostephanos Species 0.000 description 1
- 241001147476 Cyclotella Species 0.000 description 1
- 241000511240 Cylindrocapsa Species 0.000 description 1
- 241001503574 Cylindrocystis Species 0.000 description 1
- 241000565779 Cylindrospermum Species 0.000 description 1
- 241000206743 Cylindrotheca Species 0.000 description 1
- 241000904935 Cymatopleura Species 0.000 description 1
- 241001607798 Cymbella Species 0.000 description 1
- 241001368744 Cystodinium Species 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 241000721041 Dactylococcopsis Species 0.000 description 1
- 241000192091 Deinococcus radiodurans Species 0.000 description 1
- 241001529961 Denticula Species 0.000 description 1
- CKTSBUTUHBMZGZ-UHFFFAOYSA-N Deoxycytidine Natural products O=C1N=C(N)C=CN1C1OC(CO)C(O)C1 CKTSBUTUHBMZGZ-UHFFFAOYSA-N 0.000 description 1
- 241000530784 Dermocarpella Species 0.000 description 1
- 241000879539 Desmidium Species 0.000 description 1
- 241001117195 Desmococcus Species 0.000 description 1
- 241000681122 Desmonema Species 0.000 description 1
- 241001035767 Diacanthos Species 0.000 description 1
- 241001221643 Diacronema Species 0.000 description 1
- 241000904838 Diadesmis Species 0.000 description 1
- 241000199908 Diatoma Species 0.000 description 1
- 241001055981 Dichothrix Species 0.000 description 1
- 241000042508 Dicranochaete Species 0.000 description 1
- 241000790227 Dictyochloris Species 0.000 description 1
- 241000790338 Dictyococcus Species 0.000 description 1
- 241001035792 Dictyosphaerium Species 0.000 description 1
- 241000304513 Didymocystis <green alga> Species 0.000 description 1
- 241001035788 Didymogenes Species 0.000 description 1
- 241001636067 Didymosphenia Species 0.000 description 1
- 108010073112 Dihydrolipoyllysine-residue acetyltransferase Proteins 0.000 description 1
- 102000009093 Dihydrolipoyllysine-residue acetyltransferase Human genes 0.000 description 1
- 241000894358 Dilabifilum Species 0.000 description 1
- 241000391095 Dinobryon Species 0.000 description 1
- 241000199914 Dinophyceae Species 0.000 description 1
- 241001529750 Diploneis Species 0.000 description 1
- 241000569885 Docidium Species 0.000 description 1
- 241001464842 Draparnaldia Species 0.000 description 1
- 241000196133 Dryopteris Species 0.000 description 1
- 241000195634 Dunaliella Species 0.000 description 1
- 241000195633 Dunaliella salina Species 0.000 description 1
- 108700034934 EC 2.3.1.180 Proteins 0.000 description 1
- 241001271037 Ecballocystis Species 0.000 description 1
- 241001264087 Elakatothrix Species 0.000 description 1
- 241000863079 Ellerbeckia Species 0.000 description 1
- 241001607801 Encyonema Species 0.000 description 1
- 241001058905 Entocladia Species 0.000 description 1
- BRLQWZUYTZBJKN-UHFFFAOYSA-N Epichlorohydrin Chemical compound ClCC1CO1 BRLQWZUYTZBJKN-UHFFFAOYSA-N 0.000 description 1
- 241000908495 Epipyxis Species 0.000 description 1
- 241001306932 Epithemia Species 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 241000660147 Escherichia coli str. K-12 substr. MG1655 Species 0.000 description 1
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 1
- 241000761680 Eucapsis Species 0.000 description 1
- 241000306697 Eucocconeis Species 0.000 description 1
- 241001478804 Eudorina Species 0.000 description 1
- 241000195620 Euglena Species 0.000 description 1
- 241000195623 Euglenida Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241001485688 Eunotia Species 0.000 description 1
- 241000224472 Eustigmatophyceae Species 0.000 description 1
- 241000385215 Eutreptia Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 241001280345 Ferroplasma Species 0.000 description 1
- 241000393058 Ferroplasma acidarmanus Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 241001466505 Fragilaria Species 0.000 description 1
- 241001607504 Fragilariforma Species 0.000 description 1
- 241000923853 Franceia Species 0.000 description 1
- 241000023844 Frustulia Species 0.000 description 1
- 241000223218 Fusarium Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 241000354290 Geminella Species 0.000 description 1
- 241001442224 Genicularia Species 0.000 description 1
- 241000193385 Geobacillus stearothermophilus Species 0.000 description 1
- 241001517283 Glaucocystis Species 0.000 description 1
- 241001517276 Glaucocystophyceae Species 0.000 description 1
- 241000321067 Glenodiniopsis Species 0.000 description 1
- 241000924360 Glenodinium Species 0.000 description 1
- 241001556868 Gloeomonas Species 0.000 description 1
- 241001134702 Gloeothece Species 0.000 description 1
- 241000320398 Gloeotrichia Species 0.000 description 1
- 241001194596 Golenkinia Species 0.000 description 1
- 241000974867 Gomontia Species 0.000 description 1
- 241000393470 Gomphonema Species 0.000 description 1
- 241000761673 Gomphosphaeria Species 0.000 description 1
- 241000193665 Gonatozygon Species 0.000 description 1
- 241001503492 Gongrosira Species 0.000 description 1
- 241001501499 Goniochloris Species 0.000 description 1
- 241001464827 Gonium Species 0.000 description 1
- 241000160543 Gonyostomum Species 0.000 description 1
- 241000029168 Granulocystopsis Species 0.000 description 1
- 241000879545 Groenbladia Species 0.000 description 1
- 241000499473 Hafniomonas Species 0.000 description 1
- 241000350665 Hapalosiphon Species 0.000 description 1
- 241001086060 Haplotaenium Species 0.000 description 1
- 241000206759 Haptophyceae Species 0.000 description 1
- 241000947581 Haslea Species 0.000 description 1
- 241001026014 Heribaudiella Species 0.000 description 1
- 241000053425 Heteromastix Species 0.000 description 1
- 241000169078 Heterothrix Species 0.000 description 1
- 241000199483 Hibberdia Species 0.000 description 1
- 241001428192 Hildenbrandia Species 0.000 description 1
- 241001302615 Holopedium Species 0.000 description 1
- 241000565399 Hormotila Species 0.000 description 1
- 241001480714 Humicola insolens Species 0.000 description 1
- 241000091553 Hyalodiscus <Bacillariophyta> Species 0.000 description 1
- 241000879511 Hyalotheca Species 0.000 description 1
- 241001443163 Hydrococcus <sea snail> Species 0.000 description 1
- 241000568637 Hydrocoleum Species 0.000 description 1
- 241000545338 Hydrocoryne <hydrozoan> Species 0.000 description 1
- 241000196173 Hydrodictyon Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 241000359213 Hydrosera Species 0.000 description 1
- 241000199479 Hydrurus Species 0.000 description 1
- 241000659220 Hyella Species 0.000 description 1
- 241001037825 Hymenomonas Species 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- VQTUBCCKSQIDNK-UHFFFAOYSA-N Isobutene Chemical group CC(C)=C VQTUBCCKSQIDNK-UHFFFAOYSA-N 0.000 description 1
- 241000221089 Jatropha Species 0.000 description 1
- 241000051648 Johannesbaptistia Species 0.000 description 1
- 241000549394 Karayevia Species 0.000 description 1
- 241000415370 Katablepharis Species 0.000 description 1
- 241000130959 Katodinium Species 0.000 description 1
- 241000512263 Keratococcus Species 0.000 description 1
- 241000542984 Kirchneriella Species 0.000 description 1
- 241000196289 Klebsormidium Species 0.000 description 1
- 241000235649 Kluyveromyces Species 0.000 description 1
- 241000431886 Kolbesia Species 0.000 description 1
- 241000997134 Koliella Species 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 101100393312 Lactobacillus delbrueckii subsp. bulgaricus (strain ATCC 11842 / DSM 20081 / BCRC 10696 / JCM 1002 / NBRC 13953 / NCIMB 11778 / NCTC 12712 / WDCM 00102 / Lb 14) gpsA1 gene Proteins 0.000 description 1
- 240000008415 Lactuca sativa Species 0.000 description 1
- 241001167739 Lagerheimia Species 0.000 description 1
- 241000391110 Lagynion Species 0.000 description 1
- 241001503408 Lamprothamnium Species 0.000 description 1
- 241000948916 Lemanea Species 0.000 description 1
- 241000936931 Lepocinclis Species 0.000 description 1
- 241001503405 Leptosira Species 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 241000606397 Lobomonas Species 0.000 description 1
- 101710129019 Long-chain acyl-[acyl-carrier-protein] reductase Proteins 0.000 description 1
- 241000023860 Luticola Species 0.000 description 1
- 241000199476 Mallomonas Species 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 241000736252 Mantoniella Species 0.000 description 1
- 241001402923 Marssoniella Species 0.000 description 1
- 108091027974 Mature messenger RNA Proteins 0.000 description 1
- 241001491711 Melosira Species 0.000 description 1
- 241000520876 Merismopedia Species 0.000 description 1
- 241000134068 Mesostigma Species 0.000 description 1
- 241001442139 Mesotaenium Species 0.000 description 1
- 241000586743 Micractinium Species 0.000 description 1
- 241000879504 Micrasterias Species 0.000 description 1
- 241001139348 Microchaete Species 0.000 description 1
- 241000179980 Microcoleus Species 0.000 description 1
- 241000192701 Microcystis Species 0.000 description 1
- 241000417364 Microglena Species 0.000 description 1
- 241001536503 Micromonas Species 0.000 description 1
- 241000243190 Microsporidia Species 0.000 description 1
- 241001478808 Microthamnion Species 0.000 description 1
- 241000878007 Miscanthus Species 0.000 description 1
- 241000544586 Mischococcus Species 0.000 description 1
- 241000180113 Monodus Species 0.000 description 1
- 241001250715 Monomastix Species 0.000 description 1
- 241001478792 Monoraphidium Species 0.000 description 1
- 241000893951 Monostroma Species 0.000 description 1
- 241000721605 Mougeotia Species 0.000 description 1
- 241000305078 Mychonastes Species 0.000 description 1
- 241001306407 Naegeliella Species 0.000 description 1
- 241000196305 Nannochloris Species 0.000 description 1
- 241000204974 Natronobacterium Species 0.000 description 1
- 241000502321 Navicula Species 0.000 description 1
- 241000761110 Neglectella Species 0.000 description 1
- 241000038602 Neidium Species 0.000 description 1
- 241000244206 Nematoda Species 0.000 description 1
- 241000300408 Nephrocytium Species 0.000 description 1
- 241001442227 Nephroselmis Species 0.000 description 1
- 241000159606 Netrium Species 0.000 description 1
- 241000221960 Neurospora Species 0.000 description 1
- 241000196239 Nitella Species 0.000 description 1
- 241000124865 Nitellopsis Species 0.000 description 1
- 241000180701 Nitzschia <flatworm> Species 0.000 description 1
- 241000187681 Nocardia sp. Species 0.000 description 1
- 241000059630 Nodularia <Cyanobacteria> Species 0.000 description 1
- 241000192656 Nostoc Species 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical class CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 241000199478 Ochromonas Species 0.000 description 1
- 241000546131 Oedogonium Species 0.000 description 1
- 241000879540 Onychonema Species 0.000 description 1
- 241000714271 Opephora Species 0.000 description 1
- 241001245640 Ophiocytium Species 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 241000016406 Pachycladella Species 0.000 description 1
- 235000019482 Palm oil Nutrition 0.000 description 1
- 241000209117 Panicum Species 0.000 description 1
- 235000006443 Panicum miliaceum subsp. miliaceum Nutrition 0.000 description 1
- 235000009037 Panicum miliaceum subsp. ruderale Nutrition 0.000 description 1
- 241001111421 Pannus Species 0.000 description 1
- 102100024122 Pantothenate kinase 1 Human genes 0.000 description 1
- 241001606327 Paralia Species 0.000 description 1
- 241000081677 Pascherina Species 0.000 description 1
- 241000196152 Pediastrum Species 0.000 description 1
- 241001141586 Pedinella Species 0.000 description 1
- 241000196311 Pedinomonas Species 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 241001464933 Penium Species 0.000 description 1
- 108091093037 Peptide nucleic acid Proteins 0.000 description 1
- 241001365547 Peridiniopsis Species 0.000 description 1
- 241000199911 Peridinium Species 0.000 description 1
- 241000289317 Peronia Species 0.000 description 1
- 241000827182 Petroneis Species 0.000 description 1
- 241000508171 Phacotus Species 0.000 description 1
- 241000384537 Phacus Species 0.000 description 1
- 241000199919 Phaeophyceae Species 0.000 description 1
- 241001299725 Phaeothamnion Species 0.000 description 1
- 241000745991 Phalaris Species 0.000 description 1
- 241000222385 Phanerochaete Species 0.000 description 1
- 241000192608 Phormidium Species 0.000 description 1
- 241000719322 Phycopeltis Species 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 241000827163 Placoneis Species 0.000 description 1
- 241000354317 Planctonema Species 0.000 description 1
- 241001607864 Planothidium Species 0.000 description 1
- 241000192665 Plectonema Species 0.000 description 1
- 241000215422 Pleodorina Species 0.000 description 1
- 241001478797 Pleurastrum Species 0.000 description 1
- 241000179979 Pleurocapsa Species 0.000 description 1
- 241001025962 Pleurocladia Species 0.000 description 1
- 241001499701 Pleurosigma Species 0.000 description 1
- 241001535149 Pleurosira Species 0.000 description 1
- 241000879515 Pleurotaenium Species 0.000 description 1
- 241000222350 Pleurotus Species 0.000 description 1
- 241001655089 Polyblepharides Species 0.000 description 1
- 241000351424 Polyedriopsis Species 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 241000775680 Polytaenia Species 0.000 description 1
- 241000123781 Polytoma Species 0.000 description 1
- 241000195630 Polytomella Species 0.000 description 1
- 241000219000 Populus Species 0.000 description 1
- 241000206618 Porphyridium Species 0.000 description 1
- 241000196315 Prasinocladus Species 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- GOOHAUXETOMSMM-UHFFFAOYSA-N Propylene oxide Chemical compound CC1CO1 GOOHAUXETOMSMM-UHFFFAOYSA-N 0.000 description 1
- 241000091602 Psammodictyon Species 0.000 description 1
- 241000582923 Psammothidium Species 0.000 description 1
- 241000192511 Pseudanabaena Species 0.000 description 1
- 241001140502 Pseudococcomyxa Species 0.000 description 1
- 241001369990 Pseudostaurastrum Species 0.000 description 1
- 241000531151 Pyrolobus Species 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 241000404547 Quadricoccus Species 0.000 description 1
- 241001633564 Quadrigula Species 0.000 description 1
- 241000529919 Ralstonia sp. Species 0.000 description 1
- 241001011851 Raphidiopsis Species 0.000 description 1
- 241000952492 Raphidocelis Species 0.000 description 1
- 241001464838 Raphidonema Species 0.000 description 1
- 241001518925 Raphidophyceae Species 0.000 description 1
- 241000342028 Rhizoclonium Species 0.000 description 1
- 241000235402 Rhizomucor Species 0.000 description 1
- 241000235403 Rhizomucor miehei Species 0.000 description 1
- 241001501882 Rhodomonas Species 0.000 description 1
- 241001024538 Rhopalodia Species 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 241001575211 Rivularia <snail> Species 0.000 description 1
- 241000951282 Rosenvingiella Species 0.000 description 1
- 241001034931 Rossithidium Species 0.000 description 1
- 241000159610 Roya <green alga> Species 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 241000209051 Saccharum Species 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 241000426680 Salinispora arenicola Species 0.000 description 1
- 241000831652 Salinivibrio sharmensis Species 0.000 description 1
- 241000124033 Salix Species 0.000 description 1
- 241000195663 Scenedesmus Species 0.000 description 1
- 241000196303 Scherffelia Species 0.000 description 1
- 241000680878 Schizochlamydella Species 0.000 description 1
- 241001221087 Schizochlamys Species 0.000 description 1
- 241001491238 Schizomeris Species 0.000 description 1
- 241000235346 Schizosaccharomyces Species 0.000 description 1
- 241000970913 Schizothrix Species 0.000 description 1
- 241000905909 Schroederia Species 0.000 description 1
- 241001293157 Scotiellopsis Species 0.000 description 1
- 241000353062 Scourfieldia Species 0.000 description 1
- 241000192120 Scytonema Species 0.000 description 1
- 241001607804 Sellaphora Species 0.000 description 1
- 241000221095 Simmondsia Species 0.000 description 1
- 241000736692 Sirogonium Species 0.000 description 1
- 241000206733 Skeletonema Species 0.000 description 1
- 239000004902 Softening Agent Substances 0.000 description 1
- 241000420652 Sorastrum Species 0.000 description 1
- 241000798634 Sphaerodinium Species 0.000 description 1
- 241000511249 Sphaeroplea Species 0.000 description 1
- 241001495679 Sphaerozosma Species 0.000 description 1
- 241000196294 Spirogyra Species 0.000 description 1
- 241000159621 Spirotaenia Species 0.000 description 1
- 241000879542 Spondylosium Species 0.000 description 1
- 241000028617 Spumella Species 0.000 description 1
- 241001442222 Staurastrum Species 0.000 description 1
- 241000091598 Stauroneis Species 0.000 description 1
- 241001535139 Staurosira Species 0.000 description 1
- 241001531293 Stenopterobia Species 0.000 description 1
- 241001147471 Stephanodiscus Species 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 241001633038 Stichogloea Species 0.000 description 1
- 241000243446 Stigonema Species 0.000 description 1
- 241000187398 Streptomyces lividans Species 0.000 description 1
- 241001468239 Streptomyces murinus Species 0.000 description 1
- 241001669899 Stylodinium Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 241001607780 Surirella Species 0.000 description 1
- 241001512067 Symploca Species 0.000 description 1
- 241000192584 Synechocystis Species 0.000 description 1
- 241001426193 Synedra Species 0.000 description 1
- 241000199481 Synura Species 0.000 description 1
- 241001379115 Tabellaria Species 0.000 description 1
- 241000618308 Tabularia Species 0.000 description 1
- 241000142921 Tardigrada Species 0.000 description 1
- 241001370938 Teilingia Species 0.000 description 1
- 241001086137 Tetmemorus Species 0.000 description 1
- 241000513961 Tetrachlorella Species 0.000 description 1
- 241000863054 Tetracyclus Species 0.000 description 1
- 241000593219 Tetraedriella Species 0.000 description 1
- 241000891463 Tetraedron Species 0.000 description 1
- 241000196321 Tetraselmis Species 0.000 description 1
- 241001344092 Tetraspora <Myxozoa> Species 0.000 description 1
- 241000163966 Tetrastrum Species 0.000 description 1
- 241001491691 Thalassiosira Species 0.000 description 1
- 241000949391 Thorea Species 0.000 description 1
- 241001524160 Tolypella Species 0.000 description 1
- 241000157473 Tolypothrix Species 0.000 description 1
- 241000383524 Trachelomonas Species 0.000 description 1
- 241000960433 Trachydiscus Species 0.000 description 1
- 241000222354 Trametes Species 0.000 description 1
- 108700029229 Transcriptional Regulatory Elements Proteins 0.000 description 1
- 101710195626 Transcriptional activator protein Proteins 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 241000736687 Trebouxia Species 0.000 description 1
- 241000511255 Treubaria Species 0.000 description 1
- 241000199474 Tribonema Species 0.000 description 1
- 241000223259 Trichoderma Species 0.000 description 1
- 241000378866 Trichoderma koningii Species 0.000 description 1
- 241000223262 Trichoderma longibrachiatum Species 0.000 description 1
- 241000499912 Trichoderma reesei Species 0.000 description 1
- 241000223261 Trichoderma viride Species 0.000 description 1
- 241000192118 Trichodesmium Species 0.000 description 1
- 241000905502 Trochiscia Species 0.000 description 1
- 241001179808 Tryblionella Species 0.000 description 1
- DRQXUCVJDCRJDB-UHFFFAOYSA-N Turanose Natural products OC1C(CO)OC(O)(CO)C1OC1C(O)C(O)C(O)C(CO)O1 DRQXUCVJDCRJDB-UHFFFAOYSA-N 0.000 description 1
- 241000159614 Ulothrix Species 0.000 description 1
- 241000196252 Ulva Species 0.000 description 1
- 241000391106 Uroglena Species 0.000 description 1
- 241001491247 Uronema <chlorophycean alga> Species 0.000 description 1
- 241001531494 Urosolenia Species 0.000 description 1
- 241000981775 Urospora <green alga> Species 0.000 description 1
- 241000180047 Vacuolaria Species 0.000 description 1
- 241000200212 Vaucheria Species 0.000 description 1
- XTXRWKRVRITETP-UHFFFAOYSA-N Vinyl acetate Chemical compound CC(=O)OC=C XTXRWKRVRITETP-UHFFFAOYSA-N 0.000 description 1
- 241001310885 Vitreochlamys Species 0.000 description 1
- 241000195615 Volvox Species 0.000 description 1
- 241000218467 Volvulina Species 0.000 description 1
- 108700020489 Wax synthase Proteins 0.000 description 1
- 241001386273 Westella Species 0.000 description 1
- 241000200152 Woloszynskia Species 0.000 description 1
- 241000879509 Xanthidium Species 0.000 description 1
- 241000206764 Xanthophyceae Species 0.000 description 1
- 241001532060 Yucca elata Species 0.000 description 1
- 241000209149 Zea Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 241001495681 Zygnemopsis Species 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 108030000857 [Acyl-carrier-protein] S-malonyltransferases Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 101150013885 accB gene Proteins 0.000 description 1
- 101150015189 aceE gene Proteins 0.000 description 1
- 101150077561 aceF gene Proteins 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 108010069175 acyl-CoA transferase Proteins 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 150000007824 aliphatic compounds Chemical class 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- 229910052785 arsenic Inorganic materials 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 229940054340 bacillus coagulans Drugs 0.000 description 1
- 229940097012 bacillus thuringiensis Drugs 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 238000004523 catalytic cracking Methods 0.000 description 1
- 238000001833 catalytic reforming Methods 0.000 description 1
- MLYYVTUWGNIJIB-BXKDBHETSA-N cefazolin Chemical compound S1C(C)=NN=C1SCC1=C(C(O)=O)N2C(=O)[C@@H](NC(=O)CN3N=NN=C3)[C@H]2SC1 MLYYVTUWGNIJIB-BXKDBHETSA-N 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000012707 chemical precursor Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 150000001805 chlorine compounds Chemical class 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000012411 cloning technique Methods 0.000 description 1
- 101150109763 coaW gene Proteins 0.000 description 1
- 101150051152 coaX gene Proteins 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000003967 crop rotation Methods 0.000 description 1
- 239000010779 crude oil Substances 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- MXHRCPNRJAMMIM-UHFFFAOYSA-N desoxyuridine Natural products C1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 MXHRCPNRJAMMIM-UHFFFAOYSA-N 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 108010003123 dihydrolipoamide acyltransferase Proteins 0.000 description 1
- NAGJZTKCGNOGPW-UHFFFAOYSA-N dithiophosphoric acid Chemical group OP(O)(S)=S NAGJZTKCGNOGPW-UHFFFAOYSA-N 0.000 description 1
- 238000005553 drilling Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 239000000806 elastomer Substances 0.000 description 1
- 239000003974 emollient agent Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000003822 epoxy resin Substances 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 108010052305 exodeoxyribonuclease III Proteins 0.000 description 1
- 150000002190 fatty acyls Chemical group 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 230000008713 feedback mechanism Effects 0.000 description 1
- 239000002921 fermentation waste Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000010408 film Substances 0.000 description 1
- 239000003063 flame retardant Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- FTSSQIKWUOOEGC-RULYVFMPSA-N fructooligosaccharide Chemical compound OC[C@H]1O[C@@](CO)(OC[C@@]2(OC[C@@]3(OC[C@@]4(OC[C@@]5(OC[C@@]6(OC[C@@]7(OC[C@@]8(OC[C@@]9(OC[C@@]%10(OC[C@@]%11(O[C@H]%12O[C@H](CO)[C@@H](O)[C@H](O)[C@H]%12O)O[C@H](CO)[C@@H](O)[C@@H]%11O)O[C@H](CO)[C@@H](O)[C@@H]%10O)O[C@H](CO)[C@@H](O)[C@@H]9O)O[C@H](CO)[C@@H](O)[C@@H]8O)O[C@H](CO)[C@@H](O)[C@@H]7O)O[C@H](CO)[C@@H](O)[C@@H]6O)O[C@H](CO)[C@@H](O)[C@@H]5O)O[C@H](CO)[C@@H](O)[C@@H]4O)O[C@H](CO)[C@@H](O)[C@@H]3O)O[C@H](CO)[C@@H](O)[C@@H]2O)[C@@H](O)[C@@H]1O FTSSQIKWUOOEGC-RULYVFMPSA-N 0.000 description 1
- 229940107187 fructooligosaccharide Drugs 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- ZZUFCTLCJUWOSV-UHFFFAOYSA-N furosemide Chemical compound C1=C(Cl)C(S(=O)(=O)N)=CC(C(O)=O)=C1NCC1=CC=CO1 ZZUFCTLCJUWOSV-UHFFFAOYSA-N 0.000 description 1
- 235000021255 galacto-oligosaccharides Nutrition 0.000 description 1
- 150000003271 galactooligosaccharides Chemical class 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 101150095733 gpsA gene Proteins 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 101150116274 gspA gene Proteins 0.000 description 1
- 125000001475 halogen functional group Chemical group 0.000 description 1
- LNEPOXFFQSENCJ-UHFFFAOYSA-N haloperidol Chemical compound C1CC(O)(C=2C=CC(Cl)=CC=2)CCN1CCCC(=O)C1=CC=C(F)C=C1 LNEPOXFFQSENCJ-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 238000005462 in vivo assay Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002561 ketenes Chemical class 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 229910052745 lead Inorganic materials 0.000 description 1
- 235000021190 leftovers Nutrition 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 239000003915 liquefied petroleum gas Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000010871 livestock manure Substances 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 150000004668 long chain fatty acids Chemical class 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 108020004084 membrane receptors Proteins 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- TVMXDCGIABBOFY-UHFFFAOYSA-N octane Chemical compound CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- XMLQWXUVTXCDDL-UHFFFAOYSA-N oleyl alcohol Natural products CCCCCCC=CCCCCCCCCCCO XMLQWXUVTXCDDL-UHFFFAOYSA-N 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 150000002913 oxalic acids Chemical class 0.000 description 1
- 239000002540 palm oil Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 150000004965 peroxy acids Chemical class 0.000 description 1
- 239000003209 petroleum derivative Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- SXADIBFZNXBEGI-UHFFFAOYSA-N phosphoramidous acid Chemical compound NP(O)O SXADIBFZNXBEGI-UHFFFAOYSA-N 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920000058 polyacrylate Polymers 0.000 description 1
- 229920000647 polyepoxide Polymers 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000004800 polyvinyl chloride Substances 0.000 description 1
- 229920000915 polyvinyl chloride Polymers 0.000 description 1
- 241000196307 prasinophytes Species 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 239000013615 primer Substances 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 101150108780 pta gene Proteins 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 239000012925 reference material Substances 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 235000012045 salad Nutrition 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- 229930195734 saturated hydrocarbon Natural products 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000000344 soap Substances 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 229940045870 sodium palmitate Drugs 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- GGXKEBACDBNFAF-UHFFFAOYSA-M sodium;hexadecanoate Chemical compound [Na+].CCCCCCCCCCCCCCCC([O-])=O GGXKEBACDBNFAF-UHFFFAOYSA-M 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000004071 soot Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000004230 steam cracking Methods 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 101150033131 sthA gene Proteins 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 229920003051 synthetic elastomer Polymers 0.000 description 1
- 125000000383 tetramethylene group Chemical group [H]C([H])([*:1])C([H])([H])C([H])([H])C([H])([H])[*:2] 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- DCXXMTOCNZCJGO-UHFFFAOYSA-N tristearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCCCC DCXXMTOCNZCJGO-UHFFFAOYSA-N 0.000 description 1
- RULSWEULPANCDV-PIXUTMIVSA-N turanose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](C(=O)CO)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O RULSWEULPANCDV-PIXUTMIVSA-N 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 229930195735 unsaturated hydrocarbon Natural products 0.000 description 1
- 239000002966 varnish Substances 0.000 description 1
- 210000003501 vero cell Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
- 239000008096 xylene Chemical class 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
-
- C—CHEMISTRY; METALLURGY
- C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
- C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G, C10K; LIQUEFIED PETROLEUM GAS; ADDING MATERIALS TO FUELS OR FIRES TO REDUCE SMOKE OR UNDESIRABLE DEPOSITS OR TO FACILITATE SOOT REMOVAL; FIRELIGHTERS
- C10L1/00—Liquid carbonaceous fuels
- C10L1/02—Liquid carbonaceous fuels essentially based on components consisting of carbon, hydrogen, and oxygen only
- C10L1/026—Liquid carbonaceous fuels essentially based on components consisting of carbon, hydrogen, and oxygen only for compression ignition
-
- C—CHEMISTRY; METALLURGY
- C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
- C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G, C10K; LIQUEFIED PETROLEUM GAS; ADDING MATERIALS TO FUELS OR FIRES TO REDUCE SMOKE OR UNDESIRABLE DEPOSITS OR TO FACILITATE SOOT REMOVAL; FIRELIGHTERS
- C10L1/00—Liquid carbonaceous fuels
- C10L1/04—Liquid carbonaceous fuels essentially based on blends of hydrocarbons
-
- C—CHEMISTRY; METALLURGY
- C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
- C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G, C10K; LIQUEFIED PETROLEUM GAS; ADDING MATERIALS TO FUELS OR FIRES TO REDUCE SMOKE OR UNDESIRABLE DEPOSITS OR TO FACILITATE SOOT REMOVAL; FIRELIGHTERS
- C10L1/00—Liquid carbonaceous fuels
- C10L1/10—Liquid carbonaceous fuels containing additives
- C10L1/14—Organic compounds
- C10L1/18—Organic compounds containing oxygen
- C10L1/19—Esters ester radical containing compounds; ester ethers; carbonic acid esters
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
- C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
- C11C3/04—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fats or fatty oils
- C11C3/10—Ester interchange
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/36—Adaptation or attenuation of cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6458—Glycerides by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
- C12Y103/99—Oxidoreductases acting on the CH-CH group of donors (1.3) with other acceptors (1.3.99)
- C12Y103/99003—Acyl-CoA dehydrogenase (1.3.99.3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
- C12Y203/01075—Long-chain-alcohol O-fatty-acyltransferase (2.3.1.75)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
-
- Y02E50/13—
Definitions
- Petroleum is a limited, natural resource found in the Earth in liquid, gaseous, or solid forms. Petroleum is primarily composed of hydrocarbons, which are comprised mainly of carbon and hydrogen. It also contains significant amounts of other elements, such as, nitrogen, oxygen, or sulfur, in different forms.
- Petroleum is a valuable resource, but petroleum products are developed at considerable costs, both financial and environmental.
- sources of petroleum must be discovered. Petroleum exploration is an expensive and risky venture. The cost of exploring deep water wells can exceed $100 million.
- petroleum exploration carries a high environmental cost. For example, offshore exploration disturbs the surrounding marine environments.
- Petroleum extraction also carries an environmental cost. For example, petroleum extraction can result in large see pages of petroleum rising to the surface. Offshore drilling involves dredging the seabed which disrupts or destroys the surrounding marine environment.
- crude petroleum extracted from the Earth has few commercial uses. It is a mixture of hydrocarbons (e.g., paraffins (or alkanes), olefins (or alkenes), alkynes, napthenes (or cylcoalkanes), aliphatic compounds, aromatic compounds, etc.) of varying length and complexity.
- hydrocarbons e.g., paraffins (or alkanes), olefins (or alkenes), alkynes, napthenes (or cylcoalkanes), aliphatic compounds, aromatic compounds, etc.
- crude petroleum contains other organic compounds (e.g., organic compounds containing nitrogen, oxygen, sulfur, etc.) and impurities (e.g., sulfur, salt, acid, metals, etc.).
- crude petroleum must be refined and purified before it can be used commercially. Due to its high energy density and its easy transportability, most petroleum is refined into fuels, such as transportation fuels (e.g., gasoline, diesel, aviation fuel, etc.), heating oil, liquefied petroleum gas, etc.
- transportation fuels e.g., gasoline, diesel, aviation fuel, etc.
- heating oil e.g., heating oil, liquefied petroleum gas, etc.
- Crude petroleum is also a primary source of raw materials for producing petrochemicals.
- the two main classes of raw materials derived from petroleum are short chain olefins (e.g., ethylene and propylene) and aromatics (e.g., benzene and xylene isomers). These raw materials are derived from the longer chain hydrocarbons in crude petroleum by cracking the long chain hydrocarbons at considerable expense using a variety of methods, such as catalytic cracking, steam cracking, or catalytic reforming. These raw materials are used to make petrochemicals, which cannot be directly refined from crude petroleum, such as monomers, solvents, detergents, or adhesives.
- ethylene is used to produce petrochemicals such as, polyethylene, ethanol, ethylene oxide, ethylene glycol, polyester, glycol ether, ethoxylate, vinyl acetate, 1,2-dichloroethane, trichloroethylene, tetrachloroethylene, vinyl chloride, and polyvinyl chloride.
- propylene is used to produce petrochemicals such as, polyethylene, ethanol, ethylene oxide, ethylene glycol, polyester, glycol ether, ethoxylate, vinyl acetate, 1,2-dichloroethane, trichloroethylene, tetrachloroethylene, vinyl chloride, and polyvinyl chloride.
- propylene is Another example of a raw material derived from crude petroleum.
- Propylene is used to produce isopropyl alcohol, acrylonitrile, polypropylene, propylene oxide, propylene glycol, glycol ethers, butylene, isobutylene, 1,3-butadiene, synthetic elastomers, polyolefins, alpha-olefins, fatty alcohols, acrylic acid, acrylic polymers, allyl chloride, epichlorohydrin, and epoxy resins.
- Petrochemicals can be used to make specialty chemicals, such as plastics, resins, fibers, elastomers, pharmaceuticals, lubricants, or gels.
- specialty chemicals which can be produced from petrochemical raw materials are: fatty acids, hydrocarbons (e.g., long chain hydrocarbons, branched chain hydrocarbons, saturated hydrocarbons, unsaturated hydrocarbons, etc.), fatty alcohols, esters, fatty aldehydes, ketones, lubricants, etc.
- Fatty acids are used commercially as surfactants. Surfactants can be found in detergents and soaps. Fatty acids can also be used as additives in fuels, lubricating oils, paints, lacquers, candles, salad oils, shortenings, cosmetics, and emulsifiers. In addition, fatty acids are used as accelerator activators in rubber products. Fatty acids can also be used as a feedstock to produce methyl esters, amides, amines, acid chlorides, anhydrides, ketene dimers, and peroxy acids and esters.
- Esters have many commercial uses.
- biodiesel an alternative fuel, is comprised of esters (e.g., fatty acid methyl ester, fatty acid ethyl esters, etc.).
- esters are volatile with a pleasant odor which makes them useful as fragrances or flavoring agents.
- esters are used as solvents for lacquers, paints, and varnishes.
- some naturally occurring substances such as waxes, fats, and oils are comprised of esters.
- Esters are also used as softening agents in resins and plastics, plasticizers, flame retardants, and additives in gasoline and oil.
- esters can be used in the manufacture of polymers, films, textiles, dyes, and pharmaceuticals.
- Lubricants derived petroleum are typically composed of olefins, particularly polyolefins and alpha-olefins. Lubricants can either be refined from crude petroleum or manufactured using the raw materials refined from crude petroleum.
- Biofuel is a biodegradable, clean-burning combustible fuel produced from biomass, and can be made of alkanes and esters.
- An exemplary biofuel is biodiesel. Biodiesel can be used in most internal combustion diesel engines in either a pure form, which is referred to as “neat” biodiesel, or as a mixture in any concentration with regular petroleum diesel.
- Biodiesel offers advantages compared to petroleum-based diesel, including reduced emissions (e.g., carbon monoxide, sulphur, aromatic hydrocarbons, soot particles) during combustion. Biodiesel also maintains a balanced carbon dioxide cycle because it is based on renewable biological materials. Biodiesel is typically biodegradable, and imparts enhanced safety due to its high flash point and low flammability. Furthermore, biodiesel provides good lubrication properties, thereby reducing wear and tear on engines.
- reduced emissions e.g., carbon monoxide, sulphur, aromatic hydrocarbons, soot particles
- biodiesel production involves transesterification of triacylglycerides from vegetable oil feedstocks, such as rapeseed in Europe, soybean in North America, and palm oil in South East Asia.
- Industrial-scale biodiesel production is thus geographically and seasonally restricted to areas where vegetable oil feedstocks are produced.
- the transesterification process leads to a mixture of fatty esters which can be used as biodiesel.
- glycerin is an undesirable byproduct of the transesterification process.
- the fatty esters must be further purified from the heterogeneous product. This increases costs and the amount of energy required for fatty ester production and, ultimately, biodiesel production as well.
- fatty acids and derivatives thereof including, for example, fatty esters from genetically engineered microorganisms.
- fatty esters include fatty acid esters, such as those derived from short-chain alcohols, including fatty acid ethyl esters (“FAEE”) and fatty acid methyl esters (“FAME”), and those derived from long-chain fatty alcohols.
- FEE fatty acid ethyl esters
- FAME fatty acid methyl esters
- the fatty acids and/or fatty acid derivatives that are produced can be used, individually or in suitable combinations, as a biofuel (e.g., a biodiesel), an industrial chemical, or a component of, or feedstock for, a biofuel or an industrial chemical.
- the invention pertains to a method of producing one or more free fatty acids and/or one or more fatty acid derivatives such as fatty acid esters, including, for example, FAEE, FAME and/or other fatty acid ester derivatives of longer-chain alcohols.
- the method comprises providing a genetically engineered production host suitable for making fatty acids and fatty acid derivatives.
- the invention features a method of making a fatty acid or a fatty acid derivative such as a fatty ester.
- the method includes expressing in a host cell a gene encoding an ester synthase.
- the gene encoding an ester synthase is selected from the enzymes classified as EC 2.3.1.75, and any other polypeptides capable of catalyzing the conversion of an acyl thioester to fatty esters, including, without limitation, wax-ester synthases, acyl-CoA:alcohol transacylases, alcohol 0-fatty acid-acyl-transferase, acyltransferases, and fatty acyl-coA:fatty alcohol acyltransferases, or a suitable variant thereof.
- the ester synthase gene is one that encodes wax/dgat, a bifunctional ester synthase/acyl-CoA: diacylglycerol acyltransferase from Simmondsia chinensis, Acinetobacter sp. ADP1, Alcanivorax borkumensis, Pseudomonas aeruginosa, Fundibacter jadensis, Arabidopsis thaliana , or Alkaligenes eutrophus .
- the gene encoding an ester synthase is selected from the group consisting of: AtfA1 (an ester synthase derived from Alcanivorax borkumensis SK2, GenBank Accession No. YP_694462), AtfA2 (another ester synthase derived from Alcanivorax borkumensis SK2, GenBank Accession No. YP_693524), ES9 (an ester synthase from Marinobacter hydrocarbonoclasticus DSM 8798, GenBank Accession No. AB021021), ES8 (another ester synthase derived from Marinobacter hydrocarbonoclasticus DSM 8798, GenBank Accession No. AB021020), and variants thereof.
- the gene encoding the ester synthase or a suitable variant is overexpressed.
- the invention features a method of making a fatty acid derivative, for example, a fatty ester, the method comprising expressing in a host cell a gene encoding an ester synthase polypeptide comprising the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, or a variant thereof.
- the polypeptide has ester synthase and/or acyltransferase activity.
- the polypeptide has the capacity to catalyse the conversion of a thioester to a fatty acid and/or a fatty acid derivative such as a fatty ester.
- the polypeptide has the capacity to catalyze the conversion of a fatty acyl-CoA and/or a fatty acyl-ACP to a fatty acid and/or a fatty acid derivative such as a fatty ester, using an alcohol as substrate.
- the polypeptide has the capacity to catalyze the conversion of a free fatty acid to a fatty ester, using an alcohol as substrate.
- an endogenous thioesterase of the host cell if present, is unmodified.
- the host cell expresses an attenuated level of a thioesterase activity or the thioesterase is functionally deleted.
- the host cell has no detectable thioesterase activity.
- “detectable” means capable of having an existence or presence ascertained. For example, production of a product from a reactant (e.g., production of a certain type of fatty acid esters) is desirably detectable using the methods provided herein.
- the host cell expresses an attenuated level of a fatty acid degradation enzyme, such as, for example, an acyl-CoA synthase, or the fatty acid degradation enzyme is functionally deleted. In some embodiments, the host cell has no detectable fatty acid degradation enzyme activity. In particular embodiments, the host cell expresses an attenuated level of a thioesterease, a fatty acid degradation enzyme, or both. In other embodiments, the thioesterase, the fatty acid degradation enzyme, or both, are functionally deleted. In some embodiments, the host cell has no detectable thioesterase activity, acyl-CoA synthase activity, or neither.
- the host cell can convert an acyl-ACP or acyl-CoA into fatty acids and/or derivatives thereof such as esters, in the absence of a thioesterase, a fatty acid derivative enzyme, or both.
- the host cell can convert a free fatty acid to a fatty ester in the absence of a thioesterase, a fatty acid derivative enzyme, or both.
- the method further includes isolating the fatty acids or derivatives thereof from the host cell.
- the fatty acid derivative is a fatty ester. In certain embodiments, the fatty acid or fatty acid derivative is derived from a suitable alcohol substrate such as a short- or long-chain alcohol. In some embodiments, the fatty acid or fatty acid derivative is present in the extracellular environment. In certain embodiments, the fatty acid or fatty acid derivative is isolated from the extracellular environment of the host cell. In some embodiments, the fatty acid or fatty acid derivative is spontaneously secreted, partially or completely, from the host cell. In alternative embodiments, the fatty acid or derivative is transported into the extracellular environment, optionally with the aid of one or more transport proteins. In other embodiments, the fatty acid or fatty acid derivative is passively transported into the extracellular environment.
- the invention features an in vitro method of producing a fatty acid and/or a fatty acid derivative extracellulary comprising providing a substrate and a purified ester synthase comprising the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, or a variant thereof.
- the method comprising culturing a host cell under conditions that allow expression or overexpression of an ester synthase polypeptide or a variant thereof, and isolating the ester synthase from the cell.
- the method further comprising contacting a suitable substrate such with the cell-free extract under conditions that permit production of a fatty acid and/or a fatty acid derivative.
- the ester synthase polypeptide comprises the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, with one or more amino acid substitutions, additions, insertions, or deletions, and the polypeptide has ester synthase and/or acyltransferase activity.
- the ester synthase polypeptide has increased ester synthase and/or transferase activity.
- the ester synthase polypeptide is capable, or has an improved capacity, of catalyzing the conversion of thioesters, for example, fatty acyl-CoAs or fatty acyl-ACPs, to fatty acids and/or fatty acid derivatives.
- the ester synthase polypeptide is capable, or has an improved capacity, of catalyzing the conversion of thioester substrates to fatty acids and/or derivatives thereof, such as fatty esters, in the absence of a thioesterase activity, a fatty acid degradation enzyme activity, or both.
- the polypeptide converts fatty acyl-ACP and/or fatty acyl-CoA into fatty esters in vivo, in the absence of a thioesterase or an acyl-CoA synthase activity.
- the polypeptide is capable of catalyzing the conversion of a free fatty acid to a fatty ester, in the absence of a thioesterase activity, a fatty acid degradation enzyme activity, or both.
- the polypeptide can convert a free fatty acid into a fatty ester in vivo or in vitro, in the absence of a thioesterase activity, an acyl-CoA synthase activity, or both.
- the ester synthase polypeptide is a variant comprising the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, with one or more non-conserved amino acid substitutions, wherein the ester synthase polypeptide has ester synthase and/or acyltransferase activity.
- the ester synthase polypeptide has improved ester synthase and/or acyltransferase activity.
- a glycine residue at position 395 of SEQ ID NO:18 can be substituted with a basic amino acid residue, such that the resulting ester synthase variant retains or has improved ester synthase and/or acyltransferase activity.
- the glycine residue at position 395 of SEQ ID NO:18 is substituted with an arginine or a lysine residue, wherein the resulting ester synthase variant retains or has improved capacity to catalyze the conversion of a thioester into a fatty acid and/or a fatty acid derivative such as a fatty ester.
- the ester synthase variant comprises one or more of the following conserved amino acid substitutions: replacement of an aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic amino acid; replacement of a serine with a threonine; replacement of a threonine with a serine; replacement of an acidic residue, such as aspartic acid and glutamic acid, with another acidic residue; replacement of residue bearing an amide group; exchange of a basic residue, such as lysine and arginine, with another basic residue; and replacement of an aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue.
- an aliphatic amino acid such as alanine, valine, leucine, and isoleucine
- replacement of a serine with a threonine replacement of a threonine with a serine
- replacement of an acidic residue such as aspartic acid and glutamic
- the ester synthase variant has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acid substitutions, additions, insertions, or deletions.
- the polypeptide variant has ester synthase and/or acyltransferase activity.
- the ester synthase polypeptide is capable of catalyzing the conversion of thioesters to fatty acids and/or fatty acid derivatives, using alcohols as substrates.
- the polypeptide is capable of catalyzing the conversion of a fatty acyl-CoA and/or a fatty acyl-ACP to a fatty acid and/or a fatty acid ester, using a suitable alcohol substrate, such as, for instance, a methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, decanol, dodecanol, tetradecanol, or hexadecanol.
- a suitable alcohol substrate such as, for instance, a methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, decanol, dodecanol, tetradecanol, or hexadecanol.
- the ester synthase polypeptide is capable of catalyzing the conversion of a fatty acyl-ACP and/or a fatty acyl-CoA to a fatty acid and/or a fatty acid ester, in the absence of a thioesterase, a fatty acid degradation enzyme, or both.
- the polypeptide is capable of catalyzing the conversion of a free fatty acid into a fatty ester in the absence of a thioesterase, a fatty acid degradation enzyme, or both.
- the ester synthase polypeptide is about 200 amino acids to about 2,000 amino acids in length, for example, from about 250 to about 1,500 amino acid residues in length, from about 300 to about 1,200 amino acid residues in length, from about 350 to about 1,000 amino acid residues in length, from about 400 to about 800 amino acid residues in length, or from about 450 to about 600 amino acid residues in length. In certain embodiments, the ester synthase polypeptide is about 300 amino acid residues in length or longer, for example, about 400 amino acid residues in length or longer, or about 450 amino acid residues in length or longer.
- the ester synthase polypeptide is about 1,000 amino acid residues in length or shorter, for example, about 800 amino acid residues in length or shorter, about 700 amino acid residues in length or shorter, or about 600 amino acid residues in length or shorter.
- An exemplary ester synthase of the invention is about 500 amino acid residues in length.
- the method further includes modifying the expression of a gene encoding an ester synthase in the host cell.
- modifying the expression of a gene encoding an ester synthase includes expressing a heterologous gene encoding an ester synthase in the host cell and/or otherwise increasing the expression or activity of an endogenous ester synthase in the host cell.
- an endogenous thioesterase enzyme of the host cell if present, is unmodified.
- the method includes modifying the expression of a gene encoding a thioesterase in the host cell.
- modifying the expression of a gene encoding a thioesterase includes expressing a heterologous gene encoding a thioesterase in the host cell and/or increasing the expression and/or activity of an endogenous thioesterase in the host cell.
- modifying the expression of a gene encoding a thioesterase includes attenuating the expression of an endogenous gene encoding a thioesterase in the host cell, and/or decreasing the expression and/or activity of an endogenous thioesterase in the host cell.
- modifying the expression of a thioesterase in the host cell comprises functionally deleting an endogenous gene encoding a thioesterase.
- the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
- Thioesterase activity can be determined or measured using various known in vitro or in vivo assays, including without limitation an acyl-CoA hydrolysis assay, which measures the rate of cleavage of an acyl-CoA substrate using a DTNB (5,5′-Dithio-bis(2-nitro-benzoic acid)) reaction and monitors the changes of absorbance at 412 nm and a molar extinction coefficient of 13,600 M ⁇ 1 cm ⁇ 1 . It is known in the art that various ester synthases can have overlapping thioesterase activities. As used herein, however, the term “ester synthase” does not comprise enzymes that also have thioesterase activity. The ones that have both ester synthase activity and thioesterase activity are categorized as thioesterases herein.
- the invention features a method of making fatty acids and/or fatty acid derivatives, for example, a fatty ester, comprising expressing in a host cell a gene encoding an ester synthase, in the absence of a thioesterase or a thioesterase activity in the host cell.
- the host cell is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to its level in a wild type host cell.
- fatty acid degradation enzymes include, without limitation, an acyl-CoA synthase of EC 2.3.1.86 or EC 6.2.1.3.
- host cells from which the fatty acid degradation enzyme can be found include Saccharomyces cerevisiae, Candida lipolytica, Escherichia coli, Arthrobacter, Rhodotorula glutinins, Acinetobacter, Candida lipolytica, Botryococcus braunii, Vibrio fumissii, Vibrio harveyi, Micrococcus leuteus, Stenotrophomonas maltophila or Bacillus subtilis .
- the host cell is genetically engineered to express an attenuated level of an acyl-CoA synthase relative to a wild type host cell.
- the host cell expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, Pf1-4354, EAV15023, fadD1, fadD2, RPC_4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_01644857.
- the fatty acid degradation enzyme such as the acyl-CoA synthase gene, is functionally deleted such that the host cell has no detectable acyl-CoA synthase activity.
- an endogenous thioesterase enzyme of the host cell if present, is unmodified.
- the host cell is genetically engineered to express an attenuated level of a thioesterase, an attenuated level of a fatty acid degradation enzyme, or both.
- a thioesterase, a fatty acid degradation enzyme, or both are functionally deleted.
- the host cell has no detectable thioesterase activity, acyl-CoA synthase activity or neither.
- the host cell can produce fatty acids and/or derivatives from thioester and alcohol substrates. In alternative embodiments, the host cell can produce fatty esters from free fatty acids and alcohol substrates.
- the ester synthase polypeptide is derived from, for example, a bacterium, plant, insect, yeast, fungus, or mammal. In certain embodiments, the ester synthase polypeptide is from a mammalian cell, a plant cell, an insect cell, a fungus cell, a filamentous fungi cell, a bacterial cell, a cyanobacterial cell, or a cell of any other organism described herein.
- the naturally-occurring ester synthase is from Acidobacteria, Acidothermus, Acinetobacter, Aeromonas, Alcanivorax, Alcaligenes, Alteromonas, Anaeromyxobacter, Arabidopsis, Bradyrhizobium, Erythrobacter, Frankia, Fundibacter, Hahella chejuensis, Janibacter, Limnobacter, Marinobacter, Methylibium, Microscilla, Moritella, Mus musculus, Mycobacterium, Myxococcus, Natronomonas, Nocardia, Nocardioides, Photobacterium, Proteobacterium, Plesiocystis, Polaromonas, Pseudomonas, Psychrobacter, Reinekea, Rhodoferax, Rhodococcus, Roseiflexus, Saccharopolyspora, Salinibacter, Simmodsia Solibacter, Sphingopy
- the naturally-occurring ester synthase of the invention is derived from any one of Acidobacteria bacterium, Acidothermus cellulolyticus, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter sp., Aeromonas hydrophila, Aeromonas salmonicida, Alcaligenes europhus, Alcanivorax borkumensis, Alcanivorax jadensis, Alteromonas macleodii, Anaeromyxobacter dehalogenans, Anaeromyxobacter sp., Arabidopsis thaliana, Bradyrhizobium japonicum, Cryptococcus curvatus, Erythrobacter litoralis, Erythrobacter sp., Frankia sp., Fundibacter jadensis, gamma proteobacterium, Hahella chejuensis, Homo sapiens, Janibacter sp., Limno
- the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis.
- a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, a gene encoding a transcriptional regulator of fatty acid biosynthesis are functionally deleted.
- the host cell expressing a decreased level of an outer membrane protein receptor is resistant to phage infection.
- the gene encoding an acyl-CoA dehydrogenase is fadE.
- the gene encoding an outer membrane protein receptor is one that encodes a receptor for ferrichrome, colicin M, phage T1, phage T5, or phage phi80.
- Host cells comprising such an outer membrane protein receptor include, for example, Saccharomyces cerevisiae, Candida lipolytica, Escherichia coli, Arthrobacter, Rhodotorula glutinins, Acinetobacter, Candida lipolytica, Botryococcus braunii, Vibrio fumissii, Vibrio harveyi, Micrococcus leuteus, Stenotrophomonas maltophila , or Bacillus subtilis .
- the gene encoding an outer membrane protein receptor is fhuA (also known as tonA).
- the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
- the method further includes culturing the host cell in the presence of at least one biological substrate for the ester synthase polypeptide.
- the method includes culturing the host cell under conditions that allow the expression or overexpression of the ester synthase enzyme or a suitable variant thereof.
- the method includes culturing the host cell under conditions that permit production of fatty acids or derivatives thereof via an enzymatic reaction catalyzed by the ester synthase or a variant thereof, without the participation of a thioesterase, a fatty acid degradation enzyme, or both.
- the biological substrate for the ester synthase polypeptide is a thioester.
- the biological substrate is an alcohol, such as, for example, short-chain or long-chain fatty alcohols.
- the biological substrate is a free fatty acid.
- the invention features a method of producing a fatty acid and/or fatty acid derivative, including, for example, a fatty acid ester.
- the method includes expressing in a host cell a gene encoding an ester synthase or a variant thereof comprising an amino acid sequence having at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:18, 24, 25, or 26.
- the ester synthase has the amino acid sequence of SEQ ID NO:18, 24, 25 or 26.
- the method further includes isolating the fatty acid or fatty acid derivative from the genetically engineered host cell.
- the fatty acid or fatty acid derivative is present in the extracellular environment.
- the fatty acid or fatty acid derivative is isolated from the extracellular environment of the host cell.
- the fatty acid or fatty acid derivative is spontaneously secreted into the extracellular environment, partially or completely.
- the fatty acid or fatty acid derivative is transported into the extracellular environment, with or without the aid of one or more suitable transport proteins.
- the fatty acid or fatty acid derivative is passively transported into the extracellular environment.
- an endogenous thioesterase enzyme of the host cell if present, is unmodified.
- the method includes modifying the expression of a gene encoding a thioesterase in the host cell.
- modifying the expression of a gene encoding a thioesterase includes expressing a heterologous gene encoding a thioesterase in the host cell and/or increasing the expression or activity of an endogenous thioesterase in the host cell.
- modifying the expression of a gene encoding a thioesterase includes attenuating the expression of an endogenous gene encoding a thioesterase in the host cell.
- modifying the expression of a gene encoding a thioesterase includes functionally deleting an endogenous gene encoding a thioesterase.
- the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
- the host cell is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to its level in a wild type host cell.
- exemplary fatty acid degradation enzymes include, without limitation, acyl-CoA synthase enzymes of EC 2.3.1.86 or EC 6.2.1.3.
- the host cell expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, Pf1-4354, EAV15023, fadD 1, fadD2, RPC_4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_01644857.
- the above-described fatty acid degradation enzyme such as, for example, an acyl-CoA synthase, is functionally deleted such that the host cell does not have detectable acyl-CoA synthase activity.
- the host cell is genetically engineered to express an attenuated level of a thioesterease, an attenuated level of a fatty acid degradation enzyme, or both.
- a thioesterase, an acyl-CoA synthase, or both are functionally deleted.
- the host cell has no detectable thioesterase activity or fatty acid degradation enzyme activity.
- the host cell is capable of producing fatty acids and/or derivatives thereof.
- the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis.
- a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis are functionally deleted.
- the gene encoding an acyl-CoA dehydrogenase is fadE.
- the gene encoding an outer membrane protein receptor is one that encodes a receptor for ferrichrome, colicin M, phage T1, phage T5, or phage phi80.
- the gene encoding an outer membrane receptor is fhuA (also known as tonA).
- the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
- the ester synthase polypeptide is derived from a bacterium, plant, insect, yeast, fungus, or mammal.
- the polypeptide is from a mammalian cell, plant cell, insect cell, yeast cell, fungus cell, filamentous fungi cell, bacterial cell, or a cell of any other organism described herein.
- the ester synthase is from Acidobacteria, Acidothermus, Acinetobacter, Aeromonas, Alcanivorax, Alcaligenes, Alteromonas, Anaeromyxobacter, Arabidopsis, Bradyrhizobium, Erythrobacter, Frankia, Fundibacter, Hahella chejuensis, Janibacter, Limnobacter, Marinobacter, Methylibium, Microscilla, Moritella, Mus musculus, Mycobacterium, Myxococcus, Natronomonas, Nocardia, Nocardioides, Photobacterium, Proteobacterium, Plesiocystis, Polaromonas, Pseudomonas, Psychrobacter, Reinekea, Rhodoferax, Rhodococcus, Roseiflexus, Saccharopolyspora, Salinibacter, Simmodsia Solibacter, Sphingopyxis, Stigma
- the ester synthase is derived from any one of Acidobacteria bacterium, Acidothermus cellulolyticus, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter sp., Aeromonas hydrophila, Aeromonas salmonicida, Alcaligenes europhus, Alcanivorax borkumensis, Alcanivorax jadensis, Alteromonas macleodii, Anaeromyxobacter dehalogenans, Anaeromyxobacter sp., Arabidopsis thaliana, Bradyrhizobium japonicum, Cryptococcus curvatus, Erythrobacter litoralis, Erythrobacter sp., Frankia sp., Fundibacter jadensis, gamma proteobacterium, Hahella chejuensis, Homo sapiens, Janibacter sp., Limnobacter sp., marine
- the method further includes culturing the host cell in the presence of at least one biological substrate for the ester synthase polypeptide. In certain embodiments, the method includes culturing the host cell under conditions sufficient to allow expression or overexpression of an ester synthase in the host cell. In certain embodiments, an endogenous thioesterase of the host cell, if present, is unmodified. In other embodiments, the host cell expresses an attenuated level of a thioesterase, a fatty acid degradation enzyme, or both. In some embodiments, the thioesterase, the fatty acid degradation enzyme, or both, are functionally deleted.
- the host cell has no detectable thioesterase activity, acyl-CoA synthase activity, or neither activity.
- the method includes culturing the host cells under conditions sufficient to permit production of fatty acids and/or fatty acid derivatives.
- the host cell is cultured in the presence of thioester substrates.
- the host cell is cultured in the presence of an alcohol.
- the host cell is cultured in the presence of a free fatty acid.
- the invention features a method of producing a fatty acid and/or a fatty acid derivative such as, for example, a fatty ester.
- the method comprises expressing in a host cell a polynucleotide that that hybridizes to a complement of a nucleotide sequence SEQ ID NO:27, 28, 29, or 30, or to a fragment thereof, wherein the polynucleotide encodes a polypeptide having ester synthase and/or acyltransferase activity.
- the polypeptide is capable catalyzing the conversion of thioesters to fatty acids and/or fatty acid derivatives such as fatty esters, using one or more alcohols as substrates.
- suitable thioester substrates include acyl-CoAs, such as, for example, fatty acyl-CoAs, and acyl-ACPs, such as, for example, fatty acyl-ACPs.
- Suitable alcohol substrates include, for example, short or long chain alcohols, such as methanol, ethanol, propanol, butanol, pentanol, hexanol, octanol, heptanol, decanol, dodecanol, tetradecanol, or hexadecanol.
- the polypeotide is capable of catalyzing the conversion of a free fatty acid to a fatty esters.
- the polynucleotide hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions, to a complement of the nucleotide sequence of SEQ ID NO:27, 28, 29, or 30, or to a fragment thereof.
- the polynucleotide comprises a sequence that has been codon-optimized for expression or overexpression in a selected host cell.
- the method further comprises isolating the fatty acid or fatty acid derivative from the host cell.
- the fatty acid or fatty acid derivative is present in the extracellular environment.
- the fatty acid or fatty acid derivative is isolated from the extracellular environment of the host cell.
- the fatty acid or fatty acid derivative is spontaneously secreted, partially or completely, from the host cell.
- the fatty acid or fatty acid derivative is transported into the extracellular environment with or without the aid of one or more transport proteins.
- the fatty acid or fatty acid derivative is passively transported into the extracellular environment.
- an endogenous thioesterease of the host cell if present, is unmodified.
- the method includes modifying the expression of a gene encoding a thioesterase in the host cell.
- modifying the expression of a gene encoding a thioesterase includes expressing a heterologous gene encoding a thioesterase in the host cell and/or increasing the expression or activity of an endogenous thioesterase in the host cell.
- modifying the expression of a gene encoding a thioesterase includes attenuating the expression of an endogenous gene encoding a thioesterase in the host cell and/or functionally deleting an endogenous thioesterase.
- the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
- the host cell is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to its level in a wild type host cell.
- exemplary fatty acid degradation enzymes include, without limitation, acyl-CoA synthase enzymes of EC 2.3.1.86 or EC 6.2.1.3.
- the host cell expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, pf1-4354, eav15023, fadD1, fadD2, rpc_4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_01644857.
- the aforementioned fatty acid degradation enzyme such as the acyl-CoA synthase gene, is functionally deleted such that the host cell does not have a detectable acyl-CoA synthase activity.
- the host cell is genetically engineered to express an attenuated level of a thioesterase, an attenuated level of a fatty acid degradation enzyme, or both.
- a thioesterase, a fatty acid degradation enzyme, or both are functionally deleted.
- the host cell has no detectable thioesterase activity or fatty acid degradation enzyme activity.
- the host cell is capable of producing fatty acids and/or derivatives thereof.
- the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis.
- a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis are functionally deleted.
- the gene encoding an acyl-CoA dehydrogenase is fadE.
- the gene encoding an outer membrane protein receptor is one that encodes a receptor for ferrichrome, colicin M, phage T1, phage T5, or phage phi80.
- the gene encoding an outer membrane protein receptor is fhuA (also known as tonA).
- the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
- the ester synthase is derived from a bacterium, plant, insect, yeast, fungus, or mammal.
- the ester synthase polynucleotide is from a mammalian cell, plant cell, insect cell, yeast cell, fungus cell, filamentous fungi cell, bacterial cell, or a cell of any other organism described herein.
- the polynucleotide is from Acidobacteria, Acidothermus, Acinetobacter, Aeromonas, Alcanivorax, Alcaligenes, Alteromonas, Anaeromyxobacter, Arabidopsis, Bradyrhizobium, Erythrobacter, Frankia, Fundibacter, Hahella chejuensis, Janibacter, Limnobacter, Marinobacter, Methylibium, Microscilla, Moritella, Mus musculus, Mycobacterium, Myxococcus, Natronomonas, Nocardia, Nocardioides, Photobacterium, Proteobacterium, Plesiocystis, Polaromonas, Pseudomonas, Psychrobacter, Reinekea, Rhodoferax, Rhodococcus, Roseiflexus, Saccharopolyspora, Salinibacter, Simmodsia Solibacter, Sphingopyxis, St
- the ester synthase polynucleotide is derived from any one of Acidobacteria bacterium, Acidothermus cellulolyticus, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter sp., Aeromonas hydrophila, Aeromonas salmonicida, Alcaligenes europhus, Alcanivorax borkumensis, Alcanivorax jadensis, Alteromonas macleodii, Anaeromyxobacter dehalogenans, Anaeromyxobacter sp., Arabidopsis thaliana, Bradyrhizobium japonicum, Cryptococcus curvatus, Erythrobacter litoralis, Erythrobacter sp., Frankia sp., Fundibacter jadensis, gamma proteobacterium, Hahella chejuensis, Homo sapiens, Janibacter sp., Limno
- the method further comprises culturing the host cell in the presence of at least one biological substrate for the ester synthase polypeptide. In certain embodiments, the method comprises culturing the host cell under conditions that are sufficient to allow expression or overexpression of the ester synthase in the host cell. In some embodiments, the method further comprises culturing the host cell under conditions that are sufficient to permit production of fatty acids and/or fatty acid derivatives. In some embodiments, the method comprises culturing the host cell in the presence of a thioester substrate such as, for example, a fatty acyl-CoA or a fatty acyl-ACP. In certain other embodiments, the method comprises culturing the host cell in the presence of an alcohol substrate. In yet further embodiments, the method comprising culturing the host cell in the presence of a free fatty acid.
- a thioester substrate such as, for example, a fatty acyl-CoA or a fatty acyl-ACP.
- the invention features a method of producing a fatty acid or a fatty acid derivative such as, for example, a fatty ester.
- the method comprises expressing in a host cell a heterologous gene encoding an ester synthase having the amino acid sequence of SEQ ID NO:18, or a variant thereof.
- the method comprises manipulating an endogenous ester synthase gene of a host cell encoding an ester synthase of SEQ ID NO:18, or a variant thereof, using genomic alteration.
- the method further includes isolating the fatty acid and/or fatty acid derivative from the host cell.
- the invention features an in vitro method of producing fatty acids and/or fatty acid derivatives extracellularly from a substrate using a purified ester synthase having the amino acid sequence of SEQ ID NO:18, or a variant thereof.
- the host cell is cultured under conditions that allow expression or overexpression of the ester synthase or a variant thereof.
- the host cell is then be harvested and lysed.
- the resulting mixture is purified.
- Suitable substrates such as those described herein can be added to the cell-free extracts under conditions that permit production of a fatty acid and/or a fatty acid derivative.
- the ester synthase variant comprises the amino acid sequence of SEQ ID NO:18, with one or more amino acid substitutions, additions, insertions, or deletions, wherein the polypeptide has ester synthase activity and/or acyltransferase activity.
- the ester synthase variant has increased ester synthase and/or acyltransferase activity.
- the ester synthase polypeptide is capable, or has improved capacity, of catalyzing the conversion of thioesters to fatty esters, using alcohols as substrates.
- Thioester substrates include, for example, fatty thioesters such as fatty acyl-CoAs or fatty acyl-ACPs.
- the ester synthase variant is capable of, or has improved capacity of, catalyzing the conversion of free fatty acids to fatty esters, using alcohols as substrates.
- Alcohol substrates include, for example, long- or short-chain alcohols such as methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, decanol, dodecanol, tetradecanol, or hexadecanol.
- the ester synthase variant comprises the amino acid sequence of SEQ ID NO:18, with one or more non-conserved amino acid substitutions, wherein the variant retains ester synthase and/or acyltransferase activity.
- the ester synthase variant has improved ester synthase and/or acyltransferase activity.
- the glycine residue at position 395 of SEQ ID NO:18 is substituted with a basic amino acid residue, and the resultant ester synthase variant retains or has improved ester synthase and/or acyltransferase activity.
- the glycine residue at position 395 of SEQ ID NO:18 is substituted with an arginine or a lysine residue, and the resultant ester synthase variant has improved ester synthase and/or acyltransferase activity.
- the ester synthase variant comprises one or more conserved amino acid substitutions. In some embodiments, the ester synthase variant has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acid substitutions, additions, insertions, or deletions. In some embodiments, the ester synthase variant has ester synthase and/or acyltransferase activity.
- the polypeptide is capable of catalyzing the conversion of thioesters to fatty acids and/or fatty acid derivatives such as fatty esters, using alcohols as substrates.
- the polypeptide is capable of producing a fatty ester from a suitable free fatty acid and an alcohol, such as, for example, long- or short-chain alcohols, such as methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, decanol, dodecanol, tetradecanol, or hexadecanol.
- an alcohol such as, for example, long- or short-chain alcohols, such as methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, decanol, dodecanol, tetradecanol, or hexadecanol.
- an endogenous thioesterase of the host cell if present, is unmodified.
- the method includes modifying the expression of a gene encoding a thioesterase in the host cell.
- modifying the expression of a thioesterase gene includes attenuating the expression of an endogenous gene encoding a thioesterase in the host cell and/or functionally deleting such a gene.
- the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
- the host cell is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to its level in a wild type host cell.
- exemplary fatty acid degradation enzymes include, without limitation, acyl-CoA synthase enzymes of EC 2.3.1.86 or EC 6.2.1.3.
- the host cell expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, pf1-4354, EAV15023, fadD 1, fadD2, rpc_4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_01644857.
- the aforementioned fatty acid degradation enzyme such as the acyl-CoA synthase gene, is functionally deleted such that the host cell does not express an acyl-CoA synthase or does not have a detectable acyl-CoA synthase activity.
- the host cell is genetically engineered to express an attenuated level of a thioesterase, an attenuated level of a fatty acid degradation enzyme, or both.
- a thioesterase, a fatty acid degradation enzyme, or both are functionally deleted.
- the host cell has no detectable level of a thioesterase activity or an acyl-CoA synthase activity.
- the host cell is capable of producing fatty acids and/or derivatives thereof.
- the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis.
- a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis are functionally deleted.
- the gene encoding an acyl-CoA dehydrogenase is fadE.
- the gene encoding an outer membrane protein receptor is one that encodes a receptor for ferrichrome, colicin M, phage T1, phage T5, or phage phi80.
- the gene encoding an outer membrane protein receptor is fhuA (also known as tonA).
- the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
- the method further includes culturing the host cell in the presence of at least one biological substrate for the ester synthase polypeptide. In certain embodiments, the method includes culturing the host cell under conditions that allow expression or overexpression of the ester synthase in the host cell. In further embodiments, the method includes culturing the host cell under conditions that permit production of desirable fatty acids and/or fatty acid derivatives. In some embodiments, the method includes culturing the host cell in the presence of a thioester substrate. In certain other embodiments, the method includes culturing the host cell in the presence of an alcohol. In further embodiments, the method includes culturing the host cell in the presence of a free fatty acid.
- the invention features a method of producing a fatty acid and/or a fatty acid derivative such as, for example, a fatty ester.
- the method includes expressing in a host cell a gene encoding an ester synthase polypeptide comprising an amino acid sequence having at least about 35% sequence identity, for example, at least about 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:18.
- the host cell is engineered to have an attenuated level of a thioesterase, a fatty acid degradation enzyme, or both.
- the thioesterase, the fatty acid degradation enzyme, or both are functionally deleted.
- the host cell has no detectable thioesterase activity or acyl-CoA synthase activity.
- the method further includes isolating an accordingly-produced fatty acid or fatty acid derivative from the host cell.
- the invention further features an in vitro method of producing a fatty acid and/or fatty acid derivative extracellularly, using a purified ester synthase having at least about 35% sequence identity to the amino acid sequence of SEQ ID NO:18, from a suitable substrate.
- the host cell is cultured under conditions that allow expression or overexpression of the ester synthase enzyme or a variant thereof. The host cell is then harvested and lysed. Optionally, the resulting mixture is purified. Suitable substrates such as those described herein can be added to the cell-free extracts under conditions that permit production of a fatty acid and/or a fatty acid derivative.
- the amino acid sequence of an ester synthase or a suitable variant of the invention has at least about 35%, for example, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to SEQ ID NO:18.
- the amino acid sequence of the ester synthase is SEQ ID NO:18.
- An exemplary ester synthase polypeptide include an ester synthase from Limnobacter sp. MED105, with a GenBank Accession No. of ZP_01915978 (SEQ ID NO:41), which has about 51% sequence identity to the amino acid sequence of the ester synthase from Marinobacter hydrocarbonclasticus DSM 8798, SEQ ID NO:18.
- an endogenous thioesterase of the host cell if present, is unmodified.
- the method includes modifying the expression of a gene encoding a thioesterase in the host cell.
- modifying the expression of a thioesterase gene includes attenuating the expression of an endogenous gene encoding a thioesterase in the host cell and/or functionally deleting that gene.
- the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
- the host cell is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to its level in a wild type host cell.
- exemplary fatty acid degradation enzymes include, without limitation, acyl-CoA synthase enzymes of EC 2.3.1.86 or EC 6.2.1.3.
- the host cell expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, pf1-4354, EAV15023, fadD1, fadD2, rpc_4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_01644857.
- the aforementioned fatty acid degradation enzyme such as the acyl-CoA synthase gene, is functionally deleted such that the host cell does not express the acyl-CoA synthase or has no detectable acyl-CoA synthase activity.
- the host cell is genetically engineered to express an attenuated level of a thioesterase, a fatty acid degradation enzyme such as an acyl-CoA synthase, or both.
- a thioesterase, an acyl-CoA synthase, or both are functionally deleted.
- the host cell is capable of producing fatty acids and/or derivatives thereof.
- the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis.
- a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis are functionally deleted.
- the gene encoding an acyl-CoA dehydrogenase is fadE.
- the gene encoding an outer membrane protein receptor is one that encodes a receptor for ferrichrome, colicin M, phage T1, phage T5, or phage phi80.
- the gene encoding an outer membrane protein receptor is fhuA (also known as tonA).
- the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
- the invention features a method of producing a fatty acid or a fatty acid derivative such as, for example, a fatty ester.
- the method includes expressing in a host cell a polynucleotide that hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions, to a complement of SEQ ID NO:27, or to a fragment thereof, wherein the polynucleotide encodes a polypeptide having ester synthase and/or acyltransferase activity.
- the polypeptide has ester synthase activity and is capable of converting thioesters to fatty esters, using alcohols as substrates.
- Suitable thioester substrates include, for example, fatty thioesters such as fatty acyl-CoAs or fatty acyl-ACPs.
- the host cell produces fatty esters from free fatty acid and alcohol substrates.
- Suitable alcohol substrates include, for example, long or short chain alcohols such as methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, decanol, dodecanol, tetradecanol, or hexadecanol.
- an endogenous thioesterase of the host cell if present, is unmodified.
- the method includes modifying the expression of a gene encoding a thioesterase in the host cell.
- modifying the expression of a thioesterase gene includes attenuating the expression of an endogenous gene encoding a thioesterase in the host cell and/or functionally deleting that gene.
- the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
- the host cell is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to its level in a wild type host cell.
- exemplary fatty acid degradation enzymes include, without limitation, acyl-CoA synthase enzymes of EC 2.3.1.86 or EC 6.2.1.3.
