WO2013024196A1 - Producción de biodiesel a partir de glicerina - Google Patents
Producción de biodiesel a partir de glicerina Download PDFInfo
- Publication number
- WO2013024196A1 WO2013024196A1 PCT/ES2012/070628 ES2012070628W WO2013024196A1 WO 2013024196 A1 WO2013024196 A1 WO 2013024196A1 ES 2012070628 W ES2012070628 W ES 2012070628W WO 2013024196 A1 WO2013024196 A1 WO 2013024196A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- seq
- genes
- variants
- promoter
- biodiesel
- Prior art date
Links
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 title claims abstract description 132
- 239000003225 biodiesel Substances 0.000 title claims abstract description 65
- 235000011187 glycerol Nutrition 0.000 title claims abstract description 57
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 35
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 136
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 57
- 230000001580 bacterial effect Effects 0.000 claims abstract description 37
- 238000000034 method Methods 0.000 claims abstract description 37
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 33
- 239000002551 biofuel Substances 0.000 claims abstract description 32
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 32
- 241000894007 species Species 0.000 claims abstract description 24
- 239000013612 plasmid Substances 0.000 claims description 32
- 239000013604 expression vector Substances 0.000 claims description 29
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 26
- 229930195729 fatty acid Natural products 0.000 claims description 26
- 239000000194 fatty acid Substances 0.000 claims description 26
- 150000004665 fatty acids Chemical class 0.000 claims description 21
- 230000001939 inductive effect Effects 0.000 claims description 19
- 239000000203 mixture Substances 0.000 claims description 19
- 101150008263 accD gene Proteins 0.000 claims description 18
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 17
- ZOCYQVNGROEVLU-UHFFFAOYSA-N isopentadecanoic acid Chemical compound CC(C)CCCCCCCCCCCC(O)=O ZOCYQVNGROEVLU-UHFFFAOYSA-N 0.000 claims description 17
- XKLJLHAPJBUBNL-UHFFFAOYSA-N 12-methyltetradecanoic acid Chemical compound CCC(C)CCCCCCCCCCC(O)=O XKLJLHAPJBUBNL-UHFFFAOYSA-N 0.000 claims description 14
- 125000004494 ethyl ester group Chemical group 0.000 claims description 13
- 239000006227 byproduct Substances 0.000 claims description 11
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 10
- 238000010276 construction Methods 0.000 claims description 9
- 241000023308 Acca Species 0.000 claims description 8
- 101100378010 Bacillus subtilis (strain 168) accC1 gene Proteins 0.000 claims description 8
- 101100322122 Bacillus subtilis (strain 168) accC2 gene Proteins 0.000 claims description 8
- 101100127701 Bacillus subtilis (strain 168) lcfB gene Proteins 0.000 claims description 8
- 101100135734 Haloferax mediterranei (strain ATCC 33500 / DSM 1411 / JCM 8866 / NBRC 14739 / NCIMB 2177 / R-4) pccB gene Proteins 0.000 claims description 8
- 101150046124 accA gene Proteins 0.000 claims description 8
- 101150013885 accB gene Proteins 0.000 claims description 8
- 101150070497 accC gene Proteins 0.000 claims description 8
- 101150077217 yhfL gene Proteins 0.000 claims description 8
- 150000004671 saturated fatty acids Chemical class 0.000 claims description 6
- 235000003441 saturated fatty acids Nutrition 0.000 claims description 6
- 239000000654 additive Substances 0.000 claims description 5
- 230000000996 additive effect Effects 0.000 claims description 5
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 4
- 238000000605 extraction Methods 0.000 claims description 3
- 238000002955 isolation Methods 0.000 claims description 3
- 101100493774 Escherichia coli (strain K12) bcsF gene Proteins 0.000 claims 4
- 101100241170 Bacillus subtilis (strain 168) ntdA gene Proteins 0.000 claims 2
- 101100218583 Escherichia coli (strain K12) bcsC gene Proteins 0.000 claims 2
- 238000012258 culturing Methods 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 abstract description 30
- 108010016219 Acetyl-CoA carboxylase Proteins 0.000 abstract description 29
- 102000000452 Acetyl-CoA carboxylase Human genes 0.000 abstract description 29
- 108010018763 Biotin carboxylase Proteins 0.000 abstract description 29
- 238000003786 synthesis reaction Methods 0.000 abstract description 29
- 101150087812 tesA gene Proteins 0.000 abstract description 21
- 241000588724 Escherichia coli Species 0.000 abstract description 20
- 101150066782 adhB gene Proteins 0.000 abstract description 18
- 108010011449 Long-chain-fatty-acid-CoA ligase Proteins 0.000 abstract description 17
- 102000005870 Coenzyme A Ligases Human genes 0.000 abstract description 11
- 241000588625 Acinetobacter sp. Species 0.000 abstract description 5
- 101100388296 Arabidopsis thaliana DTX51 gene Proteins 0.000 abstract description 4
- 101100215626 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) ADP1 gene Proteins 0.000 abstract description 4
- 241000207738 Maritalea mobilis Species 0.000 abstract 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 33
- 241000894006 Bacteria Species 0.000 description 29
- 230000014509 gene expression Effects 0.000 description 27
- 108090000790 Enzymes Proteins 0.000 description 25
- 102000004190 Enzymes Human genes 0.000 description 24
- 229940088598 enzyme Drugs 0.000 description 24
- 210000004027 cell Anatomy 0.000 description 22
- 239000000446 fuel Substances 0.000 description 16
- 108020004705 Codon Proteins 0.000 description 15
- 235000019441 ethanol Nutrition 0.000 description 13
- 150000001413 amino acids Chemical class 0.000 description 12
- 108090000765 processed proteins & peptides Proteins 0.000 description 11
- 102000004196 processed proteins & peptides Human genes 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 11
- 229920001184 polypeptide Polymers 0.000 description 10
- 102000003960 Ligases Human genes 0.000 description 9
- 108090000364 Ligases Proteins 0.000 description 9
- 241000588902 Zymomonas mobilis Species 0.000 description 9
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 9
- 239000003208 petroleum Substances 0.000 description 9
- XDOFQFKRPWOURC-UHFFFAOYSA-N 16-methylheptadecanoic acid Chemical compound CC(C)CCCCCCCCCCCCCCC(O)=O XDOFQFKRPWOURC-UHFFFAOYSA-N 0.000 description 8
- KEMQGTRYUADPNZ-UHFFFAOYSA-N heptadecanoic acid Chemical compound CCCCCCCCCCCCCCCCC(O)=O KEMQGTRYUADPNZ-UHFFFAOYSA-N 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 239000002773 nucleotide Substances 0.000 description 7
- 125000003729 nucleotide group Chemical group 0.000 description 7
- 230000002018 overexpression Effects 0.000 description 7
- 101100162670 Bacillus subtilis (strain 168) amyE gene Proteins 0.000 description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 102100033995 Long-chain-fatty-acid-CoA ligase 1 Human genes 0.000 description 6
- 230000004048 modification Effects 0.000 description 6
- 238000012986 modification Methods 0.000 description 6
- WQEPLUUGTLDZJY-UHFFFAOYSA-N pentadecanoic acid Chemical compound CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 101150113885 yhfT gene Proteins 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 150000002632 lipids Chemical class 0.000 description 5
- 239000003921 oil Substances 0.000 description 5
- 235000019198 oils Nutrition 0.000 description 5
- 229920006395 saturated elastomer Polymers 0.000 description 5
- 101150000850 thrC gene Proteins 0.000 description 5
- 101710186512 3-ketoacyl-CoA thiolase Proteins 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 108010029485 Protein Isoforms Proteins 0.000 description 4
- 102000001708 Protein Isoforms Human genes 0.000 description 4
- 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 4
- 230000000694 effects Effects 0.000 description 4
- 239000003925 fat Substances 0.000 description 4
- -1 glucanases Proteins 0.000 description 4
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 4
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 4
- 230000001717 pathogenic effect Effects 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 3
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 3
- 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 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 101150034686 PDC gene Proteins 0.000 description 3
- 108700005078 Synthetic Genes Proteins 0.000 description 3
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 3
- 239000004473 Threonine Substances 0.000 description 3
- 125000002252 acyl group Chemical group 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 235000014633 carbohydrates Nutrition 0.000 description 3
- 238000002485 combustion reaction Methods 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 229930195733 hydrocarbon Natural products 0.000 description 3
- 150000002430 hydrocarbons Chemical class 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 230000001131 transforming effect Effects 0.000 description 3
- FTNJQNQLEGKTGD-UHFFFAOYSA-N 1,3-benzodioxole Chemical compound C1=CC=C2OCOC2=C1 FTNJQNQLEGKTGD-UHFFFAOYSA-N 0.000 description 2