- the host cell expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, pf1-4354, EAV15023, fadD1, fadD2, rpc_4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_01644857.
- the aforementioned fatty acid degradation enzyme such as the acyl-CoA synthase gene, is functionally deleted such that the host cell does not express an acyl-CoA synthase or has no detectable acyl-CoA synthase activity.
- the host cell is genetically engineered to express an attenuated level of a thioesterase, a fatty acid degradation enzyme such as an acyl-CoA synthase, or both.
- a thioesterase, an acyl-CoA synthase, or both are functionally deleted.
- the host cell is capable of producing fatty acids and/or derivatives thereof.
- the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis.
- a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis are functionally deleted.
- the gene encoding an acyl-CoA dehydrogenase is fadE.
- the gene encoding an outer membrane protein receptor is one that encodes a receptor for ferrichrome, colicin M, phage T1, phage T5, or phage phi80.
- the gene encoding an outer membrane protein receptor is fhuA (also known as tonA).
- the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
- the method further includes culturing the host cell in the presence of at least one biological substrate of the ester synthase. In certain embodiments, the method includes culturing the host cell under conditions that allow expression or overexpression of the ester synthase enzyme in the host cell. In further embodiments, the method includes culturing the host cell under conditions that permit production of fatty acids and/or fatty acid derivatives. In some embodiments, the method includes culturing the host cell in the presence of a thioester substrate. In some other embodiments, the method includes culturing the host cell in the presence of an alcohol substrate. In other embodiments, the method includes culturing the host cell in the presence of a free fatty acid.
- the method further includes isolating an accordingly-produced fatty acid or fatty acid derivative from the host cell.
- the fatty acid or fatty acid derivative is present in the extracellular environment.
- the fatty acid or fatty acid derivative is isolated from the extracellular environment of the host cell.
- the fatty acid or fatty acid derivative is spontaneously secreted, partially or completely, from the host cell.
- the fatty acid or fatty acid derivative is transported into the extracellular environment, with or without the aid of one or more suitable transport proteins.
- the fatty acid or fatty acid derivative is passively transported into the extracellular environment.
- the invention features an in vitro method of producing a fatty acid and/or fatty acid derivative extracellularly, using a purified ester synthase encoded by a polynucleotide that hybridizes to a complement of SEQ ID NO:27, or to a fragment thereof, and a suitable substrate.
- the host cell can be cultured under conditions that allow expression or overexpression of the ester synthase enzyme or a variant thereof, and the cell is then be harvested and lysed.
- the resulting mixture can be purified.
- Suitable substrates such as those described herein can be added to the cell-free extracts under conditions that permit production of a fatty acid and/or a fatty acid derivative.
- the invention features a method of producing a fatty acid and/or a fatty acid derivative such as, for example, a fatty ester.
- the method includes expressing in a host cell a recombinant vector comprising an ester synthase polynucleotide sequence having at least about 50% sequence identity to a polynucleotide sequence in FIG. 16 .
- the nucleotide sequence has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleotide sequence of SEQ ID NO:27, 28, 29, or 30.
- the nucleotide sequence is SEQ ID NO:27, 28, 29, or 30.
- a recombinant vector comprising an ester synthase polynucleotide sequence of SEQ ID NO:42 (codon-optimized from a polynucleotide encoding an ester synthase homolog from Limnobacter sp. MED105), which has about 50% sequence identity to SEQ ID NO:27, can be expressed in a host cell to produce a fatty acid and/or derivative thereof.
- the host cell is engineered to express an attenuated level of a thioesterase, a fatty acid degradation enzyme, or both.
- a thioesterase, a fatty acid degradation enzyme, or both are functionally deleted.
- the host cell is capable of producing fatty acids and/or derivatives thereof.
- the method further includes isolating the fatty acid or fatty acid derivative from the host cell.
- the fatty acid or fatty acid derivative is present in the extracellular environment.
- the fatty acid or fatty acid derivative is isolated from the extracellular environment of the host cell.
- the fatty acid or fatty acid derivative is spontaneously secreted, partially or completely, from the host cell.
- the fatty acid or fatty acid derivative is transported into the extracellular environment, with or without the aid of one or more suitable transport proteins.
- the fatty acid or fatty acid derivative is passively transported into the extracellular environment.
- the invention further features an in vitro method of producing a fatty acid and/or fatty acid derivative extracellularly, wherein a host cell comprising a recombinant vector comprising an ester synthase nucleotide sequence having at least about 50% sequence identity to a nucleotide sequence listed in FIG. 16 is cultured under conditions that allow expression or overexpression of an ester synthase. The host cell is then harvested and lysed. Optionally, the resulting mixture is purified. A suitable substrate is then added to the cell free extract under conditions that permit production of a fatty acid and/or a fatty acid derivative in vitro.
- the recombinant vector further comprises a promoter operably linked to a nucleotide sequence encoding an ester synthase or a suitable variant.
- the promoter is a developmentally-regulated, or organelle-specific, a tissue-specific, an inducible, a constitutive, or a cell-specific promoter.
- the recombinant vector comprises at least one sequence selected from the group consisting of (a) a regulatory sequence operatively coupled to the nucleotide sequence; (b) a selection marker operatively coupled to the nucleotide sequence; (c) a marker sequence operatively coupled to the nucleotide sequence; (d) a purification moiety operatively coupled to the nucleotide sequence; (e) a secretion sequence operatively coupled to the nucleotide sequence; and (f) a targeting sequence operatively coupled to the nucleotide sequence.
- the recombinant vector is a plasmid.
- the host cell expresses an ester synthase polypeptide that is encoded by the recombinant vector.
- the nucleotide sequence is stably incorporated into the genomic DNA of the host cell, and the expression of the nucleotide sequence is under the control of a regulated promoter region.
- an endogenous thioesterase of the host cell if present, is unmodified.
- the method includes modifying the expression of a gene encoding a thioesterase in the host cell.
- modifying the expression of a thioesterase gene includes attenuating the expression of an endogenous gene encoding a thioesterase in the host cell and/or functionally deleting that gene.
- the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
- the host cell is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to its level in a wild type host cell.
- exemplary fatty acid degradation enzymes include, without limitation, acyl-CoA synthase enzymes of EC 2.3.1.86, or EC 6.2.1.3.
- the host cell expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, pf1-4354, EAV15023, fadD 1, fadD2, rpc_4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_01644857.
- the aforementioned fatty acid degradation enzyme such as the acyl-CoA synthase gene, is functionally deleted such that the host cell does not express an acyl-CoA synthase, or has no acyl-CoA synthase activity.
- the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis.
- a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis are functionally deleted.
- the gene encoding an acyl-CoA dehydrogenase is fadE.
- the gene encoding an outer membrane protein receptor is one that encodes a receptor for ferrichrome, colicin M, phage T1, phage T5, or phage phi80.
- the gene encoding an outer membrane protein receptor is fhuA (also known as tonA).
- the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
- the method further includes culturing the host cell in the presence of at least one biological substrate of the ester synthase. In some embodiments, the method includes culturing the host cell under conditions that allow expression or overexpression of the ester synthase in the host cell. In further embodiments, the method includes culturing the host cell under conditions that permit production of fatty acids and/or fatty acid derivatives. In some embodiments, the method includes culturing the host cell in the presence of a thioester substrate. In other embodiments, the method includes culturing the host cell in the presence of an alcohol substrate. In further embodiments, the method includes culturing the host cell in the presence of a free fatty acid.
- the invention features a method of producing a fatty acid and/or a derivative thereof such as, for example, a fatty ester.
- the method includes expressing in a host cell a recombinant vector comprising an ester synthase nucleotide sequence having at least about 50% sequence identity to the nucleotide sequence of SEQ ID NO:27.
- the host cell is engineered to express an attenuated level of a thioesterase, a fatty acid degradation enzyme such as an acyl-CoA synthase, or both.
- an endogenous thioesterase, an acyl-CoA synthase, or both are functionally deleted.
- the host cell is capable of producing fatty acids and/or derivatives thereof.
- the method further includes isolating the fatty acid and/or fatty acid derivative from the host cell.
- the fatty acid or fatty acid derivative is present in the extracellular environment.
- the fatty acid or fatty acid derivative is isolated from the extracellular environment of the host cell.
- the fatty acid or fatty acid derivative is spontaneously secreted, partially or completely, from the host cell.
- the fatty acid or fatty acid derivative is transported into the extracellular environment, with or without the aid of one or more suitable transport proteins.
- the fatty acid or fatty acid derivative is passively transported into the extracellular environment.
- the invention features an in vitro method of producing a fatty acid and/or fatty acid derivative extracellularly, wherein a host cell comprises a recombinant vector, which in turn comprises an ester synthase polynucleotide sequence having at least about 50% identity to SEQ ID NO:27, and wherein the host cell is cultured under conditions that allow expression or overexpression of the ester synthase.
- the host cell is then harvested and lysed.
- the resulting mixture is purified.
- a suitable substrate for example, one selected from those described herein, is then added to the cell free extract under conditions that permit production of a fatty acid and/or a fatty acid derivative in vitro.
- the nucleotide sequence has at least about 55%, at least about 60%, at least about at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleotide sequence of SEQ ID NO:27.
- the nucleotide sequence is SEQ ID NO:27.
- an endogenous thioesterase of the host cell if present, is unmodified.
- the method further includes modifying the expression of a gene encoding a thioesterase in the host cell.
- modifying the expression of a thioesterase gene includes attenuating the expression of an endogenous gene encoding a thioesterase in the host cell and/or functionally deleting that gene.
- the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
- the host cell is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to its level in a wild type host cell.
- exemplary fatty acid degradation enzymes include, without limitation, acyl-CoA synthase enzymes of EC 2.3.1.86 or EC 6.2.1.3.
- the host cell expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, pf1-4354, EAV15023, fadD 1, fadD2, rpc_4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_01644857.
- the aforementioned fatty acid degradation enzyme such as the acyl-CoA synthase gene, is functionally deleted such that the host cell does not express an acyl-CoA synthase or has no acyl-CoA synthase activity.
- the host cell is genetically engineered to express attenuated level of a thioesterase, an attnenuated level of a fatty acid degradation enzyme such as an acyl-CoA synthase, or both.
- a thioesterase, an acyl-CoA synthase, or both are functionally deleted.
- the host cell is capable of producing fatty acids and/or derivatives thereof.
- the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis.
- a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis are functionally deleted.
- the gene encoding an acyl-CoA dehydrogenase is fadE.
- the gene encoding an outer membrane protein receptor is one that encodes a receptor for ferrichrome, colicin M, phage T1, phage T5, or phage phi80.
- the gene encoding an outer membrane protein receptor is fhuA (also known as tonA).
- the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
- the recombinant vector further comprises a promoter operably linked to the nucleotide sequence encoding an ester synthase or a variant thereof.
- the promoter is a developmentally-regulated, or organelle-specific, a tissue-specific, an inducible, a constitutive, or a cell-specific promoter.
- the recombinant vector comprises at least one sequence selected from the group consisting of (a) a regulatory sequence operatively coupled to the nucleotide sequence; (b) a selection marker operatively coupled to the nucleotide sequence; (c) a marker sequence operatively coupled to the nucleotide sequence; (d) a purification moiety operatively coupled to the nucleotide sequence; (e) a secretion sequence operatively coupled to the nucleotide sequence; and (f) a targeting sequence operatively coupled to the nucleotide sequence.
- the recombinant vector is a plasmid.
- the host cell expresses an ester synthase or a suitable variant that is encoded by the recombinant vector.
- the nucleotide sequence is stably incorporated into the genomic DNA of the host cell, and the expression of the nucleotide sequence is under the control of a regulated promoter region.
- the method further includes culturing the host cell in the presence of at least one biological substrate of the ester synthase. In certain embodiment, the method includes culturing the host cell under conditions that are sufficient to allow expression or overexpression of the ester synthase in the host cell. In further embodiments, the method includes culturing the host cell under conditions that are sufficient to permit production of fatty acids and/or fatty acid derivatives.
- an endogenous thioesterase of the host cell if present, is unmodified.
- the host cell expresses an attenuated level of a thioesterase.
- a thioesterase is functionally deleted.
- the host cell expresses an attenuated level of a fatty acid degradation enzyme, such as an acyl-CoA synthase.
- an endogenous fatty acid degradation enzyme, such as an acyl-CoA synthase is functionally deleted.
- the host cell is cultured in the presence of a thioester.
- the host cell is cultured in the presence of a suitable alcohol substrate.
- the host cell is cultured in the presence of a free fatty acid.
- the host cell can be selected from the group consisting of a mammalian cell, a plant cell, an insect cell, a yeast cell, a fungus cell, a filamentous fungi cell, a cyanobacterial cell, and a bacterial cell.
- the host cell is a Gram-positive bacteria cell. In other embodiments, the host cell is a Gram-negative bacteria cell.
- the host cell is selected from the genus Escherichia, Bacillus, Lactobacillus, Rhodococcus, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas, Schizosaccharomyces, Yarrowia , or Streptomyces.
- the host cell is a Bacillus lentus cell, a Bacillus brevis cell, a Bacillus stearothermophilus cell, a Bacillus licheniformis cell, a Bacillus alkalophilus cell, a Bacillus coagulans cell, a Bacillus circulans cell, a Bacillus pumilis cell, a Bacillus thuringiensis cell, a Bacillus clausii cell, a Bacillus megaterium cell, a Bacillus subtilis cell, or a Bacillus amyloliquefaciens cell.
- the host cell is a Trichoderma koningii cell, a Trichoderma viride cell, a Trichoderma reesei cell, a Trichoderma longibrachiatum cell, an Aspergillus awamori cell, an Aspergillus fumigates cell, an Aspergillus foetidus cell, an Aspergillus nidulans cell, an Aspergillus niger cell, an Aspergillus oryzae cell, a Humicola insolens cell, a Humicola lanuginose cell, a Rhodococcus opacus cell, a Rhizomucor miehei cell, or a Mucor michei cell.
- the host cell is a Streptomyces lividans cell or a Streptomyces murinus cell.
- the host cell is an Actinomycetes , a Saccharomyces cerevisiae cell, a Candida Lipolytica (or Yarrowia lipolytica ) cell, an E. coli Arthrobacter AK19 cell, a Rhodotorula glutinins cell, an Acintobacter sp. Strain M-1 cell, or a cell from other oleaginous microorganism.
- the host cell is a cell of an oleaginous yeast, for example, a Yarrowia , a Candida , a Rhodotorula , a Rhodosporidium , a Cryptococcus , a Trichosporon , or a Lipomyces .
- the host cell is a cell of Rhodosporidium toruloide, Lipomyces starkeyii, L. Lipoferus, Candida revêti, C. pulcherrima, C. Tropicalis, C. utilis, Trichosporon pullas, T. cutaneum, Rhodotorula glutinous, R.
- the host cell is a cell from Yarrowia lipolytica strain ATCC #20362, ATCC #8862, ATCC #18944, ATCC #76982 and/or LGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol., 82(1):43-9, 2002).
- oleaginous refers to those organisms that tend to store their energy source in the form of lipid (or oil) (Weete, Fungal Lipid Biochemistry, 2 nd Ed., Plenum, 1980). And for the purpose herein, oleaginous organisms can be bacteria, algae, moss, yeast, fungi, or plants that have the ability to produce oils.
- oleaginous yeast refers to those microorganisms classified as yeasts that can make oil.
- the cellular oil or triacylglycerol content of oleaginous microorganisms follows a sigmoid curve, wherein the concentration of lipid increases until it reaches a maximum at the late logarithmic or early stationary growth phase and then gradually decreases during the late stationary and death phases (Yongmaitchai & Ward, Appl. Environ. Microbiol. 57:419-25 (1991)). It is not uncommon for oleaginous microorganism to accumulate in excess of about 25% of their dry cell weight as oil.
- Examples of oleaginous yeast include, but are not limited to, the following genera: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon , and Lipomyces.
- the host cell is a cell from an eukaryotic plant, algae, cyanobacterium, green-sulfur bacterium (including, e.g., Chlorobium, Clathrochloris, Prosthecochloris ), green non-sulfur bacterium (including, e.g., Chloroflexus, chloronema, Oscillochloris, Heliothrix, Herpetosiphon, Roseiflexus, and Termomicrobium ), purple sulfur bacterium (including, e.g., Allochromatium, Chromatium, Halochromatium, Isochromatium, Marichromatium, Rhodovulum, Thermochromatium, Thiocapsa, Thiorhodococcus , and Thiocystis ), purple non-sulfur bacterium (including, e.g., Phaeospirillum, Rhodobac, Rhodobacter, Rhodomicrobium, Rhod
- the host cell is light dependent or fixes carbon via one of the known pathways including, for example, the Calvin cycle pathway, the acetyl CoA pathway, and the reductive TCA pathway. See, e.g., Fuchs, G, Alternative pathways of autotrophic CO2 fixation, p. 365-382 (1989), AUTOTROPHIC BACTERIA, Springer-Verlag, Berlin, Germany (H. G. Schlegel & B. Bowien ed.).
- the host cell has autotrophic activity.
- the host cell has photoautotrophic activity, such as in the presence of light.
- the host cell is heterotrophic or mixotrophic in the absence of light.
- the host cell is a cell from an extremphile, which are known to withstand various extreme environmental parameters such as temperature, radiation, pressure, gravity, vacuum, desiccation, salinity, pH, oxygen tension and chemicals.
- Host cells from various known extremephiles can be suitable.
- the host cell can be from a hyperthermophile such as Pyrolobus furmarii , which grow at or above 80° C.
- the host cell can be from a thermophiles such as a Synechococcus lividis , which grow between 60-80° C.
- the host cell can be from a mesophiles, which grow between 15-60° C.
- the host cell can be from a psychrophile such as a psychrobacter or certain insect, which grow at or below 15° C.
- the host cell can be from a radiation tolerant organism such as a Deinococcus radiodurans .
- the host cell can be from a pressure tolerant organism, such as a piezophile, which tolerate pressure of 130 MPa.
- the host cell can be from weight tolerant organisms such as a barophile.
- the host cell can be a hypergravity (e.g., >1 g) or a hypogravity (e.g., ⁇ 1 g) tolerant organism.
- the host cell can be from a vacuum tolerant organism such as a tardigrade, insect, microbe and seed.
- the host cell can be from a dessicant tolerant and anhydrobiotic organism such as a xerophile Artenia salina , a nematode, a certain microbe, a certain fungus and a lichen.
- the host cell is from a salt tolerant organism such as a halophiles (e.g., 2-5 M NaCl) Halobacteriacea and Dunaliella salina .
- the host cell can also be from a pH tolerant organism such as an alkaliphiles Natronobacterium.
- the host cell can alternatively be from an anaerobes, which cannot tolerate O 2 , such as a Methancococcus jannaschii , or a microaerophil, which tolerates some O 2 , such as a Clostridium , and an aerobe, which requires O 2 .
- the host cell can be from a gas tolerant organism, which tolerates pure CO 2 , including, for example, a Cyanidium caldarium .
- the host cell can be from a metal tolerant organism, including, for example a metalotolerant such as Ferroplasma acidarmanus (e.g., Cu, As, Cd, Zn), Ralstonia sp. CH34 (e.g., Zn, Co, Cd, 11, Hg, Pb).
- a metalotolerant such as Ferroplasma acidarmanus (e.g., Cu, As, Cd, Zn), Ralstonia sp. CH34 (e.g., Zn, Co, Cd, 11, Hg, Pb).
- Ferroplasma acidarmanus e.g., Cu, As, Cd, Zn
- Ralstonia sp. CH34 e.g., Zn, Co, Cd, 11, Hg, Pb.
- the host cell can be from plants, including, without limitation, plants of the genera: Arabidopsis, Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris, Populus, Saccharum, Salix, Simmondsia , and Zea .
- the host cell can be from Arabidopsis thaliana, Panicum virgatum, Miscanthus giganteus , or Zea mays .
- the host cell can be from algae and cyanobacteria, including, without limitation, the genera: Acanthoceras, Acanthococcus. Acarvochloris, Achnanthes, Achnanthidiun, Actinastrum, Actinochloris.
- Actnoc yclus. Actinotaenium, Amphichrsis, Amphidiniunm, Amphikrikos, Amplhipleura, Amphiprora, Amphithrix, Amphora, Anabaena, Anabaenopsis, Aneumnastus, Ankistrodesmius. Ankyra.
- Anomoeoneis Apatococcus, Aphanizomenon, Aphanocapsa, Aphanochaete, Aphanothece, Apiocystis, Apistonema, Arthrodesmus, Artherospira, Ascochloris, Asterionella, Asterococcus, Audouinella, Aularoseira, Bacillaria, Balbiania, Bambiusina Bangia, Basichlamys, Batrarhospermum, Binurlearia, Bitrichia, Blidingia Botrdiopsis, Botrydium, Botryococcus, Botryosphaerella, Brachiomonas, Brachysira, Brachytrichia, Brebissonia, Bulbochaete, Bumnilleria, Buinilleriopsis, Caloneis, Calothrix, Campy lodiscus, Capsosiphon, Carteria, Catena, Cavinula, Cenritract
- Exemplary microorganisms from which the host cell can be derived include, without limitation, Arabidopsis thaliana, Botryococcus braunii, Chlamydomonas reinhardtii, Dunaliela salina, Synechococcus Sp. PCC 7002 , Synechococcus Sp. PCC 7942, Synechocystis Sp.
- PCC 6803 Thermosynechococcus elongatus BP-1, Chlorobium tepidum, Chloroflexus auranticus, Chromatium vinosum, Chromatium tepidum Rhodospirillum rubrum, Rhodobacter capsulatus, Rhodopseudomonas palusris, Clostridium ljungdahlii, Clostridiuthermocellum, Panicum virgatum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, Schizosaccharomyces pombe, Pseudomonas fluorescens, Miscanthus giganteus, Zea mays , or Zymomonas mobilis.
- Yet other suitable organisms from which the host cell can be from include synthetic cells or cells produced by synthetic genomes as described in U.S. Patent Publication 2007/0264688, or 2007/0269862.
- the host cell is a CHO cell, a COS cell, a VERO cell, a BHK cell, a HeLa cell, a Cv1 cell, an MDCK cell, a 293 cell, a 3T3 cell, or a PC12 cell.
- the host cell is an E. coli cell.
- the E. coli cell is a strain B, a strain C, a strain K, or a strain W E. coli cell.
- the host cell is a cyanobacterial host cell.
- a genetically engineered host cell described herein produces fatty acids and/or fatty acid derivatives at a titer of about 50 mg/L or more, about 100 mg/L or more, about 150 mg/L or more, about 200 mg/L or more, about 500 mg/L or more, or about 1000 mg/L or more.
- the host cell overexpresses a nucleic acid sequence that encodes an ester synthase or a variant described herein.
- known genomic alteration techniques can be applied to change the features of one or more endogenous ester synthase enzymes such that they are overexpressed.
- the method further includes transforming the host cell to overexpress a nucleic acid sequence that encodes a heterologous ester synthase described herein.
- an endogenous thioesterase of the host cell if present, is unmodified.
- the host cell expresses an attenuated level of a thioesterase, a fatty acid degradation enzyme such as an acyl-CoA synthase, or both.
- a thioesterase, an acyl-CoA synthase, or both are functionally deleted.
- the host cell overproduces a substrate described herein.
- the host cell can be engineered to overproduce an alcohol substrate or a thioester substrate.
- the host cell is engineered to produce or overproduce a free fatty acid, which can be converted into fatty esters using the methods described herein.
- the method further includes modifying one or more endogenous ester synthase enzymes such that the host cell overexpresses those ester synthase enzymes.
- the method includes transforming the host cell with a nucleic acid sequence that encodes an ester synthase, and the host cell produces a fatty acid and/or a fatty acid derivative described herein.
- the method further includes culturing the host cell in the presence of at least one substrate herein, which is expressed or overexpressed by the same host cell.
- the substrate is a fatty thioester such as, for example, a fatty acyl-ACP or a fatty acyl-CoA.
- the substrate is an alcohol or a free fatty acid, as described herein.
- the substrate is an alcohol.
- the alcohol substrate is an exogenous alcohol that is introduced to the host cell.
- the alcohol substrate is produced by the genetically engineered host cell.
- the alcohol substrate such as a fatty alcohol substrate
- the alcohol substrate can be suitably produced by the host cell, which co-expresses or overexpresses one or more fatty aldehyde biosynthesis genes and/or one or more fatty alcohol biosynthesis genes.
- the alcohol substrate such as a fatty alcohol substrate is produced by a host cell, which co-expresses or overexpresses one or more acyl-ACP reductase genes and/or one or more fatty alcohol biosynthesis genes.
- the substrate is a free fatty acid.
- the free fatty acid is an undesirable side product, which can be converted by the host cell into a desirable fatty ester using the methods described herein.
- the invention features a method of producing a fatty acid or a fatty acid derivative such as, for example, a fatty ester.
- the method includes contacting a substrate with (i) an ester synthase comprising the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, or a variant thereof, or (ii) an ester synthase polypeptide encoded by a polynucleotide sequence having at least about 50% identity to the polynucleotide sequence of SEQ ID NO:27, 28, 29 or 30, or a variant thereof.
- the method further includes isolating and/or purifying the fatty acid and/or fatty acid derivative.
- the ester synthase polypeptide comprises the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, with one or more amino acid substitutions, additions, insertions or deletions, wherein the polypeptide has ester synthase and/or acyltransferase activity.
- the ester synthase polypeptide is modified wherein the polypeptide has an improved ester synthase and/or acyltransferase activity.
- the ester synthase polypeptide comprises the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, with one or more non-conserved amino acid substitutions, wherein the polypeptide has ester synthase and/or acyltransferase activity.
- the polypeptide comprises one or more conserved amino acid substitutions.
- the polypeptide has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acid substitutions, additions, insertions, or deletions.
- the polypeptide has improved ester synthase and/or acyltransferase activity.
- the polypeptide is capable, or has improved capacity, of catalyzing the conversion of thioesters or free fatty acids to fatty esters, using alcohols as substrates.
- the polypeptide has an amino acid sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:18, 24, 25, or 26. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO:18, 24, 25, or 26.
- the polynucleotide sequence has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the polynucleotide sequence of SEQ ID NO:27, 28, 29, or 30.
- the polynucleotide sequence is SEQ ID NO:27, 28, 29, or 30.
- the invention features a free fatty acid produced by a method herein.
- the free fatty acid comprises one or more points of branching, one or more points of unsaturation, and/or one or more cyclic moieties.
- the invention features a fatty acid derivative produced by a method herein.
- the fatty acid derivative is selected from a fatty acid methyl derivative, a fatty acid ethyl derivative, other fatty acid derivatives, and a combination thereof.
- the carbon chain of the fatty acid derivative comprises one or more points of branching, one or more points of unsaturation, and/or one or more cyclic moieties.
- the invention features a fatty ester produced by a method herein.
- the fatty ester comprises an A side (i.e., the carbon chain attached to the carboxylate oxygen) and a B side (i.e., the carbon chain comprising the parent carboxylate).
- the B side of the fatty ester includes a straight chain.
- the B side of the fatty ester includes a branched chain. The branched chains may have one or more points of branching.
- the B side of the fatty ester comprises at least one cyclic moiety.
- the fatty acid ester is a fatty acid ethyl ester or a fatty acid methyl ester. In some embodiments, the fatty acid ester is at least about 4, 6, 8, 10, 12, 14, 18, or 20 carbons in length.
- the carbon chains comprising the A side or B side can be of any suitable length. In one embodiment, the A side of the ester is at least about 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, or 18 carbons in length.
- the B side of the ester is at least about 4, 6, 8, 10, 12, 14, 16, 18, or 20 carbons in length.
- the fatty ester is saturated. In other embodiments, the fatty ester is unsaturated. In other embodiments, the fatty ester is monounsaturated. If unsaturated, the A side and/or B side can have one or more points of unsaturation.
- the B side can have one double bond at one or more points in the carbon chain.
- the B side can have one double bond at position 7 of the carbon chain, numbering from the reduced end of the carbon chain.
- One of ordinary skill in the art will recognize that, in a fatty methyl ester, one end of the B side will have a methyl group, and the other end of the B side will have a carboxyl group (C( ⁇ O)O—).
- the end of the B side which is a methyl group is the reduced end of the carbon chain comprising the B side, thus, the double bond is at carbon 7 counting from the methyl group terminus of the B group (e.g., at between carbons 7 and 8 of the B group).
- the double bond can have any geometry, thus, the double bond in the B group can be cis or trans. Accordingly, in some embodiments, the fatty ester comprises a double bond at position 7 in the carbon chain (between C 7 and C 8 ) from the reduced end of the fatty ester.
- the fatty ester comprises one or more points of branching. In other embodiments, the fatty ester is linear and does not comprise any branched chains.
- the invention features a genetically engineered microorganism comprising an exogenous control sequence stably incorporated into the genomic DNA of the microorganism upstream of a polynucleotide comprising a nucleotide sequence having at least about 50% identity to the nucleotide sequence of SEQ ID NO:27, 28, 29, or 30, wherein the microorganism produces an increased level of a fatty acid or fatty acid derivative relative to a wild type microorganism.
- the polynucleotide is endogenous to the microorganism.
- the polynucleotide sequence has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the polynucleotide sequence of SEQ ID NO:27, 28, 29, or 30.
- the polynucleotide sequence is SEQ ID NO:27, 28, 29, or 30.
- the microorganism is genetically engineered to express a modified level of a gene encoding an ester synthase.
- the microorganism expresses a heterologous gene encoding an ester synthase or expresses an increased level of an endogenous ester synthase.
- an endogenous thioesterase of the microorganism if present, is unmodified.
- the microorganism expresses an attenuated level of an endogenous gene encoding a thioesterase.
- an endogenous thioesterase is functionally deleted.
- the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
- the microorganism is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to its level in a wild type microorganism.
- exemplary fatty acid degradation enzymes include, without limitation, acyl-CoA synthase enzymes of EC 2.3.1.86 or EC 6.2.1.3.
- the microorganism is engineered to expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, pf1-4354, EAV15023, fadD1, fadD2, rpc_4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_01644857.
- the aforementioned fatty acid degradation enzyme such as the acyl-CoA synthase gene, is functionally deleted such that the microorganism does not comprise an acyl-CoA synthase activity or functionality.
- the microorganism is engineered to express an attenuated level of a thioesterase, a fatty acid degradation enzyme such as an acyl-CoA synthase, or both.
- a thioesterase such as an acyl-CoA synthase
- an endogenous thioesterase, a fatty acid degradation enzyme, or both are functionally deleted.
- the microorganism has no detectable thioesterase or acyl-CoA synthase activity.
- the microorganism is capable of producing fatty acids and/or derivatives thereof.
- the microorganism is genetically engineered to express, relative to a wild type microorganism, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis.
- a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis are functionally deleted.
- the gene encoding an acyl-CoA dehydrogenase is fadE.
- the gene encoding an outer membrane protein receptor is one that encodes a receptor for ferrichrome, colicin M, phage T1, phage T5, or phage phi80.
- the gene encoding an outer membrane protein receptor is fhuA (also known as tonA).
- the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
- the microorganism is a bacterium.
- the bacterium is a Gram-negative or a Gram-positive bacterium.
- the microorganism is a mycobacterium selected from the group consisting of Mycobacterium smegmatis, Mycobacterium abscessus, Mycobacterium avium, Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium marinum , and Mycobacterium ulcerans .
- the bacterium is Nocardia sp. NRRL 5646, Nocardia farcinica, Streptomyces griseus, Salinispora arenicola , or Clavibacter michiganenesis.
- the microorganism is selected from the group consisting of Acinetobacter, Alcanivorax, Alcaligenes, Arabidopsis, Fundibacter, Marinobacter, Mus musculus, Pseudomonas , or Simmodsia.
- the microorganism is selected from the group consisting of algae, cyanobacterium, green-sulfur bacterium (including, e.g., Chlorobium, Clathrochloris, Prosthecochloris ), green non-sulfur bacterium (including, e.g., Chloroflexus, chloronema, Oscillochloris, Heliothrix, Herpetosiphon, Roseiflexus , and Termomicrobium ), purple sulfur bacterium (including, e.g., Allochromatium, Chromatium, Halochromatium, Isochromatium, Marichromatium, Rhodovulum, Thermochromatium, Thiocapsa, Thiorhodococcus, and Thiocystis ), purple non-sulfur bacterium (including, e.g., Phaeospirillum, Rhodobac, Rhodobacter, Rhodomicrobium, Rhodopila,
- the microorganism is selected from the group consisting of: Arabidopsis thaliana, Botryococcus braunii, Chlamydomonas reinhardtii, Dunaliela salina, Synechococcus Sp. PCC 7002 , Synechococcus Sp. PCC 7942, Synechocystis Sp.
- PCC 6803 Thermosynechococcus elongatus BP-1, Chlorobium tepidum, Chloroflexus auranticus, Chromatium vinosum, Chromatium tepidum Rhodospirillum rubrum, Rhodobacter capsulatus, Rhodopseudomonas palusris, Clostridium ljungdahlii, Clostridiuthermocellum, Panicum virgatum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, Schizosaccharomyces pombe, Pseudomonas fluorescens, Miscanthus giganteus, Zea mays , or Zymomonas mobilis.
- the microorganism is an oleaginous yeast, for example, a Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon , or Lipomyces .
- the microorganism is a Rhodosporidium toruloide, Lipomyces starkeyii, L. Lipoferus, Candida revkaufi, C. pulcherrima, C. Tropicalis, C. utilis, Trichosporon pullas, T. cutaneum, Rhodotorula glutinous, R. Garminis , and Yarrowia lipolytica (formally classified as Candida lipolytica ).
- the microorganism is Yarrowia lipolytica strain ATCC #20362, ATCC #8862, ATCC #18944, ATCC #76982 and/or LGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol., 82(1):43-9, 2002).
- the invention features a fatty acid or fatty acid derivative produced by any of the methods or any of the microorganisms described herein, or a composition comprising a fatty acid or derivative thereof produced by any of the methods or any of the microorganisms described herein.
- the fatty acid or fatty acid derivative has a ⁇ 13 C of about ⁇ 15.4 or greater. In certain embodiments, the fatty acid or fatty acid derivative has a ⁇ 13 C of about ⁇ 15.4 to about ⁇ 10.9, or of about ⁇ 13.92 to about ⁇ 13.84.
- the fatty acid or fatty acid derivative has an f M 14 C of at least about 1.003. In certain embodiments, the fatty acid or fatty acid derivative has an f M 14 C of at least about 1.01 or at least about 1.5. In some embodiments, the fatty acid or fatty acid derivative has an f M 14 C of about 1.111 to about 1.124.
- the invention features a biofuel composition comprising the fatty acids or fatty acid derivatives produced by any of the methods or by any of the microorganisms described herein.
- a fatty acid or fatty acid derivative is produced in a host cell or a microorganism described herein from a carbon source, including, for example, an alcohol, a free fatty acid, or a thioester.
- carbon source substrates can be, for example, alcohol substrates, thioester substrates, and free fatty acids.
- Suitable alcohol substrates include short-chain alcohols such as methanol, ethanol, propanol (isopropanol), butanol, pentanol, hexanol, heptanol, and the like, as well as various long-chain alcohols such as fatty alcohols, for example, octanol, tetradecanol, hexadecanol, hexadecenol, octadecenol, and others.
- the invention pertains to converting methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, and/or other alcohol substrates to fatty acid derivatives, such as, for example, fatty esters, using the genetically engineered production host.
- ethanol is converted by a suitably-engineered production host.
- one or more fatty acid ethyl esters and/or free fatty acids are produced.
- methanol is converted by a suitably-engineered production host.
- one or more fatty acid methyl esters and/or free fatty acids are produced.
- butanol is converted by a suitably-engineered production host.
- a mixture of ethanol, methanol, and/or other suitable alcohol substrates can be converted to a mixture of fatty acids and/or fatty acid derivatives by culturing one or more of the production hosts genetically engineered according to the methods herein under conditions that allow the production of such free fatty acids and/or fatty acid derivatives. For example, one or more free fatty acids, one or more fatty acid ethyl esters, and/or one or more fatty acid methyl esters are produced.
- suitable carbon source substrates can be free fatty acids and thioesters.
- the free fatty acid can comprise a branched carbon chain.
- the free fatty acid can comprise a linear carbon chain.
- the free fatty acid can comprise a cyclic group, or one or more points of unsaturation.
- the thioester substrate is a fatty acyl-CoA or fatty acyl-ACP.
- the invention features to a method of producing fatty esters from the free fatty acids in the waste stream of a conventional fatty ester production process.
- the ester synthase polypeptide can be about 200 amino acids to about 2,000 amino acids in length, for example, from about 250 to about 1,500 amino acid residues in length, from about 300 to about 1,200 amino acid residues in length, from about 350 to about 1,000 amino acid residues in length, from about 400 to about 800 amino acid residues in length, or from about 450 to about 600 amino acid residues in length.
- the ester synthase polypeptide is about 300 amino acid residues in length or longer, for example, about 400 amino acid residues in length or longer, or about 450 amino acid residues in length or longer.
- the ester synthase polypeptide is about 1,000 amino acid residues in length or shorter, for example, about 800 amino acid residues in length or shorter, about 700 amino acid residues in length or shorter, or about 600 amino acid residues in length or shorter.
- An exemplary ester synthase of the invention is about 500 amino acid residues in length.
- FIG. 1A is a diagram illustrating a typical biosynthetic pathway known in the art (see, e.g., WO2007/136762, the disclosures of which are incorporated by reference herein), which produces fatty acid and/or fatty acid derivatives depending upon the substrates provided.
- FIG. 1B is a diagram exemplifying an alternative and more efficient pathway of the present invention.
- FIG. 2 depicts the sequence of E. coli spc ribosomal protein operon promoter Pspc (SEQ ID NO:13).
- FIG. 3 depicts a bacterial expression plasmid, pDS33.ES9 (SEQ ID NO:22), in which expression of an ester synthase gene from Marinobacter hydrocarbonoclasticus DSM 8798 (GenBank Accession No. AB021021) is under the control of the E. coli spc ribosomal protein operon promoter Pspc (SEQ ID NO:13).
- FIG. 4 depicts a bacterial expression plasmid, pDS57 (SEQ ID NO:23) wherein the expression of an ester synthase gene is under the control of a Trc promoter.
- FIG. 5 depicts the amount of free fatty acid (FFA) and fatty acyl ethyl ester (FAEE) produced by each culture in accordance with Example 6, without co-expression of an acyl-CoA synthase or a thioesterase, in the presence and absence of ethanol in microwell plates.
- FFA free fatty acid
- FAEE fatty acyl ethyl ester
- ES9 generated nearly 200 mg/L FAEE in the presence of ethanol, but only a small quantity ( ⁇ 20%) of FFA.
- the other ester synthases, including ES8, atfA1 and atfA2 produced substantially lower amounts of FAEE in the presence of ethanol, accompanied by a higher proportion of FFA.
- the culture E. coli 'tesA produced higher overall titers of FFA and FAEE in the presence of ethanol, but the amount of FFA was significantly greater than the amount of FAEE generated.
- FIG. 6 depicts the production of FFA and/or FAEE in accordance with Example 7, without co-expression of acyl-CoA synthase or a thioesterase, but in the presence of ethanol in shake flasks.
- Ester synthases ES9, ES8, atfA1 and atfA2 were all able to produce FAEE in vivo under the prescribed conditions.
- ES9 generated a high level of FAEE in the presence of ethanol, but only little FFA was produced.
- noticeably smaller amounts of FAEE were produced by atfAI, atfA2, and ES8. Consistent with the results of Example 6 and FIG. 5 , expression of E. coli 'tesA alone produced high overall titers of FFA and FAEE, but with a significantly higher proportion of FFA.
- FIG. 7 depicts the production of FFA and/or FAME in accordance with Example 8, without co-expression of acyl-CoA synthase or a thioesterase, but in the presence of methanol in shake flasks.
- the amount of FFA produced was below the limit of detection.
- ES9 produced substantial amount of FAME.
- FIG. 8 depicts a post-induction time course of fatty ester production by E. coli MG1655 DV2 ⁇ fadD expressing ES9, without co-expression of an acyl-CoA synthase or a thioesterase.
- FIG. 9 depicts the z-scores of fatty acid/fatty ester production by the strains of the ES9 position 395 mutageneisis library, as described in Example 11.
- FIG. 10A depicts in vivo production of free fatty acids and/or fatty esters by the E. coli DV2 ⁇ fadD expressing ES9, and ES9 mutants G395R, G395K, G395S, G395D, and G395E.
- FIG. 10B depicts OD600 obtained from the strains during fermentation.
- FIG. 11 depicts fatty acid methyl ester production titers by ES1, ES2, ES3, and ES4, the homologs of ES9, in accordance with Examples 12-13, which converted acyl-ACPs and methanol into methyl esters.
- FIG. 12 depicts fatty acid ethyl ester production titers by ES1, ES2, ES3, and ES4, the homologs of ES9, in accordance with Examples 12-13, which converted acyl-ACPs and ethanol into ethyl esters.
- FIG. 13 is a GC/MS trace following ester production from F. jadensis T9 strain fed with sodium palmitate and short chain alcohols.
- the top panel is a no-alcohol feeding control.
- the middle panel depicts the products made as result of ethanol feeding.
- the lower panel depicts the products made as a result of isopropanol feeding.
- FIG. 14 compares the cell growth profiles of fatty esters production processes involving recombinant host strains with or without an attenuated/deleted fadE, as described in Example 18.
- acyl-ACP and ethanol were used as substrates.
- FIG. 15 compares the total fatty ester production using recombinant host strains with or without an attenuated/deleted fadE, as described in Example 18.
- acyl-ACP and ethanol were used as substrates.
- FIG. 16 depicts various ester synthase and homolog amino acid and polynucleotide sequences. This list also includes various codon optimized polynucleotide sequences encoding the four homologs ES1, ES2, ES3, and ES4.
- FIG. 17 depicts other sequences of enzymes that can be used in connection with the present invention.
- FIG. 18 depicts the features of a pFBAIN-MOD-1 vector, which is useful for carrying out the methods described in Example 20.
- an element means one element or more than one element.
- an “aldehyde biosynthetic gene” or an “aldehyde biosynthetic polynucleotide” is a nucleic acid that encodes an aldehyde biosynthetic polypeptide.
- an “aldehyde biosynthetic polypeptide” is a polypeptide that is a part of the biosynthetic pathway of an aldehyde. Such polypeptides can act on a biological substrate to yield an aldehyde. In some instances, the aldehyde biosynthetic polypeptide has reductase activity.
- the term “attenuate” means to weaken, reduce, or diminish.
- a polypeptide can be attenuated by modifying the polypeptide to reduce its activity (e.g., by modifying a nucleotide sequence that encodes the polypeptide).
- biocrude refers to a product derived from biomass, biomass derivatives, or other biological sources that, like petroleum crude, can be converted into other fuels.
- biocrude can be converted into gasoline, diesel, jet fuel, or heating oil.
- biocrude, like petroleum crude can be converted into other industrially useful chemicals for use in, for example, pharmaceuticals, cosmetics, consumer goods, industrial processes, and the like.
- Biocrude may include, for example, hydrocarbons, hydrocarbon products, fatty acid esters, and/or aliphatic ketones.
- biocrude is comprised of hydrocarbons, for example aliphatic (e.g., alkanes, alkenes, alkynes) or aromatic hydrocarbons.
- biodiesel means a biofuel that can be a substitute of diesel, which is derived from petroleum.
- Biodiesel can be used in internal combustion diesel engines in either a pure form, which is referred to as “neat” biodiesel, or as a mixture in any concentration with petroleum-based diesel.
- biodiesel can include esters or hydrocarbons, such as aldehydes, alkanes, or alkenes.
- biofuel refers to any fuel derived from biomass, biomass derivatives, or other biological sources.
- Biofuels can be substituted for petroleum based fuels.
- biofuels are inclusive of transportation fuels (e.g., gasoline, diesel, jet fuel, etc.), heating fuels, and electricity-generating fuels.
- transportation fuels e.g., gasoline, diesel, jet fuel, etc.
- heating fuels e.g., heating fuels
- electricity-generating fuels e.g., electricity-generating fuels.
- Biofuels are a renewable energy source.
- biomass refers to a carbon source derived from biological material. Biomass can be converted into a biofuel.
- One exemplary source of biomass is plant matter. For example, corn, sugar cane, or switchgrass can be used as biomass.
- Another non-limiting example of biomass is animal matter, for example cow manure.
- Biomass also includes waste products from industry, agriculture, forestry, and households. Examples of such waste products that can be used as biomass are fermentation waste, straw, lumber, sewage, garbage, and food leftovers.
- Biomass also includes sources of carbon, such as carbohydrates (e.g., monosaccharides, disaccharides, or polysaccharides).
- carbon source refers to a substrate or compound suitable to be used as a source of carbon for prokaryotic or simple eukaryotic cell growth.
- Carbon sources can be in various forms, including, but not limited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, and gases (e.g., CO and CO 2 ).
- the carbon source can also be a product of photosynthesis, including, but not limited to, glucose.
- a preferred carbon source is biomass.
- Another preferred carbon source is glucose.
- a “cloud point lowering additive” is an additive added to a composition to decrease or lower the cloud point of a solution.
- cloud point of a fluid means the temperature at which dissolved solids are no longer completely soluble. Below this temperature, solids begin precipitating as a second phase giving the fluid a cloudy appearance.
- cloud point refers to the temperature below which a solidified material or other heavy hydrocarbon crystallizes in a crude oil, refined oil, or fuel to form a cloudy appearance.
- the presence of solidified materials influences the flowing behavior of the fluid, the tendency of the fluid to clog fuel filters, injectors, etc., the accumulation of solidified materials on cold surfaces (e.g., a pipeline or heat exchanger fouling), and the emulsion characteristics of the fluid with water.
- a nucleotide sequence is “complementary” to another nucleotide sequence if each of the bases of the two sequences matches (e.g., is capable of forming Watson Crick base pairs).
- the term “complementary strand” is used herein interchangeably with the term “complement”.
- the complement of a nucleic acid strand can be the complement of a coding strand or the complement of a non-coding strand.
- condition sufficient to allow expression means any conditions that allow a host cell or production host to produce a desired product, such as a polypeptide, acyl-CoA, fatty acid derivative (e.g., fatty acids, hydrocarbons, fatty alcohols, fatty esters, etc.), or other product described herein.
- Suitable conditions include, for example, fermentation conditions. Fermentation conditions can comprise many parameters. Exemplary conditions include temperature ranges, levels of aeration, and media composition. Each of these conditions, individually and in combination, allows the host cell to grow.
- Exemplary culture media include broths or gels.
- the medium includes a carbon source, such as glucose, fructose, cellulose, or the like, that can be metabolized by a host cell directly.
- enzymes can be used in the medium to facilitate the mobilization (e.g., the depolymerization of starch or cellulose to fermentable sugars) and subsequent metabolism of the carbon source.
- a host cell can be cultured, for example, for about 4, 8, 12, 24, 36, or 48 hours. During and/or after culturing, samples can be obtained and analyzed to determine if the conditions allow expression. For example, the host cells in the sample or the medium in which the host cells were grown can be tested for the presence of a desired product. When testing for the presence of a product, assays, such as, but not limited to, TLC, HPLC, GC/FID, GC/MS, LC/MS, MS, can be used.
- condition that permit product production refers to any fermentation conditions that allow a production host to produce a desired product, such as acyl-CoA or fatty acid derivatives (e.g., fatty acids, hydrocarbons, fatty alcohols, waxes, or fatty esters). Fermentation conditions usually comprise many parameters. Exemplary conditions include, but are not limited to, temperature ranges, levels of aeration, and media composition. Each of these conditions, individually and/or in combination, allows the production host to grow.
- Exemplary media include broths and/or gels.
- a suitable medium includes a carbon source (e.g., glucose, fructose, cellulose, etc.) that can be metabolized by the microorganism directly.
- a carbon source e.g., glucose, fructose, cellulose, etc.
- enzymes can be used in the medium to facilitate the mobilization (e.g., the depolymerization of starch or cellulose to fermentable sugars) and subsequent metabolism of the carbon source.
- the production host can be cultured for about 4, 8, 12, 24, 36, or 48 hours. During culturing or after culturing, samples can be obtained and analyzed to determine if the fermentation conditions have permitted product production. For example, the production hosts in the sample or the medium in which the production hosts are grown can be tested for the presence of the desired product. Exemplary assays, such as TLC, HPLC, GC/FID, GC/MS, LC/MS, MS, as well as those provided herein, can be used identify and quantify the presence of a product.
- “conservative amino acid substitution” means an amino acid residue is replaced with an amino acid residue having a similar side chain.
- Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), beta-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine
- polypeptides described herein may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on the polypeptide functions. Whether or not a particular substitution will be tolerated (e.g., will not adversely affect desired biological properties, such as decarboxylase activity) can be determined as described in Bowie et al., Science 247:1306 1310 (1990).
- control element means a transcriptional control element.
- Control elements include promoters and enhancers.
- the term “promoter element,” “promoter,” or “promoter sequence” refers to a DNA sequence that functions as a switch that activates the expression of a gene. If the gene is activated, it is said to be transcribed or participating in transcription. Transcription involves the synthesis of mRNA from the gene. A promoter, therefore, serves as a transcriptional regulatory element and also provides a site for initiation of transcription of the gene into mRNA. Control elements interact specifically with cellular proteins involved in transcription (Maniatis et al., Science 236:1237 (1987)).
- the term “functional deletion,” or “knockout” means modifying or inactivating a polynucleotide sequence that encodes a target protein in order to reduce or eliminate the function of the target protein.
- a polynucleotide deletion can be performed by methods well known in the art (See, e.g., Datsenko et al., Proc. Nat. Acad. Sci. USA, 97:6640-45 (2000) or International Patent Application Nos. PCT/US2007/011923 and PCT/US2008/058788).
- endogenous means a polynucleotide that is in the cell and was not introduced into the cell using recombinant genetic engineering techniques. For example, a gene that was present in the cell when the cell was originally isolated from nature. A polynucleotide is still considered endogenous if the control sequences, such as a promoter or enhancer sequences which activate transcription or translation, have been altered through recombinant techniques.
- ester synthase means a peptide capable of producing fatty esters. More specifically, an ester synthase is a peptide which converts a thioester to a fatty ester. In a preferred embodiment, the ester synthase converts a thioester (e.g., acyl-CoA) to a fatty ester.
- a thioester e.g., acyl-CoA
- an ester synthase uses a thioester and an alcohol as substrates to produce a fatty ester.
- Ester synthases are capable of using short and long chain thioesters as substrates.
- ester synthases are capable of using short and long chain alcohols as substrates.
- ester synthases are wax synthases, wax-ester synthases, acyl CoA:alcohol transacylases, acyltransferases, and fatty acyl-coenzyme A:fatty alcohol acyltransferases.
- Exemplary ester synthases are classified in enzyme classification number EC 2.3.1.75. A number of these enzymes, as well as other useful enzymes for making the products described herein, have been disclosed in, for example, International Patent Application Nos. PCT/US2007/011923 and PCT/US2008/058788, which are incorporated herein by reference.
- exogenous means a polynucleotide that does not originate from a particular cell as found in nature.
- exogenous polynucleotide could refer to a polynucleotide that was inserted within the genomic polynucleotide sequence of a microorganism or to an extra chromosomal polynucleotide that was introduced into the microorganism.
- a non-naturally-occurring polynucleotide is considered to be exogenous to a cell once introduced into the cell.
- a polynucleotide that is naturally-occurring can also be exogenous to a particular cell. For example, an entire polynucleotide isolated from a first cell can be an exogenous polynucleotide with respect to a second cell if that polynucleotide from the first cell is introduced into the second cell.
- fatty acid means a carboxylic acid having the formula RCOOH.
- R represents an aliphatic group, preferably an alkyl group.
- R can comprise between about 4 and about 22 carbon atoms.
- Fatty acids can be saturated, monounsaturated, or polyunsaturated.
- the fatty acid is made from a fatty acid biosynthetic pathway.
- fatty acid biosynthetic pathway means a biosynthetic pathway that produces fatty acids.
- the fatty acid biosynthetic pathway includes fatty acid enzymes that can be engineered, as described herein, to produce fatty acids, and in some embodiments can be expressed with additional enzymes to produce fatty acids having desired carbon chain characteristics.
- fatty acid degradation enzyme means an enzyme involved in the breakdown or conversion of a fatty acid or fatty acid derivative into another product.
- a nonlimiting example of a fatty acid degradation enzyme is an acyl-CoA synthase.
- acyl-CoA synthase A number of these enzymes, as well as other useful enzymes for making the products described herein, have been disclosed in, for example, International Patent Application Nos. PCT/US2007/011923 and PCT/US2008/058788, which are incorporated herein by reference. Additional examples of fatty acid degradation enzymes are described herein.
- fatty acid derivative means products made in part from the fatty acid biosynthetic pathway of the production host organism. “Fatty acid derivative” also includes products made in part from acyl-ACP or acyl-ACP derivatives.
- the fatty acid biosynthetic pathway includes fatty acid synthase enzymes which can be engineered as described herein to produce fatty acid derivatives, and in some examples can be expressed with additional enzymes to produce fatty acid derivatives having desired carbon chain characteristics.
- Exemplary fatty acid derivatives include for example, fatty acids, acyl-CoAs, fatty aldehydes, short and long chain alcohols, hydrocarbons, fatty alcohols, ketones, and esters (e.g., waxes, fatty acid esters, or fatty esters).
- fatty acid derivative enzymes means all enzymes that may be expressed or overexpressed in the production of fatty acid derivatives. These enzymes are collectively referred to herein as fatty acid derivative enzymes. These enzymes may be part of the fatty acid biosynthetic pathway.
- Non-limiting examples of fatty acid derivative enzymes include fatty acid synthases, thioesterases, acyl-CoA synthases, acyl-CoA reductases, alcohol dehydrogenases, alcohol acyltransferases, carboxylic acid reductases, fatty alcohol-forming acyl-CoA reductase, ester synthases, aldehyde biosynthetic polypeptides, and alkane biosynthetic polypeptides.
- Fatty acid derivative enzymes convert a substrate into a fatty acid derivative.
- the substrate may be a fatty acid derivative which the fatty acid derivative enzyme converts into a different fatty acid derivative.
- fatty acid enzyme means any enzyme involved in fatty acid biosynthesis. Fatty acid enzymes can be expressed or overexpressed in host cells to produce fatty acids. Non-limiting examples of fatty acid enzymes include fatty acid synthases and thioesterases. A number of these enzymes, as well as other useful enzymes for making the products described herein, have been disclosed in, for example, International Patent Application Nos. PCT/US2007/011923 and PCT/US2008/058788, which are incorporated herein by reference.
- fatty alcohol means an alcohol having the formula ROH.
- the fatty alcohol is any alcohol made from a fatty acid or fatty acid derivative.
- the R group is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbons in length.
- R can be straight or branched chain.
- the branched chains may have one or more points of branching.
- the branched chains may include cyclic branches.
- R can be saturated or unsaturated. If unsaturated, the R can have one or more points of unsaturation.
- the fatty alcohol is produced biosynthetically.
- Fatty alcohols have many uses.
- fatty alcohols can be used to produce many specialty chemicals.
- fatty alcohols are used as a biofuel; as solvents for fats, waxes, gums, and resins; in pharmaceutical salves, emollients and lotions; as lubricating-oil additives; in detergents and emulsifiers; as textile antistatic and finishing agents; as plasticizers; as nonionic surfactants; and in cosmetics, for examples as thickeners.
- fatty ester means an ester.
- a fatty ester is any ester made from a fatty acid to produce, for example, a fatty acid ester.
- a fatty ester contains an A side (i.e., the carbon chain attached to the carboxylate oxygen) and a B side (i.e., the carbon chain comprising the parent carboxylate).
- the A side is contributed by an alcohol
- the B side is contributed by a fatty acid. Any alcohol can be used to form the A side of the fatty esters.
- the alcohol can be derived from the fatty acid biosynthetic pathway.
- the alcohol can be produced through non-fatty acid biosynthetic pathways.
- the alcohol can be provided exogenously.
- the alcohol can be supplied in the fermentation broth in instances where the fatty ester is produced by an organism that can also produce the fatty acid.
- a carboxylic acid such as a fatty acid or acetic acid, can be supplied exogenously in instances where the fatty ester is produced by an organism that can also produce alcohol.
- the carbon chains comprising the A side or B side can be of any length.
- the A side of the ester is at least about 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, or 18 carbons in length.
- the B side of the ester is at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 carbons in length.
- the A side and/or the B side can be straight or branched chain.
- the branched chains may have one or more points of branching.
- the branched chains may include cyclic branches.
- the A side and/or B side can be saturated or unsaturated. If unsaturated, the A side and/or B side can have one or more points of unsaturation.
- the fatty ester is produced biosynthetically.
- first the fatty acid is “activated.”
- “activated” fatty acids are acyl-CoA, acyl-ACP, and acyl phosphate.
- Acyl-CoA can be a direct product of fatty acid biosynthesis or degradation.
- acyl-CoA can be synthesized from a free fatty acid, a CoA, or an adenosine nucleotide triphosphate (ATP).
- ATP adenosine nucleotide triphosphate
- An example of an enzyme which produces acyl-CoA is acyl-CoA synthase.
- nucleophiles are alcohols, thiols, or phosphates.
- the fatty ester is a wax.
- the wax can be derived from a long chain alcohol and a long chain fatty acid.
- the fatty ester can be derived from a fatty acyl-thioester and an alcohol.
- the fatty ester is a fatty acid thioester, for example fatty acyl Coenzyme A (CoA).
- the fatty ester is a fatty acyl panthothenate, an acyl carrier protein (ACP), or a fatty phosphate ester.
- Fatty esters have many uses. For example, fatty esters can be used as biofuels, surfactants, or formulated into additives that provide lubrication and other benefits to fuels and industrial chemicals.
- f M fraction of modern carbon
- SRMs National Institute of Standards and Technology (NIST) Standard Reference Materials 4990B and 4990C, known as oxalic acids standards HOxI and HOxII, respectively.
- the fundamental definition relates to 0.95 times the 14 C/ 12 C isotope ratio HOxI (referenced to AD 1950). This is roughly equivalent to decay-corrected pre-Industrial Revolution wood.
- f M is approximately 1.1.
- the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
- the length of a reference sequence that is aligned for comparison purposes is at least about 30%, preferably at least about 40%, more preferably at least about 50%, even more preferably at least about 60%, and even more preferably at least about 70%, at least about 80%, at least about 90%, or about 100% of the length of the reference sequence.
- the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
- amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”.
- the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
- the comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm.
- the percent homology between two amino acid sequences is determined using the Needleman and Wunsch (1970), J. Mol. Biol. 48:444 453, algorithm that has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
- the percent homology between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna.
- a particularly preferred set of parameters are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
- a “host cell” is a cell used to produce a product described herein (e.g., an aldehyde or alkane).
- a host cell can be modified to express or overexpress selected genes or to have attenuated expression of selected genes.
- Non-limiting examples of host cells include plant, animal, human, bacteria, cyanobacteria, yeast, or filamentous fungi cells.
- hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions describes conditions for hybridization and washing.
- Guidance for performing hybridization reactions can be found, for example, in Current Protocols in Molecular Biology , John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described in that reference and either method can be used.
- Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6 ⁇ sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2 ⁇ SSC, 0.1% SDS at least at 50° C.
- SSC sodium chloride/sodium citrate
- the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions in 6 ⁇ SSC at about 45° C., followed by one or more washes in 0.2 ⁇ SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions in 6 ⁇ SSC at about 45° C., followed by one or more washes in 0.2. ⁇ SSC, 0.1% SDS at 65° C.; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2 ⁇ SSC, 1% SDS at 65° C. Very high stringency conditions (4) are the preferred conditions unless otherwise specified.
- isolated refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the nucleic acid.
- an “isolated nucleic acid” includes nucleic acid fragments, such as fragments that are not naturally occurring.
- isolated is also used herein to refer to polypeptides, which are isolated from other cellular proteins, and encompasses both purified endogenous polypeptides and recombinant polypeptides.
- isolated as used herein also refers to a nucleic acid or polypeptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques.
- isolated as used herein also refers to a nucleic acid or polypeptide that is substantially free of chemical precursors or other chemicals when chemically synthesized.
- the “level of expression of a gene in a cell” refers to the level of mRNA, pre-mRNA nascent transcript(s), transcript processing intermediates, mature mRNA(s), and/or degradation products encoded by the gene in the cell.
- microorganism means prokaryotic and eukaryotic microbial species from the domains Archaea, Bacteria and Eucarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista.
- microbial cell means a cell from a microorganism.
- nucleic acid refers to a polynucleotide, such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
- DNA deoxyribonucleic acid
- RNA ribonucleic acid
- the term also includes analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides, ESTs, chromosomes, cDNAs, mRNAs, and rRNAs.
- nucleic acid may be used interchangeably with “polynucleotide,” “DNA,” “nucleic acid molecule,” “nucleotide sequence,” and/or “gene” unless otherwise indicated herein or otherwise clearly contradicted by context.
- operably linked means that a selected nucleotide sequence (e.g., encoding a polypeptide described herein) is in proximity with a promoter to allow the promoter to regulate expression of the selected nucleotide sequence.
- the promoter is located upstream of the selected nucleotide sequence in terms of the direction of transcription and translation.
- operably linked is meant that a nucleotide sequence and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).
- overexpress means to express or cause to be expressed or produced a nucleic acid, polypeptide, or hydrocarbon in a cell at a greater concentration than is normally expressed in a corresponding wild-type cell.
- a polypeptide can be “overexpressed” in a recombinant host cell when the polypeptide is present in a greater concentration in the recombinant host cell compared to its concentration in a non-recombinant host cell of the same species.
- partition coefficient is defined as the equilibrium concentration of a compound in an organic phase divided by the concentration at equilibrium in an aqueous phase (e.g., fermentation broth).
- the organic phase is formed by the aldehyde or alkane during the production process.
- an organic phase can be provided, such as by providing a layer of octane, to facilitate product separation.
- the partition characteristics of a compound can be described as logP. For example, a compound with a logP of 1 would partition 10:1 to the organic phase. A compound with a logP of ⁇ 1 would partition 1:10 to the organic phase.
- polypeptide may be used interchangeably with “protein,” “peptide,” and/or “enzyme” unless otherwise indicated herein or otherwise clearly contradicted by context.
- production host means a cell used to produce the products disclosed herein.
- the production host is modified to express, overexpress, attenuate or delete expression of selected polynucleotides.
- Non-limiting examples of production hosts include plant, algal, animal, human, bacteria, yeast, and filamentous fungi cells.
- the term “purify,” “purified,” or “purification” means the removal or isolation of a molecule from its environment by, for example, isolation or separation. “Substantially purified” molecules are at least about 60% free, preferably at least about 75% free, and more preferably at least about 90% free from other components with which they are associated. As used herein, these terms also refer to the removal of contaminants from a sample. For example, the removal of contaminants can result in an increase in the percentage of a fatty acid derivative or product in a sample. For example, when a fatty acid derivatives or products are produced in a host cell, the fatty acid derivatives or products can be purified by the removal of host cell proteins. After purification, the percentage of fatty acid derivatives or products in the sample is increased.
- a purified fatty acid derivative or product is one that is substantially separated from other cellular components (e.g., nucleic acids, polypeptides, lipids, carbohydrates, or other fatty acid derivatives or products).
- a purified fatty acid derivative or purified product preparation is one in which the fatty acid derivative or product is substantially free from contaminants, such as those that might be present following fermentation.
- a fatty acid derivative or product is purified when at least about 50% by weight of a sample is composed of the fatty acid derivative or product.
- a fatty acid derivative or product is purified when at least about 60%, 70%, 80%, 85%, 90%, 92%, 95%, 98%, or 99% or more by weight of a sample is composed of the fatty acid derivative or product.
- recombinant polypeptide refers to a polypeptide that is produced by recombinant DNA techniques, wherein generally DNA encoding the expressed polypeptide or RNA is inserted into a suitable expression vector and that is in turn used to transform a host cell to produce the polypeptide or RNA.
- the term “substantially identical” is used to refer to a first amino acid or nucleotide sequence that contains a sufficient number of identical or equivalent (e.g., with a similar side chain) amino acid residues (e.g., conserved amino acid substitutions) or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have similar activities.
- synthase means an enzyme which catalyzes a synthesis process.
- synthase includes synthases, synthetases, and ligases.
- transfection means the introduction of a nucleic acid (e.g., via an expression vector) into a recipient cell by nucleic acid-mediated gene transfer.
- the term “transformation” refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous nucleic acid. This may result in the transformed cell expressing a recombinant form of a RNA or polypeptide. In the case of antisense expression from the transferred gene, the expression of a naturally-occurring form of the polypeptide is disrupted.
- transport protein means a polypeptide that facilitates the movement of one or more compounds in and/or out of a cellular organelle and/or a cell.
- these proteins as well as other useful proteins for making the products described herein, have been disclosed in, for example, International Patent Application Nos. PCT/US2007/011923 and PCT/US2008/058788, which are incorporated herein by reference.
- a “variant” of polypeptide X refers to a polypeptide having the amino acid sequence of polypeptide X in which one or more amino acid residues is altered.
- the variant may have conservative changes or nonconservative changes.
- Guidance in determining which amino acid residues may be substituted, inserted, or deleted without affecting biological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).
- variant when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to that of a gene or the coding sequence thereof. This definition may also include, for example, “allelic,” “splice,” “species,” or “polymorphic” variants.
- a splice variant may have significant identity to a reference polynucleotide, but will generally have a greater or fewer number of polynucleotides due to alternative splicing of exons during mRNA processing.
- the corresponding polypeptide may possess additional functional domains or an absence of domains.
- Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other.
- a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
- vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
- One type of useful vector is an episome (i.e., a nucleic acid capable of extra-chromosomal replication).
- Useful vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.
- Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors”.
- expression vectors of utility in recombinant DNA techniques are often in the form of “plasmids,” which refer generally to circular double stranded DNA loops that, in their vector form, are not bound to the chromosome.
- plasmid and vector are used interchangeably, as the plasmid is the most commonly used form of vector.
- other forms of expression vectors that serve equivalent functions and that become known in the art subsequently hereto.
- wax means a composition comprised of fatty esters.
- the fatty ester in the wax is comprised of medium to long carbon chains.
- a wax may comprise other components (e.g., hydrocarbons, sterol esters, aliphatic aldehydes, alcohols, ketones, beta-diketones, triacylglycerols, etc.).
- gene names include all genes encoding the same polypeptide and homologous polypeptides having the same physiological function.
- Polypeptide names include all polypeptides that have the same activity (e.g., that catalyze the same fundamental chemical reaction).
- accession numbers referenced herein are derived from the NCBI database (National Center for Biotechnology Information) maintained by the National Institute of Health, U.S.A. Unless otherwise indicated, the accession numbers are as provided in the database as of April 2009.
- EC numbers are established by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) (available at http://www.chem.qmul.ac.uk/iubmb/enzyme/).
- NC-IUBMB Nomenclature Committee of the International Union of Biochemistry and Molecular Biology
- the EC numbers referenced herein are derived from the KEGG Ligand database, maintained by the Kyoto Encyclopedia of Genes and Genomics, sponsored in part by the University of Tokyo. Unless otherwise indicated, the EC numbers are as provided in the database as of April 2009.
- the invention is based, at least in part, on the discovery that fatty acids and/or fatty acid derivatives such as fatty esters can be produced by culturing genetically engineered host cells comprising an expressed or overexpressed ester synthase or a suitable variant thereof, but an attenuated level or in an absence of either or both a fatty acid degradation enzyme such as an acyl-CoA synthase a thioesterase, as compared to the naturally-occurring level of such enzymes.
- the instant method is more energetically favorable and efficient.
- the method described herein allows the direct conversion of a thioester, such as an acyl-ACP and/or an acyl-CoA, to a fatty acid and/or a fatty acid derivative, bypassing the requirement to first hydrolyze an ACP to make a fatty acid and then reactivate the fatty acid to produce an acyl-CoA.
- a thioester such as an acyl-ACP and/or an acyl-CoA
- Fatty esters can be produced by culturing genetically engineered host cells comprising ester synthase enzymes, nucleic acids encoding these enzymes, vectors comprising nucleic acids encoding these enzymes, or by culturing recombinant host cells transformed with the vectors, and recombinant host cells comprising polynucleotides encoding these ester synthase enzymes, which are chromosomally integrated therein.
- the method includes expressing in a host cell a gene encoding an ester synthase, selected from, for example, the enzymes classified as EC 2.3.1.75, which catalyzes the enzymatic transfer of saturated or unsaturated acyl residues, for example, those of chain-length C 18 to C 20 , to alcohols.
- the ester synthase may be one selected from the group consisting of a wax-ester synthase, an acyl-CoA:alcohol transacylase, an acyltransferase, and a fatty acyl-CoA:fatty alcohol acyltransferase.
- the ester synthase gene can be one that encodes ws/dgat, a bifunctional ester synthase/acyl-CoA/acyl-CoA: diacylglycerol acyltransferase from Simmondsia chinensis, Acinetobacter sp., Acinetobacter sp. ADP1, alcanivorax borkumensis, Pseudomonas aeruginosa, Fundibacter jadensis, Arabidopsis thaliana , or Alkaligenes eutrophus .
- the invention features a method of making a fatty acid or a fatty acid derivative such as, for example, a fatty ester, wherein the method includes expressing in a host cell a gene encoding an ester synthase selected from the group consisting of AtfA1 (YP_694462, SEQ ID NO:25), AtfA2 (YP_693524, SEQ ID NO:26), ES9 (AB021021, SEQ ID NO:18), and ES8 (AB021020, SEQ ID NO:24), or a variant thereof.
- an ester synthase selected from the group consisting of AtfA1 (YP_694462, SEQ ID NO:25), AtfA2 (YP_693524, SEQ ID NO:26), ES9 (AB021021, SEQ ID NO:18), and ES8 (AB021020, SEQ ID NO:24), or a variant thereof.
- the invention features a method of making a fatty acid or a fatty acid derivative such as, for example a fatty ester, wherein the method includes expressing in a host cell a gene encoding an ester synthase polypeptide comprising the amino acid sequence of SEQ ID NO: 18, 24, 25, or 26, or a variant thereof.
- the polypeptide or variant has ester synthase and/or acyltransferase activity.
- the polypeptide or variant catalyzes the conversion of a thioester into a fatty acid and/or a fatty acid derivative, using an alcohol as substrate.
- the polypeptide or variant catalyzes the conversion of an acyl-CoA to a fatty acid and/or a fatty acid derivative, using an alcohol as substrate.
- the polypeptide or variant catalyzes the conversion of an acyl-ACP to a fatty acid and/or a fatty acid derivative, using an alcohol as substrate.
- the polypeptide or variant also converts a free fatty acid to a fatty ester using an alcohol as substrate.
- the alcohol substrates can be short- or long-chain alcohols.
- the alcohol substrate is a methanol, and accordingly, the host cell expressing the ester synthase or variant produces a fatty acid and/or a fatty acid methyl derivative, such as a methyl ester.
- the alcohol substrate is an ethanol, and accordingly, the host cell expressing the ester synthase or variant produces a fatty acid and/or a fatty acid ethyl derivative, such as an ethyl ester.
- an endogenous thioesterase of the host cell if present, is unmodified.
- the host cell expresses an attenuated level of a thioesterase, as compared to the naturally-occurring level of that thioesterase.
- a thioesterase is functionally deleted.
- the host cell expresses an attenuated level of a fatty acid degradation enzyme, such as an acyl-CoA synthase, as compared to the naturally-occurring level of that enzyme.
- an acyl-CoA synthase is functionally deleted.
- the host cell has no detectable acyl-CoA synthase activity. In further embodiments, the host cell expresses attenuated levels of a thioesterase and a fatty acid degradation enzyme such as an acyl-CoA synthase. In yet further embodiments, a thioesterase and an acyl-CoA synthase are functionally deleted. In some embodiments, the host cell has no detectable activity of a thioesterase or an acyl-CoA synthase.
- the method comprises expressing in a host cell a gene encoding an ester synthase polypeptide comprising the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, or a variant thereof, wherein a thioesterase, an acyl-CoA synthase, or both, are functionally deleted or knocked out from the host cell.
- the host cell has no detectable thioesterase activity or acyl-CoA synthase activity.
- variants can be naturally occurring or created in vitro.
- variants can be created using genetic engineering techniques, such as site directed mutagenesis, random chemical mutagenesis, exonuclease III deletion procedures, or standard cloning techniques.
- variants, fragments, analogs, or derivatives can be created using chemical synthesis or modification procedures.
- variants are well known in the art. These include procedures in which nucleic acid sequences obtained from natural isolates are modified to generate nucleic acids that encode polypeptides having characteristics that enhance their value in industrial or laboratory applications. In such procedures, a large number of variant sequences having one or more nucleotide differences with respect to the sequence obtained from the natural isolate are generated and characterized. Typically, these nucleotide difference result in amino acid changes with respect to the polypeptides encoded by the nucleic acids from the natural isolates.
- variants can be created using error prone PCR (see, e.g., Leung et al., Technique 1:11-15 (1989); and Caldwell et al., PCR Methods Applic. 2:28-33 (1992)).
- error prone PCR PCR is performed under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product.
- nucleic acids to be mutagenized e.g., an ester synthase sequence
- PCR primers e.g., an ester synthase sequence
- reaction buffer MgCl 2 , MnCl 2 , Taq polymerase, and an appropriate concentration of dNTPs for achieving a high rate of point mutation along the entire length of the PCR product.
- the reaction can be performed using 20 fmoles of nucleic acid to be mutagenized (e.g., an ester synthase polynucleotide sequence), 30 pmole of each PCR primer, a reaction buffer comprising 50 mM KCl, 10 mM Tris HCl (pH 8.3), and 0.01% gelatin, 7 mM MgCl 2 , 0.5 mM MnCl 2 , 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP.
- PCR can be performed for 30 cycles of 94° C. for 1 mM, 45° C.
- the mutagenized nucleic acids are then cloned into an appropriate vector and the activities of the polypeptides encoded by the mutagenized nucleic acids are evaluated.