- ZONJATNKKGGVSU-UHFFFAOYSA-N 14-methylpentadecanoic acid Chemical compound CC(C)CCCCCCCCCCCCC(O)=O ZONJATNKKGGVSU-UHFFFAOYSA-N 0.000 description 2
- YZKLUEWQADEGKP-UHFFFAOYSA-N 5-(2,4-dichlorophenyl)cyclohexane-1,3-dione Chemical compound ClC1=CC(Cl)=CC=C1C1CC(=O)CC(=O)C1 YZKLUEWQADEGKP-UHFFFAOYSA-N 0.000 description 2
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 2
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 2
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 2
- 102000013142 Amylases Human genes 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 2
- 101100127693 Bacillus subtilis (strain 168) lcfA gene Proteins 0.000 description 2
- FXUKWLSZZHVEJD-UHFFFAOYSA-N C16:0-14-methyl Natural products CCC(C)CCCCCCCCCCCCC(O)=O FXUKWLSZZHVEJD-UHFFFAOYSA-N 0.000 description 2
- 239000004215 Carbon black (E152) Substances 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 108010078791 Carrier Proteins Proteins 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 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
- 101710088194 Dehydrogenase Proteins 0.000 description 2
- 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 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-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
- 101100281840 Neosartorya fumigata (strain ATCC MYA-4609 / Af293 / CBS 101355 / FGSC A1100) fumR gene Proteins 0.000 description 2
- 108010011939 Pyruvate Decarboxylase Proteins 0.000 description 2
- 102000005488 Thioesterase Human genes 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
- 150000001298 alcohols Chemical class 0.000 description 2
- 150000001299 aldehydes Chemical class 0.000 description 2
- 150000001336 alkenes Chemical class 0.000 description 2
- 235000019418 amylase Nutrition 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
- 244000052616 bacterial pathogen Species 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
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 238000011138 biotechnological process Methods 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000013611 chromosomal DNA Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000002283 diesel fuel Substances 0.000 description 2
- 230000008030 elimination Effects 0.000 description 2
- 238000003379 elimination reaction Methods 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 101150055019 fapR gene Proteins 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- IIUXHTGBZYEGHI-UHFFFAOYSA-N iso-margaric acid Natural products CC(C)CCCCCCCCCCCCCC(O)=O IIUXHTGBZYEGHI-UHFFFAOYSA-N 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 230000037353 metabolic pathway Effects 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 108020002982 thioesterase Proteins 0.000 description 2
- 239000010891 toxic waste Substances 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- 235000015112 vegetable and seed oil Nutrition 0.000 description 2
- 239000008158 vegetable oil Substances 0.000 description 2
- LNAZSHAWQACDHT-XIYTZBAFSA-N (2r,3r,4s,5r,6s)-4,5-dimethoxy-2-(methoxymethyl)-3-[(2s,3r,4s,5r,6r)-3,4,5-trimethoxy-6-(methoxymethyl)oxan-2-yl]oxy-6-[(2r,3r,4s,5r,6r)-4,5,6-trimethoxy-2-(methoxymethyl)oxan-3-yl]oxyoxane Chemical compound CO[C@@H]1[C@@H](OC)[C@H](OC)[C@@H](COC)O[C@H]1O[C@H]1[C@H](OC)[C@@H](OC)[C@H](O[C@H]2[C@@H]([C@@H](OC)[C@H](OC)O[C@@H]2COC)OC)O[C@@H]1COC LNAZSHAWQACDHT-XIYTZBAFSA-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
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 108700003860 Bacterial Genes Proteins 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 102000005575 Cellulases Human genes 0.000 description 1
- 108010084185 Cellulases Proteins 0.000 description 1
- 108091026890 Coding region Proteins 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
- 108020004414 DNA Proteins 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 102100023903 Glycerol kinase Human genes 0.000 description 1
- 108700016170 Glycerol kinases Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000937642 Homo sapiens Malonyl-CoA-acyl carrier protein transacylase, mitochondrial Proteins 0.000 description 1
- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
- 108010054278 Lac Repressors Proteins 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 101100504994 Lactococcus lactis subsp. lactis (strain IL1403) glpO gene Proteins 0.000 description 1
- 101100419195 Leptospira borgpetersenii serovar Hardjo-bovis (strain L550) rpsC2 gene Proteins 0.000 description 1
- 101100363550 Leptospira borgpetersenii serovar Hardjo-bovis (strain L550) rpsE2 gene Proteins 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
- 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 1
- 101710137760 Malonyl-CoA-acyl carrier protein transacylase, mitochondrial Proteins 0.000 description 1
- 102100027329 Malonyl-CoA-acyl carrier protein transacylase, mitochondrial Human genes 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
- 101100037096 Methanococcus maripaludis (strain S2 / LL) rpl6 gene Proteins 0.000 description 1
- 101100254826 Methanopyrus kandleri (strain AV19 / DSM 6324 / JCM 9639 / NBRC 100938) rps5 gene Proteins 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108020001027 Ribosomal DNA Proteins 0.000 description 1
- 102000002278 Ribosomal Proteins Human genes 0.000 description 1
- 108010000605 Ribosomal Proteins Proteins 0.000 description 1
- 229920002472 Starch Polymers 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
- 101100147268 Symbiobacterium thermophilum (strain T / IAM 14863) rpsD1 gene Proteins 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108700029229 Transcriptional Regulatory Elements Proteins 0.000 description 1
- 101000872823 Xenopus laevis Probable histone deacetylase 1-A Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000011481 absorbance measurement Methods 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
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 108010064926 acyl-CoA carboxylase Proteins 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
- 230000003321 amplification Effects 0.000 description 1
- 101150105363 amyE gene Proteins 0.000 description 1
- 229940025131 amylases Drugs 0.000 description 1
- 239000012736 aqueous medium 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
- 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 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
- 239000012620 biological material Substances 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000004136 fatty acid synthesis Effects 0.000 description 1
- 150000002194 fatty esters Chemical class 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000021588 free fatty acids Nutrition 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 239000002816 fuel additive Substances 0.000 description 1
- 230000002538 fungal effect Effects 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
- 239000003502 gasoline Substances 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 101150081661 glpD gene Proteins 0.000 description 1
- 101150020594 glpD1 gene Proteins 0.000 description 1
- 101150071897 glpF gene Proteins 0.000 description 1
- 101150056064 glpK gene Proteins 0.000 description 1
- 101150040073 glpK2 gene Proteins 0.000 description 1
- 101150095702 glpT gene Proteins 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 150000001261 hydroxy acids Chemical class 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000002440 industrial waste Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 150000004715 keto acids Chemical class 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 238000005461 lubrication Methods 0.000 description 1
- 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 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- NJTGANWAUPEOAX-UHFFFAOYSA-N molport-023-220-454 Chemical compound OCC(O)CO.OCC(O)CO NJTGANWAUPEOAX-UHFFFAOYSA-N 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 230000000529 probiotic effect Effects 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 101150060526 rpl1 gene Proteins 0.000 description 1
- 101150079275 rplA gene Proteins 0.000 description 1
- 101150015255 rplB gene Proteins 0.000 description 1
- 101150077293 rplC gene Proteins 0.000 description 1
- 101150028073 rplD gene Proteins 0.000 description 1
- 101150034310 rplF gene Proteins 0.000 description 1
- 101150037704 rplJ gene Proteins 0.000 description 1
- 101150018028 rpsC gene Proteins 0.000 description 1
- 101150087540 rpsD gene Proteins 0.000 description 1
- 101150114376 rpsD2 gene Proteins 0.000 description 1
- 101150027173 rpsE gene Proteins 0.000 description 1
- 101150036132 rpsG gene Proteins 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 1
- 229960000268 spectinomycin Drugs 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000005809 transesterification reaction Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
Classifications
-
- 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
- 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
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- 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.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- 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.)