- Variants can also be created using oligonucleotide directed mutagenesis to generate site-specific mutations in any cloned DNA of interest.
- Oligonucleotide mutagenesis is described in, for example, Reidhaar-Olson et al., Science 241:53-57 (1988). Briefly, in such procedures a plurality of double stranded oligonucleotides bearing one or more mutations to be introduced into the cloned DNA are synthesized and inserted into the cloned DNA to be mutagenized (e.g., an ester synthase polynucleotide sequence). Clones containing the mutagenized DNA are recovered, and the activities of the polypeptides they encode are assessed.
- Assembly PCR involves the assembly of a PCR product from a mixture of small DNA fragments. A large number of different PCR reactions occur in parallel in the same vial, with the products of one reaction priming the products of another reaction. Assembly PCR is described in, for example, U.S. Pat. No. 5,965,408.
- Still another method of generating variants is sexual PCR mutagenesis.
- sexual PCR mutagenesis forced homologous recombination occurs between DNA molecules of different, but highly related, DNA sequence in vitro as a result of random fragmentation of the DNA molecule based on sequence homology. This is followed by fixation of the crossover by primer extension in a PCR reaction.
- Sexual PCR mutagenesis is described in, for example, Stemmer, PNAS, USA 91:10747-10751 (1994).
- Variants can also be created by in vivo mutagenesis.
- random mutations in a nucleic acid sequence are generated by propagating the sequence in a bacterial strain, such as an E. coli strain, which carries mutations in one or more of the DNA repair pathways.
- a bacterial strain such as an E. coli strain
- Such “mutator” strains have a higher random mutation rate than that of a wild-type strain.
- Propagating a DNA sequence e.g., an ester synthase polynucleotide sequence
- Mutator strains suitable for use for in vivo mutagenesis are described in, for example, PCT Publication No. WO 91/16427.
- cassette mutagenesis a small region of a double stranded DNA molecule is replaced with a synthetic oligonucleotide “cassette” that differs from the native sequence.
- the oligonucleotide often contains a completely and/or partially randomized native sequence.
- Recursive ensemble mutagenesis can also be used to generate variants.
- Recursive ensemble mutagenesis is an algorithm for protein engineering (i.e., protein mutagenesis) developed to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis. Recursive ensemble mutagenesis is described in, for example, Arkin et al., PNAS, USA 89:7811-7815 (1992).
- variants are created using exponential ensemble mutagenesis.
- Exponential ensemble mutagenesis is a process for generating combinatorial libraries with a high percentage of unique and functional mutants, wherein small groups of residues are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins.
- Exponential ensemble mutagenesis is described in, for example, Delegrave et al., Biotech. Res. 11:1548-1552 (1993). Random and site-directed mutageneses are described in, for example, Arnold, Curr. Opin. Biotech. 4:450-455 (1993).
- variants are created using shuffling procedures wherein portions of a plurality of nucleic acids that encode distinct polypeptides are fused together to create chimeric nucleic acid sequences that encode chimeric polypeptides as described in, for example, U.S. Pat. Nos. 5,965,408 and 5,939,250.
- Polynucleotide variants also include nucleic acid analogs.
- Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety include deoxyuridine for deoxythymidine and 5-methyl-2′-deoxycytidine or 5-bromo-2′-doxycytidine for deoxycytidine. Modifications of the sugar moiety include modification of the 2′ hydroxyl of the ribose sugar to form 2′-O-methyl, 2′-halo, or 2′-O-allyl sugars.
- the deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six-membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained.
- morpholino nucleic acids in which each base moiety is linked to a six-membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained.
- deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.
- the ester synthase polypeptide is a variant comprising the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, with one or more amino acid substitutions, additions, insertions, or deletions, and the polypeptide has ester synthase and/or acyltransferase activity.
- the ester synthase variant catalyzes the conversion of thioesters to fatty acids and/or fatty acid derivatives, using alcohols as substrates.
- the ester synthase variant also catalyzes the conversion of free fatty acids to fatty esters.
- ester synthase polypeptide having an amino acid sequence of SEQ ID NO:18, 24, 25, or 26, as well as a polypeptide variants thereof.
- Ester synthase polypeptide variant can be variants in which one or more amino acid residues are substituted with conserved or non-conserved amino acid residues. Such substituted amino acid residues may or may not be ones encoded by the genetic code.
- the ester synthase polypeptide is a variant comprising the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, with one or more non-conserved amino acid substitutions, wherein the polypeptide variant has ester synthase and/or acyltransferase activity.
- the glycine residue at position 395 of SEQ ID NO:18 is substituted with a basic amino acid residue, wherein the resultant ester synthase variant retains or has improved ester synthase and/or acyltransferase activity.
- ester synthase polypeptide is a variant comprising the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, with one or more conserved amino acid substitutions. conserveed substitutions are those that substitute a given amino acid residue in a polypeptide by another amino acid of similar characteristics.
- Typical conserved substitutions include the following replacements: replacement of an aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic amino acid; replacement of a serine with a threonine; replacement of a threonine with a serine; replacement of an acidic residue, such as aspartic acid and glutamic acid, with another acidic residue; replacement of residue bearing an amide group; exchange of a basic residue, such as lysine and arginine, with another basic residue; and replacement of an aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue.
- replacement of an aliphatic amino acid such as alanine, valine, leucine, and isoleucine
- replacement of a serine with a threonine replacement of a threonine with a serine
- replacement of an acidic residue such as aspartic acid and glutamic acid, with another acidic residue
- the ester synthase polypeptide variant has ester synthase and/or acyltransferase activity.
- the ester synthase polypeptide variant catalyzes the conversion of a thioester into a fatty acid and/or a fatty acid derivative.
- the ester synthase polypeptide catalyzes the conversion of a free fatty acid into a fatty ester.
- polypeptide variants include those in which one or more amino acid residues include a substituent group. Still other polypeptide variants include those in which the polypeptide is associated with another compound, such as a compound to increase the half-life of the polypeptide (e.g., polyethylene glycol).
- a compound to increase the half-life of the polypeptide e.g., polyethylene glycol
- Additional polypeptide variants are those in which additional amino acid residues are fused to the polypeptide, such as a leader sequence, a secretory sequence, a proprotein sequence, or a sequence which facilitates purification, enrichment, or stabilization of the polypeptide.
- the polypeptide variants retain the same biological function as a polypeptide having an amino acid sequence of, for example, SEQ ID NO:18, 24, 25, or 26 and have amino acid sequences substantially identical thereto.
- the polypeptide variants retains the ester synthase and/or acyltransferase activity of, for example, a polypeptide of the amino acid sequence SEQ ID NO:18.
- the polypeptide variants has at least about 35%, at last about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% homology to an amino acid sequence of SEQ ID NO:18, 24, 25, or 26.
- the polypeptide variants include a fragment comprising at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200 consecutive amino acid residues thereof.
- polypeptide variants or fragments thereof can be obtained by isolating nucleic acids encoding them using techniques described herein or by expressing synthetic nucleic acids encoding them. Alternatively, polypeptide variants or fragments thereof can be obtained through biochemical enrichment or purification procedures. The sequence of polypeptide variants or fragments can be determined by proteolytic digestion, gel electrophoresis, and/or microsequencing. For example, the sequence of the polypeptide variants or fragments can then be compared to an amino acid sequence of SEQ ID NO:18, 24, 25, or 26 using any of the programs described herein.
- the polypeptide variants and fragments thereof can be assayed for ester synthase activity and/or acyltransferase activity using routine methods.
- the polypeptide variants or fragments can be contacted with a substrate (e.g., an acyl-CoA, an acyl-ACP, a free fatty acid, or an alcohol) under conditions that allow the polypeptide to function.
- a substrate e.g., an acyl-CoA, an acyl-ACP, a free fatty acid, or an alcohol
- a decrease in the level of the substrate or an increase in the level of a fatty acid and/or a fatty acid derivative product can be measured to determine the ester synthase/acyltransferase activity.
- the ester synthase polypeptide variant can comprise the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, with one or more non-conserved amino acid substitutions but continues to have ester synthase and/or acyltransferase activity.
- the glycine residue at position 395 is substituted with a basic amino acid residue, wherein the resultant ester synthase variant retains or has improved ester synthase and/or acyltransferase activity, relative to that of a native or wild-type ester synthase.
- the glycine residue at position 395 of SEQ ID NO:18 is substituted with an arginine or a lysine, wherein the resultant ester synthase variant has improved ester synthase and/or acyltransferase activity, relative to the activity of the wild type ester synthase of SEQ ID NO:18.
- the polypeptide comprises one or more conserved amino acid substitutions. In some embodiments, the polypeptide has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acid substitutions, additions, insertions, or deletions. In some embodiments, the polypeptide comprising the one or more amino acid substitutions, additions, insertions or deletions retains the same biological activity as a polypeptide of SEQ ID NO:18, 24, 25, or 26. As used herein, “retaining a biological activity” refers to the retention of a detectable level of that biological activity, rather than the retention of the same level of an activity.
- the polypeptide comprising one or more amino acid substitutions, additions, insertions or deletions has ester synthase and/or acyltransferase activity.
- the polypeptide is capable of catalyzing the conversion of thioesters to fatty acids and/or fatty acid derivatives such as fatty esters, using alcohols as substrates.
- thioester substrates include, for example, fatty thioesters such as fatty acyl-CoAs or fatty acyl-ACPs.
- the polypeptide is capable of catalyzing the conversion of a free fatty acid into a fatty ester using an alcohol as substrate.
- Suitable alcohol substrates include, for example, long- or short-chain alcohols such as methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, decanol, dodecanol, tetradecanol, or hexadecanol.
- long- or short-chain alcohols such as methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, decanol, dodecanol, tetradecanol, or hexadecanol.
- the invention pertains to methods of producing a fatty acid or a fatty acid derivative such as, for example, a fatty ester, wherein the method includes expressing in a host cell a gene or polynucleotide encoding a variant of ester synthase, ES9 (GenBank Accession No. AB021021, SEQ ID NO:18), wherein the host cell expresses an attenuated level of a thioesterase, or wherein the thioesterase is functionally deleted.
- ES9 GenBank Accession No. AB021021, SEQ ID NO:18
- the invention pertains to methods of producing a fatty acid or a fatty acid derivative such as, for example, a fatty ester, wherein the method includes expressing in a host cell a gene encoding a variant of ES9 (SEQ ID NO:18), wherein the host cell expresses an attenuated level of a fatty acid degradation enzyme such as an acyl-CoA synthase, or wherein the acyl-CoA synthase is functionally deleted.
- a fatty acid or a fatty acid derivative such as, for example, a fatty ester
- the invention pertains to methods of producing a fatty acid or a derivative thereof, wherein the method includes expressing in a host cell a gene encoding a variant of ES9 (SEQ ID NO:18), wherein the host cell express attenuated levels of a thioesterase, a fatty acid degradation enzyme such as an acyl-CoA synthase, or both, or wherein the thioesterase, the acyl-CoA synthase, or both, are functionally deleted.
- the host cell has no detectable thioesterase activity or acyl-CoA synthase activity.
- the variant of ester synthase comprises a substitution at the amino acid residue corresponding to position 395 of SEQ ID NO:18.
- the variant comprises a basic amino acid residue at position 395, replacing the glycine residue at that position of SEQ ID NO:18.
- the basic amino acid residue can be a lysine or an arginine residue.
- the variant has the same or improved ester synthase and/or acyltransferase activity, relative to that of the polypeptide of SEQ ID NO:18.
- the invention pertains to methods of producing a fatty acid or a fatty acid derivative wherein the method includes expressing in a host cell a polynucleotide encoding a variant of ester synthase ES9, wherein the host cell further expresses an attenuated level of thioesterase, an acyl-CoA synthase, or both, or wherein the thioesterase, the acyl-CoA synthase, or both, are functionally deleted.
- the variant of ester synthase comprises a substitution at the amino acid residue corresponding to position 395 of SEQ ID NO:18 and one or more other amino acid substitutions, additions, insertions or deletions, and the variant has ester synthase and/or acyltransferase activity.
- the variant catalyzes the conversion of thioester substrates and/or alcohol substrate into fatty acids and/or fatty acid derivatives. In other embodiments, the variant catalyzes the conversion of free fatty acids into fatty esters.
- the ester synthase polypeptide or a variant thereof is about 200 amino acids to about 2,000 amino acids in length, for example, from about 250 to about 1500 amino acid residues in length, from about 300 to about 1200 amino acid residues in length, from about 350 to about 1000 amino acid residues in length, from about 400 to about 800 amino acid residues in length, or from about 450 to about 600 amino acid residues in length.
- the naturally-occurring ester synthase polypeptide is about 300 amino acid residues in length or longer, for example, about 400 amino acid residues in length or longer, or about 450 amino acid residues in length or longer.
- the ester synthase polypeptide or the variant thereof is about 1000 amino acid residues in length or shorter, for example, about 800 amino acid residues in length or shorter, about 700 amino acid residues in length or shorter, or about 600 amino acid residues in length or shorter.
- An exemplary ester synthase of the invention is about 500 amino acid residues in length.
- the ester synthase polypeptides described herein can be used to produce antibodies directed against ester synthase polypeptides. Such antibodies can be used, for example, to detect expression of an ester synthase polypeptide using methods known in the art.
- the antibody can be, for example, a polyclonal antibody; a monoclonal antibody or antigen binding fragment thereof; a modified antibody such as a chimeric antibody, reshaped antibody, humanized antibody, or fragment thereof (e.g., Fab′, Fab, F(ab′)2); or a biosynthetic antibody, for example, a single chain antibody, single domain antibody (DAB), Fv, single chain Fv(scFv), or the like.
- DAB single domain antibody
- Fv single chain Fv(scFv
- compositions and methods described herein can be used to product fatty acids and/or fatty acid derivatives, for example, from thioesters and alcohols, or from free fatty acids and alcohols, which themselves can be produced from appropriate substrates in the same host cell. While not wishing to be bound by theory, it is believed that the recombinantly modified production host can be modified to increase the production of thioester substrates such as, for example, acyl-CoAs or acyl-ACPs, of free fatty acids, or of suitable alcohol substrates such as, for example, fatty alcohols.
- thioester substrates such as, for example, acyl-CoAs or acyl-ACPs
- suitable alcohol substrates such as, for example, fatty alcohols.
- the recombinantly modified production host can be modified to reduce the catabolism of fatty acids or fatty acid derivatives and/or intermediates in the fatty acid biosynthetic pathways, or to reduce feedback inhibition at specific points in the same pathway.
- additional cellular resources can be diverted to over-produce fatty acids and/or fatty acid derivatives.
- the lactate, succinate, and/or acetate pathways can be attenuated, and acetyl-CoA carboxylases (encoded by acc genes) can be overexpressed.
- Modifications to production host can be achieved, for example, through genomic alterations, addition of recombinant expression systems, or combinations thereof.
- FIG. 1A A representative fatty acid and/or fatty acid derivative biosynthetic pathway is depicted in FIG. 1A .
- the following sections describe the steps in these pathways. Different steps in the pathway are catalyzed by a variety of enzymes.
- Each step within a pathway that leads to the production of a fatty acid and/or a fatty acid derivative substrate can be modified to produce or overproduce the substrate of interest.
- known genes involved in the fatty acid biosynthetic pathway can be expressed, overexpressed, or attenuated in host cell to produce the desired substrate (see, e.g., the approach described in PCT/US08/05877, the disclosure of which is incorporated herein by reference).
- genes that may serve as the points of change include, without limitation, accA (EC 6.4.1.2), accB (EC 6.4.1.2), accC (EC 6.4.1.2, EC 6.3.4.14), accD (EC 6.4.1.2), aceE (EC 1.2.4.1), aceF (EC 2.3.1.12), fabD (EC 2.3.1.39), fabG (EC 1.1.1.100), fabH (EC 2.3.1.180), panD (EC 4.1.1.11), panK (EC 2.7.1.33), pdh (EC 1.2.4.1), udhA (EC 1.6.1.1).
- Fatty acid synthase is a group of peptides that catalyze the initiation and elongation of acyl chains (Marrakchi et al., Biochemical Society, 30:1050-1055 (2002)).
- the acyl carrier protein (ACP) along with the enzymes in the FAS pathway controls the length, degree of saturation, and branching of the fatty acids produced.
- the steps in this pathway are catalyzed by enzymes of the fatty acid biosynthesis (fab) and acetyl-CoA carboxylase (acc) gene families (see, e.g., Heath et al., Prog. Lipid Res. 40(6):467-97 (2001)).
- acc acetyl-CoA carboxylase
- Host cells can be engineered to express fatty acid derivative substrates by recombinantly expressing or overexpressing one or more fatty acid synthase genes, such as acetyl-CoA and/or malonyl-CoA synthase genes.
- fatty acid synthase genes such as acetyl-CoA and/or malonyl-CoA synthase genes.
- one or more of the following genes can be expressed or overexpressed in a host cell: pdh, panK, aceEF (encoding the E1p dehydrogenase component and the E2p dihydrolipoamide acyltransferase component of the pyruvate and 2′-oxoglutarate dehydrogenase complexes), fabH, fabD, fabG, acpP, and fabF.
- GenBank Accession numbers for these genes are listed in the parentheses after the gene names: pdh (BAB34380, AAC73227, AAC73226), panK (also known as coaA, AAC96952), aceEF (AAC73227, AAC73226),fabH (AAC74175), fabD (AAC74176), fabG (AAC74177), acpP (AAC74178), fabF (AAC74179).
- the expression levels of fadE, gpsA, ldhA, pflb, adhE, pta, poxB, ackA, and/or ackB can be reduced or functionally deleted in the engineered microorganism by transformation with conditionally replicative or non-replicative plasmids containing null or deletion mutations of the corresponding genes, or by substituting promoter or enhancer sequences.
- fadE AAC73325
- gspA AAC76632
- ldhA AAC74462
- pflb AAC73989
- adhE AAC74323
- pta AAC75357
- poxB AAC73958
- ackA AAC75356
- BAB81430 adE expression is attenuated or functionally deleted in a production host cell of the present invention.
- the resulting engineered production hosts can produce increased levels of acetyl-CoA when cultivated in an appropriate environment.
- malonyl-CoA overproduction can be effectuated by engineering the production host as described with accABCD (e.g., GenBank Accession No. AAC73296, classified as EC 6.4.1.2) included in the plasmid synthesized de novo.
- Fatty acid overproduction can be achieved by further including a DNA sequence encoding lipases (e.g., GenBank Accession Nos: CAA89087, CAA98876), in the plasmid synthesized de novo.
- the resultant host cells in some embodiments, overexpresses acetyl-CoA such that its intracellular concentration is increased by more than about 2-fold, for example, by more than about 5-fold, or more than about 10 fold, relative to the native expression levels of acetyl-CoA.
- Acetyl-CoA and malonyl-CoA overproduction can be verified using methods known in the art, for example, by using radioactive precursors, HPLC, and GC-MS following cell lysis.
- PlsB e.g., GenBank Accession No. AAC77011
- D311E mutant can be used to increase the amount of acyl-CoA available.
- sfa gene (suppressor of FabA, e.g., GenBank Accession No. AAN79592) can be effectuated in a production host to increase the production of monounsaturated fatty acids (Rock et al., J. Bacteriology 178:5382-5387 (1996)).
- acyl-CoA and malonyl-CoA are converted through a series of steps to form the acyl-ACP chains.
- Acyl-ACP is then converted via a series of alternative enzymatic steps into various end products, including fatty acid derivatives.
- typically acyl-ACP is converted to fatty esters by the combined consecutive reactions of a thioesterase, an acyl-CoA ligase/synthetase and an ester synthase.
- a limitation to the commercial use of these enzymes in a metabolic pathway is the requirement of hydrolysis of ACP to produce fatty acids that are subsequently reactivated to produce the fatty acyl-CoA substrates, which requires at least two enzymatic steps and the expenditure of metabolic energy from two phosphoanhydride bonds. It has been discovered that fatty acids and/or fatty acid derivatives such as, for example, fatty esters, can be produced in the presence of an ester synthase, bypassing the participations of a thioesterase, and/or bypassing the participation of a fatty acid degradation enzyme such as an acyl-CoA synthase. This more economical and efficient approach mitigates the loss of ATP caused by an otherwise multi-step pathway.
- a suitable ester synthase in the absence of a co-expressed thioesterase, and/or a co-expressed fatty acid degradation enzyme such as an acyl-CoA synthase, can effectively utilize thioesters such as acyl-ACPs or acyl-CoAs, free fatty acids, and/or alcohols such as short- or long-chain alcohols, as substrates to produce desirable fatty acids and/or fatty acid derivatives under appropriate conditions.
- Example 14 describes representative assays that can be used, among other suitable methods, to determine whether a fatty acid and/or fatty acid derivatives is produced and/or the amount that is produced in a given production system.
- ester synthases can be utilized to directly produce fatty acids and/or fatty acid derivatives, in the presence or in the absence of a thioesterase, and/or in the presence or in the absence of a fatty acid degradation enzyme such as, for example, an acyl-CoA synthase.
- a fatty acid degradation enzyme such as, for example, an acyl-CoA synthase.
- expression or overexpression of an ester synthase that can catalyze the direct production of fatty acids and/or fatty acid derivatives in a recombinant host strain. This can be used to supplement fatty acid derivative production where the strain also expresses a thioesterase and/or a fatty acid degradation enzyme.
- the free fatty acids can be converted into fatty esters using the ester synthases, variants, host cells, and microorganisms and methods herein.
- expression of an ester synthase that can catalyze the direct production of fatty acids and/or fatty acid derivatives such as fatty esters in a recombinant host cell can be used where there is no or low thioesterase expression.
- thioesterase refers to an enzyme that has thioesterase activity.
- Thioesterases include thioester hydrolases, which are identified as members of Enzyme Classification E.C. 3.1.2 and are obtainable from a variety of sources. Plant thioesterases are described in, for example, Voelker & Davies, J. Bact., 176 (23): 7320-27 (1994), U.S. Pat. Nos. 5,667,997 and 5,455,167.
- Thioesterases are also obtainable from microbial sources, such as those described in Akoh et al., Prog. Lipid Res., 43(6):534-52 (2004); Diczfalusy & Alexson, Arch. Biochem.
- Thioesterases are also obtainable from, for example, cyanobacterial, algal, mammalian, insect, and fungal sources.
- a thioesterase can have activity other than thioesterase activity, for example proteolytic activity or oxygen ester hydrolysis activity.
- a particularly useful thioesterase is the 'TesA (also termed “thioesterase I” or “leaderless thioesterase”) enzyme from E. coli , which is a truncated version of the full-length TesA serine thioesterase enzyme that is described in Cho & Cronan, J.
- An E. coli 'TesA polypeptide comprises 182 amino acids, and is the product of a cleavage reaction wherein the 26 amino acid leader sequence of E. coli TesA is removed.
- thioesterase activity refers to the capacity to catalyze a thioester cleavage reaction, which usually involves the hydrolysis of a thioester at a thiol group into an acid and a thiol, but can also include a transesterification step in which a thioester bond is cleaved and a new ester bond is formed.
- an acyl-ACP thioesterase is capable of catalyzing the hydrolytic cleavage of fatty acyl-acyl carrier protein thioesters and/or fatty acyl-CoA thioesters.
- enzymes having thioesterase activity include acetyl-CoA hydrolase, palmitoyl-CoA hydrolase, succinyl-CoA hydrolase, formyl-CoA hydrolase, acyl-CoA hydrolase, palmitoyl-protein thioesterase, and ubiquitin thiolesterase.
- Thioesterase activity can be established by any of the following assays:
- a Tris-HCl buffer, 0.1 M, pH 8.0; Palmitoyl-CoA, 5 ⁇ M; DTNB, 0.01 M in 0.1 M potassium phosphate buffer, pH 7.0 are used to prepare a complete assay mixture.
- the assay mixture thus contains a final concentration of 10 mot of Tris-HCl buffer, pH 8.0, 0.05 mot of DTNB, and 0.01 mot of palmitoyl-CoA.
- the complete assay mixture is then mixed with the thioesterase, in a final volume of 2.0 mL.
- the rate of cleavage of the acyl-CoA substrate is measured by monitoring the change in absorbance at 412 nm, using a molar extinction coefficient of 13,600 M ⁇ cm ⁇ 1 .
- the culture which potentially has thioesterase activity, is acidified with 1 N HCl to a final pH of about 2.5 and then extracted with an equal volume of butyl acetate.
- Free fatty acids in the organic phase are derivatized with BSTFA (N,O-bis (trimethyl (silyl) trifluoro acetamide) and 1% TMCS (trimethyl chloro silane) to generate the respective TMS (trimethyl silyl-) esters, which are then analyzed on a gas chromatograph equipped with a flame ionization detector.
- a 20 ⁇ L, of substrate solution is first prepared.
- the substrate solution contains 0.64 mM MU-6S-Palm- ⁇ -Glc, 15 mM dithiothreitol, 0.375% (w/v) Triton X-100, and 0.1 U ⁇ -glucosidase from almonds in Mcllvain's phosphate/citrate buffer, pH 4.0.
- a culture mixture potentially having thioesterase activity is then mixed with the substrate solution.
- the reaction mixture is incubated for 1 hour at 37° C.
- Exogenous almond ⁇ -glucosidase is added to hydrolyze the reaction intermediate, MU-6-thio- ⁇ -glucoside, quantitatively.
- a reaction mixture containing 10 ⁇ L a culture mixture potentially having thioesterase activity mixed with 10 ⁇ L of 3 mM 1-oleoyl-phosphatidylethanolamine, 25 ⁇ L of 100 mM Tris-HCl (pH 7.0), and 5 ⁇ L of 5 mM EDTA is prepared.
- the reaction is terminated with the addition of 1.5 mL CHCl 3 :CH 3 OH (1:2), followed by the addition of water to bring the total aqueous volume to 0.9 mL.
- the organic phase is then analyzed by thin layer chromatography together with suitable standards, using plates prepared from 40 g Silica Gel H suspended in 95 mL of 1 mM sodium tetraborate.
- the solvent system consists of CHCl 3 :CH 3 OH:H 2 O (95:35:5).
- a reaction mixture containing 10 ⁇ L of a culture mixture potentially having thioesterase activity mixed with 800 ⁇ L 12.5 mM Tris-HCl (pH 8.0) containing 0.25% Triton X-100 and 10 ⁇ L of Cbz-Phe-ONp dissolved in DMSO is prepared.
- the p-nitrophenol released via cleavage of the substrate is measured by monitoring the absorbance at 405 nm.
- a reagent solution containing 2% Triton X-100 in 50 mM sodium phosphate, pH 7.0, and 10 mM C 12 -p-nitrophenol (acyl-PNP) in acetone is first prepared. Then a C 12 -PNP working solution is prepared by mixing 600 ⁇ L 10 mM C 12 -PNP into a 9.4-mL phosphate buffer. The assay is performed by adding 40 ⁇ L of the acyl-PNP working solution to each well of a 96-well plate, followed by the rapid addition of 40 ⁇ L of the culture mixture potentially having thioesterase activity. The solution is mixed for 15 seconds, and the absorbance change is read at 405 nm in a microtiter plate reader at 25° C.
- a reaction mixture containing a culture mixture potentially having thioesterase activity, 100 ⁇ M myristoyl-CoA, 10% (v/v) methanol, and 50 mM sodium phosphate, pH 7.0 is prepared.
- the reaction mixture is incubated for 1 hour at 20° C. and terminated with the addition of 1 N HCl to decrease the pH to about 2.5.
- the mixture is extracted with an equal volume of ethyl acetate and the amount of fatty ester produced is determined via GC-MS or other standard methods such as GC-FID, LC-MS, or thin layer chromatography.
- a reaction mixture containing a culture mixture potentially having thioesterase activity, 300 ⁇ M lauroyl-CoA, 10% (v/v) methanol, and 50 mM sodium phosphate, pH 7.0 is prepared.
- the reaction mixture is incubated for 1 hour at 20° C. and terminated with the addition of 1 N HCl to decrease the pH to about 2.5.
- the mixture is extracted with an equal volume of ethyl acetate and the amount of lauryl ester produced is determined via GC-MS or other standard methods such as GC-FID, LC-MS, or thin layer chromatography.
- an endogenous thioesterase can be attenuated and/or functionally deleted from the host cells of the present invention.
- the thioesterase is selected from the group consisting of tesA, 'tesA, fatB1, fatB2, fatB3, fatA1, fatA, and atfata.
- Non-limiting examples of thioesterases and host cells are listed in Table 1.
- fatty acids and/or fatty acid derivatives are produced in a host cell that contains one or more naturally-occurring mutations that result in an increased level of fatty acids and/or fatty acid derivatives in the host cell.
- the host cell is genetically engineered to increase the level of fatty acids and/or fatty acid derivatives in the host cell relative to a corresponding wild-type host cell.
- the host cell can be genetically engineered to express a reduced level of a fatty acid degradation enzyme such as an acyl-CoA synthase relative to a corresponding wild-type host cell.
- the level of expression of one or more genes is attenuated or functionally deleted by genetically engineering a “knock out” host cell.
- acyl-CoA synthase gene can be reduced or functionally deleted in a host cell.
- Non-limiting examples of acyl-CoA synthase genes include fadD, fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD 1, fadD2, RPC_4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_01644857.
- Specific examples of acyl-CoA synthase genes include fadDD35 from M. tuberculosis H37Rv [NP_217021], fadDD22 from M.
- tuberculosis H37Rv [NP_217464], fadD from E. coli [NP_416319], fadK from E. coli [YP_416216], fadD from Acinetobacter sp.
- aeruginosa PAO1 [NP_251989], fadD1 from Ralstonia solanacearum GM1 1000 [NP_520978], fadD2 from P. aeruginosa PAO1 [NP_251990], the gene encoding the protein ZP_01644857 from Stenotrophomonas maltophilia R551-3, faa3p from Saccharomyces cerevisiae [NP_012257], faalp from Saccharomyces cerevisiae [NP_014962], lcfA from Bacillus subtilis [CAA99571], or those described in Shockey et al., Plant. Physiol.
- the alcohol substrates can be supplied to the host cells.
- the fatty alcohol substrates can be made by the host cell by co-expressing one or more fatty aldehyde biosynthesis genes and/or one or more fatty alcohol biosynthesis genes.
- Heterologous fatty aldehyde biosynthesis genes can be introduced to the host cell, or endogenous fatty aldehyde biosynthesis genes can be modified such that they are overexpressed in the host cell.
- Suitable fatty aldehyde biosynthesis genes include, without limitation, those encoding carboxylic acid reductases and variants of, for example, any one of SEQ ID NOs:54-97.
- Heterologous fatty alcohol biosynthesis genes can also be introduced to the host cell, or endogenous fatty alcohol biosynthesis genes can be modified such that they are overexpressed in the host cell.
- Suitable fatty alcohol biosynthesis genes include, without limitation, those encoding alcohol dehydrogenases and variants of, for example, any one of SEQ ID NOs:98-147.
- fatty alcohol substrates can be made by the host cell by co-expressing one or more acyl-ACP reductase genes and/or one or more fatty alcohol biosynthesis genes.
- Heterologous acyl-ACP reductase genes may be introduced to the host cell, or endogenous acyl-ACP reductase genes can be modified such that they are overexpressed in the host cell.
- Suitable acyl-ACP reductase genes include, without limitation, those encoding acyl-ACP reductases and variants of, for example, any one of SEQ ID NOs:148-156.
- the ester synthase polypeptides and variants of the present invention can catalyze the conversion of a free fatty acid into a fatty ester in the presence of suitable alcohol substrates. Accordingly, in some embodiments, when free fatty acid is not a desired product, the ester synthase polypeptides and variants of the invention can be used to reduce the amount of free fatty acid generated during the fatty ester production by converting them into fatty esters.
- Fatty acids and derivatives thereof can be produced that contain points of branching by using branched thioester and/or branched alcohol as substrates.
- E. coli naturally produces straight chain fatty acids (sFAs)
- E. coli can be engineered to produce branched chain fatty acids (brFAs) and/or brFA derivatives by introducing and expressing or overexpressing heterologous genes that provide branched precursors in the E. coli (e.g., bkd, ilv, icm, and fab gene families).
- a host cell can be engineered to express or overexpress genes encoding proteins for the elongation of brFAs (e.g., ACP, FabF, etc.) or derivatives, and/or to delete or attenuate the corresponding host cell genes that normally lead to straight-chain fatty acids and/or fatty acid derivatives.
- brFAs e.g., ACP, FabF, etc.
- the first step in forming brFAs or brFA derivatives is the production of the corresponding ⁇ -keto acids by a branched-chain amino acid aminotransferase.
- Host cells may endogenously include genes encoding such enzymes or such genes can be heterologously or recombinantly introduced.
- E. coli for example, endogenously expresses such an enzyme, IlvE (EC 2.6.1.42; GenBank Accession No. YP_026247). In some host cells, a heterologous branched-chain amino acid aminotransferase may not be expressed.
- E. coli for example, endogenously expresses such an enzyme, IlvE (EC 2.6.1.42; GenBank Accession No. YP_026247).
- a heterologous branched-chain amino acid aminotransferase may not be expressed.
- IlvE or any other branched-chain amino acid aminotransferase e.g., IlvE from Lactococcus lactis (GenBank Accession No. AAF34406), IlvE from Pseudomonas putida (GenBank Accession No. NP_745648), or IlvE from Streptomyces coelicolor (GenBank Accession No. NP_629657)
- if not endogenous can be introduced.
- the production of ⁇ -keto acids can be achieved by using the methods described in Atsumi et al., Nature 451:86-89 (2008).
- 2-ketoisovalerate can be produced by overexpressing the genes encoding IlvI, IlvH, IlvC, or IlvD.
- 2-keto-3-methyl-valerate can be produced by overexpressing the genes encoding IlvA and IlvI, IlvH (or AlsS of Bacillus subtilis ), IlvC, IlvD, or their corresponding homologs.
- 2-keto-4-methyl-pentanoate can be produced by overexpressing the genes encoding IlvI, IlvH, IlvC, IlvD and LeuA, LeuB, LeuC, LeuD, or their corresponding homologs.
- the second step is the oxidative decarboxylation of the ⁇ -keto acids to the corresponding branched-chain acyl-CoA.
- This reaction can be catalyzed by a branched-chain ⁇ -keto acid dehydrogenase complex (bkd; EC 1.2.4.4.) (Denoya et al., J. Bacteriol. 177:3504 (1995), which consists of E1 ⁇ / ⁇ (decarboxylase), E2 (dihydrolipoyl transacylase), and E3 (dihydrolipoyl dehydrogenase) subunits.
- bkd branched-chain ⁇ -keto acid dehydrogenase complex
- branched-chain ⁇ -keto acid dehydrogenase complexes are similar to pyruvate and ⁇ -ketoglutarate dehydrogenase complexes.
- Any microorganism that comprises brFAs and/or brFA cellular components and/or is capable of growing on branched-chain amino acids can be used as a source to isolate bkd genes for expression in host cells, for example, E. coli .
- E. coli has the E3 component as part of its pyruvate dehydrogenase complex (lpd, EC 1.8.1.4, GenBank Accession No. NP_414658).
- lpd EC 1.8.1.4
- GenBank Accession No. NP_414658 GenBank Accession No. NP_414658
- Table 2 lists non-limiting examples of bkd genes from several microorganisms that can be heterologously or recombinantly introduced and expressed in a host cell to provide branched-chain acyl
- isobutyryl-CoA can be made in a host cell, for example in E. coli , through the coexpression of a crotonyl-CoA reductase (Ccr, EC 1.6.5.5, 1.1.1.1) and isobutyryl-CoA mutase (large subunit IcmA, EC 5.4.99.2; small subunit IcmB, EC 5.4.99.2) (Han and Reynolds, J. Bacteriol. 179:5157 (1997)).
- Crotonyl-CoA is an intermediate in fatty acid biosynthesis in E. coli and other microorganisms.
- Non-limiting examples of ccr and icm genes from selected microorganisms are listed in Table 3.
- FabH ⁇ -ketoacyl-acyl-carrier-protein synthase III
- FabH ⁇ -ketoacyl-acyl-carrier-protein synthase III
- Non-limiting examples of FabH enzymes are listed in Table 4.
- fabH genes that are involved in fatty acid biosynthesis of any brFA-containing microorganism or organisms that feed on branched-chain amino acids can be expressed in a host cell.
- the Bkd and FabH enzymes from host cells that do not naturally make brFA may not support brFA production.
- bkd and fabH can be expressed recombinantly or heterologously.
- Vectors containing the bkd and/or fabH genes can be inserted into such a host cell.
- the endogenous level of Bkd and FabH production may not be sufficient to produce brFA.
- the endogenous bkd and/or fabH can be overexpressed.
- the endogenous bkd and/or fabH can be functionally deleted and replaced with a heterologous bkd and/or fabH.
- the heterologous bkd and/or fabH that is introduced into a host cell encode more effective BKD and/or FabH enzymes than those encoded by the endogenous bkd and/or fabH.
- other components of the fatty acid biosynthesis pathway can be expressed or overexpressed, such as acyl carrier proteins (ACPs) and 3-ketoacyl-acyl-carrier-protein synthase II (fabF, EC 2.3.1.41) (non-limiting examples are listed in Table 4).
- ACPs acyl carrier proteins
- fabF 3-ketoacyl-acyl-carrier-protein synthase II
- some genes in the endogenous fatty acid biosynthesis pathway can be attenuated in the host cell (e.g., the E. coli genes fabH (GenBank Accession No. NP_415609) and/or fabF (GenBank Accession No. NP_415613)).
- Cyclic fatty acids and/or derivatives thereof such as, for example, cyclic fatty esters
- suitable cyclic substrates genes that provide cyclic precursors (e.g., the ans, chc, and plm gene families) can be introduced into the host cell and expressed to allow initiation of fatty acid biosynthesis from cyclic precursors.
- a host cell such as E. coli
- cyFA a gene that provides the cyclic precursor cyclohexylcarbonyl-CoA (CHC-CoA) (Cropp et al., Nature Biotech.
- genes that provide CHC-CoA in E. coli include: ansJ, ansK, ansL, chcA, and ansM from the ansatrienin gene cluster of Streptomyces collinus (Chen et al., Eur. J. Biochem. 261: 98-107 (1999)) or plmJ, plmK, plmL, chcA, and plmM from the phoslactomycin B gene cluster of Streptomyces sp. HK803 (Palaniappan et al., J. Biol. Chem.
- the homologous genes can be isolated from microorganisms that make cyFA and expressed in a host cell (e.g., E. coli ).
- fabH, acp, and fabF allow initiation and elongation of ⁇ -cyclic fatty acids because they have broad substrate specificity. If the coexpression of any of these genes with the genes listed in Table 5 does not yield cyFA, then fabH, acp, and/or fabF homologs from microorganisms that make cyFAs (e.g., those listed in Table 6) can be isolated (e.g., by using degenerate PCR primers or heterologous DNA sequence probes) and coexpressed.
- the degree of saturation in fatty acids and/or derivatives thereof can be controlled by regulating the degree of saturation of intermediates.
- the sfa, gns, and fab families of genes can be expressed, overexpressed, or expressed at reduced levels, to control the saturation of fatty acids.
- Exemplary genes include, without limitation, fabB (EC 2.3.1.41), fabK (EC 1.3.1.9), fabL (EC 1.3.1.9), fabM (EC 4.2.1.17), which can be used in the methods and host cells described herein.
- host cells can be engineered to produce unsaturated fatty acids and/or fatty acid derivatives by engineering the production host to overexpress fabB or by growing the production host at low temperatures (e.g., less than 37° C.).
- FabB has preference to cis- ⁇ 3decenoyl-ACP and results in unsaturated fatty acid production in E. coli .
- Overexpression of fabB results in the production of a significant percentage of unsaturated fatty acids and/or fatty acid derivatives (de Mendoza et al., J. Biol. Chem. 258:2098-2101 (1983)).
- the gene fabB may be inserted into and expressed in host cells not naturally having the gene. These unsaturated fatty acids and/or fatty acid derivatives can then be used as intermediates in host cells.
- a repressor of fatty acid biosynthesis for example, fabR (GenBank Accession No. NP_418398), can be deleted, which will also result in increased unsaturated fatty acid and/or fatty acid derivative production in E. coli (Zhang et al., J. Biol. Chem. 277:15558 (2002)). Similar deletions may be made in other host cells.
- a further increase in unsaturated fatty acids may be achieved, for example, by overexpressing fabM (trans-2, cis-3-decenoyl-ACP isomerase, GenBank Accession No. DAA05501) and controlled expression of fabK (trans-2-enoyl-ACP reductase II, GenBank Accession No.
- NP_357969 from Streptococcus pneumoniae (Manakchi et al., J. Biol. Chem. 277: 44809 (2002)), while deleting E. coli fabI (trans-2-enoyl-ACP reductase, GenBank Accession No. NP_415804).
- E. coli fabI trans-2-enoyl-ACP reductase, GenBank Accession No. NP_415804.
- the endogenous fabF gene can be attenuated, thus increasing the percentage of palmitoleate (C16:1) produced.
- host cells can be engineered to produce saturated fatty acids by reducing the expression of an sfa, gns, or fab gene.
- a host cell can be engineered to express a decreased level of fabA and/or fabB.
- the host cell can be grown in the presence of unsaturated fatty acids.
- the host cell can be further engineered to express or overexpress a gene encoding a desaturase enzyme.
- a desaturase is B. subtilis DesA (AF037430).
- Other genes encoding desaturase enzymes are known in the art and can be used in the host cells and methods described herein, such as desaturases that use acyl-ACP, such as hexadecanoyl-ACP or octadecanoyl-ACP.
- a host cell can be any prokaryotic or eukaryotic cell.
- a polypeptide described herein can be expressed in bacterial cells (such as E. coli ), insect cells, yeast, or mammalian cells (such as Chinese hamster ovary cells (CHO) cells, COS cells, VERO cells, BHK cells, HeLa cells, Cv1 cells, MDCK cells, 293 cells, 3T3 cells, or PC12 cells).
- exemplary host cells include cells from the members of the genus Escherichia, Bacillus, Lactobacillus, Rhodococcus, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces, Schizosaccharomyces, Yarrowia , or Streptomyces .
- Yet other exemplary host cells can be a cell of Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus licheniformis, Bacillus alkalophilus, Bacillus coagulans, Bacillus circulans, Bacillus pumilis, Bacillus thuringiensis, Bacillus clausii, Bacillus megaterium, Bacillus subtilis, Bacillus amyloliquefaciens, Trichoderma koningii, Trichoderma viride, Trichoderma reesei, Trichoderma longibrachiatum, Aspergillus awamori, Aspergillus fumigates, Aspergillus foetidus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Humicola insolens, Humicola lanuginose, Rhizomucor miehei, Mucor michei, Streptomyces l
- the host cell is an E. coli cell, a Saccharomyces cerevisiae cell, or a Bacillus subtilis cell. In a more preferred embodiment, the host cell is from E. coli strains B, C, K, or W. Other suitable host cells are known to those skilled in the art.
- the methods can include the use of vectors, preferably expression vectors, containing a nucleic acid encoding an ester synthase polypeptide described herein, polypeptide variant, or a fragment thereof.
- vectors preferably expression vectors, containing a nucleic acid encoding an ester synthase polypeptide described herein, polypeptide variant, or a fragment thereof.
- viral vectors for example, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors
- non-viral vectors can be used in the methods described herein.
- the recombinant expression vectors described herein include a nucleic acid described herein in a form suitable for expression of the nucleic acid in a host cell.
- the recombinant expression vectors can include one or more control sequences, selected on the basis of the host cell to be used for expression.
- the control sequence is operably linked to the nucleic acid sequence to be expressed.
- control sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).
- Control sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).
- the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
- the expression vectors described herein can be introduced into host cells to produce polypeptides, including fusion polypeptides, encoded by the nucleic acids as described herein.
- Recombinant expression vectors can be designed for expression of a fatty acid biosynthetic polypeptide or variant in prokaryotic or eukaryotic cells (e.g., bacterial cells, such as E. coli , insect cells (e.g., using baculovirus expression vectors), yeast cells, or mammalian cells). Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example, by using T7 promoter regulatory sequences and T7 polymerase.
- Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide.
- Such fusion vectors typically serve three purposes: (1) to increase expression of the recombinant polypeptide; (2) to increase the solubility of the recombinant polypeptide; and (3) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification.
- a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide. This enables separation of the recombinant polypeptide from the fusion moiety after purification of the fusion polypeptide.
- enzymes include Factor Xa, thrombin, and enterokinase.
- Exemplary fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs, Ipswich, Mass.), and pRITS (Pharmacia, Piscataway, N.J.), which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant polypeptide.
- GST glutathione S-transferase
- maltose E binding protein or protein A, respectively
- inducible, non-fusion E. coli expression vectors examples include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. 60-89 (1990)).
- Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
- Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident ⁇ , prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.
- One strategy to maximize recombinant polypeptide expression is to express the polypeptide in a host cell with an impaired capacity to proteolytically cleave the recombinant polypeptide (see Gottesman, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. 119-128 (1990)).
- Another strategy is to alter the nucleic acid sequence to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the host cell (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)). Such alteration of nucleic acid sequences can be carried out by standard DNA synthesis techniques.
- the host cell is a yeast cell.
- the expression vector is a yeast expression vector.
- yeast expression vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943 (1982)), pJRY88 (Schultz et al., Gene 54:113-123(1987)), pYES2 (Invitrogen Corporation, San Diego, Calif.), picZ (Invitrogen Corp, San Diego, Calif.), and pRS425 (Christianson et al., Gene 110:119-122 (1992)).
- a polypeptide described herein can be expressed in insect cells using baculovirus expression vectors.
- Baculovirus vectors available for expression of proteins in cultured insect cells include, for example, the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).
- the nucleic acids described herein can be expressed in mammalian cells using a mammalian expression vector.
- mammalian expression vectors include pCDM8 (Seed, Nature 329:840 (1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187-195 (1987)).
- the expression vector's control functions can be provided by viral regulatory elements.
- commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus 40.
- Vectors can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
- transformation and “transfection” refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in, for example, Sambrook et al. (supra).
- a gene that encodes a selectable marker e.g., resistance to antibiotics
- selectable markers include those that confer resistance to drugs, such as ampicillin, kanamycin, chloramphenicol, or tetracycline.
- Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a polypeptide described herein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
- a gene that encodes a selectable marker e.g., resistance to antibiotics
- Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin, and methotrexate.
- Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a polypeptide described herein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
- Transport proteins can export polypeptides (e.g., ester synthase) and organic compounds (e.g., fatty acids, fatty acid derivatives) out of a host cell.
- polypeptides e.g., ester synthase
- organic compounds e.g., fatty acids, fatty acid derivatives
- Many transport and efflux proteins serve to excrete a wide variety of compounds and can be naturally modified to be selective for particular types of hydrocarbons.
- Non-limiting examples of suitable transport proteins are ATP-Binding Cassette (ABC) transport proteins, efflux proteins, and fatty acid transporter proteins (FATP). Additional non-limiting examples of suitable transport proteins include the ABC transport proteins from organisms such as Caenorhabditis elegans, Arabidopsis thalania, Alkaligenes eutrophus , and Rhodococcus erythropolis . Exemplary ABC transport proteins that can be used include, without limitation, CER5, AtMRP5, AmiS2, AtPGP1, AcrA, AcrB, TolC, AcrE, AcrF, tll1618, tll1619, and tll0139.
- ABC transport proteins include, without limitation, CER5, AtMRP5, AmiS2, AtPGP1, AcrA, AcrB, TolC, AcrE, AcrF, tll1618, tll1619, and tll0139.
- Host cells can also be chosen for their endogenous ability to secrete organic compounds.
- the efficiency of organic compound production and secretion into the host cell environment e.g., culture medium, fermentation broth
- the efficiency of organic compound production and secretion into the host cell environment can be expressed as a ratio of intracellular product to extracellular product. In some examples, the ratio can be about 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5.
- the production and isolation of fatty acids and/or derivatives thereof produced according to the present disclosure can be enhanced by employing beneficial fermentation techniques.
- One method for maximizing production while reducing costs is increasing the percentage of the carbon source that is converted to hydrocarbon products.
- carbon is used in cellular functions, such as producing lipids, saccharides, proteins, organic acids, and nucleic acids. Reducing the amount of carbon necessary for growth-related activities can increase the efficiency of carbon source conversion to product. This can be achieved by, for example, first growing host cells to a desired density (for example, a density achieved at the peak of the log phase of growth). At such a point, replication checkpoint genes can be harnessed to stop the growth of cells. Specifically, quorum sensing mechanisms (reviewed in Camilli et al., Science 311:1113 (2006); Venturi, FEMS Microbio. Rev. 30:274-291 (2006); and Reading et al., FEMS Microbiol. Lett. 254:1-11 (2006)) can be used to activate checkpoint genes, such as p53, p21, or other checkpoint genes.
- quorum sensing mechanisms can be used to activate checkpoint genes, such as p53, p21, or other checkpoint genes.
- UmuDC genes Genes that can be activated to stop cell replication and growth in E. coli include umuDC genes.
- the overexpression of umuDC genes stops the progression from stationary phase to exponential growth (Murli et al., J. of Bact. 182:1127 (2000)).
- UmuC is a DNA polymerase that can carry out translesion synthesis over non-coding lesions the mechanistic basis of most UV and chemical mutagenesis.
- the umuDC gene products are involved in the process of translesion synthesis and also serve as a DNA sequence damage checkpoint.
- the umuDC gene products include UmuC, UmuD, umuD′, UmuD′ 2 C, UmuD′ 2 , and UmuD 2 .
- product-producing genes can be activated, thus minimizing the need for replication and maintenance pathways to be used while a fatty aldehyde is being made.
- Host cells can also be engineered to express umuC and umuD from E. coli in pBAD24 under the prpBCDE promoter system through de novo synthesis of this gene with the appropriate end-product production genes.
- the percentage of input carbons converted to fatty acids and/or fatty acid derivatives can be a cost driver.
- the more efficient the process is i.e., the higher the percentage of input carbons converted to fatty acids and/or fatty acid derivatives), the less expensive the process will be.
- oxygen-containing carbon sources e.g., glucose and other carbohydrate based sources
- the oxygen must be released in the form of carbon dioxide.
- a carbon atom is also released leading to a maximal theoretical metabolic efficiency of about 34% (w/w) (for fatty acid derived products). This figure, however, changes for other organic compounds and carbon sources. Typical efficiencies in the literature are about less than 5%.
- Host cells engineered to produce fatty acids and/or fatty acid derivatives can have greater than about 1, 3, 5, 10, 15, 20, 25, and 30% efficiency. In one example, host cells can exhibit an efficiency of about 10% to about 25%. In other examples, such host cells can exhibit an efficiency of about 25% to about 30%. In other examples, host cells can exhibit greater than 30% efficiency.
- the host cell can be additionally engineered to express recombinant cellulosomes, such as those described in PCT application number PCT/US2007/003736. These cellulosomes can allow the host cell to use cellulosic material as a carbon source.
- the host cell can be additionally engineered to express invertases (EC 3.2.1.26) so that sucrose can be used as a carbon source.
- the host cell can be engineered using the teachings described in U.S. Pat. Nos. 5,000,000; 5,028,539; 5,424,202; 5,482,846; and 5,602,030; so that the host cell can assimilate carbon efficiently and use cellulosic materials as carbon sources.
- the fermentation chamber can enclose a fermentation that is undergoing a continuous reduction.
- a stable reductive environment can be created.
- the electron balance can be maintained by the release of carbon dioxide (in gaseous form).
- Efforts to augment the NAD/H and NADP/H balance can also facilitate in stabilizing the electron balance.
- the availability of intracellular NADPH can also be enhanced by engineering the host cell to express an NADH:NADPH transhydrogenase.
- the expression of one or more NADH:NADPH transhydrogenases converts the NADH produced in glycolysis to NADPH, which can enhance the production of fatty acids and/or fatty acid derivatives.
- the engineered host cells can be grown in batches of, for example, about 100 mL, 500 mL, 1 L, 2 L, 5 L, or 10 L; fermented; and induced to express desired biosynthetic genes based on the specific genes encoded in the appropriate plasmids.
- the engineered host cells can be grown in batches of about 10 L, 100 L, 1000 L, 10,000 L, 100,000 L, 1,000,000 L or larger; fermented; and induced to express desired biosynthetic genes based on the specific genes encoded in the appropriate plasmids or incorporated into the host cell's genome.
- a suitable production host such as E. coli cells, harboring plasmids containing the desired biosynthetic genes or having the biosynthetic genes integrated in its chromosome can be incubated in a suitable reactor, for example a 1 L reactor, for 20 hours at 37° C. in an M9 medium supplemented with 2% glucose, carbenicillin, and chloramphenicol.
- a suitable reactor for example a 1 L reactor
- M9 medium supplemented with 2% glucose, carbenicillin, and chloramphenicol.
- the production host can be induced with IPTG to activate the engineered gene systems for fatty acid/fatty acid derivative production.
- the spent medium can be extracted and the organic phase can be examined for the presence of fatty acids/fatty acid derivatives using known detection methods such as, for example, GC-MS.
- aliquots of no more than about 10% of the total cell volume can be removed each hour and allowed to sit without agitation to allow the fatty acid esters to rise to the surface and undergo a spontaneous phase separation or precipitation.
- the fatty acid/fatty acid derivative component can then be collected, and the aqueous phase returned to the reaction chamber.
- the reaction chamber can be operated continuously. When the OD 600 drops below about 0.6, the cells can be replaced with a new batch grown from a seed culture.
- the methods disclosed herein are performed using glucose as a carbon source.
- microorganisms are grown in a culture medium containing an initial glucose concentration of about 2 g/L to about 50 g/L, such as about 5 g/L to about 20 g/L.
- the glucose concentration of the culture medium decreases from the initial glucose concentration as the microorganisms consume the glucose, and a concentration of about 0 g/L to about 5 g/L glucose is maintained in the culture medium during the fatty acid/fatty acid derivative production process.
- glucose is fed to the microorganisms in a solution of about 50% to about 65% glucose.
- the feed rate of glucose is set to match the cells' growth rate to avoid excess accumulation of glucose (i.e., >0% glucose) in the fermentor. In other instances, and a low concentration of excess glucose (e.g., about 2 g/L to about 5 g/L) is maintained.
- fatty acids and/or fatty acid derivatives can be produced from carbohydrates other than glucose, including but not limited to fructose, hydrolyzed sucrose, hydrolyzed molasses and glycerol.
- the fatty acids and/or fatty acid derivatives produced during fermentation can be separated from the fermentation media.
- Any known technique for separating fatty acids and/or fatty acid derivatives from aqueous media can be used.
- One exemplary separation process is a two phase (bi-phasic) separation process. This process involves fermenting the genetically engineered host cells under conditions sufficient to produce a fatty acid and/or fatty acid derivative, allowing the fatty acid and/or fatty acid derivative to collect in an organic phase, and separating the organic phase from the aqueous fermentation broth. This method can be practiced in both a batch and continuous fermentation processes.
- Bi-phasic separation uses the relative immiscibility of fatty acids and/or fatty acid derivatives to facilitate separation. Immiscible refers to the relative inability of a compound to dissolve in water and is defined by the compound's partition coefficient.
- Immiscible refers to the relative inability of a compound to dissolve in water and is defined by the compound's partition coefficient.
- the fatty acids and/or fatty acid derivatives produced by the methods described herein can be relatively immiscible in the fermentation broth, as well as in the cytoplasm. Therefore, the fatty acids and/or fatty acid derivative can collect in an organic phase either intracellularly or extracellularly. The collection of the products in the organic phase can lessen the impact of the fatty acid and/or fatty acid derivative on cellular function and can allow the host cell to produce more product.
- the methods described herein can result in the production of homogeneous compounds wherein at least about 60%, 70%, 80%, 90%, or 95% of the fatty acids and/or fatty acid derivatives produced will have carbon chain lengths that vary by less than about 8 carbons, less than about 6 carbons, less than about 4 carbons, or less than about 2 carbons. These compounds can also be produced with a relatively uniform degree of saturation. These compounds can be used directly as fuels, fuel additives, starting materials for production of other chemical compounds (e.g., polymers, surfactants, plastics, textiles, solvents, adhesives, etc.), or personal care additives. These compounds can also be used as feedstock for subsequent reactions, for example, hydrogenation, catalytic cracking (e.g., via hydrogenation, pyrolisis, or both), to make other products.
- chemical compounds e.g., polymers, surfactants, plastics, textiles, solvents, adhesives, etc.
- the fatty acids and/or fatty acid derivatives produced using methods described herein can contain between about 50% and about 90% carbon; or between about 5% and about 25% hydrogen. In other embodiments, the fatty acids and/or fatty acid derivatives produced using methods described herein can contain between about 65% and about 85% carbon; or between about 10% and about 15% hydrogen.
- Certain methods described herein also encompass a fatty acid or a derivative thereof produced using a purified ester synthase polypeptide, a variant or a fragment thereof described herein and a substrate, provided or produced by, for example, a method described herein.
- a host cell can be engineered to express a suitable ester synthase, a variant or a fragment thereof, as described herein.
- the host cell can be cultured under conditions that allow the expression of the ester synthase polypeptide.
- Cell free extracts can then be generated using known methods.
- the host cells can be lysed using detergents or by sonication.
- the expressed polypeptides can then be purified or substantially purified using known methods.
- substrates described herein can be provided to the cell free extracts and incubated under conditions that allow conversion of the substrates to desired fatty acids and/or fatty acid derivatives.
- the fatty acids and/or fatty acid derivatives can then be separated and purified using known techniques. Accordingly, fatty acids and/or fatty acid derivatives of the present invention can be produced extacellularly and isolated in vitro.
- Bioproducts comprising biologically produced organic compounds, particularly fatty acids and fatty acid derivatives biologically produced using the fatty acid biosynthetic pathway, have not been produced from renewable sources and, as such, are new compositions of matter. These new bioproducts can be distinguished from organic compounds derived from petrochemical carbon on the basis of dual carbon-isotopic fingerprinting or 14 C dating. Additionally, the specific source of biosourced carbon (e.g., glucose vs. glycerol) can be determined by dual carbon-isotopic fingerprinting (see, e.g., U.S. Pat. No. 7,169,588, which is herein incorporated by reference).
- biosourced carbon e.g., glucose vs. glycerol
- organic compounds or chemicals comprising both biologically-based and petroleum-based carbon isotope profiles may be distinguished from organic compounds and chemicals made only of petroleum-based materials.
- the instant materials may be followed in commerce on the basis of their unique carbon isotope profile.
- Bioproducts can be distinguished from petroleum-based organic compounds by comparing the stable carbon isotope ratio ( 13 C/ 12 C) in each fuel.
- the 13 C/ 12 C ratio in a given bioproduct is a consequence of the 13 C/ 12 C ratio in atmospheric carbon dioxide at the time the carbon dioxide is fixed. It also reflects the precise metabolic pathway. Regional variations also occur. Petroleum, C 3 plants (the broadleaf), C 4 plants (the grasses), and marine carbonates all show significant differences in 13 C/ 12 C and the corresponding ⁇ 13 C values. Furthermore, lipid matter of C 3 and C 4 plants analyze differently than materials derived from the carbohydrate components of the same plants as a consequence of the metabolic pathway.
- 13 C shows large variations due to isotopic fractionation effects, the most significant of which for bioproducts is the photosynthetic mechanism.
- the major cause of differences in the carbon isotope ratio in plants is closely associated with differences in the pathway of photosynthetic carbon metabolism in the plants, particularly the reaction occurring during the primary carboxylation (i.e., the initial fixation of atmospheric CO 2 ).
- Two large classes of vegetation are those that incorporate the “C 3 ” (or Calvin-Benson) photosynthetic cycle and those that incorporate the “C 4 ” (or Hatch-Slack) photosynthetic cycle.
- C 3 plants the primary CO 2 fixation or carboxylation reaction involves the enzyme ribulose-1,5-diphosphate carboxylase, and the first stable product is a 3-carbon compound.
- C 3 plants such as hardwoods and conifers, are dominant in the temperate climate zones.
- C 4 plants an additional carboxylation reaction involving another enzyme, phosphoenol-pyruvate carboxylase, is the primary carboxylation reaction.
- the first stable carbon compound is a 4-carbon acid that is subsequently decarboxylated.
- the CO 2 thus released is refixed by the C 3 cycle.
- Examples of C 4 plants are tropical grasses, corn, and sugar cane.
- Both C 4 and C 3 plants exhibit a range of 13 C/ 12 C isotopic ratios, but typical values are about ⁇ 7 to about ⁇ 13 per mil for C 4 plants and about ⁇ 19 to about ⁇ 27 per mil for C 3 plants (see, e.g., Stuiver et al., Radiocarbon 19:355, 1977). Coal and petroleum fall generally in this latter range.
- the 13 C measurement scale was originally defined by a zero set by Pee Dee Belemnite (PDB) limestone, where values are given in parts per thousand deviations from this material.
- PDB Pee Dee Belemnite
- the “ ⁇ 13 C” values are expressed in parts per thousand (per mil), abbreviated, ⁇ , and are calculated as follows:
- compositions described herein include bioproducts produced by any of the methods described herein.
- the bioproduct can have a ⁇ 13 C of about ⁇ 28 or greater, about ⁇ 27 or greater, ⁇ 20 or greater, ⁇ 18 or greater, ⁇ 15 or greater, ⁇ 13 or greater, ⁇ 10 or greater, or ⁇ 8 or greater.
- the bioproduct can have a ⁇ 13 C of about ⁇ 30 to about ⁇ 15, about ⁇ 27 to about ⁇ 19, about ⁇ 25 to about ⁇ 21, about ⁇ 15 to about ⁇ 5, about ⁇ 13 to about ⁇ 7, or about ⁇ 13 to about ⁇ 10.
- the bioproduct can have a ⁇ 13 C of about ⁇ 10, ⁇ 11, ⁇ 12, or ⁇ 12.3.
- Bioproducts can also be distinguished from petroleum-based organic compounds by comparing the amount of 14 C in each compound. Because 14 C has a nuclear half life of 5730 years, petroleum-based fuels containing “older” carbon can be distinguished from bioproducts which contain “newer” carbon (see, e.g., Currie, “Source Apportionment of Atmospheric Particles”, Characterization of Environmental Particles , J. Buffle and H. P. van Leeuwen, Eds., 1 of Vol. I of the IUPAC Environmental Analytical Chemistry Series (Lewis Publishers, Inc) (1992) 3-74).
- the fundamental definition relates to 0.95 times the 14 C/ 12 C isotope ratio HOxI (referenced to AD 1950). This is roughly equivalent to decay-corrected pre-Industrial Revolution wood.
- f M is about 1.1.
- the invention provides a bioproduct comprising a fatty acid and/or a fatty acid derivative produced according to the methods herein, which can have an f M 14 C of at least about 1.
- the bioproduct can have an f M 14 C of at least about 1.01, an f M 14 C of about 1 to about 1.5, an f M 14 C of about 1.04 to about 1.18, or an f M 14 C of about 1.111 to about 1.124.
- a biologically-based carbon content is derived by assigning “100%” equal to 107.5 pMC and “0%” equal to 0 pMC. For example, a sample measuring 99 pMC will give an equivalent biologically based carbon content of 93%. This value is referred to as the mean biologically based carbon result and assumes all the components within the analyzed material originated either from present day biological material or petroleum-based material.
- a bioproduct comprising a fatty acid and/or fatty acid derivative produced in accordance with the methods described herein can have a pMC of at least about 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100.
- a fatty acid and/or fatty acid derivative described herein can have a pMC of between about 50 and about 100; about 60 and about 100; about 70 and about 100; about 80 and about 100; about 85 and about 100; about 87 and about 98; or about 90 and about 95.
- a bioproduct comprising a fatty acid and/or fatty acid derivative can have a pMC of about 90, 91, 92, 93, 94, or 94.2.
- the fatty acids and/or derivatives thereof produced by the methods described herein can be used as biofuels.
- the fatty acids and/or derivatives thereof as described herein can be used solely or as a component of biodiesel.
- the fatty acids and/or derivatives thereof produced according to the methods herein can also be used as, or as components or feedstocks of, various industrial chemicals, including without limitation, fuel additives.
- the resulting biofuel or industrial chemicals are bioproducts and will therefore have a ⁇ 13 C of about ⁇ 28 or greater, about ⁇ 27 or greater, ⁇ 20 or greater, ⁇ 18 or greater, ⁇ 15 or greater, ⁇ 13 or greater, ⁇ 10 or greater, or ⁇ 8 or greater.
- the bioproduct can have a ⁇ 13 C of about ⁇ 30 to about ⁇ 15, about ⁇ 27 to about ⁇ 19, about ⁇ 25 to about ⁇ 21, about ⁇ 15 to about ⁇ 5, about ⁇ 13 to about ⁇ 7, or about ⁇ 13 to about ⁇ 10.
- the resulting biofuel or industrial chemicals will have an f M 14 C of at least about 1.
- the bioproduct can have an f M 14 C of at least about 1.01, an f M 14 C of about 1 to about 1.5, an f M 14 C of about 1.04 to about 1.18, or an f M 14 C of about 1.111 to about 1.124.
- fatty acids and/or derivatives thereof produced according to the methods and compositions herein possess various advantageous characteristics for use as a fuel.
- One of ordinary skill in the art will appreciate that, depending upon the intended purpose of the fuel, different fatty acids and/or derivatives thereof may have advantages as compared to others fatty acids and/or derivatives.
- branched fatty acids and/or derivatives thereof may be more desirable as automobile fuels or components of automobile fuels that are intended for uses in cold climates.
- fuels comprising relatively homogeneous fatty acids and/or derivatives thereof that, at the same time, have the desired characteristics and qualities can be produced.
- Such fatty acid- and/or fatty acid derivative-based fuels can be characterized by carbon fingerprinting, and their lack of impurities, when compared to petroleum-derived fuels or biodiesel derived from triglyceride, is also advantageous.
- the fatty acid- and/or fatty acid derivative-based fuels can be combined with other fuels or fuel additives to produce alternative fuel compositions that have the desired properties.
- the production hosts and methods disclosed herein can be used to produce free fatty acids and/or various fatty acid derivatives. In some embodiments, the production hosts and methods disclosed herein can be used to produce a higher and/or improved titer or yield of free fatty acids and/or fatty acid derivatives, including, for example, fatty esters. In some embodiments, the percentage of free fatty acids in the product produced by the production host is at least about 1%, for example, at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25%.
- the percentage of fatty acid derivatives in the product produced by the production host is at least about 50%, for example, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In some embodiments, the ratio of fatty acid derivative to free fatty acids in the product produced by the production host is about 10:1, 9:1, 8:1, 7:1, 5:1, 2:1, or 1:1.
- the fatty acid derivative produced by the production host is a fatty acid ethyl derivative.
- An exemplary fatty acid ethyl derivative is a fatty ethyl ester.
- the fatty ethyl ester is an ethyl dodecanoate, ethyl tridecanoate, ethyl tetradecanoate, ethyl pentadecanoate, ethyl cis-9-hexadecenoate, ethyl hexadecanoate, ethyl heptadecanoate, ethyl cis-11-octadecenoate, or ethyl octadecanoate ester.
- the fatty acid derivative product that is produced by the production host is a combination of a variety of fatty acid derivatives.
- the fatty acid derivative product comprises a mixture of any combination of an ethyl dodecanoate, ethyl tridecanoate, ethyl tetradecanoate, ethyl pentadecanoate, ethyl cis-9-hexadecenoate, ethyl hexadecanoate, ethyl heptadecanoate, ethyl cis-11-octadecenoate, and ethyl octadecanoate ester.
- the fatty acid derivative produced is a fatty methyl derivative.
- An exemplary fatty methyl derivative if s fatty methyl ester.
- the fatty methyl ester produced by the production host is a methyl dedecanoate, methyl tridecanoate, methyl tetradecanoate, methyl pentadecanoate, methyl cis-9-hexadecenoate, methyl hexadecanoate, methyl heptadecanoate, methyl cis-11-octadecenoate, or methyl octadecanoate.
- the fatty acid derivative product produced by the production host is a combination of a variety of fatty acid derivative products.
- the fatty acid derivative is a mixture of one or more of methyl dedecanoate, methyl tridecanoate, methyl tetradecanoate, methyl pentadecanoate, methyl cis-9-hexadecenoate, methyl hexadecanoate, methyl heptadecanoate, methyl cis-11-octadecenoate, and methyl octadecanoate.
- the fatty acid derivative is a mixture of one or more fatty methyl esters and one or more fatty ethyl esters.
- the product is a mixture of one or more free fatty acids, and/or one or more fatty acid derivatives. In yet further embodiments, the product is a mixture of one or more free fatty acids, and/or one or more fatty methyl esters, and/or one or more fatty ethyl esters.
- the production hosts and methods disclosed herein can be used to produce different proportions of free fatty acids and fatty acid derivatives.
- the proportion of free fatty acids in the product can be modified according to the methods, compositions, vectors and cells described herein such that the proportion is higher or lower vs. the fatty acid derivatives that are produced.
- the proportion of fatty acid derivatives in the product can also be modified according to the disclosures herein, such that the proportion is higher or lower vs. the other products, for example, the free fatty acids, that are produced.
- the proportional yield of fatty acid derivative with certain carbon chain lengths can be increased or decreased.
- proportional yield refers to the amount of a desired product in relation to other products that are within the same mixture produced by a recombinant host of the present invention.
- the proportional yield of a desired product can be improved such that it is more predominant over the other components in the product mixture to reduce the burden of purification.
- the proportional yield of an undesired product i.e., a component that will need to be removed from the desired product
- Biofuels made according to the methods and compositions described herein have not heretofore been produced from renewable sources and are new compositions of matter. These new fuels can be distinguished from fuels derived from petrochemical carbon on the basis of dual carbon-isotopic fingerprinting. Additionally, the specific source of biosourced carbon (e.g., glucose vs. glycerol) can be determined by dual carbon-isotopic fingerprinting (see U.S. Pat. No. 7,169,588, which is herein incorporated by reference in its entirety, in particular, at col. 4, line 31, to col. 6, line 8).
- biosourced carbon e.g., glucose vs. glycerol
- the fatty acids and/or fatty acid derivatives and the associated biofuels, chemicals, and mixtures can be distinguished from their petrochemical derived counterparts on the basis of 14 C (f M ) and dual carbon-isotopic fingerprinting.
- the fatty acids and/or derivatives thereof as described herein have utility in the production of biofuels and chemicals.
- the products provided by the instant invention can be distinguished on the basis of dual carbon-isotopic fingerprinting from those materials derived solely from petrochemical sources. The ability to distinguish these products is beneficial in tracking these materials in commerce. For example, fuels or chemicals comprising both “new” and “old” carbon isotope profiles can be distinguished from fuels and chemicals made only of “old” materials. Thus, the instant materials can be followed or “tracked” in commerce or identified in commerce as a biofuel on the basis of their unique profile. In addition, other competing materials can be identified as being biologically derived or derived from a petrochemical source.
- a biofuel composition which includes a fatty acid derivative having ⁇ 13 C of from about ⁇ 10.9 to about ⁇ 15.4, wherein the fatty acid derivative accounts for at least about 85% of biosourced material (i.e., derived from a renewable resource such as, for example, cellulosic materials and sugars) in the composition.
- biosourced material i.e., derived from a renewable resource such as, for example, cellulosic materials and sugars
- the biofuel composition includes a fatty acid derivative having the formula:
- X CH 3 , —CH 2 OR 1 ; —C(O)OR 2 ; or —C(O)NR 3 R 4 ;
- R for each n, independently absent, an H, or a lower aliphatic
- n an integer from about 8 to about 34, preferably an integer from about 10 to about 24;
- R 1 , R 2 , R 3 , R 4 independently selected from an H or a lower alkyl.
- R represents a branched, unbranched or cyclic lower alkyl or lower alkenyl moiety.
- exemplary R groups include, without limitation, methyl, isopropyl, isobutyl, sec-butyl, cyclopentenyl, and the like.
- the fatty acid derivative is additionally characterized as having a ⁇ 13 C of from about ⁇ 10.9 to about ⁇ 15.4, and the fatty acid derivative accounts for at least about 85% of biosourced material in the composition.
- the fatty acid derivative in the biofuel composition is characterized by having a fraction of modern carbon (f M 14 C) of at least about 1.003, 1.010, or 1.5.
- fatty acids and/or derivatives thereof produced in accordance with the disclosures herein are useful as components or feedstocks for making biofuels as well as other industrial chemicals. These products are made directly from suitable substrates and not from the chemical processing of triglycerides. Accordingly, fuels and other industrial chemicals comprising the disclosed fatty acids and/or derivatives often contain fewer impurities than are normally associated with, for example, products derived from triglycerides such as fuels derived from vegetable oils and fats.
- the crude biofuels prepared in accordance with the disclosures herein contain less transesterification catalysts than petroleum-based diesel or other biodiesel produced via one or more transesterification steps.
- the biofuel can contain less than about 2.0 wt. %, for example, less than about 1.5, 1.0, 0.5, 0.3, 0.1, 0.05, or 0 wt. % of a transesterification catalyst or an impurity resulting from a transesterification catalyst.
- transesterification catalysts include, without limitation, hydroxide catalysts, such as NaOH, KOH, and LiOH; and acidic catalysts, such as mineral acid catalysts and Lewis acid catalysts.
- Non-limiting examples of catalysts and impurities resulting from transesterification catalysts include tin, lead, mercury, cadmium, zinc, titanium, zirconium, hafnium, boron, aluminum, phosphorus, arsenic, antimony, bismuth, calcium, magnesium, strontium, uranium, potassium, sodium, and lithium.
- the biofuels prepared in accordance with the disclosures herein tend to have a low gelling point, especially when the fatty acid and/or fatty acid derivative product comprises a C 16:1 ethyl ester or a C 18:1 ethyl ester, as compared to the gelling points of other types of biofuels.
- the crude biofuels prepared in accordance with the disclosures herein contain less glycerol (or glycerin) than biofuels made from triglycerides.
- the biofuels can contain less than about 2.0 wt. %, for example, less than about 1.5, 1.0, 0.5, 0.3, 0.1, 0.05, or 0 wt. % of glycerol.
- Crude biofuels herein also contain less free alcohol(s) (e.g., alcohols that are used to create the ester) than biodiesels made from triglycerides. This is due in part to the efficiency of utilization of the alcohols by the production hosts of the present disclosure.
- the biofuels can contain less than about 2.0, 1.5, 1.0, 0.5, 0.3, 0.1, 0.05, or 0 wt. % of free alcohol.
- the biofuels herein can be additionally characterized by its low concentration of sulfur as compared to petroleum-derived diesel.
- the biofuel can have less than about 2.0 wt. %, for example, less than about 1.5, 1.0, 0.5, 0.3, 0.1, 0.05, or 0 wt. % of sulfur.
- Fuel additives are used to enhance the performance of a fuel or engine.
- fuel additives can be used to alter the freezing/gelling points, cloud points, lubricity, viscosity, oxidative stability, ignition quality, octane levels, and flash points.
- all fuel additives must be registered with Environmental Protection Agency.
- the names of fuel additives and the companies that sell the fuel additives are publicly available by contacting the EPA or by viewing the agency's website.
- the fatty acids and/or fatty acid derivatives described herein can be mixed with one or more fuel additives to impart a desired quality.
- the fatty acids and/or derivatives thereof described herein can be formulated or processed into suitable fuel additives, which enhance the performance of fuels or engines.
- the fatty acids and/or derivatives described herein can be formulated into lubricity improvers, which impart desirable properties such as wear protection to the engine parts.
- additive compositions comprising the fatty acids and/or derivatives thereof produced in accordance with the disclosures herein are provided.
- the fatty acids and/or fatty acid derivatives described herein can be formulated into corrosion inhibitors.
- the fatty acids and/or fatty acid derivatives described herein can be mixed with other fuels such as one or more biodiesels derived from triglycerides, various alcohols such as ethanol and butanol, and petroleum-derived products such as gasoline or diesel. Under certain circumstances, a fatty acid and/or fatty acid derivative with a low gelling point, such as a C 16:1 ethyl ester or a C 18:1 ethyl ester, is produced. This low gelling point product can be mixed with one or more biodiesels made from triglycerides to reduce gelling point of the resulting fuel when compared to a fuel containing only the one or more biodiesels made from triglycerides.
- fuels such as one or more biodiesels derived from triglycerides, various alcohols such as ethanol and butanol, and petroleum-derived products such as gasoline or diesel.
- a fatty acid and/or fatty acid derivative with a low gelling point such as a C 16
- a fatty acid derivative such as a C 16:1 ethyl ester or a C 18:1 ethyl ester
- a petroleum-derived diesel can be mixed with a petroleum-derived diesel to provide a mixture that contains at least about, and often greater than about, 5% by weight of biodiesel.
- the fuel mixture includes at least about 10%, 15%, 20%, 30%, 40%, 50%, and 60% by weight of the fatty acid derivative.
- the fuel composition can further comprise a synthetic fuel.
- a synthetic fuel Any synthetic fuel obtained from coal, natural gas, or biomass can be suitably used.
- the synthetic fuel comprises a Fischer-Tropsch based fuel, a Bergius-based fuel, a Mobil-based fuel, a Karrick-based fuel, or a combination thereof.
- the synthetic fuel comprises a Coal-To-Liquids based fuel (CTL-based fuel), a Gas-To-Liquids based fuel (GTL-based fuel), a Biomass-To-Liquids based fuel (BTL-based fuel), a Coal and Biomass-To-Liquids based fuel (CBTL-based fuel), or a combination thereof.
- CTL-based fuel Coal-To-Liquids based fuel
- GTL-based fuel Gas-To-Liquids based fuel
- BTL-based fuel Biomass-To-Liquids based fuel
- CBTL-based fuel Coal and Biomass-To-Liquids based fuel
- the synthetic fuel comprises a Fischer-Tropsch-based fuel.
- the amount of synthetic fuel in the fuel composition disclosed herein may be from about 5% to about 90%, from about 5% to about 80%, from about 5% to about 70%, from about 5% to about 60%, or from about 5% to about 50%.
- a biofuel composition can be made that includes at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% of a fatty acid derivative that includes a carbon chain that is 8:0, 10:0, 12:0, 14:0, 14:1, 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:0, 20:1, 20:2, 20:3, 22:0, 22:1 or 22:3.
- Such biofuel compositions can additionally include at least one additive selected from a cloud point lowering additive that can lower the cloud point to less than about 5° C., or less than about 0° C.; a surfactant; a microemulsion; at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% diesel fuel from triglycerides; a petroleum-derived gasoline; or a diesel fuel from petroleum.
- a cloud point lowering additive that can lower the cloud point to less than about 5° C., or less than about 0° C.
- a surfactant a microemulsion
- a microemulsion at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% diesel fuel from triglycerides
- a petroleum-derived gasoline or a diesel fuel from petroleum.
- the fuel composition comprising fatty acids and/or derivatives thereof such as fatty esters produced in accordance with the methods, vectors, cells and compositions herein further comprises one or more diesel fuel additives.
- Suitable additives are desirably those that afford improved performance but also compatibility with the components in the fuel composition and devices that are typically associated with diesel engines.
- suitable fuel additives include ignition improvers or cetane number improvers, detergents, dispersants, antiwear agents, viscosity index modifiers, friction modifiers, lubricity improvers, stabilizers, antioxidants, corrosion inhibitors, biocides, metal deactivators, and minor amounts of other optional additives, including, without limitation, antifoaming agents and seal fixes.
- ignition improvers or cetane number improvers are often added to improve diesel engine performance.
- exemplary cetane number improvers include 2′-ethylhexyl nitrate, and other alkyl nitrates.
- Cetane number improvers can be added to a fuel composition in an amount that is about 0.01 wt. % to about 1.0 wt. %, e.g., about 0.05 wt. % to about 0.5 wt. %, based on the total weight of the fuel composition.
- various detergents and/or dispersants can be included in the fuel composition comprising the fatty acids and/or fatty acid derivatives produced in accordance with the present disclosures to associate and disperse or remove harmful deposits from diesel engine parts.
- Suitable detergents typically comprise a polar head comprising a metal salt of an acidic organic compound and a long hydrophobic tail.
- Exemplary detergents include borated carbonate salts, borated sulfonate salts, which are preferably overbased. See, e.g., U.S. Pat. Nos. 4,744,920, 4,965,003, the disclosures of which are incorporated herein.
- Exemplary dispersants include, without limitation, carboxylic dispersants, succinimide dispersants, amine dispersants, and Mannich dispersants. See, e.g., U.S. Pat. Nos. 3,172,892, 3,438,757, 3,980,569, and 6,165,235, the disclosures of which are incorporated by reference herein. Dispersants can be present in the fuel composition in an amount of about 0.01 wt. % to about 0.1 wt. %, e.g., 0.03 to about 0.05 wt. %, based on the total weight of the fuel composition.
- antiwear agents including for example, dihydrocarbyl dithiophosphate metal salts
- the amount of lubricity improver in the fuel composition can range from about 1 ppm to about 50,000 ppm, for example, about 10 ppm to about 20,000 ppm, or about 25 ppm to about 10,000 ppm.
- lubricity improvers include esters and fatty acids, which may or may not be the same as those produced in accordance to the methods described herein.
- the amount of stabilizers which improves the storage stability of the fuel composition, can range from about 0.001 wt. % to about 2 wt. %, e.g., about 0.01 wt. % to about 1 wt. %, based on the total weight of the fuel composition.
- An exemplary stabilizer is a tertiary alkyl primary amine.
- Antioxidants prevent the formation of gum depositions on fuel system components due to oxidation of the fuels in storage and/or inhibit the formation of peroxide compounds in fuel compositions.
- the amount of antioxidants can range from about 0.001 wt. % to about 5 wt. %, e.g., from about 0.01 wt. % to about 1 wt. %, based on the total weight of the fuel composition.
- Corrosion inhibitors protect ferrous metals in fuel handling systems, such as pipelines and storage tanks, from corrosion. Certain corrosion inhibitors are also known to impart additional lubricity, and as such are particularly suitable when additional lubricity is desired.
- the corrosion inhibitor may be present in the fuel composition in an amount of about 0.001 wt. % to about 5 wt. %, e.g., from about 0.01 wt. % to about 1 wt. %, based on the total weight of the fuel composition.
- Biocides are used to combat microbial growth in the fuel composition, which may be present in the fuel composition at a concentration of about 0.001 wt. % to about 5 wt. %, e.g., from about 0.01 wt. % to about 1 wt. %, based on the total weight of the fuel composition.
- Metal deactivators suppress the catalytic effects of some metals, particularly copper, have on fuel oxidation, which can be present in the fuel composition in an amount of about 0.001 wt. % to about 5 wt. %, e.g., at 0.01 wt. % to about 1 wt. %, based on the total weight of the fuel composition.
- viscosity improvers which are typically polymeric materials of number average molecular weights of from about 5,000 to about 250,000
- friction modifiers which are typically sulfur-containing organo-molybdenum compounds
- Foam inhibitors which typically include alkyl methacrylate polymers or dimethyl silicon polymers, can also be added to the fuel composition in an amount of less than about 10 ppm.
- seal fixes can be added to insure proper elastomer sealing and prevent premature seal failure can be included in the fuel composition.
- This example describes the construction of a genetically engineered microorganism wherein the expression of a fatty acid degradation enzyme is attenuated.
- E. coli MG1655 an E. coli K strain
- Lambda Red also known as the Red-Driven Integration
- Del-fadE-F (SEQ ID NO: 1) 5′-AAAAACAGCAACAATGTGAGCTTTGTTGTAATTATATTGTAA ACATATTGATTCCGGGGATCCGTCGACC; and Del-fadE-R (SEQ ID NO: 2) 5′-AAACGGAGCCTTTCGGCTCCGTTATTCATTTACGCGGCTTCA ACTTTCCTGTAGGCTGGAGCTGCTTC
- the Del-fadE-F and Del-fadE-R primers were used to amplify the kanamycin resistance (Km R ) cassette from plasmid pKD13 (described by Datsenko et al., supra) by PCR.
- the PCR product was then used to transform electrocompetent E. coli MG1655 cells containing pKD46 (described in Datsenko et al., supra) that had been previously induced with arabinose for 3-4 hours. Following a 3-hour outgrowth in a super optimal broth with catabolite repression (SOC) medium at 37° C., the cells were plated on Luria agar plates containing 50 ⁇ g/mL of Kanamycin.
- SOC catabolite repression
- Resistant colonies were identified and isolated after an overnight incubation at 37° C. Disruption of the fadE gene was confirmed by PCR amplification using primers fadE-L2 and fadE-R1, which were designed to flank the E. coli fadE gene.
- the fadE deletion confirmation primers were:
- fadE-L2 (SEQ ID NO: 3) 5′-CGGGCAGGTGCTATGACCAGGAC; and fadE-R1 (SEQ ID NO: 4) 5′-CGCGGCGTTGACCGGCAGCCTGG
- E. coli MG1655 ⁇ fadE E. coli MG 1655 D1.
- This example describes the construction of a genetically engineered microorganism in which the expression of a fatty acid degradation enzyme and an outer membrane protein receptor are attenuated.
- Del-fhuA-F (SEQ ID NO: 5) 5′-ATCATTCTCGTTTACGTTATCATTCACTTTACATCAGAGA TATACCAATGATTCCGGGGATCCGTCGACC; and Del-fhuA-R (SEQ ID NO: 6) 5′-GCACGGAAATCCGTGCCCCAAAAGAGAAATTAGAAACGGA AGGTTGCGGTTGTAGGCTGGAGCTGCTTC
- the Del-fhuA-F and Del-fhuA-R primers were used to amplify the kanamycin resistance (Km R ) cassette from plasmid pKD13 by PCR.
- the PCR product obtained was used to transform electrocompetent E. coli MG1655 D1 cells containing pKD46 (see Example 1). These cells had been previously induced with arabinose for about 3-4 h. Following a 3-h outgrowth in SOC medium at 37° C., the cells were plated on Luria agar plates containing 50 ⁇ g/mL of kanamycin. Resistant colonies were identified and isolated after an overnight incubation at 37° C. Disruption of the fhuA gene was confirmed in some of the colonies by PCR amplification using primers flanking the E. coli fhuA gene:
- fhuA-verF (SEQ ID NO: 7) 5′-CAACAGCAACCTGCTCAGCAA; and fhuA-verR (SEQ ID NO: 8) 5′-AAGCTGGAGCAGCAAAGCGTT
- E. coli MG1655 ⁇ fadE_ ⁇ fhuA was named E. coli MG1655 DV2.
- This example describes the construction of a genetically engineered microorganism in which the expression of an acyl-CoA dehydrogenase, an outer membrane protein receptor, a pyruvate formate lyase and a lactate dehydrogenase are attenuated.
- E. coli MG1655 which encodes a pyruvate formate lyase (GenBank Accession No. AAC73989) was deleted from E. coli MG1655 DV2 (see, Example 2) using the Lamda Red System according to Datsenko et al., supra, but with the following modifications:
- the primers used to create the deletion strain were:
- Del-pflB-F (SEQ ID NO: 9) 5′-GCCGCAGCCTGATGGACAAAGCGTTCATTATGGTGCTGCCGGTC GCGATGATTCCGGGGATCCGTCGACC Del-pflB-R: (SEQ ID NO: 10) 5′-ATCTTCAACGGTAACTTCTTTACCGCCATGCGTGTCCCAGGTGT CTGTAGGCTGGAGCTGCTTCG
- the Del-pflB-F and Del-pflB-R primers were used to amplify the kanamycin resistance (Km R ) cassette from plasmid pKD13 by PCR.
- the PCR product was then used to transform electrocompetent E. coli MG1655 DV2 cells (see Example 2).
- the ldhA gene of E. coli MG1655 which encodes a lactate dehydrogenase, specifically an NAD-linked fermentative D-lactate dehydrogenase (see, e.g., Mat-Jan et al., J. Bacteriol. 171(1):342-8 (1989); Bunch et al., Microbiol. 143(1):187-95 (1997) (GenBank Accession No. AAC74462) was also deleted from E. coli MG1655 DV2 (see, Example 2) using the Lambda Red System according to Datsenko et al., supra, but with the following modifications.
- Del-ldhA-F (SEQ ID NO: 11) 5′-CTCCCCTGGAATGCAGGGGAGCGGCAAGATTAAACCAGTTCGTTC GGGCAGTGTAGGCTGGAGCTGCTTCG-3′
- Del-ldhA-R (SEQ ID NO: 12) 5′-TATTTTTAGTAGCTTAAATGTGATTCAACATCACTGGAGAAAGTC TTATGCATATGAATATCCTCCTTAGTTCC-3′
- the Del-ldhA-F and Del-ldhA-R primers were used to amplify the chloramphenicol acetyltransferase resistance (Cm R ) cassette from plasmid pKD3 (see, Datsenko et al., supra) by PCR.
- the PCR product was also used to transform electrocompetent E. coli MG1655 DV2 cells (see, Example 2).
- the E. coli MG1655 DV2 (see Example 2) cells had been previously induced with arabinose for about 3-4 h. Following a 3-h outgrowth in SOC medium at 37° C., the cells were plated on Luria agar plates containing 50 ⁇ g/mL of kanamycin and 30 ⁇ g/mL chloroamphenicol. Colonies that were resistant to both kanamycin and chlroamphenicol were identified and isolated after an overnight incubation at 37° C. Disruption of the pflB gene was confirmed using primers flanking the E. coli pflB gene, and disruption of the ldhA gene was verified using primers flanking the E. coli ldhA gene.
- pflB-verF (SEQ ID NO: 14) 5′-GGACTAAACGTCCTACAAAC
- PflB-verR (SEQ ID NO: 15) 5′-TTCATCTGTTTGAGATCGAG
- ldhA-verF (SEQ ID NO: 16) 5′-CCCGAGCGGTAGCCAGATGCCCGCCAGCG ldhA-verR: (SEQ ID NO: 17) 5′-GCTGCGGGTTAGCGCACATCATACGGGTC
- E. coli MG1655 ⁇ fadE_ ⁇ fhuA_ ⁇ pflB_ ⁇ ldhA was named E. coli MG1655 DV4.
- This example describes the construction of a genetically engineered microorganism in which the expression of a fatty acid degradation enzyme such as an acyl-CoA synthase, an outer membrane protein receptor, and an acyl-CoA dehydrogenase are attenuated.
- a fatty acid degradation enzyme such as an acyl-CoA synthase, an outer membrane protein receptor, and an acyl-CoA dehydrogenase are attenuated.
- E. coli MG1655 The fadD gene of E. coli MG1655 was deleted from strain E. coli MG1655 DV2 (see, Example 2), using the Lambda Red system described by Datsenko et al, supra, but with the following modifications:
- the primers used to create the deletion of fadD were:
- fad1 (SEQ ID NO: 19) 5′-TAACCGGCGTCTGACGACTGACTTAACGCTCAGGCTTTATT GTCCACTTTGTGTAGGCTGGAGCTGCTTCG-3′; and fad2: (SEQ ID NO: 20) 5′-ATTTGGGGTTGCGATGACGACGAACACGCATTTTAGAGGTG AAGAATTGCATATGAATATCCTCCTTTAGTTCC-3′
- the fad1 and fad2 primers were used to amplify the chloramphenicol acetyltransferase resistance (Cm R ) casette from plasmid pKD3 (described by Datsenko et al., supra) by PCR.
- the PCR product was used to transform electrocompetent E. coli MG1655 DV2 (see, Example 2).
- the transformed cells were plated on Luria agar plates containing 30 ⁇ g/mL of chloramphenicol and grown overnight at 37° C. Individual colonies were isolated, transferred to fresh Luria agar plates, and grown at 42° C.
- fadF (SEQ ID NO: 21) 5′-CGTCCGTGGTAATCATTTGG-3′; and fadR: (SEQ ID NO: 31) 5′-TCGCAACCTTTTCGTTGG-3′
- the Cm R marker was removed using a FLP helper plasmid as described by Datsenko et al., supra.
- the resulting MG1655 E. coli strain was named E. coli MG1655 ⁇ fadE ⁇ fhuA ⁇ fadD, or E. coli MG1655 DV2 ⁇ fadD.
- the DV2 ⁇ fadD strain was unable to grow on M9+ oleate agar plates (which supply oleate as carbon source). It was also unable to grow in M9+ oleate liquid media.
- This example describes the construction of a bacterial expression plasmid, pDS33.ES9, ( FIG. 3 ) in which expression of an ester synthase gene from M. hydrocarbonoclasticus DSM 8798 (GenBank Accession No. AB021021) is under the control of the E. coli spc ribosomal protein operon promoter Pspc (SEQ ID NO:13, FIG. 4 ).
- the Pspc promoter as shown in FIG. 2 , was amplified from E. coli MG1655 chromosomal DNA with the following primers:
- PspcIFF SEQ ID NO: 32
- PspcIFR SEQ ID NO: 33
- Plasmid pDS23 does not contain the lacI q , lacZ, or Trc DNA.
- ES9 ester synthase from M. hydrocarbonoclasticus DSM 8798 (GenBank Accession No. AB021021), referred to as ES9 herein, was synthesized by DNA2.0 (Menlo Park, Calif.). This synthesized ES9 ester synthase gene was subcloned into pColaDuet-1 to form plasmid pHZ1.97-ES9. The internal BspHI site in the ES9 ester synthase gene was removed by site directed mutagenesis using the QuikChange® Multi Kit (Stratagene, Carlsbad, Calif.) and the following mutagenic primer:
- ES9BspF (SEQ ID NO: 36) 5′-CCCAGATCAGTTTTATGATTGCCTCGCTGG-3′
- This primer introduced a silent mutation into the ES9 ester synthase gene.
- the resulting plasmid named pDS32, then was used as a template to amplify the ES9 ester synthase gene with the following primers:
- ES9BspHF (SEQ ID NO: 37) 5′-ATCATGAAACGTCTCGGAAC-3′; and ES9XhoR (SEQ ID NO: 38) 5′-CCTCGAGTTACTTGCGGGTTCGGGCGCG-3′
- pDS33.ES9 SEQ ID NO:22.
- the sequence of pDS33.ES9 is shown in FIG. 3 with the residues representing the Pspc promoter in italics and the residues of the ES9 ester synthase gene in bold type.
- This example describes the construction of a bacterial expression plasmid, pDS57 (SEQ ID NO:23) in which expression of an ester synthase gene is under the control of the Ptrc promoter.
- the ES9 ester synthase gene was amplified from plasmid pDS33.ES9 (SEQ ID NO:22, Example 5) using primers ES9BspHF (SEQ ID NO:37) and ES9XhoR (SEQ ID NO:38).
- the PCR product was digested with BspHI and XhoI and ligated into NcoI/XhoI restricted OP80 to form plasmid pDS57 (SEQ ID NO:23), wherein the ES9 ester synthase gene is under control of the Trc promoter.
- SEQ ID NO:23 The sequence of pDS57 is shown in FIG. 4 with the Trc promoter in italics and the ES9 ester synthase gene in bold type.
- This example illustrates that expression of ester synthase alone, without co-expression of an acyl-CoA synthase or a thioesterase, can be used to produce fatty esters in vivo.
- a pCL plasmid containing no insert negative control
- a plasmid containing E. coli thioesterase 'tesA (3) a plasmid containing a polynucleotide encoding Alcanivorax borkumensis SK2 ester synthase atfA1(GenBank Accession No. YP_694462); (4) a plasmid containing a polynucleotide encoding Alcanivorax borkumensis SK2 ester synthase atfA2 (GenBank Accession No.
- YP_693524 (5) a plasmid containing a polynucleotide encoding Marinobacter hydrocarbonoclasticus DSM 8798 ester synthase ES8 (GenBank Accession No. ABO21020); and (6) a plasmid containing a polynucleotide encoding Marinobacter hydrocarbonoclasticus DSM 8798 ester synthase ES9 (GenBank Accession No. ABO21021) were grown in 24-well plates in LB broth supplemented with 100 ⁇ g/mL spectinomycin.
- the ester synthase plasmids were constructed as described in Example 5 to generate plasmids pDS41.S (containing atfA1 gene), pDS31atfa2 (containing atfA2 gene), pDS31.ES8 (containing the gene encoding ES8), and pDS33.ES9 (containing the gene encoding ES9).
- the cultures were diluted 1:25 in Che-9 2N-BT (2% glucose, nitrogen limited medium, 0.2 M Bis-Tris, pH 7.0, 0.1% Triton) containing spectinomycin and grown overnight.
- the cultures were diluted in 4N-BT (4% glucose, nitrogen limited medium, 0.2M Bis-Tris, pH 7.0, 0.1% Triton) to a final OD600 (optical density at 600 nm) of about 0.2.
- IPTG was added to a final concentration of 1 mM, along with either water or ethanol (2% (v/v)).
- ES9 generated nearly 200 mg/L FAEE in the presence of ethanol, but only a small quantity ( ⁇ 20%) of FFA.
- the other ester synthases including ES8, atfA1 and atfA2, produced substantially lower amounts of FAEE in the presence of ethanol, accompanied by a higher proportion of FFA.
- the culture E. coli 'tesA produced higher overall titers of FFA and FAEE in the presence of ethanol, but the amount of FFA was significantly greater than the amount of FAEE generated.
- ester synthase in E. coli MG1655 DV2 ⁇ fadD leads to the production of esters in the presence of ethanol.
- expression of ester synthase ES9 resulted in the highest ester production in the presence of ethanol as compared to that of the other ester synthases.
- this example indicated that expression of 'TesA alone in E. coli MG1655 DV2 ⁇ fadD resulted in the production of a low level of esters and a high level of free fatty acids in the presence of ethanol.
- a plasmid containing an E. coli 'tesA (2) a plasmid containing a polynucleotide encoding Alcanivorax borkumensis SK2 ester synthase atfA1 (GenBank Accession No. YP_694462); (3) a plasmid containing a polynucleotide encoding Alcanivorax borkumensis SK2 ester synthase atfA2 (GenBank Accession No.
- YP_693524 (4) a plasmid containing a polynucleotide encoding Marinobacter hydrocarbonoclasticus DSM 8798 ester synthase ES8 (GenBank Accession No. ABO21020); and (5) a plasmid containing a polynucleotide encoding Marinobacter hydrocarbonoclasticus DSM 8798 ester synthase ES9 (GenBank Accession No. ABO21021) were grown in shake flask cultures and induced with 1 mM IPTG and 2% (v/v) ethanol. At 24-h post induction, aliquots from each culture were extracted and analyzed using GC-FID for fatty acid and ester content. The amounts of free fatty acid (FFA) and fatty acyl ethyl ester (FAEE) produced in each culture were shown in FIG. 6 .
- FFAEE free fatty acid
- ester synthase ES9 generated a high level of FAEE in the presence of ethanol, accompanied by a low level of FFA.
- noticeably smaller amounts of FAEE were produced by ester synthases atfAI, atfA2, and ester synthase ES8. Consistent with the results of Example 7 and FIG. 5 , above, expression of E. coli 'tesA alone produced high overall titers of FFA and FAEE, but with a significantly higher proportion of FFA.
- a plasmid containing a polynucleotide encoding Alcanivorax borkumensis SK2 ester synthase atfA1 (GenBank Accession No. YP_694462); (2) a plasmid containing a polynucleotide encoding Alcanivorax borkumensis SK2 ester synthase atfA2 (GenBank Accession No. YP_693524); (3) a plasmid containing a polynucleotide Marinobacter hydrocarbonoclasticus DSM 8798 ester synthase ES8 (GenBank Accession No.
- the amount of overall product or titer observed for FFA and FAME was reduced in the presence of methanol as compared to the amount of overall titer observed in the presence of ethanol (see FIG. 6 ).
- the amount of FFA produced was below the limit of detection, therefore it is possible that FFA was produced in the presence of methanol, albeit at low concentrations.
- E. coli MG1655 DV2 ⁇ fadD carrying a plasmid containing a polynucleotide encoding M. hydrocarbonclasticus DSM 8798 ester synthase ES9 (GenBank Accession No. ABO21020), was grown in shake flask cultures and induced with 1 mM IPTG and either (1) 2% (v/v) ethanol or (2) 2% (v/v) methanol. Aliquots from each culture were extracted at 18, 24, 48, and 68 hours post induction, and analyzed by GC-FID for fatty acid and ester content.
- FAME fatty acyl methyl ester
- FAEE fatty acyl ethyl ester
- ester synthase ES9 in E. coli MG1655 DV2 ⁇ fadD resulted in high production levels of esters and low production levels of free fatty acids in the presence of methanol and ethanol. Furthermore, this example demonstrates that ester production in E. coli MG1655 DV2 ⁇ fadD expressing ester synthase ES9 was significantly higher in the presence of ethanol than in the presence of methanol.
- This example describes the construction of an ester synthase mutant library, wherein each member contains an amino acid substitution for glycine at position 395 of ES9.
- the glycine residue at position 395 of ES9 was mutated to each of the 20 conventional amino acid residues in order to evaluate the effect of amino acid substitution at this position.
- the mutants were created using Stratagene's QuikChange® Multi Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.).
- Primer G395Xf as shown in Table 7, was used in conjuction with a vector-specific primer to generate a pool of 3′ ester synthase fragments encoding all 20 possible amino acid residues at position 395.
- a reverse primer complementary to the 5′-portion of G395Xf, designated as G395Xr was used in conjuction with a vector-specific primer to generate the 5′ ester synthase fragments.
- Each of the 3′-fragments was then assembled with the 5′ fragments via overlap extension to reconstitute a randomized pool of gene sequences encoding a sequence with 20 different amino acid residues at position 395.
- a first gene containing the promoter and open reading frame for chloramphenicol resistance (SEQ ID NO:157) was obtained from plasmid pKD3 (see, Datsenko et al., supra).
- a portion of a second gene containing the promoter and the first 531 bases of the kanamycin resistance gene (SEQ ID NO:158) was obtained from plasmid pKD13 (see, Datsenko et al., supra).
- a resistance cassette was constructed by first joining the first gene with the second gene, with the first gene being upstream of the second gene.
- a 50-base pair homologous sequence to the 3′-end of the lacZ gene sequence (SEQ ID NO:159) was added downstream from the chloramphenicol resistance gene, and a lacI gene sequence (SEQ ID NO:160) was added upstream from the partial kanamycin resistance gene.
- This resistance cassette was then used to transform an E. coli MG1655 DV4 strain, which had been previously transformed with pKD46 as described below.
- a plasmid pKD46 (see, Datsenko et al., supra) was transformed into E. coli MG1655 DV4 (see, Example 3).
- This DV4/pKD46 strain was grown on a solid media containing 100 ⁇ g/mL cabenicillin overnight at 32° C. A single colony was then used to inoculate an overnight culture of LB containing 100 ⁇ g/mL carbenicillin After 16-h of growth at 32° C., the DV4/pKD46 culture was diluted 1 to 500 into an LB medium containing 100 ⁇ g/mL carbenicillin, arabinose and MgCl 2 to induce expression of the recombinase genes from pKD46. Growth was monitored by measuring OD600, and when it reached about 0.6, the cells were made electrocompetent and transformed with the resistance cassette.
- the transformed cells were allowed to recover for 3 h in non-selective SOC media before plating on solid chloramphenicol-selective media, containing 34 ⁇ g/mL chloramphenicol, X-gal, and IPTG. Correct insertions were confirmed using colony PCR, blue/white screening and DNA sequencing.
- This strain is named E. coli MG1655 DV4/1/2 Km R Cm R /pKD46. It is then subject to arabinose treatment for 3-4 h before the ester synthase mutant sequences were transformed
- a seed culture of each clone was grown in 96-well plates in LB broth supplemented with 50 ⁇ g/mL kanamycin at 32° C. After a 6-h growth, the cultures were diluted 1 to 10 into 2N-BT (2% glucose, nitrogen limited medium, 0.2M Bis-Tris, pH 7.0, 0.1% Triton) plus 50 ⁇ g/mL kanamycin and grown overnight at 32° C. After overnight growth, the cultures were diluted again 1 to 10 into 4N-BT (4% glucose, nitrogen limited medium, 0.2 mM Bis-Tris, pH 7.0, 0.1% Triton) and grown at 32° C. for 6 h.
- 2N-BT 2% glucose, nitrogen limited medium, 0.2M Bis-Tris, pH 7.0, 0.1% Triton
- IPTG and ethanol were then added to each culture to achieve a final concentration of 1 mM and 2% (v/v), respectively. 22-h post induction, the cultures were acidified using 40 ⁇ L 1 M HCl and extracted with 400 ⁇ L n-butylactate. The amounts of fatty species (including, e.g., fatty acids and fatty esters) in the extractants were measured using GC-FID.
- FIG. 9 which depicts the z scores of the production titers from the mutant and wild type strains, and wherein the median titer of the production strain expressing wild type ES9 was set at 0, the samples wherein the glycine residue at position 395 of ES9 was replaced by either an argine or a lysine residue resulted in the most significant increases in fatty species production.
- the z score for the G395R mutant strain was above 10, and the z score for the G395K mutant strain was about 9.
- p-values were calculated to assess statistical significance in terms of the improvements of titer.
- the productivity improvement of the G395R mutant strain had a p-value of 1.74E-19, and the productivity improvement of the G395K mutant strain had a p-value of 5.42E-05, and those improvements were statistically significant.
- ES9BspHF (SEQ ID NO: 37) 5′-ATCATGAAACGTCTCGGAAC-3′; and ES9XhoR (SEQ ID NO: 38) 5′-CCTCGAGTTACTTGCGGGTTCGGGCGCG-3′
- ester synthase fragments that were obtained from the E. coli MG1655 DV4/1/2 Km R Cm R /pKD46 transformants using these primers were wild type ES9, and mutants G395R, G395S, G395K, G395S, and G395D. These fragments were then cloned downstream of the IPTG inducible promoter in a pCL1920 vector using NcoI and HindIII restriction sites.
- the resulting plasmids were transformed into electrocompetent E. coli MG1655 DV2 ⁇ fadD (see, Example 4) and plated on LB plates containing 100 mg/L spectinomycin, incubated overnight at 37° C., and purified.
- Purified DV2 ⁇ fadD strains expressing ES9, and mutants G395R, G395S, G395K, G395S, and G395D were grown in 15 mL shake flasks and induced with 1 mM IPTG and 2% (v/v) methanol. Post induction, samples were taken from each shake flask at the 15.5 h, 40 h and 65 h, and the production levels of free fatty acids and FAME were determined at these time points using GC-FID. The results are shown in FIG. 10A .
- strains expressing ES9 which has a glycine residue at position 395, as well as strains expressing the G395R, G395K, G395D and G395E mutants of ES9, all produced substantial FAME and free fatty acids in the presence of methanol.
- the strain expressing the G395S mutant produced little if any FAME and/or free fatty acids.
- ester synthase homologs with altered activity and/or specificity towards acyl-ACPs over acyl-CoA or beta-hydroxy acyl-ACP.
- ester synthase homologs were identified based on bioinformatic analyses and described in the table below. These ester synthase homologues were evaluated for activity:
- the nucleotide sequences of the four ester synthase homologs above were E. coli codon-optimized (SEQ ID NOs: 40, 42, 44, 46) and synthesized using DNA2.0 (DNAstar, Menlo Park, Calif.) and then cloned downstream of the IPTG inducible Ptrc promoter in a pCL1920 vector using the NcoI and HindIII sites.
- the resulting plasmids were transformed into E. coli MG1655 DV2 ⁇ fadD for evaluation in shake flasks.
- This example describes a fermentation process to produce a fatty acid and/or fatty acid derivative composition using the genetically modified microorganisms described herein.
- a fermentation and recovery process was used to produce biodiesel of commercial grade quality by fermentation of carbohydrates.
- a fermentation process was developed to produce a mix of fatty acid ethyl esters (FAEE) and fatty acid methyl esters (FAME) for use as a biodiesel using the genetically engineered microorganisms described Examples 1-12.
- the fermentation is carried out in any manner known to those of ordinary skill in the art.
- the fermentation is carried out in a 2- to 10-L lab scale fermentor.
- These exemplary protocols can be scaled up as any other E. coli fermentation, using methods known to one of ordinary skill in the art.
- a fermentation run was carried out in a 2-L fermentor.
- E. coli cells from a frozen stock were grown in a defined medium consisting of 4.54 g/L K 2 HPO 4 trihydrate, 4 g/L of (NH 4 ) 2 SO 4 , 0.15 g/L of MgSO 4 heptahydrate, 20 g/L glucose, 200 mM Bis-Tris buffer (pH 7.2), 1.25 mL/L trace mineral solution, and 1.25 mL/L vitamin solution.
- the trace mineral solution comprised 27 g/L FeCl 3 •6H 2 O, 2 g/L ZnCl 2 •4H 2 O, 2 g/L CaCl 2 •6H 2 O, 2 g/L Na 2 MoO 4 •2H 2 O, 1.9 g/L CuSO 4 •5H 2 O, 0.5 g/L H 3 BO 3 , and 100 mL/L concentrated HCl.
- the vitamin solution comprised 0.42 g/L riboflavin, 5.4 g/L pantothenic acid, 6 g/L niacin, 1.4 g/L pyridoxine, 0.06 g/L biotin, and 0.04 g/L folic acid.
- Fifty (50) mL of a culture described herein was grown overnight and subsequently used to inoculate 1 L of a medium containing 0.5 g/L (NH 4 ) 2 SO 4 , 2.0 g/L KH 2 PO 4 , 0.15 g/L MgSO 4 heptahydrate, 0.034 g/L ferric citrate, 2.5 g/L bacto casamino acids, 10 g/L glucose, 1.25 mL/L trace mineral solution 2, and 1.25 mL/L vitamin solution.
- a medium containing 0.5 g/L (NH 4 ) 2 SO 4 , 2.0 g/L KH 2 PO 4 , 0.15 g/L MgSO 4 heptahydrate, 0.034 g/L ferric citrate, 2.5 g/L bacto casamino acids, 10 g/L glucose, 1.25 mL/L trace mineral solution 2, and 1.25 mL/L vitamin solution.
- the trace mineral solution 2 contained 2 g/L ZnCl 2 4H 2 O, 2 g/L CaCl 2 •6H 2 O, 2 g/L Na 2 MoO 4 •2H 2 O, 1.9 g/L CuSO 4 •5H 2 O, 0.5 g/L H 3 BO 3 , and 100 mL/L of concentrated HCl.
- the vitamin solution was the same as described for the inoculum culture medium.
- the fermentor was equipped with temperature-, pH-, agitation-, aeration-, and dissolved oxygen-controls.
- the preferred fermentation conditions include a temperature of 32° C., pH of 6.8, and a dissolved oxygen (DO) level of about 30% of saturation.
- the pH was maintained by additions of NH 4 OH, which also acted as a nitrogen source for cell growth.
- a feed consisting of 600 g/L glucose, 3.9 g/L MgSO 4 heptahydrate, 1.6 g/L KH 2 PO 4 , 2.5 g/L casamino acids, 0.05 g/L Ferric citrate, 20 mL/L trace minerals solution 2, and 2 ml/L vitamin solution was supplied to the fermentor.
- the feed rate was set up to allow for a cell growth rate of 0.3 h ⁇ 1 , for up to a maximum of 10 g glucose/L/h, at which point it was fixed. This rate was maintained for the remainder of the fermentation run as long as glucose did not accumulate in the fermentor. By avoiding glucose accumulation, it was possible to reduce or eliminate the formation of by-products such as acetate, formate, and ethanol, which are otherwise commonly produced by E. coli . In the early phases of growth, the production of FAEE was induced by the addition of 1 mM IPTG and 10 ml/L of pure ethanol. The fermentation was continued for a period of 3 days.
- Ethanol was added several times during the run to replenish what was consumed by the cells for the production of fatty ethyl esters, but mostly what was lost by evaporation in the off-gas.
- the additions helped to maintain the concentration of ethanol in the fermentation broth at a level of between 10 and 20 mL/L, in order to promote efficient production without inhibiting cell growth.
- the progression of the fermentation run was followed by measurements of OD600, glucose consumption, and ester production.
- This fermentation protocol was also scaled up to a 700-L fermentor.
- the analytical methods utilized following the fermentation runs are described in the following examples.
- HPLC High Pressure Liquid Chromatography
- fatty acid methyl or ethyl ester was analyzed by gas chromatography with flame ionization detector (GC-FID). Samples from the fermentation broth were extracted with ethyl acetate in a volume ratio of 1:1. After vigorous vortexing, the samples were centrifuged and the respective organic phases ware analyzed by GC.
- the analysis conditions were as follows:
- GC-MS mass spectrometry detector
- Oven temperature 3.00 min hold at 100° C., ramp at 20° C./min to 320° C. and hold for 5 min, with a total run time of 19 min;
- Capillary column DB-5 MS UI, capillary 30.0 m ⁇ 250 ⁇ M ⁇ 0.250 ⁇ m ⁇ 0.25 ⁇ m nominal;
- Carrier gas/flow rate/pressure Helium/1.2 mL/min/12.56 psi;
- Inlet condition 320° C. splitless; Total flow rate: 16.2 mL/min;
- fatty acid ethyl esters can be produced by fermenting an E. coli strain expressing an ester synthase, in the absence of a thioesterase and/or an acyl-CoA synthase.
- E. coli MG1655 DV2 and DV2 ⁇ fadD were transformed with plasmid pDS57 (SEQ ID NO:23), as described in Example 5.
- the resulting DV2 pDS57 and DV2 ⁇ fadD pDS57 strains were grown in 2- and 5-L fermentors according to the fermentation protocol of Example 14. Representative results of the analysis of FAEE, FFA, and the total yield of FAEE for each strain are shown in Table 10. The yield is expressed as the grams of product obtained per 100 grams of carbon source used.
- fatty acid esters can be produced by feeding fatty acids and alcohols to a microbial strain containing a native (i.e., non-recombinant) ester synthase.
- a strain of Funibacter jadensis was used as an example of a microbial strain containing such an ester synthase. It was cultured in a seawater medium.
- a seawater medium was prepared and autoclaved: 23.6 g/L NaCl, 0.64 g/L KCl, 4.53 g/L MgCl 2 ⁇ 6H 2 O, 5.94 g/L MgSO 4 ⁇ 7H 2 O.
- the pH was adjusted to 7.2 with NaHCO 3 .
- the strain Funibacter jadensis T9 (DSM 12178) was obtained from DSMZ (http://www.dsmz.de). The strain was grown in 25 mL of the seawater medium, supplemented with 100 ⁇ L of sodium palmitate (1% in H 2 O) and one of four alcohols listed in Table 11. After 18 h of growth at 30° C., the culture broths were extracted with 20 mL of ethyl acetate. Then the respective ethyl acetate phases of ethyl acetate extractants were analyzed with GC/MS. The types of esters that were produced were listed in Table 11.
- FIG. 13 depicts the GC/MS spectra of ethyl palmitate and isopropyl palmitate produced by F. jadensis T9 strain fed with ethanol and isopropanol, respectively, in the middle and lower panels.
- This example demonstrates the production of fatty acid derivatives in a wild-type E. coli host cell in the presence of an unattenuated level of endogenous fadE.
- the gene fadE encodes an acyl-CoA dehydrogenase, which catalyzes the initial step in the degradation of fatty acyl-CoAs. Therefore, fadE is often considered necessary when fatty acid derivatives are made using acyl-CoAs as substrates.
- the fatty acid derivative production cells of the present invention can use acyl-ACPs as substrates. Thus the presence of an acyl-CoA dehydrogenase will not necessarily cause degradation of the substrates.
- This example demonstrates that, indeed, the endogenous expression of fadE can be left intact without affecting the production of fatty acid derivatives.
- Plasmid pDS57 (SEQ ID NO:23, see, Example 6), which places ES9 under the transcriptional control of the trc promoter, was transformed into an electrocompetent E. coli strain MG1655.
- the same plasmid was transformed into strain E. coli MG1655 D1 (or E. coli MG1655 ⁇ fadE, see, Example 1) to serve as a comparison.
- the strains were grown in shake flasks and were evaluated using the standard shake flask fermentation protocol, such as the one described in Example 7, supra. When strains were induced with 1 mM IPTG, they were also supplemented with ethanol, to a final volume of 2% (v/v).
- strains Four time points were taken and the strains were analyzed for growth, and for fatty acid ethyl ester production. The results are depicted in FIGS. 11 and 12 . Both strains had nearly identical growth profiles ( FIG. 11 ) and fatty acid ethyl ester production profiles ( FIG. 12 ). Strain MG1655 pDS57 was able to produce about 2 g/L FAEE, demonstrating that ES9 is capable of producing ester directly in a wild-type strain without an attenuated fadE.
- This example provides an exemplary method for the expression of an ester synthase polypeptide in a Saccharomyces cerevisiae .
- Endogenous thioesterases and/or acyl-CoA synthases or other fatty acid degradation enzymes, if present, can be attenuated or functionally deleted using the appropriate methods as described herein.
- the yeast episomal plasmid (YEp)-type vector pRS425 contains sequences from the Saccharomyces cerevisiae 2 micron endogenous plasmid, a LEU2 selectable marker, and sequences based on the backbone of a multifunctional phagemid, pBluescript II SK(+).
- the Saccharomyces cerevisiae 's strong, constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter is cloned between the SacII and SpeI sites of pRS425 using the method described by Jia et al., Physiol.
- a NotI site is introduced into the BamHI site of pGPD-425, giving a NotI site flanked by BamHI sites, and this plasmid is called pY75 (SEQ ID NO:48).
- An ester synthase gene of interest such as, for example, one encoding a polypeptide selected from SEQ ID NOs:18, 24, 25, and 26, or a variant thereof, is first treated such that the DNA fragments can be ligated into the unique Nod site of pY75 (SEQ ID NO:48).
- the pY75 vector Prior to its use for cloning, the pY75 vector is linearized with NotI, filled in with T4 DNA polymerase and dephosphorylated with shrimp alkaline phosphatase (New England Biolabs, Ipswich, Mass.). Plasmid DNA is then isolated using standard techniques.
- pY75_ES Restriction digests with EcoRI is conducted to identify plasmid clones in which the start codon is in proximately to the 3′-end of the GPD promoter of pY75 (sense orientation of the ester synthase gene).
- the plasmid is referred to as pY75_ES.
- Plasmid DNA of pY75_ES and plasmid DNA of pY75 empty vector are transformed into the Saccharomyces cerevisiae strain INVSC1 (Invitrogen, Carlsbad, Calif.) using standard methods such as those described in Gietz et al., Meth. Enzymol. 350:87-96 (2002).
- Recombinant yeast colonies are selected on DOBA media supplemented with CSM-leu (Qbiogene, Carlsbad, Calif.). The cultures are fed with suitable alcohol substrates.
- the resultant fatty acids and/or fatty acid derivatives are isolated from the host cell using the methods herein.
- the amount of fatty acids and/or fatty acid derivatives produced is determined using known techniques such as, for example, GC/MS.
- This example provides an exemplary method for the expression of an ester synthase in a Yarrowia lipolytica.
- a pFBAIN-MOD-1 vector is constructed in accordance with the methods of PCT Publication No. WO2008/147935, the disclosures of which are incorporated by reference herein.
- a pictorial representation of the features of this vector is presented herein as FIG. 18 .
- An ester synthase of interest is ligated to the pFBAIN-MOD-1 predigested with NcoI and NotI (SEQ ID NO:51).
- the ligation reaction contains 10 ⁇ L 2 ⁇ ligation buffer, 1 ⁇ L T4 DNA ligase (Promega, Madison, Wis.), 3 ⁇ L ( ⁇ 300 ng) of the an ester synthase polynucleotide fragment and 1 ⁇ L of pFBAIN-MOD-1 ( ⁇ 150 ng).
- the reaction mixture is then incubated at room temperature for 2 h and used to transform E. coli Top10 Competent cells (Invitrogen, Carlsbad, Calif.). Plasmid DNAs from the transformants are recovered using a Qiagen Miniprep kit (Valencia, Calif.). Correct clones are identified by restriction mapping and the final constructs are designated pFBAIN_ES.
- Y_FOA R A clone of pFBAIN_ES and a control plasmid pFBAIN-MOD-1 are transformed into a Yarrowia lipolytica strain, for example, Y_FOA R .
- Y_FOA R is prepared by obtaining Yarrowia lipolytica ATCC#20362 cells and plating them on a YPD agar plate (containing 10 g/L of yeast extract (DIFCO Labs, Detroit, Mich.), 20 g/L of bacto pepton (DIFCO), and 20 g/L of glucose).
- MM Minimum Medium
- MM Minimum Medium
- YNB yeast nitrogen base
- ammonia sulfate without amino acids
- 20 g/L glucose 250 mg/L 5-FOA
- Plates can be incubated at 28° C., and the resultant colonies are patched separately onto MM plates containing 200 mg/mL 5-FOA and MM plates lacking uracil and uridine.
- Ura3 auxotrophy is thus obtained and the resulting strain is the Y_FOA R strain.
- the cells from the transformation are plated onto MM plates lacking uracil (0.17% yeast nitrogen base (DIFCO Labs) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, pH6.1, 20 g/L agar) and maintained at 30° C. for 2 d.
- uracil 0.17% yeast nitrogen base (DIFCO Labs) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, pH6.1, 20 g/L agar
- a few transformants are then used to inoculate individual 25 mL cultures in MM medium (0.17% YNB (DIFCO) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, pH6.1).
- Each culture is allowed to grow for 2 d at 30° C., then switched to 25 mL HGM (High Glucose Medium containing 80 g/L glucose, 2.58 g/L KH 2 PO 4 , 5.36 g/L K 2 HPO 4 ) and allowed to grow for 5 d at 30° C.
- HGM High Glucose Medium containing 80 g/L glucose, 2.58 g/L KH 2 PO 4 , 5.36 g/L K 2 HPO 4
- the cultures are fed with suitable alcohol substrates.
- Total fatty species are then extracted and the production of fatty acids and/or fatty acid derivatives can be ascertained and/or measured using the methods described herein.
- This example describes the expression of an ester synthase in a cyanobacterial host cell.
- Suitable vectors are prepared in order to produce fatty acids and/or derivatives thereof in cyanobacterial host cells including, for example, cells of Synechococcus sp. PCC7002 , Synechococcus elongatus PCC7942, or Synechocystis sp. PCC6803.
- An ester synthase of interest for example, an atfA1 (from A. borkumensis SK2, GenBank Accession No. YP_694462), AtfA2 (from A. borkumensis SK2, GenBank Accession No. YP_693524), ES9 (from M. hydrocarbonoclasticus DSM 8789, GenBank Accession No.
- ES8 from M. hydrocarbonoclasticus DSM 8789, GenBank Accession No. AB021020
- variants thereof optionally integrated in the chromosome under the control of a suitable promoter (e.g., PTrc), are then cloned into those vectors.
- a suitable promoter e.g., PTrc
- a vector is constructed to accomplish homologous recombination in Synechococcus sp. PCC7002 pAQ1 [GenBank Accession No. NC_0050525] using 500 bp homologous regions corresponding to positions 3301-3800 and 3801-4300.
- a selectable marker such as a spectinomycin resistance cassette containing aminoglycoside 3′-adenyltrasnferase gene, aad, a promoter, and a terminator is derived from the plasmid PCL1920 (in accordance with Lerner et al., Nucleic Acids Res. 18:4631 (1990)). That selectable marker is inserted into the homologous regions.
- a plasmid such as pACYC177, is prepared in accordance with Chang, et al. J. Bacteriol. 134: 1141-1156 (1978).
- the promoter and ribosome binding site of aminoglycoside phosphotransferase, aph, are added followed by appropriate unique cloning sites that are, for example, NdeI and EcoRI recognition sequences.
- This complete integration cassette is synthesized and cloned into a pUC19 vector (New England Biolabs, Ipswich, Mass.).
- the resulting plasmid, pLS9-7002 allows cloning and expression of a foreign gene and delivery and stable in vivo integration into Synechococcus sp. PCC7002 pAQ1.
- a plasmid or synthetic operon containing an ester synthase gene (e.g., one encoding ES9) is created, and cloned into the NdeI and EcoRI sites of pLS9-7002 downstream of the aph promoter and ribosome binding site.
- the resultant plasmid is transformed into Synechococcus sp. PCC7002 using a method described by Stevens et al., PNAS 77:6052-56 (1980).
- another vector is constructed for homologous recombination into the Synechococcus elongatus PCC7942 genome (GenBank Accession No. CP_000100) using 800 bp homologous regions corresponding to positions 2577844-2578659 and 2578660-2579467.
- This chromosomal location is known as neutral site one (NS1) (Mackey et al., Meth. Mol. Biol. 362:115-129 (2007).
- a selectable marker such as, for example, a spectinomycin resistance cassette is derived and introduced as described above.
- This integration cassette is synthesized and cloned into pUC19 (New England Biolabs).
- the resultant plasmid, pLS9-7942-NS1 allows cloning and expression of a foreign gene and delivery and stable integration into the Synechococcus elongatus PCC7942 genome.
- a plasmid or synthetic operon comprising an ester synthase gene of interest is created as described above, which is then cloned into the NdeI or EcoRI site of pLS9-7942-NS1.
- the resultant plasmid is transformed into S. elongatus PCC7942 in accordance with a method described by Mackey et al., supra.
- yet another vector is constructed for homologous recombination into the Synechocystis sp. PCC6803 genome (GenBank Accession BA_000022) using 1300 to 1700 bp homologous regions corresponding to positions 2299015-2300690, and 2300691-2302056, respectively.
- This chromosomal location is known as neutral site RS1/2 (Shao et al., Appl. Environ. Microbiol. 68:5026-33 (2002)).
- a plasmid, such as pACYC177 is prepared in accordance with Chang, et al. J. Bacteriol. 134: 1141-1156 (1978).
- a kanamycin resistance cassette (containing aminoglycoside phosphotransferase, aph, promoter, gene and terminator) is derived from the pACYC177 plasmid, and it is added between the homologous regions. Additionally, appropriate unique cloning sites, for example, NdeI and XbaI recognitions sites are added.
- This integration cassette is synthesized and cloned into pUC19 (New England Biolabs).
- the resultant plasmid, pLS9-6803-RS allows cloning and expression of a foreign gene and delivery and stable integration into the Synechocystis sp. PCC6803 genome.
- a plasmid or synthetic operon containing an ester synthase gene is created as described above, which is then cloned into the NdeI or XbaI site of pLS9-6803-RS.
- the resultant plasmid is transformed into Synechocystis sp. PCC6803 in accordance with a method described by Zang et al. J. Microbiol., 45:241-45 (2007).
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Emergency Medicine (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Physics & Mathematics (AREA)
- Virology (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Fats And Perfumes (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Liquid Carbonaceous Fuels (AREA)
Abstract
Description
- This application claims the benefit of U.S. Provisional Application No. 61/245,943, filed Sep. 25, 2009, the contents of which are hereby incorporated in their entirety herein.
- Petroleum is a limited, natural resource found in the Earth in liquid, gaseous, or solid forms. Petroleum is primarily composed of hydrocarbons, which are comprised mainly of carbon and hydrogen. It also contains significant amounts of other elements, such as, nitrogen, oxygen, or sulfur, in different forms.
- Petroleum is a valuable resource, but petroleum products are developed at considerable costs, both financial and environmental. First, sources of petroleum must be discovered. Petroleum exploration is an expensive and risky venture. The cost of exploring deep water wells can exceed $100 million. In addition to the economic cost, petroleum exploration carries a high environmental cost. For example, offshore exploration disturbs the surrounding marine environments.
- After a productive well is discovered, the petroleum must be extracted from the Earth at great expense. Even under the best circumstances, only 50% of the petroleum in a well can be extracted. Petroleum extraction also carries an environmental cost. For example, petroleum extraction can result in large see pages of petroleum rising to the surface. Offshore drilling involves dredging the seabed which disrupts or destroys the surrounding marine environment.
- After extraction, petroleum must be transported over great distances from petroleum producing regions to petroleum consuming regions. In addition to the shipping costs, there is also the environmental risk of devastating oil spills.
- In its natural form, crude petroleum extracted from the Earth has few commercial uses. It is a mixture of hydrocarbons (e.g., paraffins (or alkanes), olefins (or alkenes), alkynes, napthenes (or cylcoalkanes), aliphatic compounds, aromatic compounds, etc.) of varying length and complexity. In addition, crude petroleum contains other organic compounds (e.g., organic compounds containing nitrogen, oxygen, sulfur, etc.) and impurities (e.g., sulfur, salt, acid, metals, etc.).
- Hence, crude petroleum must be refined and purified before it can be used commercially. Due to its high energy density and its easy transportability, most petroleum is refined into fuels, such as transportation fuels (e.g., gasoline, diesel, aviation fuel, etc.), heating oil, liquefied petroleum gas, etc.
- Crude petroleum is also a primary source of raw materials for producing petrochemicals. The two main classes of raw materials derived from petroleum are short chain olefins (e.g., ethylene and propylene) and aromatics (e.g., benzene and xylene isomers). These raw materials are derived from the longer chain hydrocarbons in crude petroleum by cracking the long chain hydrocarbons at considerable expense using a variety of methods, such as catalytic cracking, steam cracking, or catalytic reforming. These raw materials are used to make petrochemicals, which cannot be directly refined from crude petroleum, such as monomers, solvents, detergents, or adhesives.
- One example of a raw material derived from crude petroleum is ethylene. Ethylene is used to produce petrochemicals such as, polyethylene, ethanol, ethylene oxide, ethylene glycol, polyester, glycol ether, ethoxylate, vinyl acetate, 1,2-dichloroethane, trichloroethylene, tetrachloroethylene, vinyl chloride, and polyvinyl chloride. Another example of a raw material derived from crude petroleum is propylene. Propylene is used to produce isopropyl alcohol, acrylonitrile, polypropylene, propylene oxide, propylene glycol, glycol ethers, butylene, isobutylene, 1,3-butadiene, synthetic elastomers, polyolefins, alpha-olefins, fatty alcohols, acrylic acid, acrylic polymers, allyl chloride, epichlorohydrin, and epoxy resins.
- Petrochemicals can be used to make specialty chemicals, such as plastics, resins, fibers, elastomers, pharmaceuticals, lubricants, or gels. Examples of specialty chemicals which can be produced from petrochemical raw materials are: fatty acids, hydrocarbons (e.g., long chain hydrocarbons, branched chain hydrocarbons, saturated hydrocarbons, unsaturated hydrocarbons, etc.), fatty alcohols, esters, fatty aldehydes, ketones, lubricants, etc.
- Specialty chemicals have many commercial uses. Fatty acids are used commercially as surfactants. Surfactants can be found in detergents and soaps. Fatty acids can also be used as additives in fuels, lubricating oils, paints, lacquers, candles, salad oils, shortenings, cosmetics, and emulsifiers. In addition, fatty acids are used as accelerator activators in rubber products. Fatty acids can also be used as a feedstock to produce methyl esters, amides, amines, acid chlorides, anhydrides, ketene dimers, and peroxy acids and esters.
- Esters have many commercial uses. For example, biodiesel, an alternative fuel, is comprised of esters (e.g., fatty acid methyl ester, fatty acid ethyl esters, etc.). Some low molecular weight esters are volatile with a pleasant odor which makes them useful as fragrances or flavoring agents. In addition, esters are used as solvents for lacquers, paints, and varnishes. Furthermore, some naturally occurring substances, such as waxes, fats, and oils are comprised of esters. Esters are also used as softening agents in resins and plastics, plasticizers, flame retardants, and additives in gasoline and oil. In addition, esters can be used in the manufacture of polymers, films, textiles, dyes, and pharmaceuticals.
- In addition, crude petroleum is a source of lubricants. Lubricants derived petroleum are typically composed of olefins, particularly polyolefins and alpha-olefins. Lubricants can either be refined from crude petroleum or manufactured using the raw materials refined from crude petroleum.
- Obtaining these specialty chemicals from crude petroleum requires a significant financial investment as well as a great deal of energy. It is also an inefficient process because frequently the long chain hydrocarbons in crude petroleum are cracked to produce smaller monomers. These monomers are then used as the raw material to manufacture the more complex specialty chemicals.
- In addition to the problems with exploring, extracting, transporting, and refining petroleum, petroleum is a limited and dwindling resource. One estimate of current world petroleum consumption is 30 billion barrels per year. By some estimates, it is predicted that at current production levels, the world's petroleum reserves could be depleted before the year 2050.
- Finally, the burning of petroleum based fuels releases greenhouse gases (e.g., carbon dioxide) and other forms of air pollution (e.g., carbon monoxide, sulfur dioxide, etc.). As the world's demand for fuel increases, the emission of greenhouse gases and other forms of air pollution also increases. The accumulation of greenhouse gases in the atmosphere leads to an increase in global warming. Hence, in addition to damaging the environment locally (e.g., oil spills, dredging of marine environments, etc.), burning petroleum also damages the environment globally.
- Due to the inherent challenges posed by petroleum, there is a need for a renewable petroleum source which does not need to be explored, extracted, transported over long distances, or substantially refined like petroleum. There is also a need for a renewable petroleum source that can be produced economically. In addition, there is a need for a renewable petroleum source that does not create the type of environmental damage produced by the petroleum industry and the burning of petroleum based fuels. For similar reasons, there is also a need for a renewable source of chemicals that are typically derived from petroleum.
- Renewable energy sources, such as sunlight, water, wind, and biomass, are a potential alternative to petroleum fuels. Biofuel is a biodegradable, clean-burning combustible fuel produced from biomass, and can be made of alkanes and esters. An exemplary biofuel is biodiesel. Biodiesel can be used in most internal combustion diesel engines in either a pure form, which is referred to as “neat” biodiesel, or as a mixture in any concentration with regular petroleum diesel.
- Biodiesel offers advantages compared to petroleum-based diesel, including reduced emissions (e.g., carbon monoxide, sulphur, aromatic hydrocarbons, soot particles) during combustion. Biodiesel also maintains a balanced carbon dioxide cycle because it is based on renewable biological materials. Biodiesel is typically biodegradable, and imparts enhanced safety due to its high flash point and low flammability. Furthermore, biodiesel provides good lubrication properties, thereby reducing wear and tear on engines.
- Current methods of making biodiesel involve transesterification of triacylglycerides from vegetable oil feedstocks, such as rapeseed in Europe, soybean in North America, and palm oil in South East Asia. Industrial-scale biodiesel production is thus geographically and seasonally restricted to areas where vegetable oil feedstocks are produced. The transesterification process leads to a mixture of fatty esters which can be used as biodiesel. However, glycerin is an undesirable byproduct of the transesterification process. To be usable as biodiesel, the fatty esters must be further purified from the heterogeneous product. This increases costs and the amount of energy required for fatty ester production and, ultimately, biodiesel production as well. Furthermore, vegetable oil feedstocks are inefficient sources of energy because they require extensive acreage for cultivation. For example, the yield of biodiesel from rapeseed is only 1300 L/hectare because only the seed oil is used for biodiesel production, while the rest of the rapeseed biomass is discarded. Additionally, cultivating some vegetable oil feedsocks, such as rapeseed and soybean, requires frequent crop rotation to prevent nutrient depletion of the land.
- Thus there is a need for an economically- and energy-efficient biofuel, and methods of making biofuels from renewable energy sources such as biomass.
- This disclosure relates to the production of fatty acids and derivatives thereof including, for example, fatty esters from genetically engineered microorganisms. Examples of fatty esters include fatty acid esters, such as those derived from short-chain alcohols, including fatty acid ethyl esters (“FAEE”) and fatty acid methyl esters (“FAME”), and those derived from long-chain fatty alcohols. The fatty acids and/or fatty acid derivatives that are produced can be used, individually or in suitable combinations, as a biofuel (e.g., a biodiesel), an industrial chemical, or a component of, or feedstock for, a biofuel or an industrial chemical. In some aspects, the invention pertains to a method of producing one or more free fatty acids and/or one or more fatty acid derivatives such as fatty acid esters, including, for example, FAEE, FAME and/or other fatty acid ester derivatives of longer-chain alcohols. In related aspects, the method comprises providing a genetically engineered production host suitable for making fatty acids and fatty acid derivatives.
- Accordingly, in one aspect, the invention features a method of making a fatty acid or a fatty acid derivative such as a fatty ester. The method includes expressing in a host cell a gene encoding an ester synthase. In some embodiments, the gene encoding an ester synthase is selected from the enzymes classified as EC 2.3.1.75, and any other polypeptides capable of catalyzing the conversion of an acyl thioester to fatty esters, including, without limitation, wax-ester synthases, acyl-CoA:alcohol transacylases, alcohol 0-fatty acid-acyl-transferase, acyltransferases, and fatty acyl-coA:fatty alcohol acyltransferases, or a suitable variant thereof. In other embodiments, the ester synthase gene is one that encodes wax/dgat, a bifunctional ester synthase/acyl-CoA: diacylglycerol acyltransferase from Simmondsia chinensis, Acinetobacter sp. ADP1, Alcanivorax borkumensis, Pseudomonas aeruginosa, Fundibacter jadensis, Arabidopsis thaliana, or Alkaligenes eutrophus. In some embodiments, the gene encoding an ester synthase is selected from the group consisting of: AtfA1 (an ester synthase derived from Alcanivorax borkumensis SK2, GenBank Accession No. YP_694462), AtfA2 (another ester synthase derived from Alcanivorax borkumensis SK2, GenBank Accession No. YP_693524), ES9 (an ester synthase from Marinobacter hydrocarbonoclasticus DSM 8798, GenBank Accession No. AB021021), ES8 (another ester synthase derived from Marinobacter hydrocarbonoclasticus DSM 8798, GenBank Accession No. AB021020), and variants thereof. In a particular embodiment, the gene encoding the ester synthase or a suitable variant is overexpressed.
- In another aspect, the invention features a method of making a fatty acid derivative, for example, a fatty ester, the method comprising expressing in a host cell a gene encoding an ester synthase polypeptide comprising the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, or a variant thereof. In certain embodiments, the polypeptide has ester synthase and/or acyltransferase activity. In some embodiments, the polypeptide has the capacity to catalyse the conversion of a thioester to a fatty acid and/or a fatty acid derivative such as a fatty ester. In a particular embodiment, the polypeptide has the capacity to catalyze the conversion of a fatty acyl-CoA and/or a fatty acyl-ACP to a fatty acid and/or a fatty acid derivative such as a fatty ester, using an alcohol as substrate. In alternative embodiments, the polypeptide has the capacity to catalyze the conversion of a free fatty acid to a fatty ester, using an alcohol as substrate.
- In certain embodiments, an endogenous thioesterase of the host cell, if present, is unmodified. In certain other embodiments, the host cell expresses an attenuated level of a thioesterase activity or the thioesterase is functionally deleted. In some embodiments, the host cell has no detectable thioesterase activity. As used herein the term“detectable” means capable of having an existence or presence ascertained. For example, production of a product from a reactant (e.g., production of a certain type of fatty acid esters) is desirably detectable using the methods provided herein. In certain embodiments, the host cell expresses an attenuated level of a fatty acid degradation enzyme, such as, for example, an acyl-CoA synthase, or the fatty acid degradation enzyme is functionally deleted. In some embodiments, the host cell has no detectable fatty acid degradation enzyme activity. In particular embodiments, the host cell expresses an attenuated level of a thioesterease, a fatty acid degradation enzyme, or both. In other embodiments, the thioesterase, the fatty acid degradation enzyme, or both, are functionally deleted. In some embodiments, the host cell has no detectable thioesterase activity, acyl-CoA synthase activity, or neither. In some embodiments, the host cell can convert an acyl-ACP or acyl-CoA into fatty acids and/or derivatives thereof such as esters, in the absence of a thioesterase, a fatty acid derivative enzyme, or both. Alternatively, the host cell can convert a free fatty acid to a fatty ester in the absence of a thioesterase, a fatty acid derivative enzyme, or both. In certain embodiments, the method further includes isolating the fatty acids or derivatives thereof from the host cell.
- In certain embodiments, the fatty acid derivative is a fatty ester. In certain embodiments, the fatty acid or fatty acid derivative is derived from a suitable alcohol substrate such as a short- or long-chain alcohol. In some embodiments, the fatty acid or fatty acid derivative is present in the extracellular environment. In certain embodiments, the fatty acid or fatty acid derivative is isolated from the extracellular environment of the host cell. In some embodiments, the fatty acid or fatty acid derivative is spontaneously secreted, partially or completely, from the host cell. In alternative embodiments, the fatty acid or derivative is transported into the extracellular environment, optionally with the aid of one or more transport proteins. In other embodiments, the fatty acid or fatty acid derivative is passively transported into the extracellular environment.
- In another aspect, the invention features an in vitro method of producing a fatty acid and/or a fatty acid derivative extracellulary comprising providing a substrate and a purified ester synthase comprising the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, or a variant thereof. In some embodiments, the method comprising culturing a host cell under conditions that allow expression or overexpression of an ester synthase polypeptide or a variant thereof, and isolating the ester synthase from the cell. In some embodiments, the method further comprising contacting a suitable substrate such with the cell-free extract under conditions that permit production of a fatty acid and/or a fatty acid derivative.
- In some embodiments, the ester synthase polypeptide comprises the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, with one or more amino acid substitutions, additions, insertions, or deletions, and the polypeptide has ester synthase and/or acyltransferase activity. In certain embodiments, the ester synthase polypeptide has increased ester synthase and/or transferase activity. For example, the ester synthase polypeptide is capable, or has an improved capacity, of catalyzing the conversion of thioesters, for example, fatty acyl-CoAs or fatty acyl-ACPs, to fatty acids and/or fatty acid derivatives. In particular embodiments, the ester synthase polypeptide is capable, or has an improved capacity, of catalyzing the conversion of thioester substrates to fatty acids and/or derivatives thereof, such as fatty esters, in the absence of a thioesterase activity, a fatty acid degradation enzyme activity, or both. For example, the polypeptide converts fatty acyl-ACP and/or fatty acyl-CoA into fatty esters in vivo, in the absence of a thioesterase or an acyl-CoA synthase activity. In alternative embodiments, the polypeptide is capable of catalyzing the conversion of a free fatty acid to a fatty ester, in the absence of a thioesterase activity, a fatty acid degradation enzyme activity, or both. For example, the polypeptide can convert a free fatty acid into a fatty ester in vivo or in vitro, in the absence of a thioesterase activity, an acyl-CoA synthase activity, or both.
- In some embodiments, the ester synthase polypeptide is a variant comprising the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, with one or more non-conserved amino acid substitutions, wherein the ester synthase polypeptide has ester synthase and/or acyltransferase activity. In certain embodiments, the ester synthase polypeptide has improved ester synthase and/or acyltransferase activity. For example, a glycine residue at
position 395 of SEQ ID NO:18 can be substituted with a basic amino acid residue, such that the resulting ester synthase variant retains or has improved ester synthase and/or acyltransferase activity. In an exemplary embodiment, the glycine residue atposition 395 of SEQ ID NO:18 is substituted with an arginine or a lysine residue, wherein the resulting ester synthase variant retains or has improved capacity to catalyze the conversion of a thioester into a fatty acid and/or a fatty acid derivative such as a fatty ester. - In some embodiments, the ester synthase variant comprises one or more of the following conserved amino acid substitutions: replacement of an aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic amino acid; replacement of a serine with a threonine; replacement of a threonine with a serine; replacement of an acidic residue, such as aspartic acid and glutamic acid, with another acidic residue; replacement of residue bearing an amide group; exchange of a basic residue, such as lysine and arginine, with another basic residue; and replacement of an aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue. In some embodiments, the ester synthase variant has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acid substitutions, additions, insertions, or deletions. In some embodiments, the polypeptide variant has ester synthase and/or acyltransferase activity. For example, the ester synthase polypeptide is capable of catalyzing the conversion of thioesters to fatty acids and/or fatty acid derivatives, using alcohols as substrates. In a non-limiting example, the polypeptide is capable of catalyzing the conversion of a fatty acyl-CoA and/or a fatty acyl-ACP to a fatty acid and/or a fatty acid ester, using a suitable alcohol substrate, such as, for instance, a methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, decanol, dodecanol, tetradecanol, or hexadecanol. In another non-limiting example, the ester synthase polypeptide is capable of catalyzing the conversion of a fatty acyl-ACP and/or a fatty acyl-CoA to a fatty acid and/or a fatty acid ester, in the absence of a thioesterase, a fatty acid degradation enzyme, or both. In a further embodiment, the polypeptide is capable of catalyzing the conversion of a free fatty acid into a fatty ester in the absence of a thioesterase, a fatty acid degradation enzyme, or both.
- In some embodiments, the ester synthase polypeptide is about 200 amino acids to about 2,000 amino acids in length, for example, from about 250 to about 1,500 amino acid residues in length, from about 300 to about 1,200 amino acid residues in length, from about 350 to about 1,000 amino acid residues in length, from about 400 to about 800 amino acid residues in length, or from about 450 to about 600 amino acid residues in length. In certain embodiments, the ester synthase polypeptide is about 300 amino acid residues in length or longer, for example, about 400 amino acid residues in length or longer, or about 450 amino acid residues in length or longer. In certain related embodiments, the ester synthase polypeptide is about 1,000 amino acid residues in length or shorter, for example, about 800 amino acid residues in length or shorter, about 700 amino acid residues in length or shorter, or about 600 amino acid residues in length or shorter. An exemplary ester synthase of the invention is about 500 amino acid residues in length.
- In some embodiments, the method further includes modifying the expression of a gene encoding an ester synthase in the host cell. In certain embodiments, modifying the expression of a gene encoding an ester synthase includes expressing a heterologous gene encoding an ester synthase in the host cell and/or otherwise increasing the expression or activity of an endogenous ester synthase in the host cell.
- In certain embodiments, an endogenous thioesterase enzyme of the host cell, if present, is unmodified. In other embodiments, the method includes modifying the expression of a gene encoding a thioesterase in the host cell. In certain embodiments, modifying the expression of a gene encoding a thioesterase includes expressing a heterologous gene encoding a thioesterase in the host cell and/or increasing the expression and/or activity of an endogenous thioesterase in the host cell. In alternate embodiments, modifying the expression of a gene encoding a thioesterase includes attenuating the expression of an endogenous gene encoding a thioesterase in the host cell, and/or decreasing the expression and/or activity of an endogenous thioesterase in the host cell. In certain embodiments, modifying the expression of a thioesterase in the host cell comprises functionally deleting an endogenous gene encoding a thioesterase. In some embodiments, there is no detectable thioesterase activity in the host cell. In particular embodiments, the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
- Thioesterase activity can be determined or measured using various known in vitro or in vivo assays, including without limitation an acyl-CoA hydrolysis assay, which measures the rate of cleavage of an acyl-CoA substrate using a DTNB (5,5′-Dithio-bis(2-nitro-benzoic acid)) reaction and monitors the changes of absorbance at 412 nm and a molar extinction coefficient of 13,600 M−1cm−1. It is known in the art that various ester synthases can have overlapping thioesterase activities. As used herein, however, the term “ester synthase” does not comprise enzymes that also have thioesterase activity. The ones that have both ester synthase activity and thioesterase activity are categorized as thioesterases herein.
- In another aspect, the invention features a method of making fatty acids and/or fatty acid derivatives, for example, a fatty ester, comprising expressing in a host cell a gene encoding an ester synthase, in the absence of a thioesterase or a thioesterase activity in the host cell. In certain embodiments, the host cell is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to its level in a wild type host cell. Examples of fatty acid degradation enzymes include, without limitation, an acyl-CoA synthase of EC 2.3.1.86 or EC 6.2.1.3. Examples of host cells from which the fatty acid degradation enzyme can be found include Saccharomyces cerevisiae, Candida lipolytica, Escherichia coli, Arthrobacter, Rhodotorula glutinins, Acinetobacter, Candida lipolytica, Botryococcus braunii, Vibrio fumissii, Vibrio harveyi, Micrococcus leuteus, Stenotrophomonas maltophila or Bacillus subtilis. In some embodiments, the host cell is genetically engineered to express an attenuated level of an acyl-CoA synthase relative to a wild type host cell. In particular embodiments, the host cell expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, Pf1-4354, EAV15023, fadD1, fadD2, RPC_4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_01644857. In certain embodiments, the fatty acid degradation enzyme, such as the acyl-CoA synthase gene, is functionally deleted such that the host cell has no detectable acyl-CoA synthase activity.