-
- 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
- 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
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
-
- 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
- C12N9/88—Lyases (4.)
-
- 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
- C12N9/93—Ligases (6)
-
- 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
- C12P7/6409—Fatty acids
-
- 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
-
- 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
- C10L2200/00—Components of fuel compositions
- C10L2200/04—Organic compounds
- C10L2200/0461—Fractions defined by their origin
- C10L2200/0469—Renewables or materials of biological origin
- C10L2200/0476—Biodiesel, i.e. defined lower alkyl esters of fatty acids first generation biodiesel
-
- 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
Definitions
- the present invention discloses a process for the synthesis of ethyl esters of fatty acids, also known as biodiesel (FAEE) from glycerin or glycerol. Therefore, the present invention can be included in the field of industrial biorefineries. STATE OF THE TECHNIQUE
- Crude glycerin or glycerol is a byproduct of the biodiesel manufacturing process in industrial biorefineries.
- the main function of biodiesel is its use as a fuel in substitution of petroleum-derived fuels, due to the inherent problems generated by the use of petroleum-derived fuels, in terms of pollution, greenhouse effect and global warming, thus existing the need for a renewable source of oil that can also be produced economically.
- biodiesel can be used in most diesel internal combustion engines, either in pure form, what is known as “clean biodiesel,” or as a mixture at any concentration with petroleum-derived diesel fuel.
- Biodiesel offers advantages over petroleum diesel derivatives, including the reduction of gas emissions during combustion, in addition to being able to maintain a balanced carbon dioxide cycle, since it is based on the use of renewable biological materials, It is biodegradable and gives greater security due to its high flash point and low flammability.
- Another advantage is the less wear of the engines, thanks to the good lubrication properties it presents.
- biodiesel itself is obtained through an alternative process that includes using said glycerin (glycerol) as a substrate in bacterial fermentation processes.
- Biodiesel is mainly obtained, from vegetable oils or animal fats, through industrial esterification and transesterification processes (Figure 1) (Karmakar A., et al 2010).
- Figure 1 Korean A., et al 2010
- a plant-based biodiesel plant that generates around 250,000 tons of biodiesel / year produces 10% glycerin, that is, about 25,000 tons / year of glycerin.
- biotechnological processes aimed at the conversion of crude glycerin into products with high added value is a "necessity".
- biotechnological processes could be incorporated into existing biodiesel plants, where the glycerin generated as a byproduct of the manufacturing of said biodiesel would be processed in situ, or give rise to biorefineries that integrate multiple processes, in a concept similar to that used for industrial refineries, which produce multiple fuels and products from oil.
- Kalscheuer R. et al. Demonstrated that by the heterologous expression in E. coli of the enzymes pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (AdhB) of Zymomonas mobilis and the nonspecific synthetase (Atfl) of Acinetobacter cepa baylyi ADP1, production is achieved of FAEE (Kalscheuer R. et al 2008). Said synthesis of ethanol was combined with the subsequent esterification thereof with the acyl residues of Coenzyme A fatty acids when the bacteria were grown in aerobic conditions, in the presence of glucose and oleic acid.
- the purpose of obtaining said bacterial strains, capable of synthesizing FAEE from glycerol, is that said strains are capable of secreting synthesized FAEE abroad.
- the main bacterial strains used so far in the state of the art to obtain FAEE are mainly pathogenic bacteria (Z. mobilis and E. coli), so, once synthesized the FAEE, the remaining bacteria that remain in the environment, being toxic, can be a problem of toxic waste management, since, for example, E. coli is responsible for the enteric contamination of drinking water (coliforms) .
- the present invention describes a method of producing biodiesel (FAEE) from glycerin as a carbon source, by transforming non-pathogenic bacteria, of the genus Bacillus, with different heterologous synthetic mutated genes, involved in the synthesis of said FAEE.
- Bacillus genus bacteria are approved for consumption in humans and are used on a large scale in the industry as biofactories for the production of different enzymes and proteins (Westers et al. 2004). More specifically, the invention discloses bacteria of the Bacillus subtilis species transformed with heterologous synthetic mutated genes, specifically designed to increase their expression in the bacteria of said species.
- pyruvate decarboxylase (pdc) enzyme genes SEQ ID NO: 1 and alcohol dehydrogenase (adhB) (SEQ ID NO: 3) belonging to Zymomonas mobilis, in addition to the active portion of the enzyme thioesterase A ('tesA) (SEQ ID NO: 5) of Escherichia coli and triacylglycerols synthetase and waxes ⁇ atfl ) (SEQ ID NO: 7) of Acinetobacter sp. ADP1.
- subtilis giving rise to the sequences SEQ ID NO: 18 for the pdc gene, SEQ ID NO: 19 for the adhB gene, SEQ ID NO: 20 or 21, respectively, for gene 'tes A and SEQ ID NO: 22 for the atfl gene, respectively.
- at least one of the B. subtilis genes encoding the acyl-CoA synthetase enzymes can be overexpressed therein. (IcfA, yhfL and yhfT) (EC 6.2.1.3) and for the four subunits of the enzyme acetyl-CoA carboxylase (accDABC) (EC 6.4.1.2). It is interesting to indicate that, in addition, bacteria of the genus B. subtilis are able to grow using glycerol as the sole source of carbon and energy.
- B. subtilis is as much or more efficient than E. coli (Westers et al, 2004).
- B. subtilis is not pathogenic and is in fact used as a probiotic for animals for human consumption.
- the FAEE would be secreted out of the cell and the remaining bacteria, instead of being a toxic waste management problem, would have added value, while E. coli or K. pneumoniae could not be used for this purpose, being pathogenic .
- B. subtilis efficiently secretes hydrolytic enzymes, such as glucanases, amylases, lipases, cellulases, etc., which allows such bacteria to be adapted for the degradation of other cheap compounds, such as carbon sources, which are not directly assimilable by E. coli or others organisms.
- the invention describes strains of the species B. subtilis capable of increasing the production of biodiesel from the by-product itself, glycerin, as a carbon source, being transformed with heterologous synthetic mutated genes, capable of inducing a more efficient expression in B. subtilis, obtained based on the genes: pdc (SEQ ID NO: 1) and adhB (SEQ ID NO: 3) originating from Z. mobilis, 'tesA (SEQ ID NO: 5) originating from E.
- strains of the present invention over-express at least one of the genes of the ACC (acetyl-CoA carboxylase) (E.C. 6.4.1.2) and acyl CoA synthetase (E.C. 6.2.1.3) enzyme complexes.
- the present invention describes, in turn, the use of said strains for the synthesis of biodiesel and a method of obtaining biodiesel, from glycerin as a carbon source, based on the use of said strains.
- the present invention describes bacterial strains of the species B. subtilis, preferably strains CECT 7968, CECT 7969 and NCIMB 42026, which have been transformed with heterologous synthetic mutated genes optimized for a more efficient expression in B. subtilis and that in addition, to increase the yield in the production of biodiesel through the use of said bacterial strains, they overexpress at least one of the bacterial genes that code for the ACC enzyme complexes (EC 6.4.1.2) and acyl CoA synthetase (EC 6.2.1.3), thus giving rise to a greater production of biodiesel from the by-product itself, glycerin, as a carbon source.
- ACC enzyme complexes EC 6.4.1.2
- acyl CoA synthetase EC 6.2.1.3
- the heterologous mutated synthetic genes, with which the B. subtilis bacteria of the present invention are transformed are: pdc (SEQ ID NO: 2 or SEQ ID NO.18) and adhB (SEQ ID NO: 4 or SEQ ID NO: 19) obtained from the pdc (SEQ ID NO: 1) and adhB (SEQ ID NO: 3) genes of Z. mobilis, 'tesA (SEQ ID NO: 6 or SEQ ID NO: 17 or SEQ ID NOs: 20-21) obtained from the ' tesA (SEQ ID NO: 5) gene of E.
- the bacterial strains described in the present invention overexpress at least one of the genes encoding the ACC (acetyl-CoA carboxylase) enzyme complexes (EC 6.4.1.2) and acyl CoA synthetase (EC 6.2.1.3) .