- In certain embodiments, an endogenous thioesterase enzyme of the host cell, if present, is unmodified. In other embodiments, the host cell is genetically engineered to express an attenuated level of a thioesterase, an attenuated level of a fatty acid degradation enzyme, or both. In some embodiments, a thioesterase, a fatty acid degradation enzyme, or both, are functionally deleted. In some embodiments, the host cell has no detectable thioesterase activity, acyl-CoA synthase activity or neither. In some embodiments, the host cell can produce fatty acids and/or derivatives from thioester and alcohol substrates. In alternative embodiments, the host cell can produce fatty esters from free fatty acids and alcohol substrates.
- In some embodiments, the ester synthase polypeptide is derived from, for example, a bacterium, plant, insect, yeast, fungus, or mammal. In certain embodiments, the ester synthase polypeptide is from a mammalian cell, a plant cell, an insect cell, a fungus cell, a filamentous fungi cell, a bacterial cell, a cyanobacterial cell, or a cell of any other organism described herein. In certain embodiments, the naturally-occurring ester synthase is from Acidobacteria, Acidothermus, Acinetobacter, Aeromonas, Alcanivorax, Alcaligenes, Alteromonas, Anaeromyxobacter, Arabidopsis, Bradyrhizobium, Erythrobacter, Frankia, Fundibacter, Hahella chejuensis, Janibacter, Limnobacter, Marinobacter, Methylibium, Microscilla, Moritella, Mus musculus, Mycobacterium, Myxococcus, Natronomonas, Nocardia, Nocardioides, Photobacterium, Proteobacterium, Plesiocystis, Polaromonas, Pseudomonas, Psychrobacter, Reinekea, Rhodoferax, Rhodococcus, Roseiflexus, Saccharopolyspora, Salinibacter, Simmodsia Solibacter, Sphingopyxis, Stigmatella, Streptomyces, Tenacibaculum, or Ustilago.
- In particular embodiments, the naturally-occurring ester synthase of the invention is derived from any one of Acidobacteria bacterium, Acidothermus cellulolyticus, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter sp., Aeromonas hydrophila, Aeromonas salmonicida, Alcaligenes europhus, Alcanivorax borkumensis, Alcanivorax jadensis, Alteromonas macleodii, Anaeromyxobacter dehalogenans, Anaeromyxobacter sp., Arabidopsis thaliana, Bradyrhizobium japonicum, Cryptococcus curvatus, Erythrobacter litoralis, Erythrobacter sp., Frankia sp., Fundibacter jadensis, gamma proteobacterium, Hahella chejuensis, Homo sapiens, Janibacter sp., Limnobacter sp., marine gamma proteobacterium, Marinobacter algicola, Marinobacter aquaeolei, Marinobacter hydrocarbinoclasticus, Marinobacter sp., Methylibium petroleiphilum, Microscilla marina, Moritella sp., Mortierella alpina, Mus musculus, Mycobacterium abscessus, Mycobacterium avium, Mycobacterium bovis, Mycobacterium gilvum, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium smegmatis, Mycobacterium sp., Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycobacterium vanbaalenii, Myxococcus xanthus, Natronomonas pharaonis, Nocardia farcinica, Nocardioides sp., Photobacterium profundum, Plesiocystis pacifica, Polaromonas naphthalenivorans, Polaromonas sp., Pseudomonas aeruginosa, Psychrobacter arcticus, Psychrobacter cryohalolentis, Psychrobacter sp., Reinekea sp., Rhodococcus opacus, Rhodoferax ferrireducens, Rhodococcus sp., Rhodoferax ferrireducens, Roseiflexus sp., Roseiflexus castenholzii, Saccharomyces cerevisiae, Saccharopolyspora erythraea, Salinibacter ruber, Simmodsia chinensis, Solibacter usitatus, Sphingopyxis alaskensis, Stigmatella aurantiaca, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, Tenacibaculum sp., and Ustilago maydis. In particular embodiments, the gene encoding the ester synthase or a variant thereof is overexpressed.
- In other embodiments, the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis. In certain embodiments, one or more of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, a gene encoding a transcriptional regulator of fatty acid biosynthesis are functionally deleted. In certain embodiments, the host cell expressing a decreased level of an outer membrane protein receptor is resistant to phage infection. In some embodiments, the gene encoding an acyl-CoA dehydrogenase is fadE. In some embodiments, the gene encoding an outer membrane protein receptor is one that encodes a receptor for ferrichrome, colicin M, phage T1, phage T5, or phage phi80. Host cells comprising such an outer membrane protein receptor include, for example, Saccharomyces cerevisiae, Candida lipolytica, Escherichia coli, Arthrobacter, Rhodotorula glutinins, Acinetobacter, Candida lipolytica, Botryococcus braunii, Vibrio fumissii, Vibrio harveyi, Micrococcus leuteus, Stenotrophomonas maltophila, or Bacillus subtilis. In certain embodiments, the gene encoding an outer membrane protein receptor is fhuA (also known as tonA). In yet other embodiments, the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
- In certain embodiments, the method further includes culturing the host cell in the presence of at least one biological substrate for the ester synthase polypeptide. In some embodiments, the method includes culturing the host cell under conditions that allow the expression or overexpression of the ester synthase enzyme or a suitable variant thereof. In further embodiments, the method includes culturing the host cell under conditions that permit production of fatty acids or derivatives thereof via an enzymatic reaction catalyzed by the ester synthase or a variant thereof, without the participation of a thioesterase, a fatty acid degradation enzyme, or both. In certain embodiments, the biological substrate for the ester synthase polypeptide is a thioester. In other embodiments, the biological substrate is an alcohol, such as, for example, short-chain or long-chain fatty alcohols. In yet another embodiment, the biological substrate is a free fatty acid.
- In another aspect, the invention features a method of producing a fatty acid and/or fatty acid derivative, including, for example, a fatty acid ester. The method includes expressing in a host cell a gene encoding an ester synthase or a variant thereof comprising an amino acid sequence having at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:18, 24, 25, or 26. In some embodiments, the ester synthase has the amino acid sequence of SEQ ID NO:18, 24, 25 or 26.
- In some embodiments, the method further includes isolating the fatty acid or fatty acid derivative from the genetically engineered host cell. In some embodiments, the fatty acid or fatty acid derivative is present in the extracellular environment. In certain embodiments, the fatty acid or fatty acid derivative is isolated from the extracellular environment of the host cell. In certain embodiments, the fatty acid or fatty acid derivative is spontaneously secreted into the extracellular environment, partially or completely. In some embodiments, the fatty acid or fatty acid derivative is transported into the extracellular environment, with or without the aid of one or more suitable transport proteins. In other embodiments, the fatty acid or fatty acid derivative is passively transported into the extracellular environment.
- In certain embodiments, an endogenous thioesterase enzyme of the host cell, if present, is unmodified. In certain embodiments, the method includes modifying the expression of a gene encoding a thioesterase in the host cell. In some embodiments, modifying the expression of a gene encoding a thioesterase includes expressing a heterologous gene encoding a thioesterase in the host cell and/or increasing the expression or activity of an endogenous thioesterase in the host cell. In alternate embodiments, modifying the expression of a gene encoding a thioesterase includes attenuating the expression of an endogenous gene encoding a thioesterase in the host cell. In some embodiments, modifying the expression of a gene encoding a thioesterase includes functionally deleting an endogenous gene encoding a thioesterase. In particular embodiments, the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
- In some embodiments, the host cell is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to its level in a wild type host cell. Exemplary fatty acid degradation enzymes include, without limitation, acyl-CoA synthase enzymes of EC 2.3.1.86 or EC 6.2.1.3. In particular embodiments, the host cell expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, Pf1-4354, EAV15023,
fadD 1, fadD2, RPC_4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_01644857. In certain embodiments, the above-described fatty acid degradation enzyme such as, for example, an acyl-CoA synthase, is functionally deleted such that the host cell does not have detectable acyl-CoA synthase activity. - In certain embodiments, the host cell is genetically engineered to express an attenuated level of a thioesterease, an attenuated level of a fatty acid degradation enzyme, or both. In some embodiments, a thioesterase, an acyl-CoA synthase, or both, are functionally deleted. In some embodiments, the host cell has no detectable thioesterase activity or fatty acid degradation enzyme activity. In some embodiments, the host cell is capable of producing fatty acids and/or derivatives thereof.
- In other embodiments, the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis. In some embodiments, one or more of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis are functionally deleted. In some embodiments, the gene encoding an acyl-CoA dehydrogenase is fadE. In some embodiments, the gene encoding an outer membrane protein receptor is one that encodes a receptor for ferrichrome, colicin M, phage T1, phage T5, or phage phi80. In certain embodiments, the gene encoding an outer membrane receptor is fhuA (also known as tonA). In yet other embodiments, the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
- In some embodiments, the ester synthase polypeptide is derived from a bacterium, plant, insect, yeast, fungus, or mammal. In certain embodiments, the polypeptide is from a mammalian cell, plant cell, insect cell, yeast cell, fungus cell, filamentous fungi cell, bacterial cell, or a cell of any other organism described herein. In certain embodiments, the ester synthase is from Acidobacteria, Acidothermus, Acinetobacter, Aeromonas, Alcanivorax, Alcaligenes, Alteromonas, Anaeromyxobacter, Arabidopsis, Bradyrhizobium, Erythrobacter, Frankia, Fundibacter, Hahella chejuensis, Janibacter, Limnobacter, Marinobacter, Methylibium, Microscilla, Moritella, Mus musculus, Mycobacterium, Myxococcus, Natronomonas, Nocardia, Nocardioides, Photobacterium, Proteobacterium, Plesiocystis, Polaromonas, Pseudomonas, Psychrobacter, Reinekea, Rhodoferax, Rhodococcus, Roseiflexus, Saccharopolyspora, Salinibacter, Simmodsia Solibacter, Sphingopyxis, Stigmatella, Streptomyces, Tenacibaculum, or Ustilago.
- In a particular embodiment, the ester synthase is derived from any one of Acidobacteria bacterium, Acidothermus cellulolyticus, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter sp., Aeromonas hydrophila, Aeromonas salmonicida, Alcaligenes europhus, Alcanivorax borkumensis, Alcanivorax jadensis, Alteromonas macleodii, Anaeromyxobacter dehalogenans, Anaeromyxobacter sp., Arabidopsis thaliana, Bradyrhizobium japonicum, Cryptococcus curvatus, Erythrobacter litoralis, Erythrobacter sp., Frankia sp., Fundibacter jadensis, gamma proteobacterium, Hahella chejuensis, Homo sapiens, Janibacter sp., Limnobacter sp., marine gamma proteobacterium, Marinobacter algicola, Marinobacter aquaeolei, Marinobacter hydrocarbinoclasticus, Marinobacter sp., Methylibium petroleiphilum, Microscilla marina, Moritella sp., Mortierella alpina, Mus musculus, Mycobacterium abscessus, Mycobacterium avium, Mycobacterium bovis, Mycobacterium gilvum, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium smegmatis, Mycobacterium sp., Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycobacterium vanbaalenii, Myxococcus xanthus, Natronomonas pharaonis, Nocardia farcinica, Nocardioides sp., Photobacterium profundum, Plesiocystis pacifica, Polaromonas naphthalenivorans, Polaromonas sp., Pseudomonas aeruginosa, Psychrobacter arcticus, Psychrobacter cryohalolentis, Psychrobacter sp., Reinekea sp., Rhodococcus opacus, Rhodoferax ferrireducens, Rhodococcus sp., Rhodoferax ferrireducens, Roseiflexus sp., Roseiflexus castenholzii, Saccharomyces cerevisiae, Saccharopolyspora erythraea, Salinibacter ruber, Simmodsia chinensis, Solibacter usitatus, Sphingopyxis alaskensis, Stigmatella aurantiaca, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, Tenacibaculum sp., and Ustilago maydis. In particular embodiments, the gene encoding the ester synthase or a variant thereof is overexpressed.
- In some embodiments, the method further includes culturing the host cell in the presence of at least one biological substrate for the ester synthase polypeptide. In certain embodiments, the method includes culturing the host cell under conditions sufficient to allow expression or overexpression of an ester synthase in the host cell. In certain embodiments, an endogenous thioesterase of the host cell, if present, is unmodified. In other embodiments, the host cell expresses an attenuated level of a thioesterase, a fatty acid degradation enzyme, or both. In some embodiments, the thioesterase, the fatty acid degradation enzyme, or both, are functionally deleted. In some embodiments, the host cell has no detectable thioesterase activity, acyl-CoA synthase activity, or neither activity. In some embodiments, the method includes culturing the host cells under conditions sufficient to permit production of fatty acids and/or fatty acid derivatives. In certain embodiments, the host cell is cultured in the presence of thioester substrates. In other embodiments, the host cell is cultured in the presence of an alcohol. In yet another embodiment, the host cell is cultured in the presence of a free fatty acid.
- In another aspect, the invention features a method of producing a fatty acid and/or a fatty acid derivative such as, for example, a fatty ester. The method comprises expressing in a host cell a polynucleotide that that hybridizes to a complement of a nucleotide sequence SEQ ID NO:27, 28, 29, or 30, or to a fragment thereof, wherein the polynucleotide encodes a polypeptide having ester synthase and/or acyltransferase activity. For example, the polypeptide is capable catalyzing the conversion of thioesters to fatty acids and/or fatty acid derivatives such as fatty esters, using one or more alcohols as substrates. In a particular embodiment, suitable thioester substrates include acyl-CoAs, such as, for example, fatty acyl-CoAs, and acyl-ACPs, such as, for example, fatty acyl-ACPs. Suitable alcohol substrates include, for example, short or long chain alcohols, such as methanol, ethanol, propanol, butanol, pentanol, hexanol, octanol, heptanol, decanol, dodecanol, tetradecanol, or hexadecanol. In some embodiments, the polypeotide is capable of catalyzing the conversion of a free fatty acid to a fatty esters.
- In some embodiments, the polynucleotide hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions, to a complement of the nucleotide sequence of SEQ ID NO:27, 28, 29, or 30, or to a fragment thereof. In certain embodiments, the polynucleotide comprises a sequence that has been codon-optimized for expression or overexpression in a selected host cell.
- In some embodiments, the method further comprises isolating the fatty acid or fatty acid derivative from the host cell. In some embodiments, the fatty acid or fatty acid derivative is present in the extracellular environment. In certain embodiments, the fatty acid or fatty acid derivative is isolated from the extracellular environment of the host cell. In some embodiments, the fatty acid or fatty acid derivative is spontaneously secreted, partially or completely, from the host cell. In alternative embodiments, the fatty acid or fatty acid derivative is transported into the extracellular environment with or without the aid of one or more transport proteins. In other embodiments, the fatty acid or fatty acid derivative is passively transported into the extracellular environment.
- In certain embodiments, an endogenous thioesterease of the host cell, if present, is unmodified. In certain other embodiments, the method includes modifying the expression of a gene encoding a thioesterase in the host cell. In certain embodiments, modifying the expression of a gene encoding a thioesterase includes expressing a heterologous gene encoding a thioesterase in the host cell and/or increasing the expression or activity of an endogenous thioesterase in the host cell. In alternate embodiments, modifying the expression of a gene encoding a thioesterase includes attenuating the expression of an endogenous gene encoding a thioesterase in the host cell and/or functionally deleting an endogenous thioesterase. In particular embodiments, the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
- In some embodiments, the host cell is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to its level in a wild type host cell. Exemplary fatty acid degradation enzymes include, without limitation, acyl-CoA synthase enzymes of EC 2.3.1.86 or EC 6.2.1.3. In particular embodiments, the host cell expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, pf1-4354, eav15023, fadD1, fadD2, rpc_4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_01644857. In certain embodiments, the aforementioned fatty acid degradation enzyme, such as the acyl-CoA synthase gene, is functionally deleted such that the host cell does not have a detectable acyl-CoA synthase activity.
- In some embodiments, the host cell is genetically engineered to express an attenuated level of a thioesterase, an attenuated level of a fatty acid degradation enzyme, or both. In some embodiments, a thioesterase, a fatty acid degradation enzyme, or both, are functionally deleted. In certain embodiments, the host cell has no detectable thioesterase activity or fatty acid degradation enzyme activity. In some embodiments, the host cell is capable of producing fatty acids and/or derivatives thereof.
- In other embodiments, the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis. In other embodiments, one or more of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis are functionally deleted. In some embodiments, the gene encoding an acyl-CoA dehydrogenase is fadE. In some embodiments, the gene encoding an outer membrane protein receptor is one that encodes a receptor for ferrichrome, colicin M, phage T1, phage T5, or phage phi80. In certain embodiments, the gene encoding an outer membrane protein receptor is fhuA (also known as tonA). In yet other embodiments, the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
- In some embodiments, the ester synthase is derived from a bacterium, plant, insect, yeast, fungus, or mammal. In certain embodiments, the ester synthase polynucleotide is from a mammalian cell, plant cell, insect cell, yeast cell, fungus cell, filamentous fungi cell, bacterial cell, or a cell of any other organism described herein. In certain embodiments, the polynucleotide is from Acidobacteria, Acidothermus, Acinetobacter, Aeromonas, Alcanivorax, Alcaligenes, Alteromonas, Anaeromyxobacter, Arabidopsis, Bradyrhizobium, Erythrobacter, Frankia, Fundibacter, Hahella chejuensis, Janibacter, Limnobacter, Marinobacter, Methylibium, Microscilla, Moritella, Mus musculus, Mycobacterium, Myxococcus, Natronomonas, Nocardia, Nocardioides, Photobacterium, Proteobacterium, Plesiocystis, Polaromonas, Pseudomonas, Psychrobacter, Reinekea, Rhodoferax, Rhodococcus, Roseiflexus, Saccharopolyspora, Salinibacter, Simmodsia Solibacter, Sphingopyxis, Stigmatella, Streptomyces, Tenacibaculum, or Ustilago.
- In a particular embodiment, the ester synthase polynucleotide is derived from any one of Acidobacteria bacterium, Acidothermus cellulolyticus, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter sp., Aeromonas hydrophila, Aeromonas salmonicida, Alcaligenes europhus, Alcanivorax borkumensis, Alcanivorax jadensis, Alteromonas macleodii, Anaeromyxobacter dehalogenans, Anaeromyxobacter sp., Arabidopsis thaliana, Bradyrhizobium japonicum, Cryptococcus curvatus, Erythrobacter litoralis, Erythrobacter sp., Frankia sp., Fundibacter jadensis, gamma proteobacterium, Hahella chejuensis, Homo sapiens, Janibacter sp., Limnobacter sp., marine gamma proteobacterium, Marinobacter algicola, Marinobacter aquaeolei, Marinobacter hydrocarbinoclasticus, Marinobacter sp., Methylibium petroleiphilum, Microscilla marina, Moritella sp., Mortierella alpina, Mus musculus, Mycobacterium abscessus, Mycobacterium avium, Mycobacterium bovis, Mycobacterium gilvum, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium smegmatis, Mycobacterium sp., Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycobacterium vanbaalenii, Myxococcus xanthus, Natronomonas pharaonis, Nocardia farcinica, Nocardioides sp., Photobacterium profundum, Plesiocystis pacifica, Polaromonas naphthalenivorans, Polaromonas sp., Pseudomonas aeruginosa, Psychrobacter arcticus, Psychrobacter cryohalolentis, Psychrobacter sp., Reinekea sp., Rhodococcus opacus, Rhodoferax ferrireducens, Rhodococcus sp., Rhodoferax ferrireducens, Roseiflexus sp., Roseiflexus castenholzii, Saccharomyces cerevisiae, Saccharopolyspora erythraea, Salinibacter ruber, Simmodsia chinensis, Solibacter usitatus, Sphingopyxis alaskensis, Stigmatella aurantiaca, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, Tenacibaculum sp. and Ustilago maydis. In particular embodiments, the polynucleotide encoding the ester synthase or a variant thereof is overexpressed.
- In some embodiments, the method further comprises culturing the host cell in the presence of at least one biological substrate for the ester synthase polypeptide. In certain embodiments, the method comprises culturing the host cell under conditions that are sufficient to allow expression or overexpression of the ester synthase in the host cell. In some embodiments, the method further comprises culturing the host cell under conditions that are sufficient to permit production of fatty acids and/or fatty acid derivatives. In some embodiments, the method comprises culturing the host cell in the presence of a thioester substrate such as, for example, a fatty acyl-CoA or a fatty acyl-ACP. In certain other embodiments, the method comprises culturing the host cell in the presence of an alcohol substrate. In yet further embodiments, the method comprising culturing the host cell in the presence of a free fatty acid.
- In another aspect, the invention features a method of producing a fatty acid or a fatty acid derivative such as, for example, a fatty ester. The method comprises expressing in a host cell a heterologous gene encoding an ester synthase having the amino acid sequence of SEQ ID NO:18, or a variant thereof. Alternatively, the method comprises manipulating an endogenous ester synthase gene of a host cell encoding an ester synthase of SEQ ID NO:18, or a variant thereof, using genomic alteration. In some embodiments, the method further includes isolating the fatty acid and/or fatty acid derivative from the host cell.
- In yet another aspect, the invention features an in vitro method of producing fatty acids and/or fatty acid derivatives extracellularly from a substrate using a purified ester synthase having the amino acid sequence of SEQ ID NO:18, or a variant thereof. In one embodiment, the host cell is cultured under conditions that allow expression or overexpression of the ester synthase or a variant thereof. The host cell is then be harvested and lysed. Optionally, the resulting mixture is purified. Suitable substrates such as those described herein can be added to the cell-free extracts under conditions that permit production of a fatty acid and/or a fatty acid derivative.
- In some embodiments, the ester synthase variant comprises the amino acid sequence of SEQ ID NO:18, with one or more amino acid substitutions, additions, insertions, or deletions, wherein the polypeptide has ester synthase activity and/or acyltransferase activity. In certain embodiments, the ester synthase variant has increased ester synthase and/or acyltransferase activity. For example, the ester synthase polypeptide is capable, or has improved capacity, of catalyzing the conversion of thioesters to fatty esters, using alcohols as substrates. Thioester substrates include, for example, fatty thioesters such as fatty acyl-CoAs or fatty acyl-ACPs. In some embodiments, the ester synthase variant is capable of, or has improved capacity of, catalyzing the conversion of free fatty acids to fatty esters, using alcohols as substrates. Alcohol substrates include, for example, long- or short-chain alcohols such as methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, decanol, dodecanol, tetradecanol, or hexadecanol.
- In some embodiments, the ester synthase variant comprises the amino acid sequence of SEQ ID NO:18, with one or more non-conserved amino acid substitutions, wherein the variant retains ester synthase and/or acyltransferase activity. In some embodiments, the ester synthase variant has improved ester synthase and/or acyltransferase activity. In an exemplary embodiment, the glycine residue at
position 395 of SEQ ID NO:18 is substituted with a basic amino acid residue, and the resultant ester synthase variant retains or has improved ester synthase and/or acyltransferase activity. In some embodiments, the glycine residue atposition 395 of SEQ ID NO:18 is substituted with an arginine or a lysine residue, and the resultant ester synthase variant has improved ester synthase and/or acyltransferase activity. - In some embodiments, the ester synthase variant comprises one or more conserved amino acid substitutions. In some embodiments, the ester synthase variant has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acid substitutions, additions, insertions, or deletions. In some embodiments, the ester synthase variant has ester synthase and/or acyltransferase activity. For example, the polypeptide is capable of catalyzing the conversion of thioesters to fatty acids and/or fatty acid derivatives such as fatty esters, using alcohols as substrates. In alternative embodiments, the polypeptide is capable of producing a fatty ester from a suitable free fatty acid and an alcohol, such as, for example, long- or short-chain alcohols, such as methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, decanol, dodecanol, tetradecanol, or hexadecanol.
- In certain embodiments, an endogenous thioesterase of the host cell, if present, is unmodified. In other embodiments, the method includes modifying the expression of a gene encoding a thioesterase in the host cell. In certain embodiments, modifying the expression of a thioesterase gene includes attenuating the expression of an endogenous gene encoding a thioesterase in the host cell and/or functionally deleting such a gene. In particular embodiments, the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
- In some embodiments, the host cell is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to its level in a wild type host cell. Exemplary fatty acid degradation enzymes include, without limitation, acyl-CoA synthase enzymes of EC 2.3.1.86 or EC 6.2.1.3. In particular embodiments, the host cell expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, pf1-4354, EAV15023,
fadD 1, fadD2, rpc_4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_01644857. In certain embodiments, the aforementioned fatty acid degradation enzyme, such as the acyl-CoA synthase gene, is functionally deleted such that the host cell does not express an acyl-CoA synthase or does not have a detectable acyl-CoA synthase activity. - In certain embodiments, the host cell is genetically engineered to express an attenuated level of a thioesterase, an attenuated level of a fatty acid degradation enzyme, or both. In an exemplary embodiment, a thioesterase, a fatty acid degradation enzyme, or both, are functionally deleted. In some embodiments, the host cell has no detectable level of a thioesterase activity or an acyl-CoA synthase activity. In certain embodiments, the host cell is capable of producing fatty acids and/or derivatives thereof.
- In other embodiments, the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis. In some embodiments, one or more of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis are functionally deleted. In some embodiments, the gene encoding an acyl-CoA dehydrogenase is fadE. In some embodiments, the gene encoding an outer membrane protein receptor is one that encodes a receptor for ferrichrome, colicin M, phage T1, phage T5, or phage phi80. In certain embodiments, the gene encoding an outer membrane protein receptor is fhuA (also known as tonA). In yet other embodiments, the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
- In some embodiments, the method further includes culturing the host cell in the presence of at least one biological substrate for the ester synthase polypeptide. In certain embodiments, the method includes culturing the host cell under conditions that allow expression or overexpression of the ester synthase in the host cell. In further embodiments, the method includes culturing the host cell under conditions that permit production of desirable fatty acids and/or fatty acid derivatives. In some embodiments, the method includes culturing the host cell in the presence of a thioester substrate. In certain other embodiments, the method includes culturing the host cell in the presence of an alcohol. In further embodiments, the method includes culturing the host cell in the presence of a free fatty acid.
- In another aspect, the invention features a method of producing a fatty acid and/or a fatty acid derivative such as, for example, a fatty ester. The method includes expressing in a host cell a gene encoding an ester synthase polypeptide comprising an amino acid sequence having at least about 35% sequence identity, for example, at least about 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:18. In a further embodiment, the host cell is engineered to have an attenuated level of a thioesterase, a fatty acid degradation enzyme, or both. Alternatively, the thioesterase, the fatty acid degradation enzyme, or both, are functionally deleted. In some embodiments, the host cell has no detectable thioesterase activity or acyl-CoA synthase activity. In some embodiments, the method further includes isolating an accordingly-produced fatty acid or fatty acid derivative from the host cell.
- In alternative embodiments, the invention further features an in vitro method of producing a fatty acid and/or fatty acid derivative extracellularly, using a purified ester synthase having at least about 35% sequence identity to the amino acid sequence of SEQ ID NO:18, from a suitable substrate. In an exemplary embodiment, the host cell is cultured under conditions that allow expression or overexpression of the ester synthase enzyme or a variant thereof. The host cell is then harvested and lysed. Optionally, the resulting mixture is purified. Suitable substrates such as those described herein can be added to the cell-free extracts under conditions that permit production of a fatty acid and/or a fatty acid derivative.
- In some embodiments, the amino acid sequence of an ester synthase or a suitable variant of the invention has at least about 35%, for example, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to SEQ ID NO:18. In some embodiments, the amino acid sequence of the ester synthase is SEQ ID NO:18. An exemplary ester synthase polypeptide include an ester synthase from Limnobacter sp. MED105, with a GenBank Accession No. of ZP_01915978 (SEQ ID NO:41), which has about 51% sequence identity to the amino acid sequence of the ester synthase from Marinobacter hydrocarbonclasticus DSM 8798, SEQ ID NO:18.
- In certain embodiments, an endogenous thioesterase of the host cell, if present, is unmodified. In other embodiments, the method includes modifying the expression of a gene encoding a thioesterase in the host cell. In certain embodiments, modifying the expression of a thioesterase gene includes attenuating the expression of an endogenous gene encoding a thioesterase in the host cell and/or functionally deleting that gene. In particular embodiments, the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
- In some embodiments, the host cell is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to its level in a wild type host cell. Exemplary fatty acid degradation enzymes include, without limitation, acyl-CoA synthase enzymes of EC 2.3.1.86 or EC 6.2.1.3. In particular embodiments, the host cell expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, pf1-4354, EAV15023, fadD1, fadD2, rpc_4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_01644857. In certain embodiments, the aforementioned fatty acid degradation enzyme, such as the acyl-CoA synthase gene, is functionally deleted such that the host cell does not express the acyl-CoA synthase or has no detectable acyl-CoA synthase activity.
- In certain embodiments, the host cell is genetically engineered to express an attenuated level of a thioesterase, a fatty acid degradation enzyme such as an acyl-CoA synthase, or both. In an exemplary embodiment, a thioesterase, an acyl-CoA synthase, or both, are functionally deleted. In some embodiments, the host cell is capable of producing fatty acids and/or derivatives thereof.
- In other embodiments, the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis. In some embodiments, one or more of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis are functionally deleted. In some embodiments, the gene encoding an acyl-CoA dehydrogenase is fadE. In some embodiments, the gene encoding an outer membrane protein receptor is one that encodes a receptor for ferrichrome, colicin M, phage T1, phage T5, or phage phi80. In certain embodiments, the gene encoding an outer membrane protein receptor is fhuA (also known as tonA). In yet other embodiments, the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
- In another aspect, the invention features a method of producing a fatty acid or a fatty acid derivative such as, for example, a fatty ester. The method includes expressing in a host cell a polynucleotide that hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions, to a complement of SEQ ID NO:27, or to a fragment thereof, wherein the polynucleotide encodes a polypeptide having ester synthase and/or acyltransferase activity. For example, the polypeptide has ester synthase activity and is capable of converting thioesters to fatty esters, using alcohols as substrates. Suitable thioester substrates include, for example, fatty thioesters such as fatty acyl-CoAs or fatty acyl-ACPs. In other embodiments, the host cell produces fatty esters from free fatty acid and alcohol substrates. Suitable alcohol substrates include, for example, long or short chain alcohols such as methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, decanol, dodecanol, tetradecanol, or hexadecanol.
- In certain embodiments, an endogenous thioesterase of the host cell, if present, is unmodified. In other embodiments, the method includes modifying the expression of a gene encoding a thioesterase in the host cell. In some embodiments, modifying the expression of a thioesterase gene includes attenuating the expression of an endogenous gene encoding a thioesterase in the host cell and/or functionally deleting that gene. In particular embodiments, the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
- In some embodiments, the host cell is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to its level in a wild type host cell. Exemplary fatty acid degradation enzymes include, without limitation, acyl-CoA synthase enzymes of EC 2.3.1.86 or EC 6.2.1.3. In particular embodiments, the host cell expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, pf1-4354, EAV15023, fadD1, fadD2, rpc_4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_01644857. In certain embodiments, the aforementioned fatty acid degradation enzyme, such as the acyl-CoA synthase gene, is functionally deleted such that the host cell does not express an acyl-CoA synthase or has no detectable acyl-CoA synthase activity.
- In certain embodiments, the host cell is genetically engineered to express an attenuated level of a thioesterase, a fatty acid degradation enzyme such as an acyl-CoA synthase, or both. In some embodiment, a thioesterase, an acyl-CoA synthase, or both, are functionally deleted. In some embodiments, the host cell is capable of producing fatty acids and/or derivatives thereof.
- In other embodiments, the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis. In some embodiments, one or more of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis are functionally deleted. In some embodiments, the gene encoding an acyl-CoA dehydrogenase is fadE. In some embodiments, the gene encoding an outer membrane protein receptor is one that encodes a receptor for ferrichrome, colicin M, phage T1, phage T5, or phage phi80. In certain embodiments, the gene encoding an outer membrane protein receptor is fhuA (also known as tonA). In yet other embodiments, the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
- In some embodiments, the method further includes culturing the host cell in the presence of at least one biological substrate of the ester synthase. In certain embodiments, the method includes culturing the host cell under conditions that allow expression or overexpression of the ester synthase enzyme in the host cell. In further embodiments, the method includes culturing the host cell under conditions that permit production of fatty acids and/or fatty acid derivatives. In some embodiments, the method includes culturing the host cell in the presence of a thioester substrate. In some other embodiments, the method includes culturing the host cell in the presence of an alcohol substrate. In other embodiments, the method includes culturing the host cell in the presence of a free fatty acid.
- In some embodiments, the method further includes isolating an accordingly-produced fatty acid or fatty acid derivative from the host cell. In some embodiments, the fatty acid or fatty acid derivative is present in the extracellular environment. In certain embodiments, the fatty acid or fatty acid derivative is isolated from the extracellular environment of the host cell. In some embodiments, the fatty acid or fatty acid derivative is spontaneously secreted, partially or completely, from the host cell. In alternative embodiments, the fatty acid or fatty acid derivative is transported into the extracellular environment, with or without the aid of one or more suitable transport proteins. In other embodiments, the fatty acid or fatty acid derivative is passively transported into the extracellular environment.
- In an alternative embodiment, the invention features an in vitro method of producing a fatty acid and/or fatty acid derivative extracellularly, using a purified ester synthase encoded by a polynucleotide that hybridizes to a complement of SEQ ID NO:27, or to a fragment thereof, and a suitable substrate. For example, the host cell can be cultured under conditions that allow expression or overexpression of the ester synthase enzyme or a variant thereof, and the cell is then be harvested and lysed. Optionally, the resulting mixture can be purified. Suitable substrates such as those described herein can be added to the cell-free extracts under conditions that permit production of a fatty acid and/or a fatty acid derivative.
- In another aspect, the invention features a method of producing a fatty acid and/or a fatty acid derivative such as, for example, a fatty ester. The method includes expressing in a host cell a recombinant vector comprising an ester synthase polynucleotide sequence having at least about 50% sequence identity to a polynucleotide sequence in
FIG. 16 . In some embodiments, the nucleotide sequence has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleotide sequence of SEQ ID NO:27, 28, 29, or 30. In some embodiments, the nucleotide sequence is SEQ ID NO:27, 28, 29, or 30. In an exemplary embodiment, a recombinant vector comprising an ester synthase polynucleotide sequence of SEQ ID NO:42 (codon-optimized from a polynucleotide encoding an ester synthase homolog from Limnobacter sp. MED105), which has about 50% sequence identity to SEQ ID NO:27, can be expressed in a host cell to produce a fatty acid and/or derivative thereof. In certain embodiments, the host cell is engineered to express an attenuated level of a thioesterase, a fatty acid degradation enzyme, or both. In some embodiments, a thioesterase, a fatty acid degradation enzyme, or both, are functionally deleted. In certain embodiments, the host cell is capable of producing fatty acids and/or derivatives thereof. - In some embodiments, the method further includes isolating the fatty acid or fatty acid derivative from the host cell. In some embodiments, the fatty acid or fatty acid derivative is present in the extracellular environment. In certain embodiments, the fatty acid or fatty acid derivative is isolated from the extracellular environment of the host cell. In some embodiments, the fatty acid or fatty acid derivative is spontaneously secreted, partially or completely, from the host cell. In alternative embodiments, the fatty acid or fatty acid derivative is transported into the extracellular environment, with or without the aid of one or more suitable transport proteins. In other embodiments, the fatty acid or fatty acid derivative is passively transported into the extracellular environment.
- In another aspect, the invention further features an in vitro method of producing a fatty acid and/or fatty acid derivative extracellularly, wherein a host cell comprising a recombinant vector comprising an ester synthase nucleotide sequence having at least about 50% sequence identity to a nucleotide sequence listed in
FIG. 16 is cultured under conditions that allow expression or overexpression of an ester synthase. The host cell is then harvested and lysed. Optionally, the resulting mixture is purified. A suitable substrate is then added to the cell free extract under conditions that permit production of a fatty acid and/or a fatty acid derivative in vitro. - In some embodiments, the recombinant vector further comprises a promoter operably linked to a nucleotide sequence encoding an ester synthase or a suitable variant. In certain embodiments, the promoter is a developmentally-regulated, or organelle-specific, a tissue-specific, an inducible, a constitutive, or a cell-specific promoter.
- In other embodiments, the recombinant vector comprises at least one sequence selected from the group consisting of (a) a regulatory sequence operatively coupled to the nucleotide sequence; (b) a selection marker operatively coupled to the nucleotide sequence; (c) a marker sequence operatively coupled to the nucleotide sequence; (d) a purification moiety operatively coupled to the nucleotide sequence; (e) a secretion sequence operatively coupled to the nucleotide sequence; and (f) a targeting sequence operatively coupled to the nucleotide sequence.
- In some embodiments, the recombinant vector is a plasmid.
- In some embodiments, the host cell expresses an ester synthase polypeptide that is encoded by the recombinant vector. In some embodiments, the nucleotide sequence is stably incorporated into the genomic DNA of the host cell, and the expression of the nucleotide sequence is under the control of a regulated promoter region.
- In certain embodiments, an endogenous thioesterase of the host cell, if present, is unmodified. In other embodiments, the method includes modifying the expression of a gene encoding a thioesterase in the host cell. In some embodiments, modifying the expression of a thioesterase gene includes attenuating the expression of an endogenous gene encoding a thioesterase in the host cell and/or functionally deleting that gene. In particular embodiments, the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
- In some embodiments, the host cell is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to its level in a wild type host cell. Exemplary fatty acid degradation enzymes include, without limitation, acyl-CoA synthase enzymes of EC 2.3.1.86, or EC 6.2.1.3. In particular embodiments, the host cell expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, pf1-4354, EAV15023,
fadD 1, fadD2, rpc_4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_01644857. In certain embodiments, the aforementioned fatty acid degradation enzyme, such as the acyl-CoA synthase gene, is functionally deleted such that the host cell does not express an acyl-CoA synthase, or has no acyl-CoA synthase activity. - In other embodiments, the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis. In some embodiments, one or more of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis are functionally deleted. In some embodiments, the gene encoding an acyl-CoA dehydrogenase is fadE. In some embodiments, the gene encoding an outer membrane protein receptor is one that encodes a receptor for ferrichrome, colicin M, phage T1, phage T5, or phage phi80. In certain embodiments, the gene encoding an outer membrane protein receptor is fhuA (also known as tonA). In yet other embodiments, the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
- In some embodiments, the method further includes culturing the host cell in the presence of at least one biological substrate of the ester synthase. In some embodiments, the method includes culturing the host cell under conditions that allow expression or overexpression of the ester synthase in the host cell. In further embodiments, the method includes culturing the host cell under conditions that permit production of fatty acids and/or fatty acid derivatives. In some embodiments, the method includes culturing the host cell in the presence of a thioester substrate. In other embodiments, the method includes culturing the host cell in the presence of an alcohol substrate. In further embodiments, the method includes culturing the host cell in the presence of a free fatty acid.
- In another aspect, the invention features a method of producing a fatty acid and/or a derivative thereof such as, for example, a fatty ester. The method includes expressing in a host cell a recombinant vector comprising an ester synthase nucleotide sequence having at least about 50% sequence identity to the nucleotide sequence of SEQ ID NO:27. In certain embodiments, the host cell is engineered to express an attenuated level of a thioesterase, a fatty acid degradation enzyme such as an acyl-CoA synthase, or both. In an exemplary embodiment, an endogenous thioesterase, an acyl-CoA synthase, or both are functionally deleted. In some embodiments, the host cell is capable of producing fatty acids and/or derivatives thereof.
- In some embodiments, the method further includes isolating the fatty acid and/or fatty acid derivative from the host cell. In some embodiments, the fatty acid or fatty acid derivative is present in the extracellular environment. In certain embodiments, the fatty acid or fatty acid derivative is isolated from the extracellular environment of the host cell. In some embodiments, the fatty acid or fatty acid derivative is spontaneously secreted, partially or completely, from the host cell. In alternative embodiments, the fatty acid or fatty acid derivative is transported into the extracellular environment, with or without the aid of one or more suitable transport proteins. In other embodiments, the fatty acid or fatty acid derivative is passively transported into the extracellular environment.
- In yet another aspect, the invention features an in vitro method of producing a fatty acid and/or fatty acid derivative extracellularly, wherein a host cell comprises a recombinant vector, which in turn comprises an ester synthase polynucleotide sequence having at least about 50% identity to SEQ ID NO:27, and wherein the host cell is cultured under conditions that allow expression or overexpression of the ester synthase. The host cell is then harvested and lysed. Optionally, the resulting mixture is purified. A suitable substrate, for example, one selected from those described herein, is then added to the cell free extract under conditions that permit production of a fatty acid and/or a fatty acid derivative in vitro.
- In some embodiments, the nucleotide sequence has at least about 55%, at least about 60%, at least about at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleotide sequence of SEQ ID NO:27. In some embodiments, the nucleotide sequence is SEQ ID NO:27.
- In certain embodiments, an endogenous thioesterase of the host cell, if present, is unmodified. In certain other embodiments, the method further includes modifying the expression of a gene encoding a thioesterase in the host cell. In certain embodiments, modifying the expression of a thioesterase gene includes attenuating the expression of an endogenous gene encoding a thioesterase in the host cell and/or functionally deleting that gene. In particular embodiments, the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
- In some embodiments, the host cell is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to its level in a wild type host cell. Exemplary fatty acid degradation enzymes include, without limitation, acyl-CoA synthase enzymes of EC 2.3.1.86 or EC 6.2.1.3. In particular embodiments, the host cell expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, pf1-4354, EAV15023,
fadD 1, fadD2, rpc_4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_01644857. In certain embodiments, the aforementioned fatty acid degradation enzyme, such as the acyl-CoA synthase gene, is functionally deleted such that the host cell does not express an acyl-CoA synthase or has no acyl-CoA synthase activity. - In certain embodiments, the host cell is genetically engineered to express attenuated level of a thioesterase, an attnenuated level of a fatty acid degradation enzyme such as an acyl-CoA synthase, or both. In some embodiments, a thioesterase, an acyl-CoA synthase, or both, are functionally deleted. In certain embodiments, the host cell is capable of producing fatty acids and/or derivatives thereof.
- In other embodiments, the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis. In some embodiments, one or more of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis are functionally deleted. In some embodiments, the gene encoding an acyl-CoA dehydrogenase is fadE. In some embodiments, the gene encoding an outer membrane protein receptor is one that encodes a receptor for ferrichrome, colicin M, phage T1, phage T5, or phage phi80. In certain embodiments, the gene encoding an outer membrane protein receptor is fhuA (also known as tonA). In yet other embodiments, the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
- In some embodiments, the recombinant vector further comprises a promoter operably linked to the nucleotide sequence encoding an ester synthase or a variant thereof. In certain embodiments, the promoter is a developmentally-regulated, or organelle-specific, a tissue-specific, an inducible, a constitutive, or a cell-specific promoter.
- In other embodiments, the recombinant vector comprises at least one sequence selected from the group consisting of (a) a regulatory sequence operatively coupled to the nucleotide sequence; (b) a selection marker operatively coupled to the nucleotide sequence; (c) a marker sequence operatively coupled to the nucleotide sequence; (d) a purification moiety operatively coupled to the nucleotide sequence; (e) a secretion sequence operatively coupled to the nucleotide sequence; and (f) a targeting sequence operatively coupled to the nucleotide sequence.
- In some embodiments, the recombinant vector is a plasmid.
- In some embodiments, the host cell expresses an ester synthase or a suitable variant that is encoded by the recombinant vector. In some embodiments, the nucleotide sequence is stably incorporated into the genomic DNA of the host cell, and the expression of the nucleotide sequence is under the control of a regulated promoter region.
- In some embodiments, the method further includes culturing the host cell in the presence of at least one biological substrate of the ester synthase. In certain embodiment, the method includes culturing the host cell under conditions that are sufficient to allow expression or overexpression of the ester synthase in the host cell. In further embodiments, the method includes culturing the host cell under conditions that are sufficient to permit production of fatty acids and/or fatty acid derivatives.
- In certain embodiments, an endogenous thioesterase of the host cell, if present, is unmodified. In other embodiments, the host cell expresses an attenuated level of a thioesterase. In an exemplary embodiment, a thioesterase is functionally deleted. In further embodiments, the host cell expresses an attenuated level of a fatty acid degradation enzyme, such as an acyl-CoA synthase. In other embodiments, an endogenous fatty acid degradation enzyme, such as an acyl-CoA synthase, is functionally deleted. In some embodiments, the host cell is cultured in the presence of a thioester. In other embodiments, the host cell is cultured in the presence of a suitable alcohol substrate. In yet further embodiments, the host cell is cultured in the presence of a free fatty acid.
- In any of the aspects of the invention described herein, the host cell can be selected from the group consisting of a mammalian cell, a plant cell, an insect cell, a yeast cell, a fungus cell, a filamentous fungi cell, a cyanobacterial cell, and a bacterial cell.
- In some embodiments, the host cell is a Gram-positive bacteria cell. In other embodiments, the host cell is a Gram-negative bacteria cell.
- In some embodiments, the host cell is selected from the genus Escherichia, Bacillus, Lactobacillus, Rhodococcus, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas, Schizosaccharomyces, Yarrowia, or Streptomyces.
- In certain embodiments, the host cell is a Bacillus lentus cell, a Bacillus brevis cell, a Bacillus stearothermophilus cell, a Bacillus licheniformis cell, a Bacillus alkalophilus cell, a Bacillus coagulans cell, a Bacillus circulans cell, a Bacillus pumilis cell, a Bacillus thuringiensis cell, a Bacillus clausii cell, a Bacillus megaterium cell, a Bacillus subtilis cell, or a Bacillus amyloliquefaciens cell.
- In other embodiments, the host cell is a Trichoderma koningii cell, a Trichoderma viride cell, a Trichoderma reesei cell, a Trichoderma longibrachiatum cell, an Aspergillus awamori cell, an Aspergillus fumigates cell, an Aspergillus foetidus cell, an Aspergillus nidulans cell, an Aspergillus niger cell, an Aspergillus oryzae cell, a Humicola insolens cell, a Humicola lanuginose cell, a Rhodococcus opacus cell, a Rhizomucor miehei cell, or a Mucor michei cell.
- In yet other embodiments, the host cell is a Streptomyces lividans cell or a Streptomyces murinus cell.
- In yet other embodiments, the host cell is an Actinomycetes, a Saccharomyces cerevisiae cell, a Candida Lipolytica (or Yarrowia lipolytica) cell, an E. coli Arthrobacter AK19 cell, a Rhodotorula glutinins cell, an Acintobacter sp. Strain M-1 cell, or a cell from other oleaginous microorganism.
- In some embodiments, the host cell is a cell of an oleaginous yeast, for example, a Yarrowia, a Candida, a Rhodotorula, a Rhodosporidium, a Cryptococcus, a Trichosporon, or a Lipomyces. In certain embodiments, the host cell is a cell of Rhodosporidium toruloide, Lipomyces starkeyii, L. Lipoferus, Candida revkaufi, C. pulcherrima, C. Tropicalis, C. utilis, Trichosporon pullas, T. cutaneum, Rhodotorula glutinous, R. Garminis, and Yarrowia lipolytica (formally classified as Candida lipolytica). In particular embodiments, the host cell is a cell from Yarrowia lipolytica strain ATCC #20362, ATCC #8862, ATCC #18944, ATCC #76982 and/or LGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol., 82(1):43-9, 2002).
- As used herein, the term “oleaginous” refers to those organisms that tend to store their energy source in the form of lipid (or oil) (Weete, Fungal Lipid Biochemistry, 2nd Ed., Plenum, 1980). And for the purpose herein, oleaginous organisms can be bacteria, algae, moss, yeast, fungi, or plants that have the ability to produce oils.
- The term “oleaginous yeast” refers to those microorganisms classified as yeasts that can make oil. Generally, the cellular oil or triacylglycerol content of oleaginous microorganisms follows a sigmoid curve, wherein the concentration of lipid increases until it reaches a maximum at the late logarithmic or early stationary growth phase and then gradually decreases during the late stationary and death phases (Yongmaitchai & Ward, Appl. Environ. Microbiol. 57:419-25 (1991)). It is not uncommon for oleaginous microorganism to accumulate in excess of about 25% of their dry cell weight as oil. Examples of oleaginous yeast include, but are not limited to, the following genera: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces.
- In particular embodiments, the host cell is a cell from an eukaryotic plant, algae, cyanobacterium, green-sulfur bacterium (including, e.g., Chlorobium, Clathrochloris, Prosthecochloris), green non-sulfur bacterium (including, e.g., Chloroflexus, chloronema, Oscillochloris, Heliothrix, Herpetosiphon, Roseiflexus, and Termomicrobium), purple sulfur bacterium (including, e.g., Allochromatium, Chromatium, Halochromatium, Isochromatium, Marichromatium, Rhodovulum, Thermochromatium, Thiocapsa, Thiorhodococcus, and Thiocystis), purple non-sulfur bacterium (including, e.g., Phaeospirillum, Rhodobac, Rhodobacter, Rhodomicrobium, Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodovibrio, Roseospira), extremophile, yeast, fungus, engineered organisms thereof, or a synthetic organism.
- In some embodiments, the host cell is light dependent or fixes carbon via one of the known pathways including, for example, the Calvin cycle pathway, the acetyl CoA pathway, and the reductive TCA pathway. See, e.g., Fuchs, G, Alternative pathways of autotrophic CO2 fixation, p. 365-382 (1989), AUTOTROPHIC BACTERIA, Springer-Verlag, Berlin, Germany (H. G. Schlegel & B. Bowien ed.). In some embodiments, the host cell has autotrophic activity. In some embodiments, the host cell has photoautotrophic activity, such as in the presence of light. In some embodiments, the host cell is heterotrophic or mixotrophic in the absence of light. In certain embodiments, the host cell is a cell from an extremphile, which are known to withstand various extreme environmental parameters such as temperature, radiation, pressure, gravity, vacuum, desiccation, salinity, pH, oxygen tension and chemicals. Host cells from various known extremephiles can be suitable. For example, the host cell can be from a hyperthermophile such as Pyrolobus furmarii, which grow at or above 80° C. In another example, the host cell can be from a thermophiles such as a Synechococcus lividis, which grow between 60-80° C. In a further example, the host cell can be from a mesophiles, which grow between 15-60° C. In yet a further example, the host cell can be from a psychrophile such as a psychrobacter or certain insect, which grow at or below 15° C. Moreover, the host cell can be from a radiation tolerant organism such as a Deinococcus radiodurans. In some embodiments, the host cell can be from a pressure tolerant organism, such as a piezophile, which tolerate pressure of 130 MPa. Alternatively, the host cell can be from weight tolerant organisms such as a barophile. In some other embodiments, the host cell can be a hypergravity (e.g., >1 g) or a hypogravity (e.g., <1 g) tolerant organism. In further embodiments, the host cell can be from a vacuum tolerant organism such as a tardigrade, insect, microbe and seed. In yet further embodiments, the host cell can be from a dessicant tolerant and anhydrobiotic organism such as a xerophile Artenia salina, a nematode, a certain microbe, a certain fungus and a lichen. In certain other embodiments, the host cell is from a salt tolerant organism such as a halophiles (e.g., 2-5 M NaCl) Halobacteriacea and Dunaliella salina. The host cell can also be from a pH tolerant organism such as an alkaliphiles Natronobacterium. Bacillus firmus OF4, Spirulina spp. (e.g., pH>9) or an acidophiles such as a Cyanidium caldarium, Ferroplasma sp. (e.g., low pH). The host cell can alternatively be from an anaerobes, which cannot tolerate O2, such as a Methancococcus jannaschii, or a microaerophil, which tolerates some O2, such as a Clostridium, and an aerobe, which requires O2. Furthermore, the host cell can be from a gas tolerant organism, which tolerates pure CO2, including, for example, a Cyanidium caldarium. The host cell can be from a metal tolerant organism, including, for example a metalotolerant such as Ferroplasma acidarmanus (e.g., Cu, As, Cd, Zn), Ralstonia sp. CH34 (e.g., Zn, Co, Cd, 11, Hg, Pb). See. e.g., Gross, Michael. Life on the Edge: Amazing Creatures Thriving in Extreme Environments. New York: Plenum (1998); Seckbach, J. “Search for Life in the Universe with Terrestrial Microbes Which Thrive Under Extreme Conditions.” In Cristiano Batalli Cosmovici, Stuart Bowyer, and Dan Wertheimer, eds., Astronomical and Biochemical Origins and the Search or Life in the Universe, p. 511. Milan: Editrice Compositori (1997).
- In certain embodiments, the host cell can be from plants, including, without limitation, plants of the genera: Arabidopsis, Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris, Populus, Saccharum, Salix, Simmondsia, and Zea. For example, the host cell can be from Arabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, or Zea mays. In other embodiments, the host cell can be from algae and cyanobacteria, including, without limitation, the genera: Acanthoceras, Acanthococcus. Acarvochloris, Achnanthes, Achnanthidiun, Actinastrum, Actinochloris. Actnoc)yclus. Actinotaenium, Amphichrsis, Amphidiniunm, Amphikrikos, Amplhipleura, Amphiprora, Amphithrix, Amphora, Anabaena, Anabaenopsis, Aneumnastus, Ankistrodesmius. Ankyra. Anomoeoneis, Apatococcus, Aphanizomenon, Aphanocapsa, Aphanochaete, Aphanothece, Apiocystis, Apistonema, Arthrodesmus, Artherospira, Ascochloris, Asterionella, Asterococcus, Audouinella, Aularoseira, Bacillaria, Balbiania, Bambiusina Bangia, Basichlamys, Batrarhospermum, Binurlearia, Bitrichia, Blidingia Botrdiopsis, Botrydium, Botryococcus, Botryosphaerella, Brachiomonas, Brachysira, Brachytrichia, Brebissonia, Bulbochaete, Bumnilleria, Buinilleriopsis, Caloneis, Calothrix, Campy lodiscus, Capsosiphon, Carteria, Catena, Cavinula, Cenritractus, Centroniella, Ceratiunt, Chaetoceros, Chaetochloris, Chaetomorpha, Chaetonella, Chaetonemna, Chaetopeltis, Chaetophora, Chaetosphaeridium, Chamaesiphion, Chara, Characiochloris, Characiopsis, Characium, Charales, Chilomonas, Chlainomonas, Chlamydoblepharis, Chlamydocapsa, Chlamydomonas, Chlamydomonopsis, Chlamydomnyxa, Chlamydonephris, Chlorangiella, Chlorangiopsis, Chlorella, Chlorobotrys, Chlorobrachis, Chlorochytrium, Chloroccun, Chlorogloea, Chlorogloeopsis, Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema, Cholorphyta, Cholorosaccus, Cholorosarcina, Choricystis, Chromophyton, Chromulina, Chroococcidiopsis, Chrococcus, Chroodactylon, Chroomonas, Chroothece, Chrysamoeba, Chysapsis, Chrysidiastrum, Chrysocapsa, Chrysocapsella, Chrysochaete, Chrysohromulina, Chrysococcus, Chrysocrinus, Chrynsolepidomonas, Chrysolykos, Chrysonebula, Chrysophyta, Chrysopyxis, Chrysosaccus, Chrysophaerella, Chrysotephanosphaera, Clodophora, Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis, Coelastrella, Coelastrum, Coelosphaerium, Coenochloris, Coenococcus, Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis, Compsopogon, Conjugatophyta, Conoehaete, Coronastrum, Cosmarium, Cosmnioneis, Cosmocladium, Crateriportula, Craticula, Crinalium, Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta, Ctenophora, Cyanodictyon, Cyanonephron, Cyanophora, Cyanophyta, Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella, Cylindrocapsa, Cylindrocystis, Cylindrospermum, Cylindrotheca, Cymatopleura, Cymbella, Cymbeilonitzschia, Cystodinium Dactylococcopsis, Debarya, Denticula, Dermatochrysis, Dermorarpa, Dermocarpella, Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon, Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula, Dichothrix, Dichtotomococcrus, Dicranochaete, Dictyochloris, Dictyococcus, Dictyosphaerium, Didymocystis, Didymogenes, Didymosphenia, Dilabifilum, Dimorphoccus, Dinobryon, Dinocuccus, Diplochloris, Diploneis, Diplostauron, Distrionella, Docidium, Draparnaldia, Dunaliella, Dysmorphaocuccus, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema, Enteromorpha, Entocladia, Entomoeis, Entophysalis, Ephichrysis, Epipyxis, Epithemia, Eremosphaura, Euastropsis, Euatstrum, Eucapsis, Eucocconeis, Eudorina, Euglena, Euglenophyta, Eunotia, Eustigmatophyta, Eutreptia, Fallcia, Ficherella, Fragilaria, Fragilariforma, Franceia, Frustulia, Curcilla, Geminella, Genicularia, Glaucocystis, Glaucophyta, Glenodiniopsis, Glenodinium, Gloeomonas, Gloeoplax, Gloeothece, Geloeotila, Gloeotrichia, Gloiodictyon, Golenkinia, Golenkiniopsis, Gomontia, Gomphocymbella, Gomphonema, Gomphosphaeria, Gonatozygon, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum, Granulochloris, Granulocystopsis, Groenbladia, Gymnodiunium, Gymnozyga, Gyrosignma, Haematocuccus, Hafniomonas, Hallassia, Hammatoidea, Hannaea, Hantzchia, Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Hemidinuim, Hemitonia, Heribaudiella, Heteromastix, Heterothrix, Hibberdia, Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema, Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium, Hyalotheca, Hydrianum, Hydrococcus, Hydrocoleum, Hydrocoryne, Hydrodictyon, Hydrosera, Hydrurus, Hyella, Hymenomonas, Isthmochloron, Johannesbaptistia, Juranyiella, Karayevia, Kathablepharis, Katodinium, Kaphyrion, Keratococcus, Kirchneriella, Klebsormidium, Kolbesia, Koliella, Komarekia, Korshikoviella, Kraskella, Lagerheimia, Lagynion, Lamprothamnium, Lemanea, Lepocinclis, Leptosira, Lobococcus, Lobocystis, Lobomonas, Luticola, Lynbya, Malleochloris, Mallomonas, Mantoniella, Marssoniella, Martyana, Mastigocloleus, Gastogloia, Melosira, Merismopedia, Mesostigma, Mesotaenium, Micr actinium, Micrasterias, Microchaete, Microcoleus, Microcystis, Microglena, Micromonas, Microspora, Microthamnion, Mischococcus, Monocrysis, Monodus, Monomastix, Monoraphidium, Monostroma, Mougeotia, Mougeotiopsis, Myochloris, Myromecia, Myxocarcina, Naegeliella, Nannochloris, Nautoccus, Navicula, Neglectella, Neidium, Nephroclamys, Nephrocytium, Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia, Nodularia, Nostoc, Ochromonas, Oedogonium, Oligochaetophora, Onychonema, Oocadrium, Oocrystis, Opephora, Ophiocytium, Orthoseira, Oscillartoria, Oxyneis, Pachycladella, Palmella, Palmodictyon, Pnadorina, Pannus, Paralia, Pascherina, Paulshulzia, Pediastrum, Pedinella, Pedinomonas, Pedinopera, Pelagodictyon, Penium, Peranema, Peridiniopsis, Peridinium, Peronia, Petroneis, Phacotus, Phacus, Phaeaster, Phaeodermatium, Phaeophyta, Phaeoshaera, Phaeothamnion, Phormidium, Phycopeltis, Phyllariochloris, Phyllocadium, Phyllomitas, Pinnilaria, Pitophora, Placoneis, Planctonema, Planktophaeria, Planothidium, Plectonema, Pleodorina, Pleurastrum, Pleurocapsa, Pleurocladia, Pleurodiscus, Pleurosigma, Pleurosira, Pleurotaenium, Pocillomanas, Podohedea, Polyblepharides, Polychaetophora, Polyedriella, Polyedriopsis, Polygoniochloris, Polyepidomanas, Polytaenia, Polytoma, Polytomella, Porphyridium, Posteriochromonas, Prasinochloris, Prasinocladus, Prasinophyta, Praisola, Prochlorphyta, Psammodictyon, Psammothidium, Pseudanabaena, Pseudenoclonium, Psuedocarteria, Pseudochate, Pseaudocharacium, Pseudococcomyxa, Pseudodictyosphaerium, Pseudokephyrion, Pseudoncobrysa, Pseudoquadrigula, Pseudophaerocystis, Pseudostaurastrum, Pseudostraurosira, Pyrrophyta, Quadrichloris, Quadricoccus, Quadrigula, Radiocuccus, Radioβlum, Raphidiopsis, Raphidocelis, Raphidonema, Raphidophyta, Peimeria, Rhadorderma, Rhabomonas, Rhizoclonium, Rhodomonas, Rhodiphyta, Rhoicosenia, Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus, Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix, Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia, Scytonema, Slenastrum, Selenochloris, Sellaphora, Semiorbis, Siderocelis, Diderocystopsis, Dimonsenia, Siphononema, Sirocladium, Sirogonium, Skeletonema, Sorastrum, Spermatozopis, Sphaerellocystis, Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma, Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum, Spondylosium, Sporotetras, Spumella, Staurastrum, Stauerodesmus, Stauroneis, Staurosira, Staurrosiella, Stenopterobia, Stephanocostis, Stephanodiscus, Stephanoporos, Stephanoshaera, Stichoccus, Stichogloea, Sigeoclonium, Stigonema, Stipitocuccus, Stokesiella, Stombomonas, Stylochrysalis, Stylodinium, Styloyxis, Stylosphaeridium, Surirella, Sykidion, Symploca, Synechococcus, Synechocystis, Synedra, Synochromonas, Synura, Tabellaria, Tabularia, Teilingia, Temnogametum, Tetmemorus, Tetrachlorella, Tetracyclus, Tetrademus, Tetraedriella, tetraedron, Tetraselmis, Tetraspora, Tetrastrum, Thalassiosira, Thanmiochaete, Thoakochloris, Thorea, Tolypella, Tolypothrix, Trachelomonas, Trachydiscus, Trebouxia, Trentepholia, Treubaria, Tribonema, Trichodesmium, Tricodiscus, Trochiscia, Tryblionella, Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uva, Vacuolaria, Vaucheria, Volvox, Volvulina, Westella, Woloszynskia, Xanthidium, Xanthophyta, Exencoccus, Zygenema, Zygnemopsis, and Zygonium.
- Exemplary microorganisms from which the host cell can be derived include, without limitation, Arabidopsis thaliana, Botryococcus braunii, Chlamydomonas reinhardtii, Dunaliela salina, Synechococcus Sp. PCC 7002, Synechococcus Sp. PCC 7942, Synechocystis Sp. PCC 6803, Thermosynechococcus elongatus BP-1, Chlorobium tepidum, Chloroflexus auranticus, Chromatium vinosum, Chromatium tepidum Rhodospirillum rubrum, Rhodobacter capsulatus, Rhodopseudomonas palusris, Clostridium ljungdahlii, Clostridiuthermocellum, Panicum virgatum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, Schizosaccharomyces pombe, Pseudomonas fluorescens, Miscanthus giganteus, Zea mays, or Zymomonas mobilis.
- Yet other suitable organisms from which the host cell can be from include synthetic cells or cells produced by synthetic genomes as described in U.S. Patent Publication 2007/0264688, or 2007/0269862.
- In other embodiments, the host cell is a CHO cell, a COS cell, a VERO cell, a BHK cell, a HeLa cell, a Cv1 cell, an MDCK cell, a 293 cell, a 3T3 cell, or a PC12 cell.
- In yet other embodiments, the host cell is an E. coli cell. In certain embodiments, the E. coli cell is a strain B, a strain C, a strain K, or a strain W E. coli cell.
- In other embodiment, the host cell is a cyanobacterial host cell.
- In some embodiments, a genetically engineered host cell described herein produces fatty acids and/or fatty acid derivatives at a titer of about 50 mg/L or more, about 100 mg/L or more, about 150 mg/L or more, about 200 mg/L or more, about 500 mg/L or more, or about 1000 mg/L or more.
- In certain embodiments, the host cell overexpresses a nucleic acid sequence that encodes an ester synthase or a variant described herein. In certain embodiments, known genomic alteration techniques can be applied to change the features of one or more endogenous ester synthase enzymes such that they are overexpressed. In some embodiments, the method further includes transforming the host cell to overexpress a nucleic acid sequence that encodes a heterologous ester synthase described herein.
- In certain embodiments, an endogenous thioesterase of the host cell, if present, is unmodified. In other embodiments, the host cell expresses an attenuated level of a thioesterase, a fatty acid degradation enzyme such as an acyl-CoA synthase, or both. In an exemplary embodiment, a thioesterase, an acyl-CoA synthase, or both, are functionally deleted.
- In certain embodiments, the host cell overproduces a substrate described herein. For example, the host cell can be engineered to overproduce an alcohol substrate or a thioester substrate. In other embodiments, the host cell is engineered to produce or overproduce a free fatty acid, which can be converted into fatty esters using the methods described herein. In some embodiments, the method further includes modifying one or more endogenous ester synthase enzymes such that the host cell overexpresses those ester synthase enzymes. In alternate embodiments, the method includes transforming the host cell with a nucleic acid sequence that encodes an ester synthase, and the host cell produces a fatty acid and/or a fatty acid derivative described herein.
- In other embodiments, the method further includes culturing the host cell in the presence of at least one substrate herein, which is expressed or overexpressed by the same host cell. In some embodiments, the substrate is a fatty thioester such as, for example, a fatty acyl-ACP or a fatty acyl-CoA. In other embodiments, the substrate is an alcohol or a free fatty acid, as described herein.
- In some embodiments, the substrate is an alcohol. In some embodiments, the alcohol substrate is an exogenous alcohol that is introduced to the host cell. In other embodiments, the alcohol substrate is produced by the genetically engineered host cell. For example, the alcohol substrate, such as a fatty alcohol substrate, can be suitably produced by the host cell, which co-expresses or overexpresses one or more fatty aldehyde biosynthesis genes and/or one or more fatty alcohol biosynthesis genes. In an alternative embodiment, the alcohol substrate such as a fatty alcohol substrate is produced by a host cell, which co-expresses or overexpresses one or more acyl-ACP reductase genes and/or one or more fatty alcohol biosynthesis genes. Methods of producing fatty alcohols in a recombinant host cell or microorganism have been described in, for example, International Patent Publication No. WO/2010/042664, the disclosure of which is incorporated herein by reference.
- In yet a further embodiment, the substrate is a free fatty acid. In certain embodiments, the free fatty acid is an undesirable side product, which can be converted by the host cell into a desirable fatty ester using the methods described herein.
- In another aspect, the invention features a method of producing a fatty acid or a fatty acid derivative such as, for example, a fatty ester. The method includes contacting a substrate with (i) an ester synthase comprising the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, or a variant thereof, or (ii) an ester synthase polypeptide encoded by a polynucleotide sequence having at least about 50% identity to the polynucleotide sequence of SEQ ID NO:27, 28, 29 or 30, or a variant thereof. In some embodiments, the method further includes isolating and/or purifying the fatty acid and/or fatty acid derivative.
- In some embodiments, the ester synthase polypeptide comprises the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, with one or more amino acid substitutions, additions, insertions or deletions, wherein the polypeptide has ester synthase and/or acyltransferase activity. In certain embodiments, the ester synthase polypeptide is modified wherein the polypeptide has an improved ester synthase and/or acyltransferase activity.
- In some embodiments, the ester synthase polypeptide comprises the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, with one or more non-conserved amino acid substitutions, wherein the polypeptide has ester synthase and/or acyltransferase activity. In some embodiments, the polypeptide comprises one or more conserved amino acid substitutions. In some embodiments, the polypeptide has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acid substitutions, additions, insertions, or deletions. In other embodiments, the polypeptide has improved ester synthase and/or acyltransferase activity. For example, the polypeptide is capable, or has improved capacity, of catalyzing the conversion of thioesters or free fatty acids to fatty esters, using alcohols as substrates.
- In some embodiments, the polypeptide has an amino acid sequence that is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:18, 24, 25, or 26. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO:18, 24, 25, or 26.
- In some embodiments, the polynucleotide sequence has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the polynucleotide sequence of SEQ ID NO:27, 28, 29, or 30. In some embodiment, the polynucleotide sequence is SEQ ID NO:27, 28, 29, or 30.
- In another aspect, the invention features a free fatty acid produced by a method herein. In some embodiments, the free fatty acid comprises one or more points of branching, one or more points of unsaturation, and/or one or more cyclic moieties.
- In another aspect, the invention features a fatty acid derivative produced by a method herein. In an exemplary embodiment, the fatty acid derivative is selected from a fatty acid methyl derivative, a fatty acid ethyl derivative, other fatty acid derivatives, and a combination thereof. In some embodiments, the carbon chain of the fatty acid derivative comprises one or more points of branching, one or more points of unsaturation, and/or one or more cyclic moieties.
- In another aspect, the invention features a fatty ester produced by a method herein. In some embodiments, the fatty ester comprises an A side (i.e., the carbon chain attached to the carboxylate oxygen) and a B side (i.e., the carbon chain comprising the parent carboxylate). In some embodiments, the B side of the fatty ester includes a straight chain. In other embodiments, the B side of the fatty ester includes a branched chain. The branched chains may have one or more points of branching. In still other embodiments, the B side of the fatty ester comprises at least one cyclic moiety.
- In some embodiments, the fatty acid ester is a fatty acid ethyl ester or a fatty acid methyl ester. In some embodiments, the fatty acid ester is at least about 4, 6, 8, 10, 12, 14, 18, or 20 carbons in length. The carbon chains comprising the A side or B side can be of any suitable length. In one embodiment, the A side of the ester is at least about 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, or 18 carbons in length. The B side of the ester is at least about 4, 6, 8, 10, 12, 14, 16, 18, or 20 carbons in length. In some embodiments, the fatty ester is saturated. In other embodiments, the fatty ester is unsaturated. In other embodiments, the fatty ester is monounsaturated. If unsaturated, the A side and/or B side can have one or more points of unsaturation.
- In some embodiments, the B side can have one double bond at one or more points in the carbon chain. In particular embodiments, the B side can have one double bond at
position 7 of the carbon chain, numbering from the reduced end of the carbon chain. One of ordinary skill in the art will recognize that, in a fatty methyl ester, one end of the B side will have a methyl group, and the other end of the B side will have a carboxyl group (C(═O)O—). The end of the B side which is a methyl group is the reduced end of the carbon chain comprising the B side, thus, the double bond is atcarbon 7 counting from the methyl group terminus of the B group (e.g., at betweencarbons position 7 in the carbon chain (between C7 and C8) from the reduced end of the fatty ester. - In some embodiments, the fatty ester comprises one or more points of branching. In other embodiments, the fatty ester is linear and does not comprise any branched chains.
- In another aspect, the invention features a genetically engineered microorganism comprising an exogenous control sequence stably incorporated into the genomic DNA of the microorganism upstream of a polynucleotide comprising a nucleotide sequence having at least about 50% identity to the nucleotide sequence of SEQ ID NO:27, 28, 29, or 30, wherein the microorganism produces an increased level of a fatty acid or fatty acid derivative relative to a wild type microorganism. In some embodiments, the polynucleotide is endogenous to the microorganism. In some embodiments, the polynucleotide sequence has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the polynucleotide sequence of SEQ ID NO:27, 28, 29, or 30. In some embodiment, the polynucleotide sequence is SEQ ID NO:27, 28, 29, or 30.
- In other embodiments, the microorganism is genetically engineered to express a modified level of a gene encoding an ester synthase. In certain embodiments, the microorganism expresses a heterologous gene encoding an ester synthase or expresses an increased level of an endogenous ester synthase. In certain embodiments, an endogenous thioesterase of the microorganism, if present, is unmodified. In other embodiments, the microorganism expresses an attenuated level of an endogenous gene encoding a thioesterase. Alternatively, an endogenous thioesterase is functionally deleted. In particular embodiments, the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
- In some embodiments, the microorganism is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to its level in a wild type microorganism. Exemplary fatty acid degradation enzymes include, without limitation, acyl-CoA synthase enzymes of EC 2.3.1.86 or EC 6.2.1.3. In particular embodiments, the microorganism is engineered to expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, pf1-4354, EAV15023, fadD1, fadD2, rpc_4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_01644857. In certain embodiments, the aforementioned fatty acid degradation enzyme, such as the acyl-CoA synthase gene, is functionally deleted such that the microorganism does not comprise an acyl-CoA synthase activity or functionality.
- In certain other embodiments, the microorganism is engineered to express an attenuated level of a thioesterase, a fatty acid degradation enzyme such as an acyl-CoA synthase, or both. In further embodiments, an endogenous thioesterase, a fatty acid degradation enzyme, or both, are functionally deleted. In particular embodiments, the microorganism has no detectable thioesterase or acyl-CoA synthase activity. In some embodiments, the microorganism is capable of producing fatty acids and/or derivatives thereof.
- In other embodiments, the microorganism is genetically engineered to express, relative to a wild type microorganism, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis. In some embodiments, one or more of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis are functionally deleted. In some embodiments, the gene encoding an acyl-CoA dehydrogenase is fadE. In some embodiments, the gene encoding an outer membrane protein receptor is one that encodes a receptor for ferrichrome, colicin M, phage T1, phage T5, or phage phi80. In certain embodiments, the gene encoding an outer membrane protein receptor is fhuA (also known as tonA). Yet in other embodiments, the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
- In some embodiments, the microorganism is a bacterium. In certain embodiments, the bacterium is a Gram-negative or a Gram-positive bacterium.
- In some embodiments, the microorganism is a mycobacterium selected from the group consisting of Mycobacterium smegmatis, Mycobacterium abscessus, Mycobacterium avium, Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium ulcerans. In other embodiments, the bacterium is Nocardia sp.
NRRL 5646, Nocardia farcinica, Streptomyces griseus, Salinispora arenicola, or Clavibacter michiganenesis. - In some embodiments, the microorganism is selected from the group consisting of Acinetobacter, Alcanivorax, Alcaligenes, Arabidopsis, Fundibacter, Marinobacter, Mus musculus, Pseudomonas, or Simmodsia.