- Another object described in the present invention relates to the use of the strains described above, preferably strains CECT 7968, CECT 7969 and NCIMB 42026, for the production of biodiesel from glycerin as a carbon source.
- Another object of the present invention relates to a biodiesel production process, from glycerin as a carbon source, by using the bacterial strains described in the present invention (CECT 7968, CECT 7969 and NCIMB 42026).
- the biodiesel composition thus produced can be used as a diesel fuel alone, or mixed with petroleum diesel according to the usual proportions, resulting in cleaner emission profiles.
- a fuel composition including, for example, a diesel composition, comprising a fatty ester produced by a method or a genetically modified microorganism as described in the present invention.
- the fuel composition further comprises one or more suitable fuel additives.
- the fuel obtained by the process described in the present invention using the strains CECT 7968, CECT 7969 and NCIMB 42026 is preferably formed by ethyl esters of branched and fully saturated fatty acids, odd number of carbons, said ethyl being preferably ethyl -esters of fatty acids: iso-C15 (13-methyltetradecanoic acid), anteiso-C15: 0 (12-methyltetradecanoic acid), n-C15: 0 (n-pentadecanoic acid); iso-C17: 0 (15-methylhexadecanoic acid), anteiso-C17: 0 (14-methylhexadecanoic acid) and n-C17: 0 (n-heptadecanoic acid).
- the fuel obtained by the procedure described previously can also be formed by ethyl esters of branched fatty acids of even number of carbons (although these types of fatty acids are minor), said ethyl esters being preferably: iso-C16: 0 (14 -methylpentadecanoic acid), n-C16: 0 (n- hexadecanoic acid), iso-C18: 0 (16-methylheptadecanoic acid) and n-C18: 0 (n-octadecanoic acid); as described, for both cases (ethyl esters of odd number or even carbon), in Example 3.
- biodiesel is a biofuel that can be a substitute for petroleum-derived diesel.
- Biodiesel can be used in diesel internal combustion engines, either in pure form, what is known as “clean", or as a mixture of any concentration with petroleum-derived diesel.
- the biodiesel may include esters or hydrocarbons, such as aldehydes, alkenes or alkenes.
- biofuel refers to any type of fuel derived from biomass or biological sources, in general. Biofuels can replace petroleum-based fuels.
- biofuels include all transport fuels (for example, gasoline, diesel, jet fuel, etc.), heating fuels, and fuels for generating electricity. Biofuels are a renewable energy source.
- carbon source refers to a substrate or compound suitable for use as a supplier of carbon atoms for the growth of simple prokaryotic or eukaryotic cells.
- Carbon sources can be in various forms, including, but not limited to, organic fatty acids, such as succinate, lactate and acetate, polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides. and gases (for example, CO and C0 2 ).
- the carbon source can also be a product of photosynthesis, including, but not limited to, glucose.
- a preferred carbon source is glycerol or glycerin.
- promoter element refers to a DNA sequence that functions as a switch that activates the expression of a gene, thanks to its interaction with RNA polymerase If the gene is active, it is said to be transcribed or participates in the transcript. Transcription involves the mRNA synthesis of the gene.
- a promoter therefore, acts as a transcriptional regulatory element and also provides a site for the initiation of gene transcription in mRNA. Depending on the level of expression, promoters can be grouped into strong or weak promoters.
- a strong promoter is one that, by possessing a great affinity for RNA polymerase, allows a large amount of RNA to be formed that will ultimately result in proteins (when its expression is allowed).
- a weak promoter has a low affinity for RNA polymerase, and consequently, under its control the gene expression is much lower.
- promoters can be classified as constitutive or adjustable promoters.
- a promoter is said to be constitutive when it allows the expression of the gene or genes that are under its control to occur continuously.
- adjustable promoters only allow the expression of the genes that control when their product is essential.
- They can be inducible if they only allow the expression of the gene or genes that they regulate after a certain stimulus (in the presence of induction conditions), and repressible, when the expression of the genes under their control is continuous and only stops after a certain stimulus or under certain conditions (under repression conditions).