- In particular embodiments, the microorganism is selected from the group consisting of algae, cyanobacterium, green-sulfur bacterium (including, e.g., Chlorobium, Clathrochloris, Prosthecochloris), green non-sulfur bacterium (including, e.g., Chloroflexus, chloronema, Oscillochloris, Heliothrix, Herpetosiphon, Roseiflexus, and Termomicrobium), purple sulfur bacterium (including, e.g., Allochromatium, Chromatium, Halochromatium, Isochromatium, Marichromatium, Rhodovulum, Thermochromatium, Thiocapsa, Thiorhodococcus, and Thiocystis), purple non-sulfur bacterium (including, e.g., Phaeospirillum, Rhodobac, Rhodobacter, Rhodomicrobium, Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodovibrio, Roseospira), or extremophile.
- In certain other embodiments, the microorganism is selected from the group consisting of: Arabidopsis thaliana, Botryococcus braunii, Chlamydomonas reinhardtii, Dunaliela salina, Synechococcus Sp. PCC 7002, Synechococcus Sp. PCC 7942, Synechocystis Sp. PCC 6803, Thermosynechococcus elongatus BP-1, Chlorobium tepidum, Chloroflexus auranticus, Chromatium vinosum, Chromatium tepidum Rhodospirillum rubrum, Rhodobacter capsulatus, Rhodopseudomonas palusris, Clostridium ljungdahlii, Clostridiuthermocellum, Panicum virgatum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, Schizosaccharomyces pombe, Pseudomonas fluorescens, Miscanthus giganteus, Zea mays, or Zymomonas mobilis.
- In some embodiments, the microorganism is an oleaginous yeast, for example, a Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, or Lipomyces. In certain embodiments, the microorganism is a Rhodosporidium toruloide, Lipomyces starkeyii, L. Lipoferus, Candida revkaufi, C. pulcherrima, C. Tropicalis, C. utilis, Trichosporon pullas, T. cutaneum, Rhodotorula glutinous, R. Garminis, and Yarrowia lipolytica (formally classified as Candida lipolytica). In particular embodiments, the microorganism is Yarrowia lipolytica strain ATCC #20362, ATCC #8862, ATCC #18944, ATCC #76982 and/or LGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol., 82(1):43-9, 2002).
- In another aspect, the invention features a fatty acid or fatty acid derivative produced by any of the methods or any of the microorganisms described herein, or a composition comprising a fatty acid or derivative thereof produced by any of the methods or any of the microorganisms described herein.
- In some embodiments, the fatty acid or fatty acid derivative has a δ13C of about −15.4 or greater. In certain embodiments, the fatty acid or fatty acid derivative has a δ13C of about −15.4 to about −10.9, or of about −13.92 to about −13.84.
- In some embodiments, the fatty acid or fatty acid derivative has an fM 14C of at least about 1.003. In certain embodiments, the fatty acid or fatty acid derivative has an fM 14C of at least about 1.01 or at least about 1.5. In some embodiments, the fatty acid or fatty acid derivative has an fM 14C of about 1.111 to about 1.124.
- In another aspect, the invention features a biofuel composition comprising the fatty acids or fatty acid derivatives produced by any of the methods or by any of the microorganisms described herein.
- In any of the aspects described herein, a fatty acid or fatty acid derivative is produced in a host cell or a microorganism described herein from a carbon source, including, for example, an alcohol, a free fatty acid, or a thioester.
- In any of the aspects described herein, carbon source substrates can be, for example, alcohol substrates, thioester substrates, and free fatty acids. Suitable alcohol substrates include short-chain alcohols such as methanol, ethanol, propanol (isopropanol), butanol, pentanol, hexanol, heptanol, and the like, as well as various long-chain alcohols such as fatty alcohols, for example, octanol, tetradecanol, hexadecanol, hexadecenol, octadecenol, and others. Furthermore, the invention pertains to converting methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, and/or other alcohol substrates to fatty acid derivatives, such as, for example, fatty esters, using the genetically engineered production host. In certain embodiments, ethanol is converted by a suitably-engineered production host. Preferably, one or more fatty acid ethyl esters and/or free fatty acids are produced. In certain other embodiments, methanol is converted by a suitably-engineered production host. Preferably, one or more fatty acid methyl esters and/or free fatty acids are produced. In yet other embodiments, butanol is converted by a suitably-engineered production host. In yet other embodiments, a mixture of ethanol, methanol, and/or other suitable alcohol substrates can be converted to a mixture of fatty acids and/or fatty acid derivatives by culturing one or more of the production hosts genetically engineered according to the methods herein under conditions that allow the production of such free fatty acids and/or fatty acid derivatives. For example, one or more free fatty acids, one or more fatty acid ethyl esters, and/or one or more fatty acid methyl esters are produced.
- In any of the aspects described herein, suitable carbon source substrates can be free fatty acids and thioesters. For example, the free fatty acid can comprise a branched carbon chain. Alternatively, the free fatty acid can comprise a linear carbon chain. In some embodiments, the free fatty acid can comprise a cyclic group, or one or more points of unsaturation. In some embodiments, the thioester substrate is a fatty acyl-CoA or fatty acyl-ACP. In yet another aspect, the invention features to a method of producing fatty esters from the free fatty acids in the waste stream of a conventional fatty ester production process.
- In any of the aspects described herein, the ester synthase polypeptide can be about 200 amino acids to about 2,000 amino acids in length, for example, from about 250 to about 1,500 amino acid residues in length, from about 300 to about 1,200 amino acid residues in length, from about 350 to about 1,000 amino acid residues in length, from about 400 to about 800 amino acid residues in length, or from about 450 to about 600 amino acid residues in length. In certain embodiments, the ester synthase polypeptide is about 300 amino acid residues in length or longer, for example, about 400 amino acid residues in length or longer, or about 450 amino acid residues in length or longer. In certain related embodiments, the ester synthase polypeptide is about 1,000 amino acid residues in length or shorter, for example, about 800 amino acid residues in length or shorter, about 700 amino acid residues in length or shorter, or about 600 amino acid residues in length or shorter. An exemplary ester synthase of the invention is about 500 amino acid residues in length.
- The drawings and examples provided herein are intended solely to illustrate the features of the present invention. They are not intended to be limiting.
-
FIG. 1A is a diagram illustrating a typical biosynthetic pathway known in the art (see, e.g., WO2007/136762, the disclosures of which are incorporated by reference herein), which produces fatty acid and/or fatty acid derivatives depending upon the substrates provided.FIG. 1B is a diagram exemplifying an alternative and more efficient pathway of the present invention. -
FIG. 2 depicts the sequence of E. coli spc ribosomal protein operon promoter Pspc (SEQ ID NO:13). -
FIG. 3 depicts a bacterial expression plasmid, pDS33.ES9 (SEQ ID NO:22), in which expression of an ester synthase gene from Marinobacter hydrocarbonoclasticus DSM 8798 (GenBank Accession No. AB021021) is under the control of the E. coli spc ribosomal protein operon promoter Pspc (SEQ ID NO:13). -
FIG. 4 depicts a bacterial expression plasmid, pDS57 (SEQ ID NO:23) wherein the expression of an ester synthase gene is under the control of a Trc promoter. -
FIG. 5 depicts the amount of free fatty acid (FFA) and fatty acyl ethyl ester (FAEE) produced by each culture in accordance with Example 6, without co-expression of an acyl-CoA synthase or a thioesterase, in the presence and absence of ethanol in microwell plates. ES9 generated nearly 200 mg/L FAEE in the presence of ethanol, but only a small quantity (<20%) of FFA. The other ester synthases, including ES8, atfA1 and atfA2, produced substantially lower amounts of FAEE in the presence of ethanol, accompanied by a higher proportion of FFA. The culture E. coli 'tesA produced higher overall titers of FFA and FAEE in the presence of ethanol, but the amount of FFA was significantly greater than the amount of FAEE generated. -
FIG. 6 depicts the production of FFA and/or FAEE in accordance with Example 7, without co-expression of acyl-CoA synthase or a thioesterase, but in the presence of ethanol in shake flasks. Ester synthases ES9, ES8, atfA1 and atfA2 were all able to produce FAEE in vivo under the prescribed conditions. ES9 generated a high level of FAEE in the presence of ethanol, but only little FFA was produced. Compared to ES9, noticeably smaller amounts of FAEE were produced by atfAI, atfA2, and ES8. Consistent with the results of Example 6 andFIG. 5 , expression of E. coli 'tesA alone produced high overall titers of FFA and FAEE, but with a significantly higher proportion of FFA. -
FIG. 7 depicts the production of FFA and/or FAME in accordance with Example 8, without co-expression of acyl-CoA synthase or a thioesterase, but in the presence of methanol in shake flasks. The amount of FFA produced was below the limit of detection. Lower levels of overall titers, as compared to those obtained in the presence of ethanol, were also observed in cultures expressing atfA1, atfA2, ES8, and ES9. However, ES9 produced substantial amount of FAME. -
FIG. 8 depicts a post-induction time course of fatty ester production by E. coli MG1655 DV2 ΔfadD expressing ES9, without co-expression of an acyl-CoA synthase or a thioesterase. -
FIG. 9 depicts the z-scores of fatty acid/fatty ester production by the strains of theES9 position 395 mutageneisis library, as described in Example 11. -
FIG. 10A depicts in vivo production of free fatty acids and/or fatty esters by the E. coli DV2 Δ fadD expressing ES9, and ES9 mutants G395R, G395K, G395S, G395D, and G395E.FIG. 10B depicts OD600 obtained from the strains during fermentation. -
FIG. 11 depicts fatty acid methyl ester production titers by ES1, ES2, ES3, and ES4, the homologs of ES9, in accordance with Examples 12-13, which converted acyl-ACPs and methanol into methyl esters. -
FIG. 12 depicts fatty acid ethyl ester production titers by ES1, ES2, ES3, and ES4, the homologs of ES9, in accordance with Examples 12-13, which converted acyl-ACPs and ethanol into ethyl esters. -
FIG. 13 is a GC/MS trace following ester production from F. jadensis T9 strain fed with sodium palmitate and short chain alcohols. The top panel is a no-alcohol feeding control. The middle panel depicts the products made as result of ethanol feeding. The lower panel depicts the products made as a result of isopropanol feeding. -
FIG. 14 compares the cell growth profiles of fatty esters production processes involving recombinant host strains with or without an attenuated/deleted fadE, as described in Example 18. In this example, acyl-ACP and ethanol were used as substrates. -
FIG. 15 compares the total fatty ester production using recombinant host strains with or without an attenuated/deleted fadE, as described in Example 18. In this example, acyl-ACP and ethanol were used as substrates. -
FIG. 16 depicts various ester synthase and homolog amino acid and polynucleotide sequences. This list also includes various codon optimized polynucleotide sequences encoding the four homologs ES1, ES2, ES3, and ES4. -
FIG. 17 depicts other sequences of enzymes that can be used in connection with the present invention. -
FIG. 18 depicts the features of a pFBAIN-MOD-1 vector, which is useful for carrying out the methods described in Example 20. - Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein, including GenBank database sequences, are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
- Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
- The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
- As used herein, an “aldehyde biosynthetic gene” or an “aldehyde biosynthetic polynucleotide” is a nucleic acid that encodes an aldehyde biosynthetic polypeptide.
- As used herein, an “aldehyde biosynthetic polypeptide” is a polypeptide that is a part of the biosynthetic pathway of an aldehyde. Such polypeptides can act on a biological substrate to yield an aldehyde. In some instances, the aldehyde biosynthetic polypeptide has reductase activity.
- As used herein, the term “attenuate” means to weaken, reduce, or diminish. For example, a polypeptide can be attenuated by modifying the polypeptide to reduce its activity (e.g., by modifying a nucleotide sequence that encodes the polypeptide).
- As used herein, the term “biocrude” refers to a product derived from biomass, biomass derivatives, or other biological sources that, like petroleum crude, can be converted into other fuels. For example, biocrude can be converted into gasoline, diesel, jet fuel, or heating oil. Moreover, biocrude, like petroleum crude, can be converted into other industrially useful chemicals for use in, for example, pharmaceuticals, cosmetics, consumer goods, industrial processes, and the like.
- Biocrude may include, for example, hydrocarbons, hydrocarbon products, fatty acid esters, and/or aliphatic ketones. In a preferred embodiment, biocrude is comprised of hydrocarbons, for example aliphatic (e.g., alkanes, alkenes, alkynes) or aromatic hydrocarbons.
- As used herein, the term “biodiesel” means a biofuel that can be a substitute of diesel, which is derived from petroleum. Biodiesel can be used in internal combustion diesel engines in either a pure form, which is referred to as “neat” biodiesel, or as a mixture in any concentration with petroleum-based diesel. In one embodiment, biodiesel can include esters or hydrocarbons, such as aldehydes, alkanes, or alkenes.
- As used herein, the term “biofuel” refers to any fuel derived from biomass, biomass derivatives, or other biological sources. Biofuels can be substituted for petroleum based fuels. For example, biofuels are inclusive of transportation fuels (e.g., gasoline, diesel, jet fuel, etc.), heating fuels, and electricity-generating fuels. Biofuels are a renewable energy source.
- As used herein, the term “biomass” refers to a carbon source derived from biological material. Biomass can be converted into a biofuel. One exemplary source of biomass is plant matter. For example, corn, sugar cane, or switchgrass can be used as biomass. Another non-limiting example of biomass is animal matter, for example cow manure. Biomass also includes waste products from industry, agriculture, forestry, and households. Examples of such waste products that can be used as biomass are fermentation waste, straw, lumber, sewage, garbage, and food leftovers. Biomass also includes sources of carbon, such as carbohydrates (e.g., monosaccharides, disaccharides, or polysaccharides).
- As used herein, the phrase “carbon source” refers to a substrate or compound suitable to be used as a source of carbon for prokaryotic or simple eukaryotic cell growth. Carbon sources can be in various forms, including, but not limited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, and gases (e.g., CO and CO2). These include, for example, various monosaccharides, such as glucose, fructose, mannose, and galactose; oligosaccharides, such as fructo-oligosaccharide and galacto-oligosaccharide; polysaccharides such as xylose and arabinose; disaccharides, such as sucrose, maltose, and turanose; cellulosic material, such as methyl cellulose and sodium carboxymethyl cellulose; saturated or unsaturated fatty acid esters, such as succinate, lactate, and acetate; alcohols, such as methanol, ethanol, propanol, or mixtures thereof. The carbon source can also be a product of photosynthesis, including, but not limited to, glucose. A preferred carbon source is biomass. Another preferred carbon source is glucose.
- As used herein, a “cloud point lowering additive” is an additive added to a composition to decrease or lower the cloud point of a solution.
- As used herein, the phrase “cloud point of a fluid” means the temperature at which dissolved solids are no longer completely soluble. Below this temperature, solids begin precipitating as a second phase giving the fluid a cloudy appearance. In the petroleum industry, cloud point refers to the temperature below which a solidified material or other heavy hydrocarbon crystallizes in a crude oil, refined oil, or fuel to form a cloudy appearance. The presence of solidified materials influences the flowing behavior of the fluid, the tendency of the fluid to clog fuel filters, injectors, etc., the accumulation of solidified materials on cold surfaces (e.g., a pipeline or heat exchanger fouling), and the emulsion characteristics of the fluid with water.
- A nucleotide sequence is “complementary” to another nucleotide sequence if each of the bases of the two sequences matches (e.g., is capable of forming Watson Crick base pairs). The term “complementary strand” is used herein interchangeably with the term “complement”. The complement of a nucleic acid strand can be the complement of a coding strand or the complement of a non-coding strand.
- The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (e.g., meaning “including, but not limited to,”) unless otherwise noted.
- As used herein, the term “conditions sufficient to allow expression” means any conditions that allow a host cell or production host to produce a desired product, such as a polypeptide, acyl-CoA, fatty acid derivative (e.g., fatty acids, hydrocarbons, fatty alcohols, fatty esters, etc.), or other product described herein. Suitable conditions include, for example, fermentation conditions. Fermentation conditions can comprise many parameters. Exemplary conditions include temperature ranges, levels of aeration, and media composition. Each of these conditions, individually and in combination, allows the host cell to grow.
- Exemplary culture media include broths or gels. Generally, the medium includes a carbon source, such as glucose, fructose, cellulose, or the like, that can be metabolized by a host cell directly. In addition, enzymes can be used in the medium to facilitate the mobilization (e.g., the depolymerization of starch or cellulose to fermentable sugars) and subsequent metabolism of the carbon source.
- To determine if conditions are sufficient to allow expression, a host cell can be cultured, for example, for about 4, 8, 12, 24, 36, or 48 hours. During and/or after culturing, samples can be obtained and analyzed to determine if the conditions allow expression. For example, the host cells in the sample or the medium in which the host cells were grown can be tested for the presence of a desired product. When testing for the presence of a product, assays, such as, but not limited to, TLC, HPLC, GC/FID, GC/MS, LC/MS, MS, can be used.
- As used herein, “conditions that permit product production” refers to any fermentation conditions that allow a production host to produce a desired product, such as acyl-CoA or fatty acid derivatives (e.g., fatty acids, hydrocarbons, fatty alcohols, waxes, or fatty esters). Fermentation conditions usually comprise many parameters. Exemplary conditions include, but are not limited to, temperature ranges, levels of aeration, and media composition. Each of these conditions, individually and/or in combination, allows the production host to grow.
- Exemplary media include broths and/or gels. Generally, a suitable medium includes a carbon source (e.g., glucose, fructose, cellulose, etc.) that can be metabolized by the microorganism directly. In addition, enzymes can be used in the medium to facilitate the mobilization (e.g., the depolymerization of starch or cellulose to fermentable sugars) and subsequent metabolism of the carbon source.
- To determine if the fermentation conditions permit product production, the production host can be cultured for about 4, 8, 12, 24, 36, or 48 hours. During culturing or after culturing, samples can be obtained and analyzed to determine if the fermentation conditions have permitted product production. For example, the production hosts in the sample or the medium in which the production hosts are grown can be tested for the presence of the desired product. Exemplary assays, such as TLC, HPLC, GC/FID, GC/MS, LC/MS, MS, as well as those provided herein, can be used identify and quantify the presence of a product.
- As used herein, “conservative amino acid substitution” means an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), beta-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).
- It is understood that the polypeptides described herein may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on the polypeptide functions. Whether or not a particular substitution will be tolerated (e.g., will not adversely affect desired biological properties, such as decarboxylase activity) can be determined as described in Bowie et al., Science 247:1306 1310 (1990).
- As used herein, “control element” means a transcriptional control element. Control elements include promoters and enhancers. The term “promoter element,” “promoter,” or “promoter sequence” refers to a DNA sequence that functions as a switch that activates the expression of a gene. If the gene is activated, it is said to be transcribed or participating in transcription. Transcription involves the synthesis of mRNA from the gene. A promoter, therefore, serves as a transcriptional regulatory element and also provides a site for initiation of transcription of the gene into mRNA. Control elements interact specifically with cellular proteins involved in transcription (Maniatis et al., Science 236:1237 (1987)).
- As used herein, the term “functional deletion,” or “knockout” means modifying or inactivating a polynucleotide sequence that encodes a target protein in order to reduce or eliminate the function of the target protein. A polynucleotide deletion can be performed by methods well known in the art (See, e.g., Datsenko et al., Proc. Nat. Acad. Sci. USA, 97:6640-45 (2000) or International Patent Application Nos. PCT/US2007/011923 and PCT/US2008/058788).
- As used herein, the term “endogenous” means a polynucleotide that is in the cell and was not introduced into the cell using recombinant genetic engineering techniques. For example, a gene that was present in the cell when the cell was originally isolated from nature. A polynucleotide is still considered endogenous if the control sequences, such as a promoter or enhancer sequences which activate transcription or translation, have been altered through recombinant techniques.
- As used herein, the term “ester synthase” means a peptide capable of producing fatty esters. More specifically, an ester synthase is a peptide which converts a thioester to a fatty ester. In a preferred embodiment, the ester synthase converts a thioester (e.g., acyl-CoA) to a fatty ester.
- In an alternate embodiment, an ester synthase uses a thioester and an alcohol as substrates to produce a fatty ester. Ester synthases are capable of using short and long chain thioesters as substrates. In addition, ester synthases are capable of using short and long chain alcohols as substrates.
- Non-limiting examples of ester synthases are wax synthases, wax-ester synthases, acyl CoA:alcohol transacylases, acyltransferases, and fatty acyl-coenzyme A:fatty alcohol acyltransferases. Exemplary ester synthases are classified in enzyme classification number EC 2.3.1.75. A number of these enzymes, as well as other useful enzymes for making the products described herein, have been disclosed in, for example, International Patent Application Nos. PCT/US2007/011923 and PCT/US2008/058788, which are incorporated herein by reference.
- As used herein, the term “exogenous” means a polynucleotide that does not originate from a particular cell as found in nature. For example, “exogenous polynucleotide” could refer to a polynucleotide that was inserted within the genomic polynucleotide sequence of a microorganism or to an extra chromosomal polynucleotide that was introduced into the microorganism. Thus, a non-naturally-occurring polynucleotide is considered to be exogenous to a cell once introduced into the cell. A polynucleotide that is naturally-occurring can also be exogenous to a particular cell. For example, an entire polynucleotide isolated from a first cell can be an exogenous polynucleotide with respect to a second cell if that polynucleotide from the first cell is introduced into the second cell.
- As used herein, the term “fatty acid” means a carboxylic acid having the formula RCOOH. R represents an aliphatic group, preferably an alkyl group. R can comprise between about 4 and about 22 carbon atoms. Fatty acids can be saturated, monounsaturated, or polyunsaturated. In a preferred embodiment, the fatty acid is made from a fatty acid biosynthetic pathway.
- As used herein, the term “fatty acid biosynthetic pathway” means a biosynthetic pathway that produces fatty acids. The fatty acid biosynthetic pathway includes fatty acid enzymes that can be engineered, as described herein, to produce fatty acids, and in some embodiments can be expressed with additional enzymes to produce fatty acids having desired carbon chain characteristics.
- As used herein, the term “fatty acid degradation enzyme” means an enzyme involved in the breakdown or conversion of a fatty acid or fatty acid derivative into another product. A nonlimiting example of a fatty acid degradation enzyme is an acyl-CoA synthase. A number of these enzymes, as well as other useful enzymes for making the products described herein, have been disclosed in, for example, International Patent Application Nos. PCT/US2007/011923 and PCT/US2008/058788, which are incorporated herein by reference. Additional examples of fatty acid degradation enzymes are described herein.
- As used herein, the term “fatty acid derivative” means products made in part from the fatty acid biosynthetic pathway of the production host organism. “Fatty acid derivative” also includes products made in part from acyl-ACP or acyl-ACP derivatives. The fatty acid biosynthetic pathway includes fatty acid synthase enzymes which can be engineered as described herein to produce fatty acid derivatives, and in some examples can be expressed with additional enzymes to produce fatty acid derivatives having desired carbon chain characteristics. Exemplary fatty acid derivatives include for example, fatty acids, acyl-CoAs, fatty aldehydes, short and long chain alcohols, hydrocarbons, fatty alcohols, ketones, and esters (e.g., waxes, fatty acid esters, or fatty esters).
- As used herein, the term “fatty acid derivative enzymes” means all enzymes that may be expressed or overexpressed in the production of fatty acid derivatives. These enzymes are collectively referred to herein as fatty acid derivative enzymes. These enzymes may be part of the fatty acid biosynthetic pathway. Non-limiting examples of fatty acid derivative enzymes include fatty acid synthases, thioesterases, acyl-CoA synthases, acyl-CoA reductases, alcohol dehydrogenases, alcohol acyltransferases, carboxylic acid reductases, fatty alcohol-forming acyl-CoA reductase, ester synthases, aldehyde biosynthetic polypeptides, and alkane biosynthetic polypeptides. Fatty acid derivative enzymes convert a substrate into a fatty acid derivative. In some examples, the substrate may be a fatty acid derivative which the fatty acid derivative enzyme converts into a different fatty acid derivative. A number of these enzymes, as well as other useful enzymes for making the products described herein, have been disclosed in, for example, International Patent Application Nos. PCT/US2007/011923 and PCT/US2008/058788, which are incorporated herein by reference.
- As used herein, “fatty acid enzyme” means any enzyme involved in fatty acid biosynthesis. Fatty acid enzymes can be expressed or overexpressed in host cells to produce fatty acids. Non-limiting examples of fatty acid enzymes include fatty acid synthases and thioesterases. A number of these enzymes, as well as other useful enzymes for making the products described herein, have been disclosed in, for example, International Patent Application Nos. PCT/US2007/011923 and PCT/US2008/058788, which are incorporated herein by reference.
- As used herein, the term “fatty alcohol” means an alcohol having the formula ROH. In a preferred embodiment, the fatty alcohol is any alcohol made from a fatty acid or fatty acid derivative.
- In one embodiment, the R group is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbons in length. R can be straight or branched chain. The branched chains may have one or more points of branching. In addition, the branched chains may include cyclic branches. Furthermore, R can be saturated or unsaturated. If unsaturated, the R can have one or more points of unsaturation.
- In one embodiment, the fatty alcohol is produced biosynthetically. Fatty alcohols have many uses. For example, fatty alcohols can be used to produce many specialty chemicals. For example, fatty alcohols are used as a biofuel; as solvents for fats, waxes, gums, and resins; in pharmaceutical salves, emollients and lotions; as lubricating-oil additives; in detergents and emulsifiers; as textile antistatic and finishing agents; as plasticizers; as nonionic surfactants; and in cosmetics, for examples as thickeners.
- As used herein, the term “fatty ester” means an ester. In a preferred embodiment, a fatty ester is any ester made from a fatty acid to produce, for example, a fatty acid ester. In one embodiment, a fatty ester contains an A side (i.e., the carbon chain attached to the carboxylate oxygen) and a B side (i.e., the carbon chain comprising the parent carboxylate). In a preferred embodiment, when the fatty ester is derived from the fatty acid biosynthetic pathway, the A side is contributed by an alcohol, and the B side is contributed by a fatty acid. Any alcohol can be used to form the A side of the fatty esters. For example, the alcohol can be derived from the fatty acid biosynthetic pathway. Alternatively, the alcohol can be produced through non-fatty acid biosynthetic pathways. Moreover, the alcohol can be provided exogenously. For example, the alcohol can be supplied in the fermentation broth in instances where the fatty ester is produced by an organism that can also produce the fatty acid. Alternatively, a carboxylic acid, such as a fatty acid or acetic acid, can be supplied exogenously in instances where the fatty ester is produced by an organism that can also produce alcohol.
- The carbon chains comprising the A side or B side can be of any length. In one embodiment, the A side of the ester is at least about 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, or 18 carbons in length. The B side of the ester is at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 carbons in length. The A side and/or the B side can be straight or branched chain. The branched chains may have one or more points of branching. In addition, the branched chains may include cyclic branches. Furthermore, the A side and/or B side can be saturated or unsaturated. If unsaturated, the A side and/or B side can have one or more points of unsaturation.
- In one embodiment, the fatty ester is produced biosynthetically. In this embodiment, first the fatty acid is “activated.” Non-limiting examples of “activated” fatty acids are acyl-CoA, acyl-ACP, and acyl phosphate. Acyl-CoA can be a direct product of fatty acid biosynthesis or degradation. In addition, acyl-CoA can be synthesized from a free fatty acid, a CoA, or an adenosine nucleotide triphosphate (ATP). An example of an enzyme which produces acyl-CoA is acyl-CoA synthase.
- After the fatty acid is activated, it can be readily transferred to a recipient nucleophile. Exemplary nucleophiles are alcohols, thiols, or phosphates.
- In one embodiment, the fatty ester is a wax. The wax can be derived from a long chain alcohol and a long chain fatty acid. In another embodiment, the fatty ester can be derived from a fatty acyl-thioester and an alcohol. In another embodiment, the fatty ester is a fatty acid thioester, for example fatty acyl Coenzyme A (CoA). In other embodiments, the fatty ester is a fatty acyl panthothenate, an acyl carrier protein (ACP), or a fatty phosphate ester. Fatty esters have many uses. For example, fatty esters can be used as biofuels, surfactants, or formulated into additives that provide lubrication and other benefits to fuels and industrial chemicals.
- As used herein, “fraction of modern carbon” or “fM” has the same meaning as defined by National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs) 4990B and 4990C, known as oxalic acids standards HOxI and HOxII, respectively. The fundamental definition relates to 0.95 times the 14C/12C isotope ratio HOxI (referenced to AD 1950). This is roughly equivalent to decay-corrected pre-Industrial Revolution wood. For the current living biosphere (e.g., plant material), fM is approximately 1.1.
- Calculations of “homology” between two sequences can be performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence that is aligned for comparison purposes is at least about 30%, preferably at least about 40%, more preferably at least about 50%, even more preferably at least about 60%, and even more preferably at least about 70%, at least about 80%, at least about 90%, or about 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
- The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent homology between two amino acid sequences is determined using the Needleman and Wunsch (1970), J. Mol. Biol. 48:444 453, algorithm that has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent homology between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna. CMP matrix and a gap weight of about 40, 50, 60, 70, or 80 and a length weight of about 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about which parameters should be applied to determine if a molecule is within a homology limitation of the claims) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
- Other methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in, for example, Smith & Waterman, Adv. Appl. Math. 2:482 (1981); Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988); Higgins & Sharp, Gene 73:237 244 (1988); Higgins & Sharp, CABIOS 5:151-153 (1989); Corpet et al., Nucleic Acids Research 16:10881-10890 (1988); Huang et al., CABIOS 8:155-165 (1992); and Pearson et al., Methods in Molecular Biology 24:307-331 (1994), and Altschul et al., J. Mol. Biol. 215:403-410 (1990).
- As used herein, a “host cell” is a cell used to produce a product described herein (e.g., an aldehyde or alkane). A host cell can be modified to express or overexpress selected genes or to have attenuated expression of selected genes. Non-limiting examples of host cells include plant, animal, human, bacteria, cyanobacteria, yeast, or filamentous fungi cells.
- As used herein, the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described in that reference and either method can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2.×SSC, 0.1% SDS at 65° C.; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are the preferred conditions unless otherwise specified.
- The term “isolated” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the nucleic acid. Moreover, an “isolated nucleic acid” includes nucleic acid fragments, such as fragments that are not naturally occurring. The term “isolated” is also used herein to refer to polypeptides, which are isolated from other cellular proteins, and encompasses both purified endogenous polypeptides and recombinant polypeptides. The term “isolated” as used herein also refers to a nucleic acid or polypeptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques. The term “isolated” as used herein also refers to a nucleic acid or polypeptide that is substantially free of chemical precursors or other chemicals when chemically synthesized.
- As used herein, the “level of expression of a gene in a cell” refers to the level of mRNA, pre-mRNA nascent transcript(s), transcript processing intermediates, mature mRNA(s), and/or degradation products encoded by the gene in the cell.
- As used herein, the term “microorganism” means prokaryotic and eukaryotic microbial species from the domains Archaea, Bacteria and Eucarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. The term “microbial cell”, as used herein, means a cell from a microorganism.
- As used herein, the term “nucleic acid” refers to a polynucleotide, such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term also includes analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides, ESTs, chromosomes, cDNAs, mRNAs, and rRNAs. The term “nucleic acid” may be used interchangeably with “polynucleotide,” “DNA,” “nucleic acid molecule,” “nucleotide sequence,” and/or “gene” unless otherwise indicated herein or otherwise clearly contradicted by context.
- As used herein, the term “operably linked” means that a selected nucleotide sequence (e.g., encoding a polypeptide described herein) is in proximity with a promoter to allow the promoter to regulate expression of the selected nucleotide sequence. In addition, the promoter is located upstream of the selected nucleotide sequence in terms of the direction of transcription and translation. By “operably linked” is meant that a nucleotide sequence and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).
- The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise.
- As used herein, “overexpress” means to express or cause to be expressed or produced a nucleic acid, polypeptide, or hydrocarbon in a cell at a greater concentration than is normally expressed in a corresponding wild-type cell. For example, a polypeptide can be “overexpressed” in a recombinant host cell when the polypeptide is present in a greater concentration in the recombinant host cell compared to its concentration in a non-recombinant host cell of the same species.
- As used herein, “partition coefficient” or “P,” is defined as the equilibrium concentration of a compound in an organic phase divided by the concentration at equilibrium in an aqueous phase (e.g., fermentation broth). In one embodiment of a bi-phasic system described herein, the organic phase is formed by the aldehyde or alkane during the production process. However, in some examples, an organic phase can be provided, such as by providing a layer of octane, to facilitate product separation. When describing a two phase system, the partition characteristics of a compound can be described as logP. For example, a compound with a logP of 1 would partition 10:1 to the organic phase. A compound with a logP of −1 would partition 1:10 to the organic phase. By choosing an appropriate fermentation broth and organic phase, an organic fatty acid derivative or product with a high logP value can separate into the organic phase even at very low concentrations in the fermentation vessel.
- As used herein, the term “polypeptide” may be used interchangeably with “protein,” “peptide,” and/or “enzyme” unless otherwise indicated herein or otherwise clearly contradicted by context.
- As used herein, the term “production host” means a cell used to produce the products disclosed herein. The production host is modified to express, overexpress, attenuate or delete expression of selected polynucleotides. Non-limiting examples of production hosts include plant, algal, animal, human, bacteria, yeast, and filamentous fungi cells.
- As used herein, the term “purify,” “purified,” or “purification” means the removal or isolation of a molecule from its environment by, for example, isolation or separation. “Substantially purified” molecules are at least about 60% free, preferably at least about 75% free, and more preferably at least about 90% free from other components with which they are associated. As used herein, these terms also refer to the removal of contaminants from a sample. For example, the removal of contaminants can result in an increase in the percentage of a fatty acid derivative or product in a sample. For example, when a fatty acid derivatives or products are produced in a host cell, the fatty acid derivatives or products can be purified by the removal of host cell proteins. After purification, the percentage of fatty acid derivatives or products in the sample is increased.
- The terms “purify,” “purified,” and “purification” do not require absolute purity. They are relative terms. Thus, for example, when the fatty acid derivatives or products are produced in host cells, a purified fatty acid derivative or product is one that is substantially separated from other cellular components (e.g., nucleic acids, polypeptides, lipids, carbohydrates, or other fatty acid derivatives or products). In another example, a purified fatty acid derivative or purified product preparation is one in which the fatty acid derivative or product is substantially free from contaminants, such as those that might be present following fermentation. In some embodiments, a fatty acid derivative or product is purified when at least about 50% by weight of a sample is composed of the fatty acid derivative or product. In other embodiments, a fatty acid derivative or product is purified when at least about 60%, 70%, 80%, 85%, 90%, 92%, 95%, 98%, or 99% or more by weight of a sample is composed of the fatty acid derivative or product.
- As used herein, the term “recombinant polypeptide” refers to a polypeptide that is produced by recombinant DNA techniques, wherein generally DNA encoding the expressed polypeptide or RNA is inserted into a suitable expression vector and that is in turn used to transform a host cell to produce the polypeptide or RNA.
- As used herein, the term “substantially identical” (or “substantially homologous”) is used to refer to a first amino acid or nucleotide sequence that contains a sufficient number of identical or equivalent (e.g., with a similar side chain) amino acid residues (e.g., conserved amino acid substitutions) or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have similar activities.
- As used herein, the term “synthase” means an enzyme which catalyzes a synthesis process. As used herein, the term synthase includes synthases, synthetases, and ligases.
- As used herein, the term “transfection” means the introduction of a nucleic acid (e.g., via an expression vector) into a recipient cell by nucleic acid-mediated gene transfer.
- As used herein, the term “transformation” refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous nucleic acid. This may result in the transformed cell expressing a recombinant form of a RNA or polypeptide. In the case of antisense expression from the transferred gene, the expression of a naturally-occurring form of the polypeptide is disrupted.
- As used herein, the term “transport protein” means a polypeptide that facilitates the movement of one or more compounds in and/or out of a cellular organelle and/or a cell. A number of these proteins, as well as other useful proteins for making the products described herein, have been disclosed in, for example, International Patent Application Nos. PCT/US2007/011923 and PCT/US2008/058788, which are incorporated herein by reference.
- As used herein, a “variant” of polypeptide X refers to a polypeptide having the amino acid sequence of polypeptide X in which one or more amino acid residues is altered. The variant may have conservative changes or nonconservative changes. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without affecting biological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).
- The term “variant,” when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to that of a gene or the coding sequence thereof. This definition may also include, for example, “allelic,” “splice,” “species,” or “polymorphic” variants. A splice variant may have significant identity to a reference polynucleotide, but will generally have a greater or fewer number of polynucleotides due to alternative splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or an absence of domains. Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
- As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of useful vector is an episome (i.e., a nucleic acid capable of extra-chromosomal replication). Useful vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of “plasmids,” which refer generally to circular double stranded DNA loops that, in their vector form, are not bound to the chromosome. In the present specification, “plasmid” and “vector” are used interchangeably, as the plasmid is the most commonly used form of vector. However, also included are such other forms of expression vectors that serve equivalent functions and that become known in the art subsequently hereto.
- As used herein, the term “wax” means a composition comprised of fatty esters. In a preferred embodiment, the fatty ester in the wax is comprised of medium to long carbon chains. In addition to fatty esters, a wax may comprise other components (e.g., hydrocarbons, sterol esters, aliphatic aldehydes, alcohols, ketones, beta-diketones, triacylglycerols, etc.).
- Throughout the specification, a reference may be made using an abbreviated gene name or polypeptide name, but it is understood that such an abbreviated gene or polypeptide name represents the genus of genes or polypeptides. Such gene names include all genes encoding the same polypeptide and homologous polypeptides having the same physiological function. Polypeptide names include all polypeptides that have the same activity (e.g., that catalyze the same fundamental chemical reaction).
- The accession numbers referenced herein are derived from the NCBI database (National Center for Biotechnology Information) maintained by the National Institute of Health, U.S.A. Unless otherwise indicated, the accession numbers are as provided in the database as of April 2009.
- EC numbers are established by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) (available at http://www.chem.qmul.ac.uk/iubmb/enzyme/). The EC numbers referenced herein are derived from the KEGG Ligand database, maintained by the Kyoto Encyclopedia of Genes and Genomics, sponsored in part by the University of Tokyo. Unless otherwise indicated, the EC numbers are as provided in the database as of April 2009.
- Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
- Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.
- Unless otherwise stated, amounts listed in percentage (%) are in weight percent, based on the total weight of the composition.
- All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
- Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.
- The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
- Other features and advantages of the invention will be apparent from the following detailed description and from the claims.
- The invention is based, at least in part, on the discovery that fatty acids and/or fatty acid derivatives such as fatty esters can be produced by culturing genetically engineered host cells comprising an expressed or overexpressed ester synthase or a suitable variant thereof, but an attenuated level or in an absence of either or both a fatty acid degradation enzyme such as an acyl-CoA synthase a thioesterase, as compared to the naturally-occurring level of such enzymes. Compared to the previously disclosed methods of producing fatty acids and/or fatty acid derivatives through microbial fermentation procedures, which typically involve, in addition to an ester synthase, at least one of a thioesterase and a fatty acid degradation enzyme such as an acyl-CoA synthase, which preferably require the presence of increased activities or levels of these enzymes, the instant method is more energetically favorable and efficient. For example, the method described herein allows the direct conversion of a thioester, such as an acyl-ACP and/or an acyl-CoA, to a fatty acid and/or a fatty acid derivative, bypassing the requirement to first hydrolyze an ACP to make a fatty acid and then reactivate the fatty acid to produce an acyl-CoA.
- Fatty esters can be produced by culturing genetically engineered host cells comprising ester synthase enzymes, nucleic acids encoding these enzymes, vectors comprising nucleic acids encoding these enzymes, or by culturing recombinant host cells transformed with the vectors, and recombinant host cells comprising polynucleotides encoding these ester synthase enzymes, which are chromosomally integrated therein. Specifically, the method includes expressing in a host cell a gene encoding an ester synthase, selected from, for example, the enzymes classified as EC 2.3.1.75, which catalyzes the enzymatic transfer of saturated or unsaturated acyl residues, for example, those of chain-length C18 to C20, to alcohols. The ester synthase may be one selected from the group consisting of a wax-ester synthase, an acyl-CoA:alcohol transacylase, an acyltransferase, and a fatty acyl-CoA:fatty alcohol acyltransferase. For example, the ester synthase gene can be one that encodes ws/dgat, a bifunctional ester synthase/acyl-CoA/acyl-CoA: diacylglycerol acyltransferase from Simmondsia chinensis, Acinetobacter sp., Acinetobacter sp. ADP1, alcanivorax borkumensis, Pseudomonas aeruginosa, Fundibacter jadensis, Arabidopsis thaliana, or Alkaligenes eutrophus. In some embodiments, the invention features a method of making a fatty acid or a fatty acid derivative such as, for example, a fatty ester, wherein the method includes expressing in a host cell a gene encoding an ester synthase selected from the group consisting of AtfA1 (YP_694462, SEQ ID NO:25), AtfA2 (YP_693524, SEQ ID NO:26), ES9 (AB021021, SEQ ID NO:18), and ES8 (AB021020, SEQ ID NO:24), or a variant thereof.
- In certain embodiments, the invention features a method of making a fatty acid or a fatty acid derivative such as, for example a fatty ester, wherein the method includes expressing in a host cell a gene encoding an ester synthase polypeptide comprising the amino acid sequence of SEQ ID NO: 18, 24, 25, or 26, or a variant thereof. In certain embodiments, the polypeptide or variant has ester synthase and/or acyltransferase activity. In particular embodiments, the polypeptide or variant catalyzes the conversion of a thioester into a fatty acid and/or a fatty acid derivative, using an alcohol as substrate. In one example, the polypeptide or variant catalyzes the conversion of an acyl-CoA to a fatty acid and/or a fatty acid derivative, using an alcohol as substrate. In another example, the polypeptide or variant catalyzes the conversion of an acyl-ACP to a fatty acid and/or a fatty acid derivative, using an alcohol as substrate. In yet another example, the polypeptide or variant also converts a free fatty acid to a fatty ester using an alcohol as substrate. The alcohol substrates can be short- or long-chain alcohols. In some embodiments, the alcohol substrate is a methanol, and accordingly, the host cell expressing the ester synthase or variant produces a fatty acid and/or a fatty acid methyl derivative, such as a methyl ester. In other embodiments, the alcohol substrate is an ethanol, and accordingly, the host cell expressing the ester synthase or variant produces a fatty acid and/or a fatty acid ethyl derivative, such as an ethyl ester.
- In certain embodiments, an endogenous thioesterase of the host cell, if present, is unmodified. In other embodiments, the host cell expresses an attenuated level of a thioesterase, as compared to the naturally-occurring level of that thioesterase. In alternative embodiments, a thioesterase is functionally deleted. In some embodiments, there is no dectable thioesterase activity in the host cell. In other embodiments, the host cell expresses an attenuated level of a fatty acid degradation enzyme, such as an acyl-CoA synthase, as compared to the naturally-occurring level of that enzyme. In alternative embodiment, an acyl-CoA synthase is functionally deleted. In some embodiments, the host cell has no detectable acyl-CoA synthase activity. In further embodiments, the host cell expresses attenuated levels of a thioesterase and a fatty acid degradation enzyme such as an acyl-CoA synthase. In yet further embodiments, a thioesterase and an acyl-CoA synthase are functionally deleted. In some embodiments, the host cell has no detectable activity of a thioesterase or an acyl-CoA synthase.
- In certain embodiments, the method comprises expressing in a host cell a gene encoding an ester synthase polypeptide comprising the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, or a variant thereof, wherein a thioesterase, an acyl-CoA synthase, or both, are functionally deleted or knocked out from the host cell. In some embodiments, the host cell has no detectable thioesterase activity or acyl-CoA synthase activity.
- Variant can be naturally occurring or created in vitro. In particular, variants can be created using genetic engineering techniques, such as site directed mutagenesis, random chemical mutagenesis, exonuclease III deletion procedures, or standard cloning techniques. Alternatively, such variants, fragments, analogs, or derivatives can be created using chemical synthesis or modification procedures.
- Methods of making variants are well known in the art. These include procedures in which nucleic acid sequences obtained from natural isolates are modified to generate nucleic acids that encode polypeptides having characteristics that enhance their value in industrial or laboratory applications. In such procedures, a large number of variant sequences having one or more nucleotide differences with respect to the sequence obtained from the natural isolate are generated and characterized. Typically, these nucleotide difference result in amino acid changes with respect to the polypeptides encoded by the nucleic acids from the natural isolates.
- For example, variants can be created using error prone PCR (see, e.g., Leung et al., Technique 1:11-15 (1989); and Caldwell et al., PCR Methods Applic. 2:28-33 (1992)). In error prone PCR, PCR is performed under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product. Briefly, in such procedures, nucleic acids to be mutagenized (e.g., an ester synthase sequence), are mixed with PCR primers, reaction buffer, MgCl2, MnCl2, Taq polymerase, and an appropriate concentration of dNTPs for achieving a high rate of point mutation along the entire length of the PCR product. For example, the reaction can be performed using 20 fmoles of nucleic acid to be mutagenized (e.g., an ester synthase polynucleotide sequence), 30 pmole of each PCR primer, a reaction buffer comprising 50 mM KCl, 10 mM Tris HCl (pH 8.3), and 0.01% gelatin, 7 mM MgCl2, 0.5 mM MnCl2, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP. PCR can be performed for 30 cycles of 94° C. for 1 mM, 45° C. for 1 min, and 72° C. for 1 mM However, it will be appreciated that these parameters can be varied as appropriate. The mutagenized nucleic acids are then cloned into an appropriate vector and the activities of the polypeptides encoded by the mutagenized nucleic acids are evaluated.
- Variants can also be created using oligonucleotide directed mutagenesis to generate site-specific mutations in any cloned DNA of interest. Oligonucleotide mutagenesis is described in, for example, Reidhaar-Olson et al., Science 241:53-57 (1988). Briefly, in such procedures a plurality of double stranded oligonucleotides bearing one or more mutations to be introduced into the cloned DNA are synthesized and inserted into the cloned DNA to be mutagenized (e.g., an ester synthase polynucleotide sequence). Clones containing the mutagenized DNA are recovered, and the activities of the polypeptides they encode are assessed.
- Another method for generating variants is assembly PCR. Assembly PCR involves the assembly of a PCR product from a mixture of small DNA fragments. A large number of different PCR reactions occur in parallel in the same vial, with the products of one reaction priming the products of another reaction. Assembly PCR is described in, for example, U.S. Pat. No. 5,965,408.
- Still another method of generating variants is sexual PCR mutagenesis. In sexual PCR mutagenesis, forced homologous recombination occurs between DNA molecules of different, but highly related, DNA sequence in vitro as a result of random fragmentation of the DNA molecule based on sequence homology. This is followed by fixation of the crossover by primer extension in a PCR reaction. Sexual PCR mutagenesis is described in, for example, Stemmer, PNAS, USA 91:10747-10751 (1994).
- Variants can also be created by in vivo mutagenesis. In some embodiments, random mutations in a nucleic acid sequence are generated by propagating the sequence in a bacterial strain, such as an E. coli strain, which carries mutations in one or more of the DNA repair pathways. Such “mutator” strains have a higher random mutation rate than that of a wild-type strain. Propagating a DNA sequence (e.g., an ester synthase polynucleotide sequence) in one of these strains will eventually generate random mutations within the DNA. Mutator strains suitable for use for in vivo mutagenesis are described in, for example, PCT Publication No. WO 91/16427.
- Variants can also be generated using cassette mutagenesis. In cassette mutagenesis, a small region of a double stranded DNA molecule is replaced with a synthetic oligonucleotide “cassette” that differs from the native sequence. The oligonucleotide often contains a completely and/or partially randomized native sequence.
- Recursive ensemble mutagenesis can also be used to generate variants. Recursive ensemble mutagenesis is an algorithm for protein engineering (i.e., protein mutagenesis) developed to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis. Recursive ensemble mutagenesis is described in, for example, Arkin et al., PNAS, USA 89:7811-7815 (1992).
- In some embodiments, variants are created using exponential ensemble mutagenesis. Exponential ensemble mutagenesis is a process for generating combinatorial libraries with a high percentage of unique and functional mutants, wherein small groups of residues are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins. Exponential ensemble mutagenesis is described in, for example, Delegrave et al., Biotech. Res. 11:1548-1552 (1993). Random and site-directed mutageneses are described in, for example, Arnold, Curr. Opin. Biotech. 4:450-455 (1993).
- In some embodiments, variants are created using shuffling procedures wherein portions of a plurality of nucleic acids that encode distinct polypeptides are fused together to create chimeric nucleic acid sequences that encode chimeric polypeptides as described in, for example, U.S. Pat. Nos. 5,965,408 and 5,939,250.
- Polynucleotide variants also include nucleic acid analogs. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety include deoxyuridine for deoxythymidine and 5-methyl-2′-deoxycytidine or 5-bromo-2′-doxycytidine for deoxycytidine. Modifications of the sugar moiety include modification of the 2′ hydroxyl of the ribose sugar to form 2′-O-methyl, 2′-halo, or 2′-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six-membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. (See, e.g., Summerton et al., Antisense Nucleic Acid Drug Dev. 7:187-195 (1997); and Hyrup et al., Bioorgan. Med. Chem. 4:5-23 (1996).) In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.
- In some embodiments, the ester synthase polypeptide is a variant comprising the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, with one or more amino acid substitutions, additions, insertions, or deletions, and the polypeptide has ester synthase and/or acyltransferase activity. For example, the ester synthase variant catalyzes the conversion of thioesters to fatty acids and/or fatty acid derivatives, using alcohols as substrates. In certain embodiments, the ester synthase variant also catalyzes the conversion of free fatty acids to fatty esters.
- In some instances, the methods described herein can be used to produce fatty acids or fatty acid derivatives using an ester synthase polypeptide having an amino acid sequence of SEQ ID NO:18, 24, 25, or 26, as well as a polypeptide variants thereof. Ester synthase polypeptide variant can be variants in which one or more amino acid residues are substituted with conserved or non-conserved amino acid residues. Such substituted amino acid residues may or may not be ones encoded by the genetic code.
- In some embodiments, the ester synthase polypeptide is a variant comprising the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, with one or more non-conserved amino acid substitutions, wherein the polypeptide variant has ester synthase and/or acyltransferase activity. In some embodiments, the glycine residue at
position 395 of SEQ ID NO:18 is substituted with a basic amino acid residue, wherein the resultant ester synthase variant retains or has improved ester synthase and/or acyltransferase activity. - In some embodiments, the ester synthase polypeptide is a variant comprising the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, with one or more conserved amino acid substitutions. Conserved substitutions are those that substitute a given amino acid residue in a polypeptide by another amino acid of similar characteristics. Typical conserved substitutions include the following replacements: replacement of an aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic amino acid; replacement of a serine with a threonine; replacement of a threonine with a serine; replacement of an acidic residue, such as aspartic acid and glutamic acid, with another acidic residue; replacement of residue bearing an amide group; exchange of a basic residue, such as lysine and arginine, with another basic residue; and replacement of an aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue. In some embodiment, the ester synthase polypeptide variant has ester synthase and/or acyltransferase activity. For example, the ester synthase polypeptide variant catalyzes the conversion of a thioester into a fatty acid and/or a fatty acid derivative. In another example, the ester synthase polypeptide catalyzes the conversion of a free fatty acid into a fatty ester.
- Other polypeptide variants include those in which one or more amino acid residues include a substituent group. Still other polypeptide variants include those in which the polypeptide is associated with another compound, such as a compound to increase the half-life of the polypeptide (e.g., polyethylene glycol).
- Additional polypeptide variants are those in which additional amino acid residues are fused to the polypeptide, such as a leader sequence, a secretory sequence, a proprotein sequence, or a sequence which facilitates purification, enrichment, or stabilization of the polypeptide.
- In certain instances, the polypeptide variants retain the same biological function as a polypeptide having an amino acid sequence of, for example, SEQ ID NO:18, 24, 25, or 26 and have amino acid sequences substantially identical thereto. In particular instances, the polypeptide variants retains the ester synthase and/or acyltransferase activity of, for example, a polypeptide of the amino acid sequence SEQ ID NO:18.
- In other instances, the polypeptide variants has at least about 35%, at last about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% homology to an amino acid sequence of SEQ ID NO:18, 24, 25, or 26. In another instance, the polypeptide variants include a fragment comprising at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200 consecutive amino acid residues thereof.
- The polypeptide variants or fragments thereof can be obtained by isolating nucleic acids encoding them using techniques described herein or by expressing synthetic nucleic acids encoding them. Alternatively, polypeptide variants or fragments thereof can be obtained through biochemical enrichment or purification procedures. The sequence of polypeptide variants or fragments can be determined by proteolytic digestion, gel electrophoresis, and/or microsequencing. For example, the sequence of the polypeptide variants or fragments can then be compared to an amino acid sequence of SEQ ID NO:18, 24, 25, or 26 using any of the programs described herein.
- The polypeptide variants and fragments thereof can be assayed for ester synthase activity and/or acyltransferase activity using routine methods. For example, the polypeptide variants or fragments can be contacted with a substrate (e.g., an acyl-CoA, an acyl-ACP, a free fatty acid, or an alcohol) under conditions that allow the polypeptide to function. A decrease in the level of the substrate or an increase in the level of a fatty acid and/or a fatty acid derivative product can be measured to determine the ester synthase/acyltransferase activity.
- In some embodiments, the ester synthase polypeptide variant can comprise the amino acid sequence of SEQ ID NO:18, 24, 25, or 26, with one or more non-conserved amino acid substitutions but continues to have ester synthase and/or acyltransferase activity. In some embodiments, the glycine residue at
position 395 is substituted with a basic amino acid residue, wherein the resultant ester synthase variant retains or has improved ester synthase and/or acyltransferase activity, relative to that of a native or wild-type ester synthase. In a particular instance, the glycine residue atposition 395 of SEQ ID NO:18 is substituted with an arginine or a lysine, wherein the resultant ester synthase variant has improved ester synthase and/or acyltransferase activity, relative to the activity of the wild type ester synthase of SEQ ID NO:18. - In some embodiments, the polypeptide comprises one or more conserved amino acid substitutions. In some embodiments, the polypeptide has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acid substitutions, additions, insertions, or deletions. In some embodiments, the polypeptide comprising the one or more amino acid substitutions, additions, insertions or deletions retains the same biological activity as a polypeptide of SEQ ID NO:18, 24, 25, or 26. As used herein, “retaining a biological activity” refers to the retention of a detectable level of that biological activity, rather than the retention of the same level of an activity. For example, the polypeptide comprising one or more amino acid substitutions, additions, insertions or deletions has ester synthase and/or acyltransferase activity. For example, the polypeptide is capable of catalyzing the conversion of thioesters to fatty acids and/or fatty acid derivatives such as fatty esters, using alcohols as substrates. Suitably thioester substrates include, for example, fatty thioesters such as fatty acyl-CoAs or fatty acyl-ACPs. In another example, the polypeptide is capable of catalyzing the conversion of a free fatty acid into a fatty ester using an alcohol as substrate. Suitable alcohol substrates include, for example, long- or short-chain alcohols such as methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, decanol, dodecanol, tetradecanol, or hexadecanol.
- In certain embodiments, the invention pertains to methods of producing a fatty acid or a fatty acid derivative such as, for example, a fatty ester, wherein the method includes expressing in a host cell a gene or polynucleotide encoding a variant of ester synthase, ES9 (GenBank Accession No. AB021021, SEQ ID NO:18), wherein the host cell expresses an attenuated level of a thioesterase, or wherein the thioesterase is functionally deleted. In other embodiments, the invention pertains to methods of producing a fatty acid or a fatty acid derivative such as, for example, a fatty ester, wherein the method includes expressing in a host cell a gene encoding a variant of ES9 (SEQ ID NO:18), wherein the host cell expresses an attenuated level of a fatty acid degradation enzyme such as an acyl-CoA synthase, or wherein the acyl-CoA synthase is functionally deleted. In a further embodiment, the invention pertains to methods of producing a fatty acid or a derivative thereof, wherein the method includes expressing in a host cell a gene encoding a variant of ES9 (SEQ ID NO:18), wherein the host cell express attenuated levels of a thioesterase, a fatty acid degradation enzyme such as an acyl-CoA synthase, or both, or wherein the thioesterase, the acyl-CoA synthase, or both, are functionally deleted. In some embodiments, the host cell has no detectable thioesterase activity or acyl-CoA synthase activity.
- In some embodiments, the variant of ester synthase comprises a substitution at the amino acid residue corresponding to position 395 of SEQ ID NO:18. In a particular embodiment, the variant comprises a basic amino acid residue at
position 395, replacing the glycine residue at that position of SEQ ID NO:18. For example, the basic amino acid residue can be a lysine or an arginine residue. In some embodiments, the variant has the same or improved ester synthase and/or acyltransferase activity, relative to that of the polypeptide of SEQ ID NO:18. - In certain embodiments, the invention pertains to methods of producing a fatty acid or a fatty acid derivative wherein the method includes expressing in a host cell a polynucleotide encoding a variant of ester synthase ES9, wherein the host cell further expresses an attenuated level of thioesterase, an acyl-CoA synthase, or both, or wherein the thioesterase, the acyl-CoA synthase, or both, are functionally deleted. In some embodiments, the variant of ester synthase comprises a substitution at the amino acid residue corresponding to position 395 of SEQ ID NO:18 and one or more other amino acid substitutions, additions, insertions or deletions, and the variant has ester synthase and/or acyltransferase activity. In some embodiments, the variant catalyzes the conversion of thioester substrates and/or alcohol substrate into fatty acids and/or fatty acid derivatives. In other embodiments, the variant catalyzes the conversion of free fatty acids into fatty esters.
- In some embodiments, the ester synthase polypeptide or a variant thereof is about 200 amino acids to about 2,000 amino acids in length, for example, from about 250 to about 1500 amino acid residues in length, from about 300 to about 1200 amino acid residues in length, from about 350 to about 1000 amino acid residues in length, from about 400 to about 800 amino acid residues in length, or from about 450 to about 600 amino acid residues in length. In certain embodiments, the naturally-occurring ester synthase polypeptide is about 300 amino acid residues in length or longer, for example, about 400 amino acid residues in length or longer, or about 450 amino acid residues in length or longer. In certain related embodiments, the ester synthase polypeptide or the variant thereof is about 1000 amino acid residues in length or shorter, for example, about 800 amino acid residues in length or shorter, about 700 amino acid residues in length or shorter, or about 600 amino acid residues in length or shorter. An exemplary ester synthase of the invention is about 500 amino acid residues in length.
- Additional ester synthases that can be used are described in the PCT patent application entitled “Production of Fatty Acid Derivatives”, filed concurrently herewith, under Attorney Docket No. 2001235.150WO1.
- The ester synthase polypeptides described herein can be used to produce antibodies directed against ester synthase polypeptides. Such antibodies can be used, for example, to detect expression of an ester synthase polypeptide using methods known in the art. The antibody can be, for example, a polyclonal antibody; a monoclonal antibody or antigen binding fragment thereof; a modified antibody such as a chimeric antibody, reshaped antibody, humanized antibody, or fragment thereof (e.g., Fab′, Fab, F(ab′)2); or a biosynthetic antibody, for example, a single chain antibody, single domain antibody (DAB), Fv, single chain Fv(scFv), or the like.
- Methods of making and using polyclonal and monoclonal antibodies are described, for example, in Harlow et al., Using Antibodies: A Laboratory Manual: Portable Protocol I. Cold Spring Harbor Laboratory (Dec. 1, 1998). Methods for making modified antibodies and antibody fragments (e g, chimeric antibodies, reshaped antibodies, humanized antibodies, or fragments thereof, e.g., Fab′, Fab, F(ab′)2 fragments); or biosynthetic antibodies (e.g., single chain antibodies, single domain antibodies (DABs), Fv, single chain Fv (scFv), and the like), are known in the art and can be found, for example, in Zola, Monoclonal Antibodies: Preparation and Use of Monoclonal Antibodies and Engineered Antibody Derivatives, Springer Verlag (Dec. 15, 2000; 1st edition).
- The compositions and methods described herein can be used to product fatty acids and/or fatty acid derivatives, for example, from thioesters and alcohols, or from free fatty acids and alcohols, which themselves can be produced from appropriate substrates in the same host cell. While not wishing to be bound by theory, it is believed that the recombinantly modified production host can be modified to increase the production of thioester substrates such as, for example, acyl-CoAs or acyl-ACPs, of free fatty acids, or of suitable alcohol substrates such as, for example, fatty alcohols. Moreover, the recombinantly modified production host can be modified to reduce the catabolism of fatty acids or fatty acid derivatives and/or intermediates in the fatty acid biosynthetic pathways, or to reduce feedback inhibition at specific points in the same pathway. In addition to modifying the genes described herein, additional cellular resources can be diverted to over-produce fatty acids and/or fatty acid derivatives. For example, the lactate, succinate, and/or acetate pathways can be attenuated, and acetyl-CoA carboxylases (encoded by acc genes) can be overexpressed. Modifications to production host can be achieved, for example, through genomic alterations, addition of recombinant expression systems, or combinations thereof.
- A representative fatty acid and/or fatty acid derivative biosynthetic pathway is depicted in
FIG. 1A . The following sections describe the steps in these pathways. Different steps in the pathway are catalyzed by a variety of enzymes. Each step within a pathway that leads to the production of a fatty acid and/or a fatty acid derivative substrate can be modified to produce or overproduce the substrate of interest. For example, known genes involved in the fatty acid biosynthetic pathway can be expressed, overexpressed, or attenuated in host cell to produce the desired substrate (see, e.g., the approach described in PCT/US08/05877, the disclosure of which is incorporated herein by reference). Exemplary genes that may serve as the points of change include, without limitation, accA (EC 6.4.1.2), accB (EC 6.4.1.2), accC (EC 6.4.1.2, EC 6.3.4.14), accD (EC 6.4.1.2), aceE (EC 1.2.4.1), aceF (EC 2.3.1.12), fabD (EC 2.3.1.39), fabG (EC 1.1.1.100), fabH (EC 2.3.1.180), panD (EC 4.1.1.11), panK (EC 2.7.1.33), pdh (EC 1.2.4.1), udhA (EC 1.6.1.1). - Fatty acid synthase (FAS) is a group of peptides that catalyze the initiation and elongation of acyl chains (Marrakchi et al., Biochemical Society, 30:1050-1055 (2002)). The acyl carrier protein (ACP) along with the enzymes in the FAS pathway controls the length, degree of saturation, and branching of the fatty acids produced. The steps in this pathway are catalyzed by enzymes of the fatty acid biosynthesis (fab) and acetyl-CoA carboxylase (acc) gene families (see, e.g., Heath et al., Prog. Lipid Res. 40(6):467-97 (2001)). Depending on the desired product, one or more of these genes can be attenuated or overexpresssed.
- Host cells can be engineered to express fatty acid derivative substrates by recombinantly expressing or overexpressing one or more fatty acid synthase genes, such as acetyl-CoA and/or malonyl-CoA synthase genes. For example, to increase acetyl-CoA production one or more of the following genes can be expressed or overexpressed in a host cell: pdh, panK, aceEF (encoding the E1p dehydrogenase component and the E2p dihydrolipoamide acyltransferase component of the pyruvate and 2′-oxoglutarate dehydrogenase complexes), fabH, fabD, fabG, acpP, and fabF. Exemplary GenBank Accession numbers for these genes are listed in the parentheses after the gene names: pdh (BAB34380, AAC73227, AAC73226), panK (also known as coaA, AAC96952), aceEF (AAC73227, AAC73226),fabH (AAC74175), fabD (AAC74176), fabG (AAC74177), acpP (AAC74178), fabF (AAC74179).
- Additionally, the expression levels of fadE, gpsA, ldhA, pflb, adhE, pta, poxB, ackA, and/or ackB can be reduced or functionally deleted in the engineered microorganism by transformation with conditionally replicative or non-replicative plasmids containing null or deletion mutations of the corresponding genes, or by substituting promoter or enhancer sequences. Exemplary GenBank Accession numbers for these genes are listed in the parentheses: fadE (AAC73325), gspA (AAC76632), ldhA (AAC74462), pflb (AAC73989), adhE (AAC74323), pta (AAC75357), poxB (AAC73958), ackA (AAC75356), and ackB (BAB81430). In an exemplary embodiment, fadE expression is attenuated or functionally deleted in a production host cell of the present invention. The resulting engineered production hosts can produce increased levels of acetyl-CoA when cultivated in an appropriate environment.
- Similarly, malonyl-CoA overproduction can be effectuated by engineering the production host as described with accABCD (e.g., GenBank Accession No. AAC73296, classified as EC 6.4.1.2) included in the plasmid synthesized de novo. Fatty acid overproduction can be achieved by further including a DNA sequence encoding lipases (e.g., GenBank Accession Nos: CAA89087, CAA98876), in the plasmid synthesized de novo.
- The resultant host cells, in some embodiments, overexpresses acetyl-CoA such that its intracellular concentration is increased by more than about 2-fold, for example, by more than about 5-fold, or more than about 10 fold, relative to the native expression levels of acetyl-CoA.
- Acetyl-CoA and malonyl-CoA overproduction can be verified using methods known in the art, for example, by using radioactive precursors, HPLC, and GC-MS following cell lysis.
- Additionally, the PlsB (e.g., GenBank Accession No. AAC77011) D311E mutant can be used to increase the amount of acyl-CoA available.
- Furthermore, overexpression of an sfa gene (suppressor of FabA, e.g., GenBank Accession No. AAN79592) can be effectuated in a production host to increase the production of monounsaturated fatty acids (Rock et al., J. Bacteriology 178:5382-5387 (1996)).
- In a typical microbial process model for fatty acid and fatty acid derivative synthesis, acetyl-CoA and malonyl-CoA are converted through a series of steps to form the acyl-ACP chains. Acyl-ACP is then converted via a series of alternative enzymatic steps into various end products, including fatty acid derivatives. For example, typically acyl-ACP is converted to fatty esters by the combined consecutive reactions of a thioesterase, an acyl-CoA ligase/synthetase and an ester synthase. A limitation to the commercial use of these enzymes in a metabolic pathway is the requirement of hydrolysis of ACP to produce fatty acids that are subsequently reactivated to produce the fatty acyl-CoA substrates, which requires at least two enzymatic steps and the expenditure of metabolic energy from two phosphoanhydride bonds. It has been discovered that fatty acids and/or fatty acid derivatives such as, for example, fatty esters, can be produced in the presence of an ester synthase, bypassing the participations of a thioesterase, and/or bypassing the participation of a fatty acid degradation enzyme such as an acyl-CoA synthase. This more economical and efficient approach mitigates the loss of ATP caused by an otherwise multi-step pathway. Specifically, it has been discovered that a suitable ester synthase, in the absence of a co-expressed thioesterase, and/or a co-expressed fatty acid degradation enzyme such as an acyl-CoA synthase, can effectively utilize thioesters such as acyl-ACPs or acyl-CoAs, free fatty acids, and/or alcohols such as short- or long-chain alcohols, as substrates to produce desirable fatty acids and/or fatty acid derivatives under appropriate conditions.
- One of ordinary skill in the art is capable of determining the fitness of using a particular ester synthase enzyme to directly produce fatty acids and/or fatty acid derivatives from thioester substrates. Example 14 describes representative assays that can be used, among other suitable methods, to determine whether a fatty acid and/or fatty acid derivatives is produced and/or the amount that is produced in a given production system.
- According to this aspect of the invention, then, various ester synthases can be utilized to directly produce fatty acids and/or fatty acid derivatives, in the presence or in the absence of a thioesterase, and/or in the presence or in the absence of a fatty acid degradation enzyme such as, for example, an acyl-CoA synthase. For example, expression or overexpression of an ester synthase that can catalyze the direct production of fatty acids and/or fatty acid derivatives in a recombinant host strain. This can be used to supplement fatty acid derivative production where the strain also expresses a thioesterase and/or a fatty acid degradation enzyme. For example, on occasions wherein free fatty acids are undesirable products of a fatty ester production process, the free fatty acids can be converted into fatty esters using the ester synthases, variants, host cells, and microorganisms and methods herein. Additionally, expression of an ester synthase that can catalyze the direct production of fatty acids and/or fatty acid derivatives such as fatty esters in a recombinant host cell can be used where there is no or low thioesterase expression.
- The term “thioesterase” refers to an enzyme that has thioesterase activity. Thioesterases include thioester hydrolases, which are identified as members of Enzyme Classification E.C. 3.1.2 and are obtainable from a variety of sources. Plant thioesterases are described in, for example, Voelker & Davies, J. Bact., 176 (23): 7320-27 (1994), U.S. Pat. Nos. 5,667,997 and 5,455,167. Thioesterases are also obtainable from microbial sources, such as those described in Akoh et al., Prog. Lipid Res., 43(6):534-52 (2004); Diczfalusy & Alexson, Arch. Biochem. Biophys., 334(1):104-12 (1996); Larson & Kolattukudy, Arch. Biochem. Biophys., 237(1):27-37(1985); Lawson et al., Biochemistry 33(32):9382-88 (1994); Lee et al., Eur. J. Biochem., 184 (1):21-28 (1989); Naggert et al., J. Biol. Chem., 266(17):11044-50 (1991); Nie et al., Biochemistry, 47 (29):7744-51 (2008); Seay & Lueking, Biochemistry 25(9): 2480-85 (1986); Spencer et al., J. Biol. Chem., 253(17):5922-26 (1978); and Zhuang et al., Biochemistry 47(9):2789-96 (2008). Thioesterases are also obtainable from, for example, cyanobacterial, algal, mammalian, insect, and fungal sources. A thioesterase can have activity other than thioesterase activity, for example proteolytic activity or oxygen ester hydrolysis activity. A particularly useful thioesterase is the 'TesA (also termed “thioesterase I” or “leaderless thioesterase”) enzyme from E. coli, which is a truncated version of the full-length TesA serine thioesterase enzyme that is described in Cho & Cronan, J. Biol. Chem., 268(13): 9238-45 (1992). An E. coli 'TesA polypeptide comprises 182 amino acids, and is the product of a cleavage reaction wherein the 26 amino acid leader sequence of E. coli TesA is removed.
- The term “thioesterase activity” refers to the capacity to catalyze a thioester cleavage reaction, which usually involves the hydrolysis of a thioester at a thiol group into an acid and a thiol, but can also include a transesterification step in which a thioester bond is cleaved and a new ester bond is formed. In general, an acyl-ACP thioesterase is capable of catalyzing the hydrolytic cleavage of fatty acyl-acyl carrier protein thioesters and/or fatty acyl-CoA thioesters. Examples of enzymes having thioesterase activity include acetyl-CoA hydrolase, palmitoyl-CoA hydrolase, succinyl-CoA hydrolase, formyl-CoA hydrolase, acyl-CoA hydrolase, palmitoyl-protein thioesterase, and ubiquitin thiolesterase. Thioesterase activity can be established by any of the following assays:
- Acyl-CoA Hydrolysis Assay:
- A Tris-HCl buffer, 0.1 M, pH 8.0; Palmitoyl-CoA, 5 μM; DTNB, 0.01 M in 0.1 M potassium phosphate buffer, pH 7.0 are used to prepare a complete assay mixture. The assay mixture thus contains a final concentration of 10 mot of Tris-HCl buffer, pH 8.0, 0.05 mot of DTNB, and 0.01 mot of palmitoyl-CoA. The complete assay mixture is then mixed with the thioesterase, in a final volume of 2.0 mL. The rate of cleavage of the acyl-CoA substrate is measured by monitoring the change in absorbance at 412 nm, using a molar extinction coefficient of 13,600 M− cm−1.
- In Vivo Assay:
- Following expression of the protein of interest, the culture, which potentially has thioesterase activity, is acidified with 1 N HCl to a final pH of about 2.5 and then extracted with an equal volume of butyl acetate. Free fatty acids in the organic phase are derivatized with BSTFA (N,O-bis (trimethyl (silyl) trifluoro acetamide) and 1% TMCS (trimethyl chloro silane) to generate the respective TMS (trimethyl silyl-) esters, which are then analyzed on a gas chromatograph equipped with a flame ionization detector.
- 4-MU-6S-Palm-βGlc Assay:
- A 20 μL, of substrate solution is first prepared. The substrate solution contains 0.64 mM MU-6S-Palm-β-Glc, 15 mM dithiothreitol, 0.375% (w/v) Triton X-100, and 0.1 U β-glucosidase from almonds in Mcllvain's phosphate/citrate buffer, pH 4.0. A culture mixture potentially having thioesterase activity is then mixed with the substrate solution. The reaction mixture is incubated for 1 hour at 37° C. Exogenous almond β-glucosidase is added to hydrolyze the reaction intermediate, MU-6-thio-β-glucoside, quantitatively. The hydrolysis reaction is terminated by the addition of 200 μL of 0.5 M sodium carbonate, pH 10.7, containing 0.025% Triton X-100, and the fluorescence of the released 4-methylumbelliferone (MU) is measured in a fluorometer (λex=372, λcm=445 nm).
- Lysophospholipase Assay:
- A reaction mixture containing 10 μL a culture mixture potentially having thioesterase activity mixed with 10 μL of 3 mM 1-oleoyl-phosphatidylethanolamine, 25 μL of 100 mM Tris-HCl (pH 7.0), and 5 μL of 5 mM EDTA is prepared. The reaction is terminated with the addition of 1.5 mL CHCl3:CH3OH (1:2), followed by the addition of water to bring the total aqueous volume to 0.9 mL. The organic phase is then analyzed by thin layer chromatography together with suitable standards, using plates prepared from 40 g Silica Gel H suspended in 95 mL of 1 mM sodium tetraborate. The solvent system consists of CHCl3:CH3OH:H2O (95:35:5).
- Protease Substrate Assay:
- A reaction mixture containing 10 μL of a culture mixture potentially having thioesterase activity mixed with 800 μL 12.5 mM Tris-HCl (pH 8.0) containing 0.25% Triton X-100 and 10 μL of Cbz-Phe-ONp dissolved in DMSO is prepared. The p-nitrophenol released via cleavage of the substrate is measured by monitoring the absorbance at 405 nm.
- Fatty Acyl-PNP Hydrolysis Assay:
- A reagent solution containing 2% Triton X-100 in 50 mM sodium phosphate, pH 7.0, and 10 mM C12-p-nitrophenol (acyl-PNP) in acetone is first prepared. Then a C12-PNP working solution is prepared by mixing 600 μL 10 mM C12-PNP into a 9.4-mL phosphate buffer. The assay is performed by adding 40 μL of the acyl-PNP working solution to each well of a 96-well plate, followed by the rapid addition of 40 μL of the culture mixture potentially having thioesterase activity. The solution is mixed for 15 seconds, and the absorbance change is read at 405 nm in a microtiter plate reader at 25° C.
- Ester Formation from Thioester:
- A reaction mixture containing a culture mixture potentially having thioesterase activity, 100 μM myristoyl-CoA, 10% (v/v) methanol, and 50 mM sodium phosphate, pH 7.0 is prepared. The reaction mixture is incubated for 1 hour at 20° C. and terminated with the addition of 1 N HCl to decrease the pH to about 2.5. The mixture is extracted with an equal volume of ethyl acetate and the amount of fatty ester produced is determined via GC-MS or other standard methods such as GC-FID, LC-MS, or thin layer chromatography.
- Ester Formation from Ester:
- A reaction mixture containing a culture mixture potentially having thioesterase activity, 300 μM lauroyl-CoA, 10% (v/v) methanol, and 50 mM sodium phosphate, pH 7.0 is prepared. The reaction mixture is incubated for 1 hour at 20° C. and terminated with the addition of 1 N HCl to decrease the pH to about 2.5. The mixture is extracted with an equal volume of ethyl acetate and the amount of lauryl ester produced is determined via GC-MS or other standard methods such as GC-FID, LC-MS, or thin layer chromatography.
- Thus, an endogenous thioesterase can be attenuated and/or functionally deleted from the host cells of the present invention. In certain embodiments the thioesterase is selected from the group consisting of tesA, 'tesA, fatB1, fatB2, fatB3, fatA1, fatA, and atfata. Non-limiting examples of thioesterases and host cells are listed in Table 1.
-
TABLE 1 Thioesterases Accession Number Source Organism Gene AAC73596 E. coli tesA without leader sequence AAC73555 E. coli tesB Q41635, AAA34215 Umbellularia california fatB AAC49269 Cuphea hookeriana fatB2 Q39513; AAC72881 Cuphea hookeriana fatB3 Q39473, AAC49151 Cinnamonum camphorum fatB CAA85388 Arabidopsis thaliana fatB [M141T]* NP 189147; NP 193041 Arabidopsis thaliana fatA CAC39106 Bradyrhiizobium japonicum fatA AAC72883 Cuphea hookeriana fatA AAL79361 Helianthus annus fatA1 *Mayer et al., BMC Plant Biology 7: 1-11 (2007) - In other instances, fatty acids and/or fatty acid derivatives are produced in a host cell that contains one or more naturally-occurring mutations that result in an increased level of fatty acids and/or fatty acid derivatives in the host cell. In some instances, the host cell is genetically engineered to increase the level of fatty acids and/or fatty acid derivatives in the host cell relative to a corresponding wild-type host cell. For example, the host cell can be genetically engineered to express a reduced level of a fatty acid degradation enzyme such as an acyl-CoA synthase relative to a corresponding wild-type host cell. In one embodiment, the level of expression of one or more genes (e.g., an acyl-CoA synthase gene) is attenuated or functionally deleted by genetically engineering a “knock out” host cell.
- Any known acyl-CoA synthase gene can be reduced or functionally deleted in a host cell. Non-limiting examples of acyl-CoA synthase genes include fadD, fadK, BH3103, yhfL, Pfl-4354, EAV15023,
fadD 1, fadD2, RPC_4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_01644857. Specific examples of acyl-CoA synthase genes include fadDD35 from M. tuberculosis H37Rv [NP_217021], fadDD22 from M. tuberculosis H37Rv [NP_217464], fadD from E. coli [NP_416319], fadK from E. coli [YP_416216], fadD from Acinetobacter sp. ADP1 [YP_045024], fadD from Haemophilus influenza RdkW20 [NP_438551], fadD from Rhodopseudomonas palustris Bis B 18 [YP_533919], BH3101 from Bacillus halodurans C-125 [NP_243969], Pfl-4354 from Pseudomonas fluorescens Pfo-1 [YP_350082], EAV15023 from Comamonas testosterone KF-1 [ZP_01520072], yhfL from B. subtilis [NP_388908], fadD1 from P. aeruginosa PAO1 [NP_251989], fadD1 from Ralstonia solanacearum GM1 1000 [NP_520978], fadD2 from P. aeruginosa PAO1 [NP_251990], the gene encoding the protein ZP_01644857 from Stenotrophomonas maltophilia R551-3, faa3p from Saccharomyces cerevisiae [NP_012257], faalp from Saccharomyces cerevisiae [NP_014962], lcfA from Bacillus subtilis [CAA99571], or those described in Shockey et al., Plant. Physiol. 129:1710-1722 (2002); Caviglia et al., J. Biol. Chem. 279:1163-1169 (2004); Knoll et al., J. Biol. Chem. 269(23):16348-56 (1994); Johnson et al., J. Biol. Chem. 269: 18037-18046 (1994); and Black et al., J. Biol Chem. 267: 25513-25520 (1992). - The alcohol substrates can be supplied to the host cells. In alternative embodiments, when fatty alcohols are used as substrartes, the fatty alcohol substrates can be made by the host cell by co-expressing one or more fatty aldehyde biosynthesis genes and/or one or more fatty alcohol biosynthesis genes. Heterologous fatty aldehyde biosynthesis genes can be introduced to the host cell, or endogenous fatty aldehyde biosynthesis genes can be modified such that they are overexpressed in the host cell. Suitable fatty aldehyde biosynthesis genes include, without limitation, those encoding carboxylic acid reductases and variants of, for example, any one of SEQ ID NOs:54-97. Heterologous fatty alcohol biosynthesis genes can also be introduced to the host cell, or endogenous fatty alcohol biosynthesis genes can be modified such that they are overexpressed in the host cell. Suitable fatty alcohol biosynthesis genes include, without limitation, those encoding alcohol dehydrogenases and variants of, for example, any one of SEQ ID NOs:98-147. In additional embodiments, fatty alcohol substrates can be made by the host cell by co-expressing one or more acyl-ACP reductase genes and/or one or more fatty alcohol biosynthesis genes. Heterologous acyl-ACP reductase genes may be introduced to the host cell, or endogenous acyl-ACP reductase genes can be modified such that they are overexpressed in the host cell. Suitable acyl-ACP reductase genes include, without limitation, those encoding acyl-ACP reductases and variants of, for example, any one of SEQ ID NOs:148-156. Methods, host cells, and microorganisms of producing fatty alcohols have been previously described, for example, in International Patent Publication No. WO/2010/042664, the disclosures of which are incorporated herein by reference.
- In another embodiment, the ester synthase polypeptides and variants of the present invention can catalyze the conversion of a free fatty acid into a fatty ester in the presence of suitable alcohol substrates. Accordingly, in some embodiments, when free fatty acid is not a desired product, the ester synthase polypeptides and variants of the invention can be used to reduce the amount of free fatty acid generated during the fatty ester production by converting them into fatty esters.
- Formation of Branched Fatty Acids and/or Fatty Acid Derivatives
- Fatty acids and derivatives thereof can be produced that contain points of branching by using branched thioester and/or branched alcohol as substrates. For example, although E. coli naturally produces straight chain fatty acids (sFAs), E. coli can be engineered to produce branched chain fatty acids (brFAs) and/or brFA derivatives by introducing and expressing or overexpressing heterologous genes that provide branched precursors in the E. coli (e.g., bkd, ilv, icm, and fab gene families). Additionally, a host cell can be engineered to express or overexpress genes encoding proteins for the elongation of brFAs (e.g., ACP, FabF, etc.) or derivatives, and/or to delete or attenuate the corresponding host cell genes that normally lead to straight-chain fatty acids and/or fatty acid derivatives.
- The first step in forming brFAs or brFA derivatives is the production of the corresponding α-keto acids by a branched-chain amino acid aminotransferase. Host cells may endogenously include genes encoding such enzymes or such genes can be heterologously or recombinantly introduced. E. coli, for example, endogenously expresses such an enzyme, IlvE (EC 2.6.1.42; GenBank Accession No. YP_026247). In some host cells, a heterologous branched-chain amino acid aminotransferase may not be expressed. However, E. coli IlvE or any other branched-chain amino acid aminotransferase (e.g., IlvE from Lactococcus lactis (GenBank Accession No. AAF34406), IlvE from Pseudomonas putida (GenBank Accession No. NP_745648), or IlvE from Streptomyces coelicolor (GenBank Accession No. NP_629657)), if not endogenous, can be introduced.
- In another embodiment, the production of α-keto acids can be achieved by using the methods described in Atsumi et al., Nature 451:86-89 (2008). For example, 2-ketoisovalerate can be produced by overexpressing the genes encoding IlvI, IlvH, IlvC, or IlvD. In another example, 2-keto-3-methyl-valerate can be produced by overexpressing the genes encoding IlvA and IlvI, IlvH (or AlsS of Bacillus subtilis), IlvC, IlvD, or their corresponding homologs. In a further embodiment, 2-keto-4-methyl-pentanoate can be produced by overexpressing the genes encoding IlvI, IlvH, IlvC, IlvD and LeuA, LeuB, LeuC, LeuD, or their corresponding homologs.
- The second step is the oxidative decarboxylation of the α-keto acids to the corresponding branched-chain acyl-CoA. This reaction can be catalyzed by a branched-chain α-keto acid dehydrogenase complex (bkd; EC 1.2.4.4.) (Denoya et al., J. Bacteriol. 177:3504 (1995), which consists of E1α/β (decarboxylase), E2 (dihydrolipoyl transacylase), and E3 (dihydrolipoyl dehydrogenase) subunits. These branched-chain α-keto acid dehydrogenase complexes are similar to pyruvate and α-ketoglutarate dehydrogenase complexes. Any microorganism that comprises brFAs and/or brFA cellular components and/or is capable of growing on branched-chain amino acids can be used as a source to isolate bkd genes for expression in host cells, for example, E. coli. Furthermore, E. coli has the E3 component as part of its pyruvate dehydrogenase complex (lpd, EC 1.8.1.4, GenBank Accession No. NP_414658). Thus, it may be sufficient to express only the E1 α/β and E2 bkd genes. Table 2 lists non-limiting examples of bkd genes from several microorganisms that can be heterologously or recombinantly introduced and expressed in a host cell to provide branched-chain acyl-CoA precursors.
-
TABLE 2 Bkd genes from selected microorganisms Organism Gene GenBank Accession # Streptomyces coelicolor bkdA1 (E1α) NP_628006 bkdB1 (E1β) NP_628005 bkdC1 (E2) NP_628004 Streptomyces coelicolor bkdA2 (E1α) NP_733618 bkdB2 (E1β) NP_628019 bkdC2 (E2) NP_628018 Streptomyces avermitilis bkdA (E1a) BAC72074 bkdB (E1b) BAC72075 bkdC (E2) BAC72076 Streptomyces avermitilis bkdF (E1α) BAC72088 bkdG (E1β) BAC72089 bkdH (E2) BAC72090 Bacillus subtilis bkdAA (E1α) NP_390288 bkdAB (E1β) NP_390288 bkdB (E2) NP_390288 Pseudomonas putida bkdA1 (E1α) AAA65614 bkdA2 (E1β) AAA65615 bkdC (E2) AAA65617 - In another example, isobutyryl-CoA can be made in a host cell, for example in E. coli, through the coexpression of a crotonyl-CoA reductase (Ccr, EC 1.6.5.5, 1.1.1.1) and isobutyryl-CoA mutase (large subunit IcmA, EC 5.4.99.2; small subunit IcmB, EC 5.4.99.2) (Han and Reynolds, J. Bacteriol. 179:5157 (1997)). Crotonyl-CoA is an intermediate in fatty acid biosynthesis in E. coli and other microorganisms. Non-limiting examples of ccr and icm genes from selected microorganisms are listed in Table 3.
-
TABLE 3 Ccr and icm genes from selected microorganisms Organism Gene GenBank Accession # Streptomyces coelicolor ccr NP_630556 icmA NP_629554 icmB NP_630904 Streptomyces cinnamonensis ccr AAD53915 icmA AAC08713 icmB AJ246005 - In addition to expression of the bkd genes, the initiation of brFA biosynthesis utilizes β-ketoacyl-acyl-carrier-protein synthase III (FabH, EC 2.3.1.41) with specificity for branched chain acyl-CoAs (Li et al., J. Bacteriol. 187:3795-3799 (2005)). Non-limiting examples of FabH enzymes are listed in Table 4. fabH genes that are involved in fatty acid biosynthesis of any brFA-containing microorganism or organisms that feed on branched-chain amino acids can be expressed in a host cell. The Bkd and FabH enzymes from host cells that do not naturally make brFA may not support brFA production. Thus, bkd and fabH can be expressed recombinantly or heterologously. Vectors containing the bkd and/or fabH genes can be inserted into such a host cell. Similarly, the endogenous level of Bkd and FabH production may not be sufficient to produce brFA. In this case, the endogenous bkd and/or fabH can be overexpressed. Or the endogenous bkd and/or fabH can be functionally deleted and replaced with a heterologous bkd and/or fabH. Preferably, the heterologous bkd and/or fabH that is introduced into a host cell encode more effective BKD and/or FabH enzymes than those encoded by the endogenous bkd and/or fabH. Additionally, other components of the fatty acid biosynthesis pathway can be expressed or overexpressed, such as acyl carrier proteins (ACPs) and 3-ketoacyl-acyl-carrier-protein synthase II (fabF, EC 2.3.1.41) (non-limiting examples are listed in Table 4). In addition to expressing these genes, some genes in the endogenous fatty acid biosynthesis pathway can be attenuated in the host cell (e.g., the E. coli genes fabH (GenBank Accession No. NP_415609) and/or fabF (GenBank Accession No. NP_415613)).
-
TABLE 4 FabH, ACP and fabF genes from selected microorganisms with brFAs GenBank Organism Gene Accession # Streptomyces coelicolor fabH1 NP_626634 acp NP_626635 fabF NP_626636 Streptomyces avermitilis fabH3 NP_823466 fabC3 (acp) NP_823467 fabF NP_823468 Bacillus subtilis fabH_A NP_389015 fabH_B NP_388898 acp NP_389474 fabF NP_389016 Stenotrophomonas SmalDRAFT_0818 (fabH) ZP_01643059 maltophilia SmalDRAFT_0821 (acp) ZP_01643063 SmalDRAFT_0822 (fabF) ZP_01643064 Legionella pneumophila fabH YP_123672 acp YP_123675 fabF YP_123676
Formation of Cyclic Fatty Acids and/or Fatty Acid Derivatives - Cyclic fatty acids and/or derivatives thereof such as, for example, cyclic fatty esters, can be produced by using suitable cyclic substrates. To produce cyclic substrates, genes that provide cyclic precursors (e.g., the ans, chc, and plm gene families) can be introduced into the host cell and expressed to allow initiation of fatty acid biosynthesis from cyclic precursors. For example, to convert a host cell, such as E. coli, into one capable of synthesizing ω-cyclic fatty acids (cyFA), a gene that provides the cyclic precursor cyclohexylcarbonyl-CoA (CHC-CoA) (Cropp et al., Nature Biotech. 18:980-983 (2000)) can be introduced and expressed in the host cell. Non-limiting examples of genes that provide CHC-CoA in E. coli include: ansJ, ansK, ansL, chcA, and ansM from the ansatrienin gene cluster of Streptomyces collinus (Chen et al., Eur. J. Biochem. 261: 98-107 (1999)) or plmJ, plmK, plmL, chcA, and plmM from the phoslactomycin B gene cluster of Streptomyces sp. HK803 (Palaniappan et al., J. Biol. Chem. 278:35552-57 (2003)) together with the chcB gene (Patton et al., Biochem. 39:7595-7604 (2000)) from S. collinus, S. avermitilis, or S. coelicolor (see Table 5). The genes listed in Table 4 can then be expressed to allow initiation and elongation of ω-cyclic fatty acids. Alternatively, the homologous genes can be isolated from microorganisms that make cyFA and expressed in a host cell (e.g., E. coli).
-
TABLE 5 Genes for the synthesis of CHC-CoA Organism Gene GenBank Accession # Streptomyces collinus ansJK U72144* ansL chcA ansM chcB AF268489 Streptomyces sp. HK803 pmlJK AAQ84158 pmlL AAQ84159 chcA AAQ84160 pmlM AAQ84161 Streptomyces coelicolor chcB/caiD NP_629292 Streptomyces avermitilis chcB/caiD NP_629292 *Only chcA is annotated in GenBank entry U72144, ansJKLM are according to Chen et al. (Eur. J. Biochem. 261: 98-107, 1999). - The genes listed in Table 4, supra (fabH, acp, and fabF) allow initiation and elongation of ω-cyclic fatty acids because they have broad substrate specificity. If the coexpression of any of these genes with the genes listed in Table 5 does not yield cyFA, then fabH, acp, and/or fabF homologs from microorganisms that make cyFAs (e.g., those listed in Table 6) can be isolated (e.g., by using degenerate PCR primers or heterologous DNA sequence probes) and coexpressed.
-
TABLE 6 Non-limiting examples of microorganisms that contain ω-cyclic fatty acids Organism Reference Curtobacterium pusillum ATCC19096 Alicyclobacillus acidoterrestris ATCC49025 Alicyclobacillus acidocaldarius ATCC27009 Alicyclobacillus cycloheptanicus* Moore, J. Org. Chem. 62: pp. 2173, 1997 *Uses cycloheptylcarbonyl-CoA and not cyclohexylcarbonyl-CoA as precursor for cyFA biosynthesis. - The degree of saturation in fatty acids and/or derivatives thereof can be controlled by regulating the degree of saturation of intermediates. For example, the sfa, gns, and fab families of genes can be expressed, overexpressed, or expressed at reduced levels, to control the saturation of fatty acids. Exemplary genes include, without limitation, fabB (EC 2.3.1.41), fabK (EC 1.3.1.9), fabL (EC 1.3.1.9), fabM (EC 4.2.1.17), which can be used in the methods and host cells described herein.
- For example, host cells can be engineered to produce unsaturated fatty acids and/or fatty acid derivatives by engineering the production host to overexpress fabB or by growing the production host at low temperatures (e.g., less than 37° C.). FabB has preference to cis-δ3decenoyl-ACP and results in unsaturated fatty acid production in E. coli. Overexpression of fabB results in the production of a significant percentage of unsaturated fatty acids and/or fatty acid derivatives (de Mendoza et al., J. Biol. Chem. 258:2098-2101 (1983)). The gene fabB may be inserted into and expressed in host cells not naturally having the gene. These unsaturated fatty acids and/or fatty acid derivatives can then be used as intermediates in host cells.
- In other instances, a repressor of fatty acid biosynthesis, for example, fabR (GenBank Accession No. NP_418398), can be deleted, which will also result in increased unsaturated fatty acid and/or fatty acid derivative production in E. coli (Zhang et al., J. Biol. Chem. 277:15558 (2002)). Similar deletions may be made in other host cells. A further increase in unsaturated fatty acids may be achieved, for example, by overexpressing fabM (trans-2, cis-3-decenoyl-ACP isomerase, GenBank Accession No. DAA05501) and controlled expression of fabK (trans-2-enoyl-ACP reductase II, GenBank Accession No. NP_357969) from Streptococcus pneumoniae (Manakchi et al., J. Biol. Chem. 277: 44809 (2002)), while deleting E. coli fabI (trans-2-enoyl-ACP reductase, GenBank Accession No. NP_415804). In some examples, the endogenous fabF gene can be attenuated, thus increasing the percentage of palmitoleate (C16:1) produced.
- In yet other examples, host cells can be engineered to produce saturated fatty acids by reducing the expression of an sfa, gns, or fab gene.
- For example, a host cell can be engineered to express a decreased level of fabA and/or fabB. In some instances, the host cell can be grown in the presence of unsaturated fatty acids. In other instances, the host cell can be further engineered to express or overexpress a gene encoding a desaturase enzyme. One nonlimiting example of a desaturase is B. subtilis DesA (AF037430). Other genes encoding desaturase enzymes are known in the art and can be used in the host cells and methods described herein, such as desaturases that use acyl-ACP, such as hexadecanoyl-ACP or octadecanoyl-ACP.
- Genetic Engineering of Host Cells to Produce Fatty Acids and/or Fatty Acid Derivatives
- Various host cells can be used to produce fatty acids and/or fatty acid derivatives, as described herein. A host cell can be any prokaryotic or eukaryotic cell. For example, a polypeptide described herein can be expressed in bacterial cells (such as E. coli), insect cells, yeast, or mammalian cells (such as Chinese hamster ovary cells (CHO) cells, COS cells, VERO cells, BHK cells, HeLa cells, Cv1 cells, MDCK cells, 293 cells, 3T3 cells, or PC12 cells). Other exemplary host cells include cells from the members of the genus Escherichia, Bacillus, Lactobacillus, Rhodococcus, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces, Schizosaccharomyces, Yarrowia, or Streptomyces. Yet other exemplary host cells can be a cell of Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus licheniformis, Bacillus alkalophilus, Bacillus coagulans, Bacillus circulans, Bacillus pumilis, Bacillus thuringiensis, Bacillus clausii, Bacillus megaterium, Bacillus subtilis, Bacillus amyloliquefaciens, Trichoderma koningii, Trichoderma viride, Trichoderma reesei, Trichoderma longibrachiatum, Aspergillus awamori, Aspergillus fumigates, Aspergillus foetidus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Humicola insolens, Humicola lanuginose, Rhizomucor miehei, Mucor michei, Streptomyces lividans, Streptomyces murinus, or Actinomycetes. Other host cells are cyanobacterial host cells.
- In a preferred embodiment, the host cell is an E. coli cell, a Saccharomyces cerevisiae cell, or a Bacillus subtilis cell. In a more preferred embodiment, the host cell is from E. coli strains B, C, K, or W. Other suitable host cells are known to those skilled in the art.
- Various methods well known in the art can be used to genetically engineer host cells to produce fatty acids and/or fatty acid derivatives. The methods can include the use of vectors, preferably expression vectors, containing a nucleic acid encoding an ester synthase polypeptide described herein, polypeptide variant, or a fragment thereof. Those skilled in the art will appreciate a variety of viral vectors (for example, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors) and non-viral vectors can be used in the methods described herein.
- The recombinant expression vectors described herein include a nucleic acid described herein in a form suitable for expression of the nucleic acid in a host cell. The recombinant expression vectors can include one or more control sequences, selected on the basis of the host cell to be used for expression. The control sequence is operably linked to the nucleic acid sequence to be expressed. Such control sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Control sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors described herein can be introduced into host cells to produce polypeptides, including fusion polypeptides, encoded by the nucleic acids as described herein.
- Recombinant expression vectors can be designed for expression of a fatty acid biosynthetic polypeptide or variant in prokaryotic or eukaryotic cells (e.g., bacterial cells, such as E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells, or mammalian cells). Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example, by using T7 promoter regulatory sequences and T7 polymerase.
- Expression of polypeptides in prokaryotes, for example, E. coli, is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors typically serve three purposes: (1) to increase expression of the recombinant polypeptide; (2) to increase the solubility of the recombinant polypeptide; and (3) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide. This enables separation of the recombinant polypeptide from the fusion moiety after purification of the fusion polypeptide. Examples of such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, and enterokinase. Exemplary fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs, Ipswich, Mass.), and pRITS (Pharmacia, Piscataway, N.J.), which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant polypeptide.
- Examples of inducible, non-fusion E. coli expression vectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. 60-89 (1990)). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident λ, prophage harboring a T7 gn1 gene under the transcriptional control of the
lacUV 5 promoter. - One strategy to maximize recombinant polypeptide expression is to express the polypeptide in a host cell with an impaired capacity to proteolytically cleave the recombinant polypeptide (see Gottesman, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. 119-128 (1990)). Another strategy is to alter the nucleic acid sequence to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the host cell (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)). Such alteration of nucleic acid sequences can be carried out by standard DNA synthesis techniques.
- In another embodiment, the host cell is a yeast cell. In this embodiment, the expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943 (1982)), pJRY88 (Schultz et al., Gene 54:113-123(1987)), pYES2 (Invitrogen Corporation, San Diego, Calif.), picZ (Invitrogen Corp, San Diego, Calif.), and pRS425 (Christianson et al., Gene 110:119-122 (1992)).
- Alternatively, a polypeptide described herein can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include, for example, the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).
- In yet another embodiment, the nucleic acids described herein can be expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, Nature 329:840 (1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187-195 (1987)). When used in mammalian cells, the expression vector's control functions can be provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma,
Adenovirus 2, cytomegalovirus, andSimian Virus 40. Other suitable expression systems for both prokaryotic and eukaryotic cells are described in chapters 16 and 17 of Sambrook et al., eds., Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. - Vectors can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in, for example, Sambrook et al. (supra).
- For stable transformation of bacterial cells, it is known that, depending upon the expression vector and transformation technique used, only a small fraction of cells will take-up and replicate the expression vector. In order to identify and select these transformants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) can be introduced into the host cells along with the gene of interest. Selectable markers include those that confer resistance to drugs, such as ampicillin, kanamycin, chloramphenicol, or tetracycline. Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a polypeptide described herein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
- For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) can be introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin, and methotrexate. Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a polypeptide described herein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
- Transport proteins can export polypeptides (e.g., ester synthase) and organic compounds (e.g., fatty acids, fatty acid derivatives) out of a host cell. Many transport and efflux proteins serve to excrete a wide variety of compounds and can be naturally modified to be selective for particular types of hydrocarbons.
- Non-limiting examples of suitable transport proteins are ATP-Binding Cassette (ABC) transport proteins, efflux proteins, and fatty acid transporter proteins (FATP). Additional non-limiting examples of suitable transport proteins include the ABC transport proteins from organisms such as Caenorhabditis elegans, Arabidopsis thalania, Alkaligenes eutrophus, and Rhodococcus erythropolis. Exemplary ABC transport proteins that can be used include, without limitation, CER5, AtMRP5, AmiS2, AtPGP1, AcrA, AcrB, TolC, AcrE, AcrF, tll1618, tll1619, and tll0139. Host cells can also be chosen for their endogenous ability to secrete organic compounds. The efficiency of organic compound production and secretion into the host cell environment (e.g., culture medium, fermentation broth) can be expressed as a ratio of intracellular product to extracellular product. In some examples, the ratio can be about 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5.
- The production and isolation of fatty acids and/or derivatives thereof produced according to the present disclosure can be enhanced by employing beneficial fermentation techniques. One method for maximizing production while reducing costs is increasing the percentage of the carbon source that is converted to hydrocarbon products.
- During normal cellular lifecycles, carbon is used in cellular functions, such as producing lipids, saccharides, proteins, organic acids, and nucleic acids. Reducing the amount of carbon necessary for growth-related activities can increase the efficiency of carbon source conversion to product. This can be achieved by, for example, first growing host cells to a desired density (for example, a density achieved at the peak of the log phase of growth). At such a point, replication checkpoint genes can be harnessed to stop the growth of cells. Specifically, quorum sensing mechanisms (reviewed in Camilli et al., Science 311:1113 (2006); Venturi, FEMS Microbio. Rev. 30:274-291 (2006); and Reading et al., FEMS Microbiol. Lett. 254:1-11 (2006)) can be used to activate checkpoint genes, such as p53, p21, or other checkpoint genes.
- Genes that can be activated to stop cell replication and growth in E. coli include umuDC genes. The overexpression of umuDC genes stops the progression from stationary phase to exponential growth (Murli et al., J. of Bact. 182:1127 (2000)). UmuC is a DNA polymerase that can carry out translesion synthesis over non-coding lesions the mechanistic basis of most UV and chemical mutagenesis. The umuDC gene products are involved in the process of translesion synthesis and also serve as a DNA sequence damage checkpoint. The umuDC gene products include UmuC, UmuD, umuD′, UmuD′2C, UmuD′2, and UmuD2. Simultaneously, product-producing genes can be activated, thus minimizing the need for replication and maintenance pathways to be used while a fatty aldehyde is being made. Host cells can also be engineered to express umuC and umuD from E. coli in pBAD24 under the prpBCDE promoter system through de novo synthesis of this gene with the appropriate end-product production genes.
- The percentage of input carbons converted to fatty acids and/or fatty acid derivatives can be a cost driver. The more efficient the process is (i.e., the higher the percentage of input carbons converted to fatty acids and/or fatty acid derivatives), the less expensive the process will be. For oxygen-containing carbon sources (e.g., glucose and other carbohydrate based sources), the oxygen must be released in the form of carbon dioxide. For every 2 oxygen atoms released, a carbon atom is also released leading to a maximal theoretical metabolic efficiency of about 34% (w/w) (for fatty acid derived products). This figure, however, changes for other organic compounds and carbon sources. Typical efficiencies in the literature are about less than 5%. Host cells engineered to produce fatty acids and/or fatty acid derivatives can have greater than about 1, 3, 5, 10, 15, 20, 25, and 30% efficiency. In one example, host cells can exhibit an efficiency of about 10% to about 25%. In other examples, such host cells can exhibit an efficiency of about 25% to about 30%. In other examples, host cells can exhibit greater than 30% efficiency.
- The host cell can be additionally engineered to express recombinant cellulosomes, such as those described in PCT application number PCT/US2007/003736. These cellulosomes can allow the host cell to use cellulosic material as a carbon source. For example, the host cell can be additionally engineered to express invertases (EC 3.2.1.26) so that sucrose can be used as a carbon source. Similarly, the host cell can be engineered using the teachings described in U.S. Pat. Nos. 5,000,000; 5,028,539; 5,424,202; 5,482,846; and 5,602,030; so that the host cell can assimilate carbon efficiently and use cellulosic materials as carbon sources.
- In one example, the fermentation chamber can enclose a fermentation that is undergoing a continuous reduction. In this instance, a stable reductive environment can be created. The electron balance can be maintained by the release of carbon dioxide (in gaseous form). Efforts to augment the NAD/H and NADP/H balance can also facilitate in stabilizing the electron balance. The availability of intracellular NADPH can also be enhanced by engineering the host cell to express an NADH:NADPH transhydrogenase. The expression of one or more NADH:NADPH transhydrogenases converts the NADH produced in glycolysis to NADPH, which can enhance the production of fatty acids and/or fatty acid derivatives.
- For small scale production, the engineered host cells can be grown in batches of, for example, about 100 mL, 500 mL, 1 L, 2 L, 5 L, or 10 L; fermented; and induced to express desired biosynthetic genes based on the specific genes encoded in the appropriate plasmids. For large scale production, the engineered host cells can be grown in batches of about 10 L, 100 L, 1000 L, 10,000 L, 100,000 L, 1,000,000 L or larger; fermented; and induced to express desired biosynthetic genes based on the specific genes encoded in the appropriate plasmids or incorporated into the host cell's genome.
- For example, a suitable production host, such as E. coli cells, harboring plasmids containing the desired biosynthetic genes or having the biosynthetic genes integrated in its chromosome can be incubated in a suitable reactor, for example a 1 L reactor, for 20 hours at 37° C. in an M9 medium supplemented with 2% glucose, carbenicillin, and chloramphenicol. When the OD600 of the culture reaches about 0.9, the production host can be induced with IPTG to activate the engineered gene systems for fatty acid/fatty acid derivative production. After incubation, the spent medium can be extracted and the organic phase can be examined for the presence of fatty acids/fatty acid derivatives using known detection methods such as, for example, GC-MS.
- In some instances, after the first hour of induction, aliquots of no more than about 10% of the total cell volume can be removed each hour and allowed to sit without agitation to allow the fatty acid esters to rise to the surface and undergo a spontaneous phase separation or precipitation. The fatty acid/fatty acid derivative component can then be collected, and the aqueous phase returned to the reaction chamber. The reaction chamber can be operated continuously. When the OD600 drops below about 0.6, the cells can be replaced with a new batch grown from a seed culture.
- In some instances, the methods disclosed herein are performed using glucose as a carbon source. In certain instances, microorganisms are grown in a culture medium containing an initial glucose concentration of about 2 g/L to about 50 g/L, such as about 5 g/L to about 20 g/L. In some instances, the glucose concentration of the culture medium decreases from the initial glucose concentration as the microorganisms consume the glucose, and a concentration of about 0 g/L to about 5 g/L glucose is maintained in the culture medium during the fatty acid/fatty acid derivative production process. In certain instances, glucose is fed to the microorganisms in a solution of about 50% to about 65% glucose.
- In some instances, the feed rate of glucose is set to match the cells' growth rate to avoid excess accumulation of glucose (i.e., >0% glucose) in the fermentor. In other instances, and a low concentration of excess glucose (e.g., about 2 g/L to about 5 g/L) is maintained.
- In certain instances, fatty acids and/or fatty acid derivatives can be produced from carbohydrates other than glucose, including but not limited to fructose, hydrolyzed sucrose, hydrolyzed molasses and glycerol.
- The fatty acids and/or fatty acid derivatives produced during fermentation can be separated from the fermentation media. Any known technique for separating fatty acids and/or fatty acid derivatives from aqueous media can be used. One exemplary separation process is a two phase (bi-phasic) separation process. This process involves fermenting the genetically engineered host cells under conditions sufficient to produce a fatty acid and/or fatty acid derivative, allowing the fatty acid and/or fatty acid derivative to collect in an organic phase, and separating the organic phase from the aqueous fermentation broth. This method can be practiced in both a batch and continuous fermentation processes.
- Bi-phasic separation uses the relative immiscibility of fatty acids and/or fatty acid derivatives to facilitate separation. Immiscible refers to the relative inability of a compound to dissolve in water and is defined by the compound's partition coefficient. One of ordinary skill in the art will appreciate that by choosing a fermentation broth and organic phase, such that the fatty acid and/or fatty acid derivative being produced has a high logP value, the fatty acid and/or fatty acid derivative can separate into the organic phase, even at very low concentrations, in the fermentation vessel.
- The fatty acids and/or fatty acid derivatives produced by the methods described herein can be relatively immiscible in the fermentation broth, as well as in the cytoplasm. Therefore, the fatty acids and/or fatty acid derivative can collect in an organic phase either intracellularly or extracellularly. The collection of the products in the organic phase can lessen the impact of the fatty acid and/or fatty acid derivative on cellular function and can allow the host cell to produce more product.
- The methods described herein can result in the production of homogeneous compounds wherein at least about 60%, 70%, 80%, 90%, or 95% of the fatty acids and/or fatty acid derivatives produced will have carbon chain lengths that vary by less than about 8 carbons, less than about 6 carbons, less than about 4 carbons, or less than about 2 carbons. These compounds can also be produced with a relatively uniform degree of saturation. These compounds can be used directly as fuels, fuel additives, starting materials for production of other chemical compounds (e.g., polymers, surfactants, plastics, textiles, solvents, adhesives, etc.), or personal care additives. These compounds can also be used as feedstock for subsequent reactions, for example, hydrogenation, catalytic cracking (e.g., via hydrogenation, pyrolisis, or both), to make other products.
- In some embodiments, the fatty acids and/or fatty acid derivatives produced using methods described herein can contain between about 50% and about 90% carbon; or between about 5% and about 25% hydrogen. In other embodiments, the fatty acids and/or fatty acid derivatives produced using methods described herein can contain between about 65% and about 85% carbon; or between about 10% and about 15% hydrogen.
- Producing Fatty Acids and/or Fatty Acid Derivatives Using Cell-Free Methods
- Certain methods described herein also encompass a fatty acid or a derivative thereof produced using a purified ester synthase polypeptide, a variant or a fragment thereof described herein and a substrate, provided or produced by, for example, a method described herein. For example, a host cell can be engineered to express a suitable ester synthase, a variant or a fragment thereof, as described herein. The host cell can be cultured under conditions that allow the expression of the ester synthase polypeptide. Cell free extracts can then be generated using known methods. For example, the host cells can be lysed using detergents or by sonication. The expressed polypeptides can then be purified or substantially purified using known methods. After obtaining the cell free extracts, substrates described herein can be provided to the cell free extracts and incubated under conditions that allow conversion of the substrates to desired fatty acids and/or fatty acid derivatives. The fatty acids and/or fatty acid derivatives can then be separated and purified using known techniques. Accordingly, fatty acids and/or fatty acid derivatives of the present invention can be produced extacellularly and isolated in vitro.
- Bioproducts comprising biologically produced organic compounds, particularly fatty acids and fatty acid derivatives biologically produced using the fatty acid biosynthetic pathway, have not been produced from renewable sources and, as such, are new compositions of matter. These new bioproducts can be distinguished from organic compounds derived from petrochemical carbon on the basis of dual carbon-isotopic fingerprinting or 14C dating. Additionally, the specific source of biosourced carbon (e.g., glucose vs. glycerol) can be determined by dual carbon-isotopic fingerprinting (see, e.g., U.S. Pat. No. 7,169,588, which is herein incorporated by reference).
- The ability to distinguish bioproducts from petroleum-based organic compounds is beneficial in tracking these materials in commerce. For example, organic compounds or chemicals comprising both biologically-based and petroleum-based carbon isotope profiles may be distinguished from organic compounds and chemicals made only of petroleum-based materials. Hence, the instant materials may be followed in commerce on the basis of their unique carbon isotope profile.
- Bioproducts can be distinguished from petroleum-based organic compounds by comparing the stable carbon isotope ratio (13C/12C) in each fuel. The 13C/12C ratio in a given bioproduct is a consequence of the 13C/12C ratio in atmospheric carbon dioxide at the time the carbon dioxide is fixed. It also reflects the precise metabolic pathway. Regional variations also occur. Petroleum, C3 plants (the broadleaf), C4 plants (the grasses), and marine carbonates all show significant differences in 13C/12C and the corresponding δ13C values. Furthermore, lipid matter of C3 and C4 plants analyze differently than materials derived from the carbohydrate components of the same plants as a consequence of the metabolic pathway.
- Within the precision of measurement, 13C shows large variations due to isotopic fractionation effects, the most significant of which for bioproducts is the photosynthetic mechanism. The major cause of differences in the carbon isotope ratio in plants is closely associated with differences in the pathway of photosynthetic carbon metabolism in the plants, particularly the reaction occurring during the primary carboxylation (i.e., the initial fixation of atmospheric CO2). Two large classes of vegetation are those that incorporate the “C3” (or Calvin-Benson) photosynthetic cycle and those that incorporate the “C4” (or Hatch-Slack) photosynthetic cycle.
- In C3 plants, the primary CO2 fixation or carboxylation reaction involves the enzyme ribulose-1,5-diphosphate carboxylase, and the first stable product is a 3-carbon compound. C3 plants, such as hardwoods and conifers, are dominant in the temperate climate zones.
- In C4 plants, an additional carboxylation reaction involving another enzyme, phosphoenol-pyruvate carboxylase, is the primary carboxylation reaction. The first stable carbon compound is a 4-carbon acid that is subsequently decarboxylated. The CO2 thus released is refixed by the C3 cycle. Examples of C4 plants are tropical grasses, corn, and sugar cane.
- Both C4 and C3 plants exhibit a range of 13C/12C isotopic ratios, but typical values are about −7 to about −13 per mil for C4 plants and about −19 to about −27 per mil for C3 plants (see, e.g., Stuiver et al., Radiocarbon 19:355, 1977). Coal and petroleum fall generally in this latter range. The 13C measurement scale was originally defined by a zero set by Pee Dee Belemnite (PDB) limestone, where values are given in parts per thousand deviations from this material. The “δ13C” values are expressed in parts per thousand (per mil), abbreviated, ‰, and are calculated as follows:
-
δ13C(‰)=[(13C/12C)sample−(13C/12C)standard]/(13C/12C)standard×1000 - Since the PDB reference material (RM) has been exhausted, a series of alternative RMs have been developed in cooperation with the IAEA, USGS, NIST, and other selected international isotope laboratories. Notations for the per mil deviations from PDB is δ13C. Measurements are made on CO2 by high precision stable ratio mass spectrometry (IRMS) on molecular ions of
masses - The compositions described herein include bioproducts produced by any of the methods described herein. Specifically, the bioproduct can have a δ13C of about −28 or greater, about −27 or greater, −20 or greater, −18 or greater, −15 or greater, −13 or greater, −10 or greater, or −8 or greater. For example, the bioproduct can have a δ13C of about −30 to about −15, about −27 to about −19, about −25 to about −21, about −15 to about −5, about −13 to about −7, or about −13 to about −10. In other instances, the bioproduct can have a δ13C of about −10, −11, −12, or −12.3.
- Bioproducts can also be distinguished from petroleum-based organic compounds by comparing the amount of 14C in each compound. Because 14C has a nuclear half life of 5730 years, petroleum-based fuels containing “older” carbon can be distinguished from bioproducts which contain “newer” carbon (see, e.g., Currie, “Source Apportionment of Atmospheric Particles”, Characterization of Environmental Particles, J. Buffle and H. P. van Leeuwen, Eds., 1 of Vol. I of the IUPAC Environmental Analytical Chemistry Series (Lewis Publishers, Inc) (1992) 3-74).
- The basic assumption in radiocarbon dating is that the constancy of 14C concentration in the atmosphere leads to the constancy of 14C in living organisms. However, because of atmospheric nuclear testing since 1950 and the burning of fossil fuel since 1850, 14C has acquired a second, geochemical time characteristic. Its concentration in atmospheric CO2, and hence in the living biosphere, about doubled at the peak of nuclear testing, in the mid-1960s. It has since been gradually returning to the steady-state cosmogenic (atmospheric) baseline isotope rate (14C/12C) of about 1.2×10−12, with an approximate relaxation “half-life” of 7-10 years. (This latter half-life must not be taken literally; rather, one must use the detailed atmospheric nuclear input/decay function to trace the variation of atmospheric and biospheric 14C since the onset of the nuclear age.)
- It is this latter biospheric 14C time characteristic that holds out the promise of annual dating of recent biospheric carbon. 14C can be measured by accelerator mass spectrometry (AMS), with results given in units of “fraction of modern carbon” (fM). fM is defined by National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs) 4990B and 4990C. As used herein, “fraction of modern carbon” or “fM” has the same meaning as defined by National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs) 4990B and 4990C, known as oxalic acids standards HOxI and HOxII, respectively. The fundamental definition relates to 0.95 times the 14C/12C isotope ratio HOxI (referenced to AD 1950). This is roughly equivalent to decay-corrected pre-Industrial Revolution wood. For the current living biosphere (plant material), fM is about 1.1.
- The invention provides a bioproduct comprising a fatty acid and/or a fatty acid derivative produced according to the methods herein, which can have an fM 14C of at least about 1. For example, the bioproduct can have an fM 14C of at least about 1.01, an fM 14C of about 1 to about 1.5, an fM 14C of about 1.04 to about 1.18, or an fM 14C of about 1.111 to about 1.124.
- Another measurement of 14C is known as the percent of modern carbon, pMC. For an archaeologist or geologist using 14C dates, AD 1950 equals “zero years old”. This also represents 100 pMC. “Bomb carbon” in the atmosphere reached almost twice the normal level in 1963 at the peak of thermo-nuclear weapons. Its distribution within the atmosphere has been approximated since its appearance, showing values that are greater than 100 pMC for plants and animals living since AD 1950. It has gradually decreased over time with today's value being near 107.5 pMC. This means that a fresh biomass material, such as corn, would give a 14C signature near 107.5 pMC. Petroleum-based compounds will have a pMC value of zero. Combining fossil carbon with present day carbon will result in a dilution of the present day pMC content. By presuming 107.5 pMC represents the 14C content of present day biomass materials and 0 pMC represents the 14C content of petroleum-based products, the measured pMC value for that material will reflect the proportions of the two component types. For example, a material derived 100% from present day soybeans would give a radiocarbon signature near 107.5 pMC. If that material was diluted 50% with petroleum-based products, it would give a radiocarbon signature of about 54 pMC.
- A biologically-based carbon content is derived by assigning “100%” equal to 107.5 pMC and “0%” equal to 0 pMC. For example, a sample measuring 99 pMC will give an equivalent biologically based carbon content of 93%. This value is referred to as the mean biologically based carbon result and assumes all the components within the analyzed material originated either from present day biological material or petroleum-based material.
- A bioproduct comprising a fatty acid and/or fatty acid derivative produced in accordance with the methods described herein can have a pMC of at least about 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100. In other instances, a fatty acid and/or fatty acid derivative described herein can have a pMC of between about 50 and about 100; about 60 and about 100; about 70 and about 100; about 80 and about 100; about 85 and about 100; about 87 and about 98; or about 90 and about 95. In yet other instances, a bioproduct comprising a fatty acid and/or fatty acid derivative can have a pMC of about 90, 91, 92, 93, 94, or 94.2.
- For example, the fatty acids and/or derivatives thereof produced by the methods described herein can be used as biofuels. For example, the fatty acids and/or derivatives thereof as described herein can be used solely or as a component of biodiesel. The fatty acids and/or derivatives thereof produced according to the methods herein can also be used as, or as components or feedstocks of, various industrial chemicals, including without limitation, fuel additives. The resulting biofuel or industrial chemicals are bioproducts and will therefore have a δ13C of about −28 or greater, about −27 or greater, −20 or greater, −18 or greater, −15 or greater, −13 or greater, −10 or greater, or −8 or greater. For example, the bioproduct can have a δ13C of about −30 to about −15, about −27 to about −19, about −25 to about −21, about −15 to about −5, about −13 to about −7, or about −13 to about −10. Moreover, the resulting biofuel or industrial chemicals will have an fM 14C of at least about 1. For example, the bioproduct can have an fM 14C of at least about 1.01, an fM 14C of about 1 to about 1.5, an fM 14C of about 1.04 to about 1.18, or an fM 14C of about 1.111 to about 1.124.
- The fatty acids and/or derivatives thereof produced according to the methods and compositions herein possess various advantageous characteristics for use as a fuel. One of ordinary skill in the art will appreciate that, depending upon the intended purpose of the fuel, different fatty acids and/or derivatives thereof may have advantages as compared to others fatty acids and/or derivatives. For example, branched fatty acids and/or derivatives thereof may be more desirable as automobile fuels or components of automobile fuels that are intended for uses in cold climates. Similarly, for certain applications, it may be advantageous to produce a fuel that is either more or less oxygenated or more or less saturated.
- Using the methods described herein, fuels comprising relatively homogeneous fatty acids and/or derivatives thereof that, at the same time, have the desired characteristics and qualities can be produced. Such fatty acid- and/or fatty acid derivative-based fuels can be characterized by carbon fingerprinting, and their lack of impurities, when compared to petroleum-derived fuels or biodiesel derived from triglyceride, is also advantageous. The fatty acid- and/or fatty acid derivative-based fuels can be combined with other fuels or fuel additives to produce alternative fuel compositions that have the desired properties.
- The production hosts and methods disclosed herein can be used to produce free fatty acids and/or various fatty acid derivatives. In some embodiments, the production hosts and methods disclosed herein can be used to produce a higher and/or improved titer or yield of free fatty acids and/or fatty acid derivatives, including, for example, fatty esters. In some embodiments, the percentage of free fatty acids in the product produced by the production host is at least about 1%, for example, at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25%. In some embodiments, the percentage of fatty acid derivatives in the product produced by the production host is at least about 50%, for example, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In some embodiments, the ratio of fatty acid derivative to free fatty acids in the product produced by the production host is about 10:1, 9:1, 8:1, 7:1, 5:1, 2:1, or 1:1.
- In certain embodiments, the fatty acid derivative produced by the production host is a fatty acid ethyl derivative. An exemplary fatty acid ethyl derivative is a fatty ethyl ester. In particular embodiments, the fatty ethyl ester is an ethyl dodecanoate, ethyl tridecanoate, ethyl tetradecanoate, ethyl pentadecanoate, ethyl cis-9-hexadecenoate, ethyl hexadecanoate, ethyl heptadecanoate, ethyl cis-11-octadecenoate, or ethyl octadecanoate ester. In particular embodiments, the fatty acid derivative product that is produced by the production host is a combination of a variety of fatty acid derivatives. For example, the fatty acid derivative product comprises a mixture of any combination of an ethyl dodecanoate, ethyl tridecanoate, ethyl tetradecanoate, ethyl pentadecanoate, ethyl cis-9-hexadecenoate, ethyl hexadecanoate, ethyl heptadecanoate, ethyl cis-11-octadecenoate, and ethyl octadecanoate ester. In certain other embodiment, the fatty acid derivative produced is a fatty methyl derivative. An exemplary fatty methyl derivative if s fatty methyl ester. In particular embodiments, the fatty methyl ester produced by the production host is a methyl dedecanoate, methyl tridecanoate, methyl tetradecanoate, methyl pentadecanoate, methyl cis-9-hexadecenoate, methyl hexadecanoate, methyl heptadecanoate, methyl cis-11-octadecenoate, or methyl octadecanoate. In certain embodiments, the fatty acid derivative product produced by the production host is a combination of a variety of fatty acid derivative products. For example, the fatty acid derivative is a mixture of one or more of methyl dedecanoate, methyl tridecanoate, methyl tetradecanoate, methyl pentadecanoate, methyl cis-9-hexadecenoate, methyl hexadecanoate, methyl heptadecanoate, methyl cis-11-octadecenoate, and methyl octadecanoate. In certain other embodiments, the fatty acid derivative is a mixture of one or more fatty methyl esters and one or more fatty ethyl esters. In certain embodiments, the product is a mixture of one or more free fatty acids, and/or one or more fatty acid derivatives. In yet further embodiments, the product is a mixture of one or more free fatty acids, and/or one or more fatty methyl esters, and/or one or more fatty ethyl esters.
- The production hosts and methods disclosed herein can be used to produce different proportions of free fatty acids and fatty acid derivatives. In some embodiments, the proportion of free fatty acids in the product can be modified according to the methods, compositions, vectors and cells described herein such that the proportion is higher or lower vs. the fatty acid derivatives that are produced. In certain related embodiments, the proportion of fatty acid derivatives in the product can also be modified according to the disclosures herein, such that the proportion is higher or lower vs. the other products, for example, the free fatty acids, that are produced. In certain other embodiments, the proportional yield of fatty acid derivative with certain carbon chain lengths can be increased or decreased.
- The terms “proportional yield,” as used herein, refers to the amount of a desired product in relation to other products that are within the same mixture produced by a recombinant host of the present invention. For example, the proportional yield of a desired product can be improved such that it is more predominant over the other components in the product mixture to reduce the burden of purification. In another example, the proportional yield of an undesired product (i.e., a component that will need to be removed from the desired product) can be reduced such that it is less predominant over the desired component in the product mixture to achieve the same end.
- Carbon Fingerprinting
- Biologically produced fatty acid derivatives can provide a new source of fuels, such as alcohols, diesel, and gasoline. Biofuels made according to the methods and compositions described herein have not heretofore been produced from renewable sources and are new compositions of matter. These new fuels can be distinguished from fuels derived from petrochemical carbon on the basis of dual carbon-isotopic fingerprinting. Additionally, the specific source of biosourced carbon (e.g., glucose vs. glycerol) can be determined by dual carbon-isotopic fingerprinting (see U.S. Pat. No. 7,169,588, which is herein incorporated by reference in its entirety, in particular, at col. 4, line 31, to col. 6, line 8).
- The fatty acids and/or fatty acid derivatives and the associated biofuels, chemicals, and mixtures can be distinguished from their petrochemical derived counterparts on the basis of 14C (fM) and dual carbon-isotopic fingerprinting.
- The fatty acids and/or derivatives thereof as described herein have utility in the production of biofuels and chemicals. The products provided by the instant invention can be distinguished on the basis of dual carbon-isotopic fingerprinting from those materials derived solely from petrochemical sources. The ability to distinguish these products is beneficial in tracking these materials in commerce. For example, fuels or chemicals comprising both “new” and “old” carbon isotope profiles can be distinguished from fuels and chemicals made only of “old” materials. Thus, the instant materials can be followed or “tracked” in commerce or identified in commerce as a biofuel on the basis of their unique profile. In addition, other competing materials can be identified as being biologically derived or derived from a petrochemical source.
- In some examples, a biofuel composition is made, which includes a fatty acid derivative having δ13C of from about −10.9 to about −15.4, wherein the fatty acid derivative accounts for at least about 85% of biosourced material (i.e., derived from a renewable resource such as, for example, cellulosic materials and sugars) in the composition. In other examples, the biofuel composition includes a fatty acid derivative having the formula:
-
X—(CH(R))nCH3 - wherein
- X=CH3, —CH2OR1; —C(O)OR2; or —C(O)NR3R4;
- R=for each n, independently absent, an H, or a lower aliphatic;
- n=an integer from about 8 to about 34, preferably an integer from about 10 to about 24;
- R1, R2, R3, R4=independently selected from an H or a lower alkyl.
- Typically, when R is a lower aliphatic group, R represents a branched, unbranched or cyclic lower alkyl or lower alkenyl moiety. Exemplary R groups include, without limitation, methyl, isopropyl, isobutyl, sec-butyl, cyclopentenyl, and the like. The fatty acid derivative is additionally characterized as having a δ13C of from about −10.9 to about −15.4, and the fatty acid derivative accounts for at least about 85% of biosourced material in the composition. In some examples the fatty acid derivative in the biofuel composition is characterized by having a fraction of modern carbon (fM 14C) of at least about 1.003, 1.010, or 1.5.
- Impurities
- The fatty acids and/or derivatives thereof produced in accordance with the disclosures herein are useful as components or feedstocks for making biofuels as well as other industrial chemicals. These products are made directly from suitable substrates and not from the chemical processing of triglycerides. Accordingly, fuels and other industrial chemicals comprising the disclosed fatty acids and/or derivatives often contain fewer impurities than are normally associated with, for example, products derived from triglycerides such as fuels derived from vegetable oils and fats.
- The crude biofuels prepared in accordance with the disclosures herein (prior to mixing the fatty acids and/or derivatives with other fuels such as petroleum-based fuels) contain less transesterification catalysts than petroleum-based diesel or other biodiesel produced via one or more transesterification steps. The biofuel can contain less than about 2.0 wt. %, for example, less than about 1.5, 1.0, 0.5, 0.3, 0.1, 0.05, or 0 wt. % of a transesterification catalyst or an impurity resulting from a transesterification catalyst. Examples of transesterification catalysts include, without limitation, hydroxide catalysts, such as NaOH, KOH, and LiOH; and acidic catalysts, such as mineral acid catalysts and Lewis acid catalysts. Non-limiting examples of catalysts and impurities resulting from transesterification catalysts include tin, lead, mercury, cadmium, zinc, titanium, zirconium, hafnium, boron, aluminum, phosphorus, arsenic, antimony, bismuth, calcium, magnesium, strontium, uranium, potassium, sodium, and lithium.
- The biofuels prepared in accordance with the disclosures herein (prior to mixing the fatty acids and/or fatty acid derivatives with one or more other fuels) tend to have a low gelling point, especially when the fatty acid and/or fatty acid derivative product comprises a C16:1 ethyl ester or a C18:1 ethyl ester, as compared to the gelling points of other types of biofuels.
- Similarly, the crude biofuels prepared in accordance with the disclosures herein contain less glycerol (or glycerin) than biofuels made from triglycerides. The biofuels can contain less than about 2.0 wt. %, for example, less than about 1.5, 1.0, 0.5, 0.3, 0.1, 0.05, or 0 wt. % of glycerol.
- Crude biofuels herein also contain less free alcohol(s) (e.g., alcohols that are used to create the ester) than biodiesels made from triglycerides. This is due in part to the efficiency of utilization of the alcohols by the production hosts of the present disclosure. For example, the biofuels can contain less than about 2.0, 1.5, 1.0, 0.5, 0.3, 0.1, 0.05, or 0 wt. % of free alcohol.
- The biofuels herein can be additionally characterized by its low concentration of sulfur as compared to petroleum-derived diesel. For example, the biofuel can have less than about 2.0 wt. %, for example, less than about 1.5, 1.0, 0.5, 0.3, 0.1, 0.05, or 0 wt. % of sulfur.
- Additives and Fuel Compositions
- Fuel additives are used to enhance the performance of a fuel or engine. For example, fuel additives can be used to alter the freezing/gelling points, cloud points, lubricity, viscosity, oxidative stability, ignition quality, octane levels, and flash points. In the United States, all fuel additives must be registered with Environmental Protection Agency. The names of fuel additives and the companies that sell the fuel additives are publicly available by contacting the EPA or by viewing the agency's website. One of ordinary skill in the art will appreciate that the fatty acids and/or fatty acid derivatives described herein can be mixed with one or more fuel additives to impart a desired quality.
- The fatty acids and/or derivatives thereof described herein can be formulated or processed into suitable fuel additives, which enhance the performance of fuels or engines. For example, the fatty acids and/or derivatives described herein can be formulated into lubricity improvers, which impart desirable properties such as wear protection to the engine parts. Accordingly, additive compositions comprising the fatty acids and/or derivatives thereof produced in accordance with the disclosures herein are provided. In another example, the fatty acids and/or fatty acid derivatives described herein can be formulated into corrosion inhibitors.
- The fatty acids and/or fatty acid derivatives described herein can be mixed with other fuels such as one or more biodiesels derived from triglycerides, various alcohols such as ethanol and butanol, and petroleum-derived products such as gasoline or diesel. Under certain circumstances, a fatty acid and/or fatty acid derivative with a low gelling point, such as a C16:1 ethyl ester or a C18:1 ethyl ester, is produced. This low gelling point product can be mixed with one or more biodiesels made from triglycerides to reduce gelling point of the resulting fuel when compared to a fuel containing only the one or more biodiesels made from triglycerides. Similarly, a fatty acid derivative, such as a C16:1 ethyl ester or a C18:1 ethyl ester, can be mixed with a petroleum-derived diesel to provide a mixture that contains at least about, and often greater than about, 5% by weight of biodiesel. In some examples, the fuel mixture includes at least about 10%, 15%, 20%, 30%, 40%, 50%, and 60% by weight of the fatty acid derivative.
- In some embodiments, the fuel composition can further comprise a synthetic fuel. Any synthetic fuel obtained from coal, natural gas, or biomass can be suitably used. In a further embodiments, the synthetic fuel comprises a Fischer-Tropsch based fuel, a Bergius-based fuel, a Mobil-based fuel, a Karrick-based fuel, or a combination thereof. In still further embodiments, the synthetic fuel comprises a Coal-To-Liquids based fuel (CTL-based fuel), a Gas-To-Liquids based fuel (GTL-based fuel), a Biomass-To-Liquids based fuel (BTL-based fuel), a Coal and Biomass-To-Liquids based fuel (CBTL-based fuel), or a combination thereof. In an exemplary embodiment, the synthetic fuel comprises a Fischer-Tropsch-based fuel.
- The amount of synthetic fuel in the fuel composition disclosed herein may be from about 5% to about 90%, from about 5% to about 80%, from about 5% to about 70%, from about 5% to about 60%, or from about 5% to about 50%.
- In certain embodiments, a biofuel composition can be made that includes at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% of a fatty acid derivative that includes a carbon chain that is 8:0, 10:0, 12:0, 14:0, 14:1, 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:0, 20:1, 20:2, 20:3, 22:0, 22:1 or 22:3. Such biofuel compositions can additionally include at least one additive selected from a cloud point lowering additive that can lower the cloud point to less than about 5° C., or less than about 0° C.; a surfactant; a microemulsion; at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% diesel fuel from triglycerides; a petroleum-derived gasoline; or a diesel fuel from petroleum.
- In some embodiments, the fuel composition comprising fatty acids and/or derivatives thereof such as fatty esters produced in accordance with the methods, vectors, cells and compositions herein further comprises one or more diesel fuel additives. Suitable additives are desirably those that afford improved performance but also compatibility with the components in the fuel composition and devices that are typically associated with diesel engines. Examples of other suitable fuel additives include ignition improvers or cetane number improvers, detergents, dispersants, antiwear agents, viscosity index modifiers, friction modifiers, lubricity improvers, stabilizers, antioxidants, corrosion inhibitors, biocides, metal deactivators, and minor amounts of other optional additives, including, without limitation, antifoaming agents and seal fixes.
- In particular embodiments, ignition improvers or cetane number improvers are often added to improve diesel engine performance. Exemplary cetane number improvers include 2′-ethylhexyl nitrate, and other alkyl nitrates. Cetane number improvers can be added to a fuel composition in an amount that is about 0.01 wt. % to about 1.0 wt. %, e.g., about 0.05 wt. % to about 0.5 wt. %, based on the total weight of the fuel composition.
- In certain embodiments, various detergents and/or dispersants can be included in the fuel composition comprising the fatty acids and/or fatty acid derivatives produced in accordance with the present disclosures to associate and disperse or remove harmful deposits from diesel engine parts. Suitable detergents typically comprise a polar head comprising a metal salt of an acidic organic compound and a long hydrophobic tail. Exemplary detergents include borated carbonate salts, borated sulfonate salts, which are preferably overbased. See, e.g., U.S. Pat. Nos. 4,744,920, 4,965,003, the disclosures of which are incorporated herein. Exemplary dispersants include, without limitation, carboxylic dispersants, succinimide dispersants, amine dispersants, and Mannich dispersants. See, e.g., U.S. Pat. Nos. 3,172,892, 3,438,757, 3,980,569, and 6,165,235, the disclosures of which are incorporated by reference herein. Dispersants can be present in the fuel composition in an amount of about 0.01 wt. % to about 0.1 wt. %, e.g., 0.03 to about 0.05 wt. %, based on the total weight of the fuel composition.
- In certain embodiments, antiwear agents, including for example, dihydrocarbyl dithiophosphate metal salts, can be added to the fuel composition to provide both antiwear and antioxidation benefits. See, e.g., U.S. Pat. No. 5,898,023, the disclosures of which are incorporated herein by reference.
- In particular embodiments, the amount of lubricity improver in the fuel composition can range from about 1 ppm to about 50,000 ppm, for example, about 10 ppm to about 20,000 ppm, or about 25 ppm to about 10,000 ppm. Non-limiting examples of lubricity improvers include esters and fatty acids, which may or may not be the same as those produced in accordance to the methods described herein.
- In particular embodiments, the amount of stabilizers, which improves the storage stability of the fuel composition, can range from about 0.001 wt. % to about 2 wt. %, e.g., about 0.01 wt. % to about 1 wt. %, based on the total weight of the fuel composition. An exemplary stabilizer is a tertiary alkyl primary amine.
- Antioxidants prevent the formation of gum depositions on fuel system components due to oxidation of the fuels in storage and/or inhibit the formation of peroxide compounds in fuel compositions. The amount of antioxidants can range from about 0.001 wt. % to about 5 wt. %, e.g., from about 0.01 wt. % to about 1 wt. %, based on the total weight of the fuel composition.
- Corrosion inhibitors protect ferrous metals in fuel handling systems, such as pipelines and storage tanks, from corrosion. Certain corrosion inhibitors are also known to impart additional lubricity, and as such are particularly suitable when additional lubricity is desired. The corrosion inhibitor may be present in the fuel composition in an amount of about 0.001 wt. % to about 5 wt. %, e.g., from about 0.01 wt. % to about 1 wt. %, based on the total weight of the fuel composition.
- Biocides are used to combat microbial growth in the fuel composition, which may be present in the fuel composition at a concentration of about 0.001 wt. % to about 5 wt. %, e.g., from about 0.01 wt. % to about 1 wt. %, based on the total weight of the fuel composition.
- Metal deactivators suppress the catalytic effects of some metals, particularly copper, have on fuel oxidation, which can be present in the fuel composition in an amount of about 0.001 wt. % to about 5 wt. %, e.g., at 0.01 wt. % to about 1 wt. %, based on the total weight of the fuel composition.
- In addition, viscosity improvers, which are typically polymeric materials of number average molecular weights of from about 5,000 to about 250,000, and friction modifiers, which are typically sulfur-containing organo-molybdenum compounds can be added in minor amounts. Foam inhibitors, which typically include alkyl methacrylate polymers or dimethyl silicon polymers, can also be added to the fuel composition in an amount of less than about 10 ppm. Furthermore, seal fixes can be added to insure proper elastomer sealing and prevent premature seal failure can be included in the fuel composition.
- The invention is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the invention in any way.
- This example describes the construction of a genetically engineered microorganism wherein the expression of a fatty acid degradation enzyme is attenuated.
- The fadE gene of E. coli MG1655 (an E. coli K strain) was deleted using the Lambda Red (also known as the Red-Driven Integration) system described by Datsenko et al., Proc. Natl. Acad. Sci. USA 97: 6640-6645 (2000), with the following modifications:
- The following two primers were used to create the deletion of fadE:
-
Del-fadE-F (SEQ ID NO: 1) 5′-AAAAACAGCAACAATGTGAGCTTTGTTGTAATTATATTGTAA ACATATTGATTCCGGGGATCCGTCGACC; and Del-fadE-R (SEQ ID NO: 2) 5′-AAACGGAGCCTTTCGGCTCCGTTATTCATTTACGCGGCTTCA ACTTTCCTGTAGGCTGGAGCTGCTTC - The Del-fadE-F and Del-fadE-R primers were used to amplify the kanamycin resistance (KmR) cassette from plasmid pKD13 (described by Datsenko et al., supra) by PCR. The PCR product was then used to transform electrocompetent E. coli MG1655 cells containing pKD46 (described in Datsenko et al., supra) that had been previously induced with arabinose for 3-4 hours. Following a 3-hour outgrowth in a super optimal broth with catabolite repression (SOC) medium at 37° C., the cells were plated on Luria agar plates containing 50 μg/mL of Kanamycin. Resistant colonies were identified and isolated after an overnight incubation at 37° C. Disruption of the fadE gene was confirmed by PCR amplification using primers fadE-L2 and fadE-R1, which were designed to flank the E. coli fadE gene.
- The fadE deletion confirmation primers were:
-
fadE-L2 (SEQ ID NO: 3) 5′-CGGGCAGGTGCTATGACCAGGAC; and fadE-R1 (SEQ ID NO: 4) 5′-CGCGGCGTTGACCGGCAGCCTGG - After the fadE deletion was confirmed, a single colony was used to remove the KmR marker using the pCP20 plasmid as described by Datsenko et al., supra. The resulting MG1655 E. coli strain with the fadE gene deleted and the KmR marker removed was named E. coli MG1655 ΔfadE, or E. coli MG 1655 D1.
- This example describes the construction of a genetically engineered microorganism in which the expression of a fatty acid degradation enzyme and an outer membrane protein receptor are attenuated.
- The fhuA (also known as the tonA) gene of E. coli MG1655, which encodes a ferrichrome outer membrane transporter (GenBank Accession No. NP_414692), was deleted from strain E. coli MG1655 D1 of Example 1, using the Lambda Red system according to Datsenko et al., supra, but with the following modifications:
- The primers used to create the deletion:
-
Del-fhuA-F (SEQ ID NO: 5) 5′-ATCATTCTCGTTTACGTTATCATTCACTTTACATCAGAGA TATACCAATGATTCCGGGGATCCGTCGACC; and Del-fhuA-R (SEQ ID NO: 6) 5′-GCACGGAAATCCGTGCCCCAAAAGAGAAATTAGAAACGGA AGGTTGCGGTTGTAGGCTGGAGCTGCTTC - The Del-fhuA-F and Del-fhuA-R primers were used to amplify the kanamycin resistance (KmR) cassette from plasmid pKD13 by PCR. The PCR product obtained was used to transform electrocompetent E. coli MG1655 D1 cells containing pKD46 (see Example 1). These cells had been previously induced with arabinose for about 3-4 h. Following a 3-h outgrowth in SOC medium at 37° C., the cells were plated on Luria agar plates containing 50 μg/mL of kanamycin. Resistant colonies were identified and isolated after an overnight incubation at 37° C. Disruption of the fhuA gene was confirmed in some of the colonies by PCR amplification using primers flanking the E. coli fhuA gene:
- Confirmation of the deletion was performed using the following primers:
-
fhuA-verF (SEQ ID NO: 7) 5′-CAACAGCAACCTGCTCAGCAA; and fhuA-verR (SEQ ID NO: 8) 5′-AAGCTGGAGCAGCAAAGCGTT - After the fhuA deletion was confirmed, a single colony was used to remove the KmR marker using the pCP20 plasmid as described by Datsenko et al., supra. The resultant MG1655 E. coli strain having fadE and fhuA gene deletions was named E. coli MG1655 ΔfadE_ΔfhuA, or E. coli MG1655 DV2.
- This example describes the construction of a genetically engineered microorganism in which the expression of an acyl-CoA dehydrogenase, an outer membrane protein receptor, a pyruvate formate lyase and a lactate dehydrogenase are attenuated.
- The NW gene of E. coli MG1655, which encodes a pyruvate formate lyase (GenBank Accession No. AAC73989) was deleted from E. coli MG1655 DV2 (see, Example 2) using the Lamda Red System according to Datsenko et al., supra, but with the following modifications:
- The primers used to create the deletion strain were:
-
Del-pflB-F: (SEQ ID NO: 9) 5′-GCCGCAGCCTGATGGACAAAGCGTTCATTATGGTGCTGCCGGTC GCGATGATTCCGGGGATCCGTCGACC Del-pflB-R: (SEQ ID NO: 10) 5′-ATCTTCAACGGTAACTTCTTTACCGCCATGCGTGTCCCAGGTGT CTGTAGGCTGGAGCTGCTTCG - The Del-pflB-F and Del-pflB-R primers were used to amplify the kanamycin resistance (KmR) cassette from plasmid pKD13 by PCR. The PCR product was then used to transform electrocompetent E. coli MG1655 DV2 cells (see Example 2).
- In parallel, the ldhA gene of E. coli MG1655, which encodes a lactate dehydrogenase, specifically an NAD-linked fermentative D-lactate dehydrogenase (see, e.g., Mat-Jan et al., J. Bacteriol. 171(1):342-8 (1989); Bunch et al., Microbiol. 143(1):187-95 (1997) (GenBank Accession No. AAC74462) was also deleted from E. coli MG1655 DV2 (see, Example 2) using the Lambda Red System according to Datsenko et al., supra, but with the following modifications.
- Two primers were used to create the deletion:
-
Del-ldhA-F: (SEQ ID NO: 11) 5′-CTCCCCTGGAATGCAGGGGAGCGGCAAGATTAAACCAGTTCGTTC GGGCAGTGTAGGCTGGAGCTGCTTCG-3′ Del-ldhA-R: (SEQ ID NO: 12) 5′-TATTTTTAGTAGCTTAAATGTGATTCAACATCACTGGAGAAAGTC TTATGCATATGAATATCCTCCTTAGTTCC-3′ - The Del-ldhA-F and Del-ldhA-R primers were used to amplify the chloramphenicol acetyltransferase resistance (CmR) cassette from plasmid pKD3 (see, Datsenko et al., supra) by PCR. The PCR product was also used to transform electrocompetent E. coli MG1655 DV2 cells (see, Example 2).
- The E. coli MG1655 DV2 (see Example 2) cells had been previously induced with arabinose for about 3-4 h. Following a 3-h outgrowth in SOC medium at 37° C., the cells were plated on Luria agar plates containing 50 μg/mL of kanamycin and 30 μg/mL chloroamphenicol. Colonies that were resistant to both kanamycin and chlroamphenicol were identified and isolated after an overnight incubation at 37° C. Disruption of the pflB gene was confirmed using primers flanking the E. coli pflB gene, and disruption of the ldhA gene was verified using primers flanking the E. coli ldhA gene.
- Confirmation of the deletion of pflB was performed using the following primers:
-
pflB-verF: (SEQ ID NO: 14) 5′-GGACTAAACGTCCTACAAAC PflB-verR: (SEQ ID NO: 15) 5′-TTCATCTGTTTGAGATCGAG - Confirmation of the deletion of ldhA gene was performed using the following primers:
-
ldhA-verF: (SEQ ID NO: 16) 5′-CCCGAGCGGTAGCCAGATGCCCGCCAGCG ldhA-verR: (SEQ ID NO: 17) 5′-GCTGCGGGTTAGCGCACATCATACGGGTC - After the deletions were confirmed, a single colony was used to remove the KmR and CmR markers in accordance with the method described by Datsenko et al., supra. The resultant MG1655 E. coli strain having fadE, fhuA, pflB and ldhA gene deletions was named E. coli MG1655 ΔfadE_ΔfhuA_ΔpflB_ΔldhA, or E. coli MG1655 DV4.
- This example describes the construction of a genetically engineered microorganism in which the expression of a fatty acid degradation enzyme such as an acyl-CoA synthase, an outer membrane protein receptor, and an acyl-CoA dehydrogenase are attenuated.
- The fadD gene of E. coli MG1655 was deleted from strain E. coli MG1655 DV2 (see, Example 2), using the Lambda Red system described by Datsenko et al, supra, but with the following modifications:
- The primers used to create the deletion of fadD were:
-
fad1: (SEQ ID NO: 19) 5′-TAACCGGCGTCTGACGACTGACTTAACGCTCAGGCTTTATT GTCCACTTTGTGTAGGCTGGAGCTGCTTCG-3′; and fad2: (SEQ ID NO: 20) 5′-ATTTGGGGTTGCGATGACGACGAACACGCATTTTAGAGGTG AAGAATTGCATATGAATATCCTCCTTTAGTTCC-3′ - The fad1 and fad2 primers were used to amplify the chloramphenicol acetyltransferase resistance (CmR) casette from plasmid pKD3 (described by Datsenko et al., supra) by PCR. The PCR product was used to transform electrocompetent E. coli MG1655 DV2 (see, Example 2). The transformed cells were plated on Luria agar plates containing 30 μg/mL of chloramphenicol and grown overnight at 37° C. Individual colonies were isolated, transferred to fresh Luria agar plates, and grown at 42° C. These colonies were then patched onto Luria agar plates containing 30 μg/mL of chloramphenicol and 100 μg/mL carbenicillin, and grown at 37° C. overnight. Colonies that were resistant to chloramphenicol and sensitive to carbenicillin were evaluated further by PCR to ensure that the PCR product inserted at the correct site. Specifically, disruption of the fadD gene was confirmed by PCR amplification using primers fadF and fadR, which were designed to flank the fadD gene:
-
fadF: (SEQ ID NO: 21) 5′-CGTCCGTGGTAATCATTTGG-3′; and fadR: (SEQ ID NO: 31) 5′-TCGCAACCTTTTCGTTGG-3′ - After the fadD deletion was confirmed, the CmR marker was removed using a FLP helper plasmid as described by Datsenko et al., supra. The resulting MG1655 E. coli strain was named E. coli MG1655 ΔfadEΔfhuAΔfadD, or E. coli MG1655 DV2 ΔfadD. The DV2 ΔfadD strain was unable to grow on M9+ oleate agar plates (which supply oleate as carbon source). It was also unable to grow in M9+ oleate liquid media.
- This example describes the construction of a bacterial expression plasmid, pDS33.ES9, (
FIG. 3 ) in which expression of an ester synthase gene from M. hydrocarbonoclasticus DSM 8798 (GenBank Accession No. AB021021) is under the control of the E. coli spc ribosomal protein operon promoter Pspc (SEQ ID NO:13,FIG. 4 ). - The Pspc promoter, as shown in
FIG. 2 , was amplified from E. coli MG1655 chromosomal DNA with the following primers: -
PspcIFF (SEQ ID NO: 32) 5′-AAAGGATGTCGCAAACGCTGTTTCAGTACACTCTCTCAATAC-3′; and PspcIFR (SEQ ID NO: 33) 5′-GAGCTCGGATCCATGGTTTAGTGCTCCGCTAATG-3′ - All PCR reactions described in this example were performed with Phusion™ Polymerase (NEB, Ipswich, Mass.). The Pspc promoter PCR fragment was used to replace both the lacIq and Trc promoter regions of plasmid OP80 by cloning the PCR product into BseRI/NcoI restricted OP80 using the InFusion™ Cloning kit (Clontech, Palo Alto, Calif.). The resulting plasmid, named pDS22, still possessed a lacZ sequence downstream of the multiple cloning site. Next, the lacZ sequence was removed by PCR amplification of plasmid pDS22 using the following primers:
-
pCLlacDF (SEQ ID NO: 34) 5′-GAATTCCACCCGCTGACGAGCTTAG-3′; and pCLEcoR (SEQ ID NO: 35) 5′-CGAATTCCCATATGGTACCAG-3′ - The PCR product was digested by EcoRI and self ligated to form pDS23. Plasmid pDS23 does not contain the lacIq, lacZ, or Trc DNA.
- An ester synthase from M. hydrocarbonoclasticus DSM 8798 (GenBank Accession No. AB021021), referred to as ES9 herein, was synthesized by DNA2.0 (Menlo Park, Calif.). This synthesized ES9 ester synthase gene was subcloned into pColaDuet-1 to form plasmid pHZ1.97-ES9. The internal BspHI site in the ES9 ester synthase gene was removed by site directed mutagenesis using the QuikChange® Multi Kit (Stratagene, Carlsbad, Calif.) and the following mutagenic primer:
-
ES9BspF (SEQ ID NO: 36) 5′-CCCAGATCAGTTTTATGATTGCCTCGCTGG-3′ - This primer introduced a silent mutation into the ES9 ester synthase gene. The resulting plasmid, named pDS32, then was used as a template to amplify the ES9 ester synthase gene with the following primers:
-
ES9BspHF (SEQ ID NO: 37) 5′-ATCATGAAACGTCTCGGAAC-3′; and ES9XhoR (SEQ ID NO: 38) 5′-CCTCGAGTTACTTGCGGGTTCGGGCGCG-3′ - The resulting PCR product was subject to restriction digestions with BspHI and XhoI and ligated into plasmid pDS23 digested with NcoI and XhoI to form pDS33.ES9 (SEQ ID NO:22). The sequence of pDS33.ES9 is shown in
FIG. 3 with the residues representing the Pspc promoter in italics and the residues of the ES9 ester synthase gene in bold type. - This example describes the construction of a bacterial expression plasmid, pDS57 (SEQ ID NO:23) in which expression of an ester synthase gene is under the control of the Ptrc promoter.
- The ES9 ester synthase gene was amplified from plasmid pDS33.ES9 (SEQ ID NO:22, Example 5) using primers ES9BspHF (SEQ ID NO:37) and ES9XhoR (SEQ ID NO:38).
- The PCR product was digested with BspHI and XhoI and ligated into NcoI/XhoI restricted OP80 to form plasmid pDS57 (SEQ ID NO:23), wherein the ES9 ester synthase gene is under control of the Trc promoter. The sequence of pDS57 is shown in
FIG. 4 with the Trc promoter in italics and the ES9 ester synthase gene in bold type. - This example illustrates that expression of ester synthase alone, without co-expression of an acyl-CoA synthase or a thioesterase, can be used to produce fatty esters in vivo.
- Seed cultures of E. coli MG1655 DV2 ΔfadD of Example 4, each carrying one of the following plasmids:
- (1) a pCL plasmid containing no insert (negative control);
(2) a plasmid containing E. coli thioesterase 'tesA;
(3) a plasmid containing a polynucleotide encoding Alcanivorax borkumensis SK2 ester synthase atfA1(GenBank Accession No. YP_694462);
(4) a plasmid containing a polynucleotide encoding Alcanivorax borkumensis SK2 ester synthase atfA2 (GenBank Accession No. YP_693524);
(5) a plasmid containing a polynucleotide encoding Marinobacter hydrocarbonoclasticus DSM 8798 ester synthase ES8 (GenBank Accession No. ABO21020); and
(6) a plasmid containing a polynucleotide encoding Marinobacter hydrocarbonoclasticus DSM 8798 ester synthase ES9 (GenBank Accession No. ABO21021)
were grown in 24-well plates in LB broth supplemented with 100 μg/mL spectinomycin. The ester synthase plasmids were constructed as described in Example 5 to generate plasmids pDS41.S (containing atfA1 gene), pDS31atfa2 (containing atfA2 gene), pDS31.ES8 (containing the gene encoding ES8), and pDS33.ES9 (containing the gene encoding ES9). - After 4-h of growth, the cultures were diluted 1:25 in Che-9 2N-BT (2% glucose, nitrogen limited medium, 0.2 M Bis-Tris, pH 7.0, 0.1% Triton) containing spectinomycin and grown overnight. The cultures were diluted in 4N-BT (4% glucose, nitrogen limited medium, 0.2M Bis-Tris, pH 7.0, 0.1% Triton) to a final OD600 (optical density at 600 nm) of about 0.2. After 6-h of growth, IPTG was added to a final concentration of 1 mM, along with either water or ethanol (2% (v/v)). At 22-h post-induction, 1 mL of culture was acidified with 200 μM HCl, and extracted with 500 μL, ethyl acetate. The fatty acids and esters were then treated with the derivatizing agent TMAH and quantified by GC/FID. The amount of free fatty acid (FFA) and fatty acyl ethyl ester (FAEE) produced in each culture is shown in
FIG. 5 . - As shown in
FIG. 5 , ES9 generated nearly 200 mg/L FAEE in the presence of ethanol, but only a small quantity (<20%) of FFA. Compared to ester synthase ES9, the other ester synthases, including ES8, atfA1 and atfA2, produced substantially lower amounts of FAEE in the presence of ethanol, accompanied by a higher proportion of FFA. The culture E. coli 'tesA produced higher overall titers of FFA and FAEE in the presence of ethanol, but the amount of FFA was significantly greater than the amount of FAEE generated. - The results indicate that the expression of ester synthase in E. coli MG1655 DV2 ΔfadD leads to the production of esters in the presence of ethanol. In particular, expression of ester synthase ES9 resulted in the highest ester production in the presence of ethanol as compared to that of the other ester synthases. Furthermore, this example indicated that expression of 'TesA alone in E. coli MG1655 DV2 ΔfadD resulted in the production of a low level of esters and a high level of free fatty acids in the presence of ethanol.
- This example demonstrates that expression of an ester synthase alone, without co-expression of an acyl-CoA synthase or a thioesterase, can be used to produce esters in vivo in the presence of ethanol.
- Seed cultures of E. coli MG1655 DV2 ΔfadD of Example 4, each carrying one of the following plasmids:
- (1) a plasmid containing an E. coli 'tesA;
(2) a plasmid containing a polynucleotide encoding Alcanivorax borkumensis SK2 ester synthase atfA1 (GenBank Accession No. YP_694462);
(3) a plasmid containing a polynucleotide encoding Alcanivorax borkumensis SK2 ester synthase atfA2 (GenBank Accession No. YP_693524);
(4) a plasmid containing a polynucleotide encoding Marinobacter hydrocarbonoclasticus DSM 8798 ester synthase ES8 (GenBank Accession No. ABO21020); and
(5) a plasmid containing a polynucleotide encoding Marinobacter hydrocarbonoclasticus DSM 8798 ester synthase ES9 (GenBank Accession No. ABO21021)
were grown in shake flask cultures and induced with 1 mM IPTG and 2% (v/v) ethanol. At 24-h post induction, aliquots from each culture were extracted and analyzed using GC-FID for fatty acid and ester content. The amounts of free fatty acid (FFA) and fatty acyl ethyl ester (FAEE) produced in each culture were shown inFIG. 6 . - As shown in
FIG. 6 , ester synthase ES9 generated a high level of FAEE in the presence of ethanol, accompanied by a low level of FFA. Compared to ES9, noticeably smaller amounts of FAEE were produced by ester synthases atfAI, atfA2, and ester synthase ES8. Consistent with the results of Example 7 andFIG. 5 , above, expression of E. coli 'tesA alone produced high overall titers of FFA and FAEE, but with a significantly higher proportion of FFA. - The results demonstrate that expression of ester synthase ES9 in E. coli MG1655 DV2 ΔfadD results in the production of high levels of esters and low levels of free fatty acids in the presence of ethanol. Furthermore, this example demonstrates that expression of 'TesA in E. coli MG1655 DV2 ΔfadD resulted in the production of a low level of esters and a high level of free fatty acids in the presence of ethanol.
- This example demonstrates that expression of an ester synthase alone, without co-expression of an acyl-CoA synthase or a thioesterase, can be used to produce esters in vivo in the presence of methanol.
- Seed cultures of E. coli MG1655 DV2 ΔfadD of Example 4, each carrying one of the following plasmids:
- (1) a plasmid containing a polynucleotide encoding Alcanivorax borkumensis SK2 ester synthase atfA1 (GenBank Accession No. YP_694462);
(2) a plasmid containing a polynucleotide encoding Alcanivorax borkumensis SK2 ester synthase atfA2 (GenBank Accession No. YP_693524);
(3) a plasmid containing a polynucleotide Marinobacter hydrocarbonoclasticus DSM 8798 ester synthase ES8 (GenBank Accession No. AB021020); and
(4) a plasmid containing a polynucleotide Marinobacter hydrocarbonoclasticus DSM 8798 ester synthase ES9 (GenBank Accession No. AB021021) were grown in shake flask cultures and induced with 1 mM IPTG and 2% (v/v) methanol. At 24-h post induction, aliquots from each culture were extracted and analyzed by GC-FID for fatty acid and ester content. The amounts of free fatty acid (FFA) and fatty acyl methyl ester (FAME) produced in each culture were shown inFIG. 7 . - The amount of overall product or titer observed for FFA and FAME was reduced in the presence of methanol as compared to the amount of overall titer observed in the presence of ethanol (see
FIG. 6 ). Here the amount of FFA produced was below the limit of detection, therefore it is possible that FFA was produced in the presence of methanol, albeit at low concentrations. Lower levels of overall titers, as compared to those obtained in the presence of ethanol, were also observed in cultures expressing ester synthases atfAI, atfA2, and ES8. - The results demonstrate that expression of ester synthase ES9 in E. coli MG1655 DV2 ΔfadD resulted in the production of esters in the presence of methanol.
- This example demonstrates that expression of an ester synthase alone, without co-expression of an acyl-CoA synthase or a thioesterase, can be used to produce esters in vivo in the presence of ethanol or methanol.
- E. coli MG1655 DV2 ΔfadD carrying a plasmid containing a polynucleotide encoding M. hydrocarbonclasticus DSM 8798 ester synthase ES9 (GenBank Accession No. ABO21020), was grown in shake flask cultures and induced with 1 mM IPTG and either (1) 2% (v/v) ethanol or (2) 2% (v/v) methanol. Aliquots from each culture were extracted at 18, 24, 48, and 68 hours post induction, and analyzed by GC-FID for fatty acid and ester content. The amounts of fatty acyl methyl ester (FAME) or fatty acyl ethyl ester (FAEE) produced in each culture were shown in
FIG. 8 , as well as the total amount of FAME or FAEE plus free fatty acid (FFA). - As shown in
FIG. 8 , significantly higher levels of esters were produced in the presence of ethanol (FAEE) as compared to the levels of esters produced in the presence of methanol (FAME) throughout the post-induction time course. Low levels of free fatty acids were produced both in the presence of ethanol and in the presence of methanol. - The results demonstrate that expression of ester synthase ES9 in E. coli MG1655 DV2 ΔfadD resulted in high production levels of esters and low production levels of free fatty acids in the presence of methanol and ethanol. Furthermore, this example demonstrates that ester production in E. coli MG1655 DV2 ΔfadD expressing ester synthase ES9 was significantly higher in the presence of ethanol than in the presence of methanol.
- This example describes the construction of an ester synthase mutant library, wherein each member contains an amino acid substitution for glycine at
position 395 of ES9. - When the sequence of the ester synthase ES9 of M. hydrocarbonoclasticus DSM 8798 (GenBank Accession No. ABO21021.1) was subjected to a BLAST search (http://www.ncbi.nlm.nih.gov/), several bacterial ester synthase homologues were identified. Sequence alignment of these microbial ester synthases revealed a general pattern of an acidic amino acid at
position 395 of glutamic acid or aspartic acid, notwithstanding the fact that the reported sequence of ester synthase ES9 contained a glycine atamino acid 395. - The glycine residue at
position 395 of ES9 was mutated to each of the 20 conventional amino acid residues in order to evaluate the effect of amino acid substitution at this position. The mutants were created using Stratagene's QuikChange® Multi Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.). Primer G395Xf, as shown in Table 7, was used in conjuction with a vector-specific primer to generate a pool of 3′ ester synthase fragments encoding all 20 possible amino acid residues atposition 395. A reverse primer complementary to the 5′-portion of G395Xf, designated as G395Xr, was used in conjuction with a vector-specific primer to generate the 5′ ester synthase fragments. Each of the 3′-fragments was then assembled with the 5′ fragments via overlap extension to reconstitute a randomized pool of gene sequences encoding a sequence with 20 different amino acid residues atposition 395. -
TABLE 7 Primer Primer Sequence 5′-3′ G395Xf CCATTTCCAACGTGCCCGGCCCGGAANNKACGCTGTAT TATGAAGG (SEQ ID NO: 47), wherein N is any base and K is G or T G395Xr TTCCGGGCCGGGCACGTTGGAAATGG (SEQ ID NO: 52) - The randomized pool of ester synthase mutant sequences were then electroporated into electrocompetent E. coli MG1655 DV4/1/2 KmRCmR/pKD46 cells, which were prepared in accordance with the description below.
- A first gene containing the promoter and open reading frame for chloramphenicol resistance (SEQ ID NO:157) was obtained from plasmid pKD3 (see, Datsenko et al., supra). Separately, a portion of a second gene containing the promoter and the first 531 bases of the kanamycin resistance gene (SEQ ID NO:158) was obtained from plasmid pKD13 (see, Datsenko et al., supra). A resistance cassette was constructed by first joining the first gene with the second gene, with the first gene being upstream of the second gene. A 50-base pair homologous sequence to the 3′-end of the lacZ gene sequence (SEQ ID NO:159) was added downstream from the chloramphenicol resistance gene, and a lacI gene sequence (SEQ ID NO:160) was added upstream from the partial kanamycin resistance gene. This resistance cassette was then used to transform an E. coli MG1655 DV4 strain, which had been previously transformed with pKD46 as described below.
- A plasmid pKD46 (see, Datsenko et al., supra) was transformed into E. coli MG1655 DV4 (see, Example 3). This DV4/pKD46 strain was grown on a solid media containing 100 μg/mL cabenicillin overnight at 32° C. A single colony was then used to inoculate an overnight culture of LB containing 100 μg/mL carbenicillin After 16-h of growth at 32° C., the DV4/pKD46 culture was diluted 1 to 500 into an LB medium containing 100 μg/mL carbenicillin, arabinose and MgCl2 to induce expression of the recombinase genes from pKD46. Growth was monitored by measuring OD600, and when it reached about 0.6, the cells were made electrocompetent and transformed with the resistance cassette.
- The transformed cells were allowed to recover for 3 h in non-selective SOC media before plating on solid chloramphenicol-selective media, containing 34 μg/mL chloramphenicol, X-gal, and IPTG. Correct insertions were confirmed using colony PCR, blue/white screening and DNA sequencing. This strain is named E. coli MG1655 DV4/1/2 KmRCmR/pKD46. It is then subject to arabinose treatment for 3-4 h before the ester synthase mutant sequences were transformed
- Following transformation, the cultures was allowed to recover and plated onto LB agar medium containing 50 μg/mL kanamycin and allowed to grow overnight at 32° C. 96 clones from this plate were sequenced to verify the types of intended mutations at
ES9 position 395. The capacity to make fatty acid/fatty esters of each was then assessed. - A seed culture of each clone was grown in 96-well plates in LB broth supplemented with 50 μg/mL kanamycin at 32° C. After a 6-h growth, the cultures were diluted 1 to 10 into 2N-BT (2% glucose, nitrogen limited medium, 0.2M Bis-Tris, pH 7.0, 0.1% Triton) plus 50 μg/mL kanamycin and grown overnight at 32° C. After overnight growth, the cultures were diluted again 1 to 10 into 4N-BT (4% glucose, nitrogen limited medium, 0.2 mM Bis-Tris, pH 7.0, 0.1% Triton) and grown at 32° C. for 6 h. IPTG and ethanol were then added to each culture to achieve a final concentration of 1 mM and 2% (v/v), respectively. 22-h post induction, the cultures were acidified using 40 μL 1 M HCl and extracted with 400 μL n-butylactate. The amounts of fatty species (including, e.g., fatty acids and fatty esters) in the extractants were measured using GC-FID.
- As can be seen
FIG. 9 , which depicts the z scores of the production titers from the mutant and wild type strains, and wherein the median titer of the production strain expressing wild type ES9 was set at 0, the samples wherein the glycine residue atposition 395 of ES9 was replaced by either an argine or a lysine residue resulted in the most significant increases in fatty species production. For example, the z score for the G395R mutant strain was above 10, and the z score for the G395K mutant strain was about 9. Moreover, p-values were calculated to assess statistical significance in terms of the improvements of titer. The productivity improvement of the G395R mutant strain had a p-value of 1.74E-19, and the productivity improvement of the G395K mutant strain had a p-value of 5.42E-05, and those improvements were statistically significant. - This example demonstrates that expression of an ester synthase mutant of ester synthase ES9 from Marinobacter hydrocarbonoclasticus, wherein glycine at
amino acid residue 395 of the reported sequence was replaced with a number of other amino acid residues, can be used to produce esters in vivo in the presence of a suitable alcohol substrate. - The following primers were used to remove the ester synthase mutant and wild type fragments from their E. coli MG1655 DV4/1/2 KmRCmR/pKD46 transformants of Example 11:
-
ES9BspHF (SEQ ID NO: 37) 5′-ATCATGAAACGTCTCGGAAC-3′; and ES9XhoR (SEQ ID NO: 38) 5′-CCTCGAGTTACTTGCGGGTTCGGGCGCG-3′ - The ester synthase fragments that were obtained from the E. coli MG1655 DV4/1/2 KmRCmR/pKD46 transformants using these primers were wild type ES9, and mutants G395R, G395S, G395K, G395S, and G395D. These fragments were then cloned downstream of the IPTG inducible promoter in a pCL1920 vector using NcoI and HindIII restriction sites.
- The resulting plasmids were transformed into electrocompetent E. coli MG1655 DV2 ΔfadD (see, Example 4) and plated on LB plates containing 100 mg/L spectinomycin, incubated overnight at 37° C., and purified.
- Purified DV2 ΔfadD strains expressing ES9, and mutants G395R, G395S, G395K, G395S, and G395D were grown in 15 mL shake flasks and induced with 1 mM IPTG and 2% (v/v) methanol. Post induction, samples were taken from each shake flask at the 15.5 h, 40 h and 65 h, and the production levels of free fatty acids and FAME were determined at these time points using GC-FID. The results are shown in
FIG. 10A . - Cell growth during fermentation was also followed by taking small samples of the fermentation broth and measuring OD600. The result is shown in
FIG. 10B . - As shown in
FIG. 10A , strains expressing ES9, which has a glycine residue atposition 395, as well as strains expressing the G395R, G395K, G395D and G395E mutants of ES9, all produced substantial FAME and free fatty acids in the presence of methanol. In contrast, the strain expressing the G395S mutant produced little if any FAME and/or free fatty acids. - In the presence of methanol, the production strain expressing the G395R mutant ester synthase produced about 1,800 g/L FAME/FFA, whereas the production strain expressing the G395K mutant ester synthase produced about 1,400 g/L FAME/FFA. In comparison with the amount of FAME/FFA produced by the production strain comprising the wild type ester synthase ES9, these two mutants appeared to have substantially improved capacity to produce fatty acids and derivatives thereof. Substituting the glycine residue at
position 395 of ES9 with acidic amino acid residues did not provide the production strain with an improved capacity to produce fatty acids and/or derivatives. Nor did substituting the glycine residue atposition 395 of ES9 with a cysteine residue - The results demonstrate that the glycine mutation to a basic amino acid residue such as a lysine or an arginine residue at
amino acid position 395 in the reported sequence of ester synthase ES9 provides an improved ester synthase variant that can be utilized to achieve an improved production of fatty acids and/or fatty acid derivatives. - This example describes ester synthase homologs with altered activity and/or specificity towards acyl-ACPs over acyl-CoA or beta-hydroxy acyl-ACP. Four ester synthase homologs were identified based on bioinformatic analyses and described in the table below. These ester synthase homologues were evaluated for activity:
-
TABLE 8 Given names Organism source Accession number SEQ ID ES1 Marinobacter algicola ZP_01893763 SEQ ID NO: 39 DG893 ES2 Limnobacter ZP_01915979 SEQ ID NO: 41 sp. MED105 ES3 Marine metagenome EDJ50241 SEQ ID NO: 43 ES4 Whalefall sample # 32001496676* SEQ ID NO: 45 *IMG identification number - The percent identity and percent similarity of these homologues to the reported sequence of ester synthase ES9 are listed in the table below:
-
TABLE 9 Ester Synthase ES9 % identity % similarity ES1 85 91 ES2 51 68 ES3 39 56 ES4 36 56 - The nucleotide sequences of the four ester synthase homologs above were E. coli codon-optimized (SEQ ID NOs: 40, 42, 44, 46) and synthesized using DNA2.0 (DNAstar, Menlo Park, Calif.) and then cloned downstream of the IPTG inducible Ptrc promoter in a pCL1920 vector using the NcoI and HindIII sites.
- The resulting plasmids were transformed into E. coli MG1655 DV2 ΔfadD for evaluation in shake flasks. The resultant production titers of FAME, when the cells were fed with methanol, were indicated in
FIG. 11 . The resultant production titers of FAEE, when the cells were fed ethanol, were indicated inFIG. 12 . - This example describes a fermentation process to produce a fatty acid and/or fatty acid derivative composition using the genetically modified microorganisms described herein.
- A fermentation and recovery process was used to produce biodiesel of commercial grade quality by fermentation of carbohydrates.
- A fermentation process was developed to produce a mix of fatty acid ethyl esters (FAEE) and fatty acid methyl esters (FAME) for use as a biodiesel using the genetically engineered microorganisms described Examples 1-12. The fermentation is carried out in any manner known to those of ordinary skill in the art. For example, the fermentation is carried out in a 2- to 10-L lab scale fermentor. These exemplary protocols can be scaled up as any other E. coli fermentation, using methods known to one of ordinary skill in the art.
- In one embodiment, a fermentation run was carried out in a 2-L fermentor. E. coli cells from a frozen stock were grown in a defined medium consisting of 4.54 g/L K2HPO4 trihydrate, 4 g/L of (NH4)2SO4, 0.15 g/L of MgSO4 heptahydrate, 20 g/L glucose, 200 mM Bis-Tris buffer (pH 7.2), 1.25 mL/L trace mineral solution, and 1.25 mL/L vitamin solution. The trace mineral solution comprised 27 g/L FeCl3 •6H2O, 2 g/L ZnCl2 •4H2O, 2 g/L CaCl2 •6H2O, 2 g/L Na2MoO4 •2H2O, 1.9 g/L CuSO4 •5H2O, 0.5 g/L H3BO3, and 100 mL/L concentrated HCl. The vitamin solution comprised 0.42 g/L riboflavin, 5.4 g/L pantothenic acid, 6 g/L niacin, 1.4 g/L pyridoxine, 0.06 g/L biotin, and 0.04 g/L folic acid.
- Fifty (50) mL of a culture described herein was grown overnight and subsequently used to inoculate 1 L of a medium containing 0.5 g/L (NH4)2SO4, 2.0 g/L KH2PO4, 0.15 g/L MgSO4 heptahydrate, 0.034 g/L ferric citrate, 2.5 g/L bacto casamino acids, 10 g/L glucose, 1.25 mL/L
trace mineral solution 2, and 1.25 mL/L vitamin solution. Thetrace mineral solution 2 contained 2 g/L ZnCl2 4H2O, 2 g/L CaCl2 •6H2O, 2 g/L Na2MoO4 •2H2O, 1.9 g/L CuSO4 •5H2O, 0.5 g/L H3BO3, and 100 mL/L of concentrated HCl. The vitamin solution was the same as described for the inoculum culture medium. - The fermentor was equipped with temperature-, pH-, agitation-, aeration-, and dissolved oxygen-controls. The preferred fermentation conditions include a temperature of 32° C., pH of 6.8, and a dissolved oxygen (DO) level of about 30% of saturation. The pH was maintained by additions of NH4OH, which also acted as a nitrogen source for cell growth. When the initial glucose was almost consumed, a feed consisting of 600 g/L glucose, 3.9 g/L MgSO4 heptahydrate, 1.6 g/L KH2PO4, 2.5 g/L casamino acids, 0.05 g/L Ferric citrate, 20 mL/L
trace minerals solution - Ethanol was added several times during the run to replenish what was consumed by the cells for the production of fatty ethyl esters, but mostly what was lost by evaporation in the off-gas. The additions helped to maintain the concentration of ethanol in the fermentation broth at a level of between 10 and 20 mL/L, in order to promote efficient production without inhibiting cell growth. The progression of the fermentation run was followed by measurements of OD600, glucose consumption, and ester production.
- This fermentation protocol was also scaled up to a 700-L fermentor. The analytical methods utilized following the fermentation runs are described in the following examples.
- This example describes the analytical procedures used to detect glucose consumption, and productions of free fatty acids and/or fatty acid esters. Glucose consumption throughout the fermentation was analyzed by High Pressure Liquid Chromatography (HPLC). The HPLC analysis was performed according to methods commonly used in the art for measuring sugars and organic acids, which typically involve the following equipment and conditions:
Agilent HPLC 1200 Series (Agilent Technology, Santa Clara, Calif.) with Refractive Index detector; Aminex HPX-87H column, 300 mm×7.8 mm (Catalogue: 125-0140); column temperature: 50° C.; pH range: 1-3; mobile phase: 0.005 M H2SO4 (aqueous); flow rate: 0.6 mL/min; injection volume: 5 μL; run time: 25 min. - The production of fatty acid methyl or ethyl ester was analyzed by gas chromatography with flame ionization detector (GC-FID). Samples from the fermentation broth were extracted with ethyl acetate in a volume ratio of 1:1. After vigorous vortexing, the samples were centrifuged and the respective organic phases ware analyzed by GC. The analysis conditions were as follows:
- Instrument: Trace GC Ultra, Thermo Electron Corp. (Marietta, Ohio) with Flame ionization detector (FID) detector;
Column: DB-1 (1% diphenyl siloxane; 99% dimethyl siloxane)Col UFM 1/0.1/5/0.1 DET from Thermo Electron Corp. (Part# UFMC00001010401; S/N 520070046), with Phase pH of 5, FT of 0.1 μm, length of 5 m and an internal diameter of 0.1 mm;
Inlet conditions: 300° C., split 1/300 with a split flow of 150 mL/min, total run time: 2 min;
Carrier gas/flow rate: Helium/0.5 mL/min;
Block temperature: 260° C.;
Oven temperature: 0.3 min hold at 140° C.; ramp at 100° C./min to 300° C.; hold for 0.05 min, total run time: 4 min.
Detector temperature: 300° C.;
Injection volume: 1 μL. - Production of fatty methyl or ethyl ester was followed by gas chromatography linked with a mass spectrometry detector (GC-MS). Samples from the fermentation broths were extracted with ethyl acetate or butyl acetate in a volume ratio of 1:1. After vigorous vortexing, the samples were centrifuged and the respective organic phases were analyzed by GC-MS on an Agilent 6850 (Agilent Technology, Santa Clara, Calif.) equipped with a 5975B VL MSD. Ions were extracted by single ion monitoring. The analysis conditions were as follows:
- Oven temperature: 3.00 min hold at 100° C., ramp at 20° C./min to 320° C. and hold for 5 min, with a total run time of 19 min;
Capillary column: DB-5 MS UI, capillary 30.0 m×250 μM×0.250 μm×0.25 μm nominal;
Carrier gas/flow rate/pressure: Helium/1.2 mL/min/12.56 psi;
Inlet condition: 320° C. splitless;
Total flow rate: 16.2 mL/min; - This example demonstrates that fatty acid ethyl esters can be produced by fermenting an E. coli strain expressing an ester synthase, in the absence of a thioesterase and/or an acyl-CoA synthase.
- E. coli MG1655 DV2 and DV2 ΔfadD, as described in Examples 2 and 4, respectively, were transformed with plasmid pDS57 (SEQ ID NO:23), as described in Example 5. The resulting DV2 pDS57 and DV2 ΔfadD pDS57 strains were grown in 2- and 5-L fermentors according to the fermentation protocol of Example 14. Representative results of the analysis of FAEE, FFA, and the total yield of FAEE for each strain are shown in Table 10. The yield is expressed as the grams of product obtained per 100 grams of carbon source used.
-
TABLE 10 Parameter DV2 pDS57 DV2 ΔfadD pDS57 FAEE concentration (g/L) 5.9 7.3 FFA concentration (g/L) 0.3 0.5 Yield of FAEE on glucose (%) 5.6 6.1 - The results indicate that high levels of fatty acid ethyl esters and low levels of free fatty acids can be produced by fermentation of E. coli strains DV2 and DV2 ΔfadD expressing ester synthase ES9.
- This example demonstrates that fatty acid esters can be produced by feeding fatty acids and alcohols to a microbial strain containing a native (i.e., non-recombinant) ester synthase. A strain of Funibacter jadensis was used as an example of a microbial strain containing such an ester synthase. It was cultured in a seawater medium.
- A seawater medium was prepared and autoclaved: 23.6 g/L NaCl, 0.64 g/L KCl, 4.53 g/L MgCl2×6H2O, 5.94 g/L MgSO4×7H2O. The pH was adjusted to 7.2 with NaHCO3.
- The strain Funibacter jadensis T9 (DSM 12178) was obtained from DSMZ (http://www.dsmz.de). The strain was grown in 25 mL of the seawater medium, supplemented with 100 μL of sodium palmitate (1% in H2O) and one of four alcohols listed in Table 11. After 18 h of growth at 30° C., the culture broths were extracted with 20 mL of ethyl acetate. Then the respective ethyl acetate phases of ethyl acetate extractants were analyzed with GC/MS. The types of esters that were produced were listed in Table 11.
FIG. 13 depicts the GC/MS spectra of ethyl palmitate and isopropyl palmitate produced by F. jadensis T9 strain fed with ethanol and isopropanol, respectively, in the middle and lower panels. -
TABLE 11 Alcohols Esters produced Ethanol (250 μL) Ethyl palmitate Isopropanol (250 μL) Isopropyl palmitate Butanol (250 μL) Butyl palmitate Hexadecanol (250 μL, 100% W/V in hexadecanoyl hexadecanoate DMSO) Control (250 μL H2O) palmitate - This example demonstrates the production of fatty acid derivatives in a wild-type E. coli host cell in the presence of an unattenuated level of endogenous fadE. The gene fadE encodes an acyl-CoA dehydrogenase, which catalyzes the initial step in the degradation of fatty acyl-CoAs. Therefore, fadE is often considered necessary when fatty acid derivatives are made using acyl-CoAs as substrates. However, the fatty acid derivative production cells of the present invention can use acyl-ACPs as substrates. Thus the presence of an acyl-CoA dehydrogenase will not necessarily cause degradation of the substrates. This example demonstrates that, indeed, the endogenous expression of fadE can be left intact without affecting the production of fatty acid derivatives.
- Plasmid pDS57 (SEQ ID NO:23, see, Example 6), which places ES9 under the transcriptional control of the trc promoter, was transformed into an electrocompetent E. coli strain MG1655. The same plasmid was transformed into strain E. coli MG1655 D1 (or E. coli MG1655ΔfadE, see, Example 1) to serve as a comparison. The strains were grown in shake flasks and were evaluated using the standard shake flask fermentation protocol, such as the one described in Example 7, supra. When strains were induced with 1 mM IPTG, they were also supplemented with ethanol, to a final volume of 2% (v/v). Four time points were taken and the strains were analyzed for growth, and for fatty acid ethyl ester production. The results are depicted in
FIGS. 11 and 12 . Both strains had nearly identical growth profiles (FIG. 11 ) and fatty acid ethyl ester production profiles (FIG. 12 ). Strain MG1655 pDS57 was able to produce about 2 g/L FAEE, demonstrating that ES9 is capable of producing ester directly in a wild-type strain without an attenuated fadE. - It was also determined that free fatty acid accumulation was less than 20 mg/L total for each strain.
- This example provides an exemplary method for the expression of an ester synthase polypeptide in a Saccharomyces cerevisiae. Endogenous thioesterases and/or acyl-CoA synthases or other fatty acid degradation enzymes, if present, can be attenuated or functionally deleted using the appropriate methods as described herein.
- The yeast episomal plasmid (YEp)-type vector pRS425 (Christianson et al., Gene 110:119-122 (1992)) contains sequences from the
Saccharomyces cerevisiae 2 micron endogenous plasmid, a LEU2 selectable marker, and sequences based on the backbone of a multifunctional phagemid, pBluescript II SK(+). The Saccharomyces cerevisiae's strong, constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter is cloned between the SacII and SpeI sites of pRS425 using the method described by Jia et al., Physiol. Genomics 3:83-92 (2000) to produce pGPD-425 (SEQ ID NO:50). A NotI site is introduced into the BamHI site of pGPD-425, giving a NotI site flanked by BamHI sites, and this plasmid is called pY75 (SEQ ID NO:48). - An ester synthase gene of interest, such as, for example, one encoding a polypeptide selected from SEQ ID NOs:18, 24, 25, and 26, or a variant thereof, is first treated such that the DNA fragments can be ligated into the unique Nod site of pY75 (SEQ ID NO:48). Prior to its use for cloning, the pY75 vector is linearized with NotI, filled in with T4 DNA polymerase and dephosphorylated with shrimp alkaline phosphatase (New England Biolabs, Ipswich, Mass.). Plasmid DNA is then isolated using standard techniques. Restriction digests with EcoRI is conducted to identify plasmid clones in which the start codon is in proximately to the 3′-end of the GPD promoter of pY75 (sense orientation of the ester synthase gene). The plasmid is referred to as pY75_ES.
- An empty pY75 vector is used as control. Plasmid DNA of pY75_ES and plasmid DNA of pY75 empty vector are transformed into the Saccharomyces cerevisiae strain INVSC1 (Invitrogen, Carlsbad, Calif.) using standard methods such as those described in Gietz et al., Meth. Enzymol. 350:87-96 (2002). Recombinant yeast colonies are selected on DOBA media supplemented with CSM-leu (Qbiogene, Carlsbad, Calif.). The cultures are fed with suitable alcohol substrates. The resultant fatty acids and/or fatty acid derivatives are isolated from the host cell using the methods herein. The amount of fatty acids and/or fatty acid derivatives produced is determined using known techniques such as, for example, GC/MS.
- This example provides an exemplary method for the expression of an ester synthase in a Yarrowia lipolytica.
- A pFBAIN-MOD-1 vector is constructed in accordance with the methods of PCT Publication No. WO2008/147935, the disclosures of which are incorporated by reference herein. A pictorial representation of the features of this vector is presented herein as
FIG. 18 . - An ester synthase of interest is ligated to the pFBAIN-MOD-1 predigested with NcoI and NotI (SEQ ID NO:51). The ligation reaction contains 10
μL 2× ligation buffer, 1 μL T4 DNA ligase (Promega, Madison, Wis.), 3 μL (˜300 ng) of the an ester synthase polynucleotide fragment and 1 μL of pFBAIN-MOD-1 (˜150 ng). The reaction mixture is then incubated at room temperature for 2 h and used to transform E. coli Top10 Competent cells (Invitrogen, Carlsbad, Calif.). Plasmid DNAs from the transformants are recovered using a Qiagen Miniprep kit (Valencia, Calif.). Correct clones are identified by restriction mapping and the final constructs are designated pFBAIN_ES. - A clone of pFBAIN_ES and a control plasmid pFBAIN-MOD-1 are transformed into a Yarrowia lipolytica strain, for example, Y_FOAR. Y_FOAR is prepared by obtaining Yarrowia lipolytica ATCC#20362 cells and plating them on a YPD agar plate (containing 10 g/L of yeast extract (DIFCO Labs, Detroit, Mich.), 20 g/L of bacto pepton (DIFCO), and 20 g/L of glucose). The cells are subsequently streaked onto a Minimum Medium (MM) plate (containing 75 mg/L each of uracil and uridine, 6.7 g/L YNB (yeast nitrogen base) with ammonia sulfate, without amino acids, and 20 g/L glucose) containing 250 mg/L 5-FOA (Zymo Research, Orange, Calif.). Plates can be incubated at 28° C., and the resultant colonies are patched separately onto MM plates containing 200 mg/mL 5-FOA and MM plates lacking uracil and uridine. Ura3 auxotrophy is thus obtained and the resulting strain is the Y_FOAR strain.
- The cells from the transformation are plated onto MM plates lacking uracil (0.17% yeast nitrogen base (DIFCO Labs) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, pH6.1, 20 g/L agar) and maintained at 30° C. for 2 d. A few transformants are then used to inoculate individual 25 mL cultures in MM medium (0.17% YNB (DIFCO) without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, pH6.1). Each culture is allowed to grow for 2 d at 30° C., then switched to 25 mL HGM (High Glucose Medium containing 80 g/L glucose, 2.58 g/L KH2PO4, 5.36 g/L K2HPO4) and allowed to grow for 5 d at 30° C. The cultures are fed with suitable alcohol substrates.
- Total fatty species are then extracted and the production of fatty acids and/or fatty acid derivatives can be ascertained and/or measured using the methods described herein.
- This example describes the expression of an ester synthase in a cyanobacterial host cell.
- Suitable vectors are prepared in order to produce fatty acids and/or derivatives thereof in cyanobacterial host cells including, for example, cells of Synechococcus sp. PCC7002, Synechococcus elongatus PCC7942, or Synechocystis sp. PCC6803. An ester synthase of interest, for example, an atfA1 (from A. borkumensis SK2, GenBank Accession No. YP_694462), AtfA2 (from A. borkumensis SK2, GenBank Accession No. YP_693524), ES9 (from M. hydrocarbonoclasticus DSM 8789, GenBank Accession No. AB021021), ES8 (from M. hydrocarbonoclasticus DSM 8789, GenBank Accession No. AB021020), and variants thereof, optionally integrated in the chromosome under the control of a suitable promoter (e.g., PTrc), are then cloned into those vectors.
- A vector is constructed to accomplish homologous recombination in Synechococcus sp. PCC7002 pAQ1 [GenBank Accession No. NC_0050525] using 500 bp homologous regions corresponding to positions 3301-3800 and 3801-4300. A selectable marker, such as a spectinomycin resistance
cassette containing aminoglycoside 3′-adenyltrasnferase gene, aad, a promoter, and a terminator is derived from the plasmid PCL1920 (in accordance with Lerner et al., Nucleic Acids Res. 18:4631 (1990)). That selectable marker is inserted into the homologous regions. A plasmid, such as pACYC177, is prepared in accordance with Chang, et al. J. Bacteriol. 134: 1141-1156 (1978). The promoter and ribosome binding site of aminoglycoside phosphotransferase, aph, are added followed by appropriate unique cloning sites that are, for example, NdeI and EcoRI recognition sequences. This complete integration cassette is synthesized and cloned into a pUC19 vector (New England Biolabs, Ipswich, Mass.). The resulting plasmid, pLS9-7002, allows cloning and expression of a foreign gene and delivery and stable in vivo integration into Synechococcus sp. PCC7002 pAQ1. - A plasmid or synthetic operon containing an ester synthase gene (e.g., one encoding ES9) is created, and cloned into the NdeI and EcoRI sites of pLS9-7002 downstream of the aph promoter and ribosome binding site. The resultant plasmid is transformed into Synechococcus sp. PCC7002 using a method described by Stevens et al., PNAS 77:6052-56 (1980).
- In some embodiments, another vector is constructed for homologous recombination into the Synechococcus elongatus PCC7942 genome (GenBank Accession No. CP_000100) using 800 bp homologous regions corresponding to positions 2577844-2578659 and 2578660-2579467. This chromosomal location is known as neutral site one (NS1) (Mackey et al., Meth. Mol. Biol. 362:115-129 (2007). A selectable marker, such as, for example, a spectinomycin resistance cassette is derived and introduced as described above. This integration cassette is synthesized and cloned into pUC19 (New England Biolabs). The resultant plasmid, pLS9-7942-NS1, allows cloning and expression of a foreign gene and delivery and stable integration into the Synechococcus elongatus PCC7942 genome.
- A plasmid or synthetic operon comprising an ester synthase gene of interest, is created as described above, which is then cloned into the NdeI or EcoRI site of pLS9-7942-NS1. The resultant plasmid is transformed into S. elongatus PCC7942 in accordance with a method described by Mackey et al., supra.
- In some embodiments, yet another vector is constructed for homologous recombination into the Synechocystis sp. PCC6803 genome (GenBank Accession BA_000022) using 1300 to 1700 bp homologous regions corresponding to positions 2299015-2300690, and 2300691-2302056, respectively. This chromosomal location is known as neutral site RS1/2 (Shao et al., Appl. Environ. Microbiol. 68:5026-33 (2002)). A plasmid, such as pACYC177, is prepared in accordance with Chang, et al. J. Bacteriol. 134: 1141-1156 (1978). As a selectable marker, a kanamycin resistance cassette (containing aminoglycoside phosphotransferase, aph, promoter, gene and terminator) is derived from the pACYC177 plasmid, and it is added between the homologous regions. Additionally, appropriate unique cloning sites, for example, NdeI and XbaI recognitions sites are added. This integration cassette is synthesized and cloned into pUC19 (New England Biolabs). The resultant plasmid, pLS9-6803-RS, allows cloning and expression of a foreign gene and delivery and stable integration into the Synechocystis sp. PCC6803 genome.
- A plasmid or synthetic operon containing an ester synthase gene is created as described above, which is then cloned into the NdeI or XbaI site of pLS9-6803-RS. The resultant plasmid is transformed into Synechocystis sp. PCC6803 in accordance with a method described by Zang et al. J. Microbiol., 45:241-45 (2007).
- It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
Claims (18)
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14/709,232 US10316298B2 (en) | 2009-09-25 | 2015-05-11 | Production of fatty acid derivatives |
US16/427,201 US20190316099A1 (en) | 2009-09-25 | 2019-05-30 | Production of fatty acid derivatives |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US24594309P | 2009-09-25 | 2009-09-25 | |
US12/889,066 US9133406B2 (en) | 2009-09-25 | 2010-09-23 | Production of fatty acid derivatives |
US14/709,232 US10316298B2 (en) | 2009-09-25 | 2015-05-11 | Production of fatty acid derivatives |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/889,066 Division US9133406B2 (en) | 2009-09-25 | 2010-09-23 | Production of fatty acid derivatives |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/427,201 Division US20190316099A1 (en) | 2009-09-25 | 2019-05-30 | Production of fatty acid derivatives |
Publications (3)
Publication Number | Publication Date |
---|---|
US20160333325A1 US20160333325A1 (en) | 2016-11-17 |
US20170335295A9 true US20170335295A9 (en) | 2017-11-23 |
US10316298B2 US10316298B2 (en) | 2019-06-11 |
Family
ID=43778722
Family Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/889,066 Active 2032-02-01 US9133406B2 (en) | 2009-09-25 | 2010-09-23 | Production of fatty acid derivatives |
US12/889,163 Abandoned US20110162259A1 (en) | 2009-09-25 | 2010-09-23 | Production of fatty acid derivatives |
US14/709,232 Expired - Fee Related US10316298B2 (en) | 2009-09-25 | 2015-05-11 | Production of fatty acid derivatives |
US16/427,201 Abandoned US20190316099A1 (en) | 2009-09-25 | 2019-05-30 | Production of fatty acid derivatives |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/889,066 Active 2032-02-01 US9133406B2 (en) | 2009-09-25 | 2010-09-23 | Production of fatty acid derivatives |
US12/889,163 Abandoned US20110162259A1 (en) | 2009-09-25 | 2010-09-23 | Production of fatty acid derivatives |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/427,201 Abandoned US20190316099A1 (en) | 2009-09-25 | 2019-05-30 | Production of fatty acid derivatives |
Country Status (9)
Country | Link |
---|---|
US (4) | US9133406B2 (en) |
EP (2) | EP2480680B1 (en) |
CN (2) | CN105969710A (en) |
BR (1) | BR112012006681A2 (en) |
CA (2) | CA2774975C (en) |
ES (2) | ES2707747T3 (en) |
IN (1) | IN2012DN02571A (en) |
MX (1) | MX2012003472A (en) |
WO (2) | WO2011038134A1 (en) |
Families Citing this family (88)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100274033A1 (en) * | 2009-04-27 | 2010-10-28 | Ls9, Inc. | Production of fatty acid esters |
US8110670B2 (en) | 2006-05-19 | 2012-02-07 | Ls9, Inc. | Enhanced production of fatty acid derivatives |
US20100242345A1 (en) | 2006-05-19 | 2010-09-30 | LS9, Inc | Production of fatty acids & derivatives thereof |
EP2351845A1 (en) | 2007-06-01 | 2011-08-03 | Solazyme, Inc. | Renewable chemicals and fuels from oleaginous yeast |
US20100199548A1 (en) * | 2007-07-06 | 2010-08-12 | Ls9, Inc. | Systems and methods for the production of fatty esters |
WO2009085278A1 (en) | 2007-12-21 | 2009-07-09 | Ls9, Inc. | Methods and compositions for producing olefins |
RU2542374C2 (en) | 2008-04-09 | 2015-02-20 | Солазим, Инк. | Method for chemical modification of microalgae lipids, method of producing soap and soap containing fatty acid salts of saponified microalgae lipids |
CN105112454B (en) | 2008-05-16 | 2017-01-18 | Reg生命科学有限责任公司 | Methods and compositions for producing hydrocarbons |
US20110146142A1 (en) * | 2008-08-18 | 2011-06-23 | Ls9, Inc. | Systems and methods for production of mixed fatty esters |
US20100071259A1 (en) * | 2008-08-18 | 2010-03-25 | Ls9, Inc. | Systems and methods for production of mixed fatty esters |
US20100297296A1 (en) * | 2008-10-14 | 2010-11-25 | Solazyme, Inc. | Healthier Baked Goods Containing Microalgae |
US20100303989A1 (en) | 2008-10-14 | 2010-12-02 | Solazyme, Inc. | Microalgal Flour |
US20100297323A1 (en) * | 2008-10-14 | 2010-11-25 | Solazyme, Inc. | Gluten-free Foods Containing Microalgae |
ES2742527T3 (en) | 2008-11-28 | 2020-02-14 | Corbion Biotech Inc | Manufacture of customized oils in recombinant heterotrophic microorganisms |
US8835137B2 (en) | 2008-12-23 | 2014-09-16 | Matrix Genetics, Llc | Modified photosynthetic microorganisms with reduced glycogen and their use in producing carbon-based products |
MX2011006785A (en) * | 2008-12-23 | 2011-09-27 | Ls9 Inc | Methods and compositions related to thioesterase enzymes. |
CN102459569B (en) * | 2009-04-10 | 2018-02-23 | Reg生命科学有限责任公司 | The generation of derivative of fatty acid |
WO2011038134A1 (en) * | 2009-09-25 | 2011-03-31 | Ls9, Inc. | Production of fatty acid derivatives |
WO2011063304A1 (en) * | 2009-11-20 | 2011-05-26 | Opx Biotechnologies, Inc. | Methods, systems, and compositions for microbial bio-production of biomolecules using syngas components, or sugars, as feedstocks |
US8980613B2 (en) | 2010-04-06 | 2015-03-17 | Matrix Genetics, Llc | Modified photosynthetic microorganisms for producing lipids |
CA2801057C (en) | 2010-05-28 | 2019-06-18 | Solazyme, Inc. | Tailored oils produced from recombinant heterotrophic microorganisms |
CN108823254A (en) | 2010-11-03 | 2018-11-16 | 柯碧恩生物技术公司 | With reduce pour point microbial oil, from the dielectric fluid wherein generated, and associated method |
AU2011349444B2 (en) * | 2010-12-20 | 2016-06-30 | Lumen Bioscience, Inc. | Modified photosynthetic microorganisms for producing lipids |
US9096859B2 (en) | 2011-01-26 | 2015-08-04 | The Regents Of The University Of California | Microbial conversion of plant biomass to advanced biofuels |
CN110066836A (en) | 2011-02-02 | 2019-07-30 | 柯碧恩生物技术公司 | Originate from the customization oil of recombination oleaginous microorganism |
US20140215904A1 (en) | 2011-03-30 | 2014-08-07 | Ls9, Inc. | Compositions comprising and methods for producing beta-hydroxy fatty acid esters |
EP2691510A4 (en) | 2011-03-31 | 2014-11-12 | Exxonmobil Res & Eng Co | Metabolic pathway targeting by transcription factor overexpression |
BR112013028621A2 (en) | 2011-05-06 | 2016-11-29 | Solazyme Inc | "Recombinant oilseed microalgae, method for producing microalgae biomass or a product produced from biomass, and method for producing a microalgae lipid composition". |
US20130034884A1 (en) | 2011-06-22 | 2013-02-07 | Genomatica, Inc. | Microorganisms for producing 1,4-butanediol and methods related thereto |
US9399768B2 (en) | 2011-07-27 | 2016-07-26 | Iowa State University Research Foundation, Inc. | Materials and methods for using an acyl-acyl carrier protein thioesterase and mutants and chimeras thereof in fatty acid synthesis |
US8951762B2 (en) | 2011-07-27 | 2015-02-10 | Iowa State University Research Foundation, Inc. | Materials and methods for using an acyl—acyl carrier protein thioesterase and mutants and chimeras thereof in fatty acid synthesis |
ES2396823B1 (en) | 2011-08-18 | 2014-01-28 | Iden Biotechnology S.L. | BIODIESEL PRODUCTION FROM GLYCERINE. |
PT2753701T (en) | 2011-09-27 | 2018-08-10 | Exxonmobil Res & Eng Co | Acyl-acp wax ester synthases |
WO2013096475A1 (en) * | 2011-12-19 | 2013-06-27 | Joule Unlimited Technologies, Inc. | Extracellular transport of biosynthetic hydrocarbons and other molecules |
EP2607479A1 (en) | 2011-12-22 | 2013-06-26 | Evonik Industries AG | Biotechnological production of alcohols and derivatives thereof |
US20160024532A1 (en) * | 2012-02-23 | 2016-01-28 | The Regents Of The University Of California | Atp driven direct photosynthetic production of fuels and chemicals |
US8962299B2 (en) | 2012-02-29 | 2015-02-24 | Exxonmobil Research And Engineering Company | Four-gene pathway for wax ester synthesis |
BR112014024675B1 (en) | 2012-04-02 | 2021-12-07 | Genomatica, Inc | RECOMBINANT HOST CELL |
US9163267B2 (en) | 2012-04-11 | 2015-10-20 | REG Life Sciences, LLC | Metathesis transformations of microbially-produced fatty acids and fatty acid derivatives |
CN108949660A (en) | 2012-04-13 | 2018-12-07 | Reg生命科学有限责任公司 | Alkanolamide and the micro-organisms of amide amine and application thereof |
CN110358686A (en) | 2012-04-18 | 2019-10-22 | 柯碧恩生物技术公司 | Customization oil |
US9719114B2 (en) | 2012-04-18 | 2017-08-01 | Terravia Holdings, Inc. | Tailored oils |
CA2876484A1 (en) | 2012-06-15 | 2013-12-19 | Microvi Biotech Inc. | Novel biocatalyst compositions and processes for use |
WO2014012055A1 (en) | 2012-07-13 | 2014-01-16 | Calysta Energy, Llc | Biorefinery system, methods and compositions thereof |
AU2013299414A1 (en) | 2012-08-10 | 2015-03-26 | Opx Biotechnologies, Inc. | Microorganisms and methods for the production of fatty acids and fatty acid derived products |
MX366179B (en) * | 2012-09-14 | 2019-07-01 | Reg Life Sciences Llc | Enzyme variants with improved ester synthase properties. |
CN104781411B (en) * | 2012-11-09 | 2019-10-18 | 凯利斯塔公司 | The composition and method of derivative of fatty acid are generated for biology |
EP2931742A2 (en) | 2012-12-12 | 2015-10-21 | REG Life Sciences, LLC | Acp-mediated production of fatty acid derivatives |
EP3385375B1 (en) | 2013-01-16 | 2021-06-16 | Genomatica, Inc. | Acyl-acp reductase with improved properties |
US10098371B2 (en) | 2013-01-28 | 2018-10-16 | Solazyme Roquette Nutritionals, LLC | Microalgal flour |
US9816079B2 (en) | 2013-01-29 | 2017-11-14 | Terravia Holdings, Inc. | Variant thioesterases and methods of use |
US9567615B2 (en) | 2013-01-29 | 2017-02-14 | Terravia Holdings, Inc. | Variant thioesterases and methods of use |
BR112015021683A2 (en) | 2013-03-05 | 2017-07-18 | Procter & Gamble | mixed sugar compositions |
US9447438B2 (en) | 2013-03-15 | 2016-09-20 | Cargill, Incorporated | Acetyl-coA carboxylases |
US9290749B2 (en) | 2013-03-15 | 2016-03-22 | Solazyme, Inc. | Thioesterases and cells for production of tailored oils |
US9783836B2 (en) | 2013-03-15 | 2017-10-10 | Terravia Holdings, Inc. | Thioesterases and cells for production of tailored oils |
AU2014277874B2 (en) | 2013-06-14 | 2020-12-03 | Genomatica, Inc. | Methods of producing omega-hydroxylated fatty acid derivatives |
US11408013B2 (en) | 2013-07-19 | 2022-08-09 | Cargill, Incorporated | Microorganisms and methods for the production of fatty acids and fatty acid derived products |
CN106795483A (en) * | 2013-07-19 | 2017-05-31 | 嘉吉公司 | Microorganism and method for producing aliphatic acid and fatty acid derived product |
FR3009619B1 (en) | 2013-08-07 | 2017-12-29 | Roquette Freres | BIOMASS COMPOSITIONS OF MICROALGUES RICH IN PROTEINS OF SENSORY QUALITY OPTIMIZED |
CN103571764B (en) * | 2013-08-26 | 2015-06-10 | 天津科技大学 | Saccharomyces cerevisiae engineering bacterium for highly yielding medium-chain fatty acid ethyl ester as well as construction method thereof |
EP3052636A2 (en) | 2013-10-04 | 2016-08-10 | Solazyme, Inc. | Tailored oils |
US8916659B1 (en) * | 2013-10-31 | 2014-12-23 | Exxonmobil Chemical Patents Inc. | Process and apparatus for continuous solution polymerization |
WO2015157719A1 (en) | 2014-04-10 | 2015-10-15 | REG Life Sciences, LLC | Semisynthetic routes to organic compounds |
CN106604990B (en) | 2014-06-16 | 2022-04-05 | 基因组股份公司 | Omega-hydroxylase-related fusion polypeptides with improved properties |
EP3620517A3 (en) | 2014-07-10 | 2020-07-29 | Corbion Biotech, Inc. | Ketoacyl acp synthase genes and uses thereof |
BR112017001404A2 (en) | 2014-07-24 | 2017-11-21 | Terravia Holdings Inc | Variant thioesterases and methods of use |
EP2993228B1 (en) | 2014-09-02 | 2019-10-09 | Cargill, Incorporated | Production of fatty acid esters |
WO2016036751A1 (en) * | 2014-09-03 | 2016-03-10 | Coffa Gianguido | Genetically modified microbes for the biological conversion of carbonaceous materials to fatty acids |
US10125382B2 (en) | 2014-09-18 | 2018-11-13 | Corbion Biotech, Inc. | Acyl-ACP thioesterases and mutants thereof |
CN105985987B (en) * | 2015-03-02 | 2020-01-31 | 苏州安捷生物科技有限公司 | biological method for preparing fatty alcohol |
FR3040709B1 (en) * | 2015-09-03 | 2019-06-28 | Total Marketing Services | LUBRICATION ADDITIVE FOR FUEL WITH LOW SULFUR CONTENT. |
EP3173478A1 (en) * | 2015-11-25 | 2017-05-31 | Evonik Degussa GmbH | Biotechnological production of omega-functionalised carboxylic acids and esters thereof |
WO2017101987A1 (en) | 2015-12-15 | 2017-06-22 | REG Life Sciences, LLC | Omega-hydroxylase-related fusion polypeptide variants with improved properties |
CN108203701A (en) * | 2016-12-19 | 2018-06-26 | 中国科学院深圳先进技术研究院 | Complex microbial inoculum and its preparation method and application |
CN110494566A (en) | 2017-02-02 | 2019-11-22 | 嘉吉公司 | Generate the genetically modified cell of C6-C10 derivative of fatty acid |
WO2018172331A1 (en) * | 2017-03-23 | 2018-09-27 | Wageningen Universiteit | Diterminal oxidation of alkanes |
BR112019020784A2 (en) * | 2017-04-03 | 2020-04-28 | Genomatica Inc | thioesterase variants with improved activity for the production of medium chain fatty acid derivatives |
WO2018191653A1 (en) * | 2017-04-13 | 2018-10-18 | Aemetis, Inc. | Methods and compositions for biodiesel production |
US20200063169A1 (en) * | 2017-05-08 | 2020-02-27 | The University Of British Columbia | Methods and compositions for the production of lipids |
CN107338210B (en) * | 2017-09-05 | 2021-08-17 | 天津科技大学 | Abamectin streptomycete synthetic medium and preparation method of fermentation liquor thereof |
CN108048385B (en) * | 2018-01-19 | 2021-07-27 | 中国农业大学 | Strain domestication method for improving degradation efficiency of mycotoxin degrading bacteria |
CN108522430B (en) * | 2018-03-23 | 2021-02-26 | 海南大学 | Indoor breeding method for common thrips |
EP3670495A1 (en) | 2018-12-21 | 2020-06-24 | Clariant International Ltd | Glucamide-based surfactants |
CN109536426B (en) * | 2019-01-25 | 2021-11-16 | 黑龙江大学 | Psychrophile and application thereof |
CN109762046B (en) * | 2019-01-31 | 2020-10-23 | 中国科学院南海海洋研究所 | Cyclic peptide antibiotics, preparation method thereof and application thereof in preparation of anti-mycobacterium tuberculosis drugs |
GB202019175D0 (en) * | 2020-12-04 | 2021-01-20 | Univ Tartu | Biosynthesis method |
KR102421911B1 (en) * | 2022-02-16 | 2022-07-21 | 대상 주식회사 | Novel variant of zinc-binding dehydrogenase and method for preparing L-aromatic amino acid using the same |
Family Cites Families (50)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE1248643B (en) | 1959-03-30 | 1967-08-31 | The Lubrizol Corporation, Cleveland, Ohio (V. St. A.) | Process for the preparation of oil-soluble aylated amines |
US3574576A (en) | 1965-08-23 | 1971-04-13 | Chevron Res | Distillate fuel compositions having a hydrocarbon substituted alkylene polyamine |
US3980569A (en) | 1974-03-15 | 1976-09-14 | The Lubrizol Corporation | Dispersants and process for their preparation |
US4744920A (en) | 1986-12-22 | 1988-05-17 | The Lubrizol Corporation | Borated overbased material |
US5424202A (en) | 1988-08-31 | 1995-06-13 | The University Of Florida | Ethanol production by recombinant hosts |
US5000000A (en) | 1988-08-31 | 1991-03-19 | University Of Florida | Ethanol production by Escherichia coli strains co-expressing Zymomonas PDC and ADH genes |
US5482846A (en) | 1988-08-31 | 1996-01-09 | University Of Florida | Ethanol production in Gram-positive microbes |
US5028539A (en) | 1988-08-31 | 1991-07-02 | The University Of Florida | Ethanol production using engineered mutant E. coli |
US4965003A (en) | 1989-04-21 | 1990-10-23 | Texaco Inc. | Borated detergent additive by an improved process |
WO1991016427A1 (en) | 1990-04-24 | 1991-10-31 | Stratagene | Methods for phenotype creation from multiple gene populations |
US5455167A (en) | 1991-05-21 | 1995-10-03 | Calgene Inc. | Medium-chain thioesterases in plants |
US5602030A (en) | 1994-03-28 | 1997-02-11 | University Of Florida Research Foundation | Recombinant glucose uptake system |
US6428767B1 (en) | 1995-05-12 | 2002-08-06 | E. I. Du Pont De Nemours And Company | Method for identifying the source of carbon in 1,3-propanediol |
US5965408A (en) | 1996-07-09 | 1999-10-12 | Diversa Corporation | Method of DNA reassembly by interrupting synthesis |
US5939250A (en) | 1995-12-07 | 1999-08-17 | Diversa Corporation | Production of enzymes having desired activities by mutagenesis |
US6165235A (en) | 1997-08-26 | 2000-12-26 | The Lubrizol Corporation | Low chlorine content compositions for use in lubricants and fuels |
US5898023A (en) | 1998-03-24 | 1999-04-27 | Exxon Research And Engineering Co. | Lubricant additive composition for inhibiting viscosity increase and dispersency decrease |
WO2001038553A1 (en) * | 1999-11-26 | 2001-05-31 | Kansai Chemical Engineering Co., Ltd. | Process for producing fatty acid lower alcohol ester |
EP1339835A2 (en) * | 2000-10-20 | 2003-09-03 | Millennium Pharmaceuticals, Inc. | 32229, a human acyl-coa dehydrogenase family member and uses thereof |
AR038719A1 (en) * | 2002-03-01 | 2005-01-26 | Monsanto Technology Llc | DNA SEQUENCE OF BACTERIAL SYNTHEASES OF CEROSOUS ESTERES, PROTEINS AND USES OF THE SAME |
CA2496630A1 (en) * | 2002-08-30 | 2004-03-11 | North Carolina State University | Abiz phage resistance gene |
US7056714B2 (en) * | 2003-03-11 | 2006-06-06 | University Of Iowa Research Foundation, Inc. | Carboxylic acid reductase polypeptide, nucleotide sequence encoding same and methods of use |
US20070251141A1 (en) * | 2004-02-26 | 2007-11-01 | Purdue Research Foundation | Method for Preparation, Use and Separation of Fatty Acid Esters |
US7652167B2 (en) * | 2004-07-19 | 2010-01-26 | Board Of Trustees Of Michigan State University | Process for production of organic acid esters |
GB0421937D0 (en) * | 2004-10-02 | 2004-11-03 | Univ York | Acyl CoA synthetases |
EP2543726B1 (en) * | 2005-08-20 | 2018-10-03 | Scarab Genomics, LLC | Reduced genome E. coli |
EP1968994B1 (en) | 2005-12-06 | 2013-07-03 | Synthetic Genomics, Inc. | Synthetic genomes |
AU2006346810B2 (en) | 2005-12-23 | 2013-05-02 | Synthetic Genomics, Inc. | Installation of genomes or partial genomes into cells or cell-like systems |
WO2007136762A2 (en) | 2006-05-19 | 2007-11-29 | Ls9, Inc. | Production of fatty acids and derivatives thereof |
US20100242345A1 (en) * | 2006-05-19 | 2010-09-30 | LS9, Inc | Production of fatty acids & derivatives thereof |
US8110670B2 (en) * | 2006-05-19 | 2012-02-07 | Ls9, Inc. | Enhanced production of fatty acid derivatives |
US20100274033A1 (en) * | 2009-04-27 | 2010-10-28 | Ls9, Inc. | Production of fatty acid esters |
TW200819540A (en) * | 2006-07-11 | 2008-05-01 | Genelux Corp | Methods and compositions for detection of microorganisms and cells and treatment of diseases and disorders |
TWI330178B (en) * | 2006-12-29 | 2010-09-11 | Univ Nat Taiwan | Process for transesterification of methyl acetate |
WO2008113041A2 (en) * | 2007-03-14 | 2008-09-18 | Ls9, Inc. | Process for producing low molecular weight hydrocarbons from renewable resources |
BRPI0809345A2 (en) * | 2007-03-28 | 2014-10-07 | Ls9 Inc | RECOMBINANT CELLS, COMPOSITIONS PRODUCED BY THESE, METHODS FOR PRODUCING FATTY ACID DERIVATIVES IN A RECOMBINANT CELL, FOR INCREASING THE PRODUCTION OF RECOMBINANT DUCTIDOUS CONDUCTIVE PRODUCTS FOR GRADIC ACIDS A PRODUCTION HOST CELL FOR PRODUCING FATTY ACID DERIVATIVES, FATTY ACID DERIVATIVES, BIOFUEL COMPOSITIONS. |
EP2730656A1 (en) | 2007-05-24 | 2014-05-14 | E. I. du Pont de Nemours and Company | Soybean meal from beans having DGAT genes from yarrowia lipolytica for increased seed storage lipid production and altered fatty acid profiles in soybean |
US8082274B2 (en) * | 2007-06-28 | 2011-12-20 | Microsoft Corporation | Scheduling application allowing freeform data entry |
WO2009006430A1 (en) | 2007-06-29 | 2009-01-08 | The Regents Of The University Of California | Host cells and methods for producing fatty acid derived compounds |
CA2617614C (en) * | 2007-08-10 | 2012-03-27 | Indian Oil Corporation Limited | Novel synthetic fuel and method of preparation thereof |
US7897369B2 (en) * | 2007-08-27 | 2011-03-01 | Regents Of The University Of Minnesota | Isoprenoid wax ester synthases, isoprenoid acyl CoA-synthetases, and uses thereof |
CN105112454B (en) * | 2008-05-16 | 2017-01-18 | Reg生命科学有限责任公司 | Methods and compositions for producing hydrocarbons |
US20100071259A1 (en) * | 2008-08-18 | 2010-03-25 | Ls9, Inc. | Systems and methods for production of mixed fatty esters |
US20110146142A1 (en) * | 2008-08-18 | 2011-06-23 | Ls9, Inc. | Systems and methods for production of mixed fatty esters |
MX2011003597A (en) | 2008-10-07 | 2011-06-20 | Ls9 Inc | Method and compositions for producing fatty aldehydes. |
CN102459569B (en) * | 2009-04-10 | 2018-02-23 | Reg生命科学有限责任公司 | The generation of derivative of fatty acid |
WO2011038134A1 (en) * | 2009-09-25 | 2011-03-31 | Ls9, Inc. | Production of fatty acid derivatives |
US8530221B2 (en) * | 2010-01-14 | 2013-09-10 | Ls9, Inc. | Production of branched chain fatty acids and derivatives thereof in recombinant microbial cells |
PT2753701T (en) * | 2011-09-27 | 2018-08-10 | Exxonmobil Res & Eng Co | Acyl-acp wax ester synthases |
US8962299B2 (en) * | 2012-02-29 | 2015-02-24 | Exxonmobil Research And Engineering Company | Four-gene pathway for wax ester synthesis |
-
2010
- 2010-09-23 WO PCT/US2010/050026 patent/WO2011038134A1/en active Application Filing
- 2010-09-23 US US12/889,066 patent/US9133406B2/en active Active
- 2010-09-23 CN CN201610297467.6A patent/CN105969710A/en active Pending
- 2010-09-23 EP EP10819469.7A patent/EP2480680B1/en not_active Not-in-force
- 2010-09-23 CN CN201080053578.3A patent/CN102656273B/en not_active Expired - Fee Related
- 2010-09-23 EP EP15153935.0A patent/EP2910630B1/en not_active Not-in-force
- 2010-09-23 ES ES15153935T patent/ES2707747T3/en active Active
- 2010-09-23 CA CA2774975A patent/CA2774975C/en not_active Expired - Fee Related
- 2010-09-23 MX MX2012003472A patent/MX2012003472A/en active IP Right Grant
- 2010-09-23 BR BR112012006681A patent/BR112012006681A2/en not_active Application Discontinuation
- 2010-09-23 ES ES10819469.7T patent/ES2536240T3/en active Active
- 2010-09-23 US US12/889,163 patent/US20110162259A1/en not_active Abandoned
- 2010-09-23 WO PCT/US2010/050024 patent/WO2011038132A1/en active Application Filing
- 2010-09-23 CA CA3056664A patent/CA3056664A1/en not_active Abandoned
-
2012
- 2012-03-23 IN IN2571DEN2012 patent/IN2012DN02571A/en unknown
-
2015
- 2015-05-11 US US14/709,232 patent/US10316298B2/en not_active Expired - Fee Related
-
2019
- 2019-05-30 US US16/427,201 patent/US20190316099A1/en not_active Abandoned
Non-Patent Citations (1)
Title |
---|
Guo et al., (PNAS, vol. 10, no.25, June 2004, pages 9205-9210) * |
Also Published As
Publication number | Publication date |
---|---|
WO2011038132A1 (en) | 2011-03-31 |
ES2536240T3 (en) | 2015-05-21 |
US20190316099A1 (en) | 2019-10-17 |
CN105969710A (en) | 2016-09-28 |
CA2774975C (en) | 2019-11-05 |
EP2480680B1 (en) | 2015-02-11 |
EP2480680A4 (en) | 2013-11-06 |
EP2480680A1 (en) | 2012-08-01 |
CA3056664A1 (en) | 2011-03-31 |
CN102656273B (en) | 2016-05-25 |
BR112012006681A2 (en) | 2015-09-08 |
CN102656273A (en) | 2012-09-05 |
CA2774975A1 (en) | 2011-03-31 |
US10316298B2 (en) | 2019-06-11 |
US20110072714A1 (en) | 2011-03-31 |
EP2910630B1 (en) | 2018-10-24 |
US20160333325A1 (en) | 2016-11-17 |
WO2011038134A1 (en) | 2011-03-31 |
MX2012003472A (en) | 2012-05-22 |
IN2012DN02571A (en) | 2015-08-28 |
ES2707747T3 (en) | 2019-04-04 |
EP2910630A1 (en) | 2015-08-26 |
US20110162259A1 (en) | 2011-07-07 |
US9133406B2 (en) | 2015-09-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20190316099A1 (en) | Production of fatty acid derivatives | |
JP6925311B2 (en) | Enhanced production of fatty acid derivatives | |
US11130944B2 (en) | Acyl-ACP reductase with improved properties | |
US20100257778A1 (en) | Production of fatty acid derivatives | |
US20210371889A1 (en) | Methods And Compositions For Improved Production Of Fatty Acids And Derivatives Thereof | |
WO2010118410A1 (en) | Production of fatty acid derivatives | |
CA2759273A1 (en) | Production of fatty acid esters | |
US20160002681A1 (en) | Acp-mediated production of fatty acid derivatives | |
KR20090029708A (en) | Production of fatty acids and derivatives thereof | |
GAERTNER | Patent 2774975 Summary |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: REG LIFE SCIENCES, LLC, CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:BRIGITTE A. HAJOS, PATENT AGENT, REGISTRATION NUMBER 50,971, APPLICANT AUTHORITY - LEGAL REPRESENTATIVE UNDER 35 U.S.C. 117, FOR ALFRED GAERTNER, DECEASED;REEL/FRAME:035865/0975 Effective date: 20150617 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NOTICE OF ALLOWANCE MAILED -- APPLICATION RECEIVED IN OFFICE OF PUBLICATIONS |
|
STCF | Information on status: patent grant |
Free format text: PATENTED CASE |
|
AS | Assignment |
Owner name: GENOMATICA, INC., CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:REG LIFE SCIENCES, LLC;REEL/FRAME:050695/0661 Effective date: 20190529 |
|
FEPP | Fee payment procedure |
Free format text: ENTITY STATUS SET TO SMALL (ORIGINAL EVENT CODE: SMAL); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
FEPP | Fee payment procedure |
Free format text: MAINTENANCE FEE REMINDER MAILED (ORIGINAL EVENT CODE: REM.); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
LAPS | Lapse for failure to pay maintenance fees |
Free format text: PATENT EXPIRED FOR FAILURE TO PAY MAINTENANCE FEES (ORIGINAL EVENT CODE: EXP.); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
STCH | Information on status: patent discontinuation |
Free format text: PATENT EXPIRED DUE TO NONPAYMENT OF MAINTENANCE FEES UNDER 37 CFR 1.362 |
|
FP | Lapsed due to failure to pay maintenance fee |
Effective date: 20230611 |