- glycosylin and “glycerol” are synonymous and used interchangeably.
- region is described as a group of genes regulated by the same transcription factor. Regulated genes can be grouped, in the case of operons, or dispersed throughout the genome.
- operon is defined as a functional genetic unit formed by a group or complex of genes that are transcribed from the same promoter and its expression is coordinated by the same systems.
- heterologous is defined relative to genes, proteins, tissues, cells, etc., as belonging to an individual of a different genus or species from that of the individual from which it is isolated.
- host cell or organism or “host cell or organism” refers to a cell or organism that can be genetically modified to express, over-express or infra-express, selected specific genes.
- host cells or organisms include plant cells, animals, human, bacterial or fungal.
- Preferred host cells in the present invention are bacterial cells.
- the term “over-express” or “over-expression” refers to a higher level of expression or concentration of a nucleic acid, polypeptide, protein, enzyme, carbohydrate, fat, hydrocarbon, etc. , in a given cell compared to the level of expression or concentration of said nucleic acid, polypeptide, protein, enzyme, carbohydrate, fat, hydrocarbon, in the same type of cell but belonging to a wild phenotype.
- a polypeptide can be "overexpressed” in a recombinant host cell when the polypeptide is present in a higher concentration than in the non-recombinant cell of the same species.
- recombinant polypeptide refers to a polypeptide that is produced by recombinant DNA techniques, where generally, the DNA or RNA encoding the expressed polypeptide is inserted into a suitable expression vector that at its It is once used to transform a host cell to produce the polypeptide or RNA.
- imitated genes refers to the introduction of mutations in the coding region of genes in order to increase protein production.
- the criteria used to carry out the mutations are described in Example 2 of the present invention.
- the term “synthetic” refers to gene or protein sequences obtained artificially by genetic engineering procedures.
- the term “comprises” or “comprising”, throughout the description of the present patent, means the presence of the characteristics, elements, integers, stages or components, especially DNA sequences or protein sequences, which are mentioned in the claims, but that does not exclude the presence or addition of one or more other characteristics, elements, integers, stages, components, sequences or groups thereof.
- the term “comprises” or “comprising” is also intended to include embodiments encompassed by the terms “consisting essentially of” and “consisting of”. Likewise, the term “consists essentially of” is intended to include embodiments comprised of the term “consisting of”.
- variant used in the present invention is understood as a nucleotide sequence of the genes described in the present invention or even as a sequence of amino acids of a protein or polypeptide, encoded by the nucleotide sequences mentioned above, modified in one or more of its nucleotides or even amino acids.
- variant preferably refers to a modification of one or more nucleotides in a given gene sequence.
- the variant may have "conservative" changes, in which the nucleotide or substituted amino acid has structural or chemical properties similar to the nucleotide or substituted amino acid.
- the variant may also have "non-conservative" changes or a deletion and / or insertion of one or more nucleotides or amino acids.
- the variant is preferably a conservative variant.
- a "functional variant” for the purposes of the present invention is understood as a variant that retains the functional capacity of the original nucleotide or amino acid sequence without modifications or mutations and from which it is derived.
- polynucleotide or polypeptide variants are functional variants that have the same function as the original sequences without modifications or mutations, from which they derive.
- additive refers to a chemical added to a product to improve its properties and ensure the proper functioning of the engines.
- Figure 1 Diagram of the synthesis of biodiesel from vegetable oils and / or animal fats.
- FIG. 1 Diagram of the modified metabolic pathways in B. subtilis to increase the synthesis of biodiesel from glycerin.
- the genes shown within squares are heterologous synthetic mutated genes, which belong to species other than B. subtilis and, which have been obtained synthetically, optimizing the use of codons specific to increase the expression of said genes in B. subtilis.
- the genes that appear included in an ellipse are the own genes present in B. subtilis that, alternatively, can be overexpressed in these bacteria, by means of specific promoters to increase and enhance the production of biodiesel .
- glpF glycerol facilitator
- glpT glycerol-3P transporter
- glpK glycerol kinase
- glpD glycerol-3P dehydrogenase
- pdc pyruvate decarboxylase
- adhB alcohol dehydrogenase
- atfl wax-triacylglyceride synthetase
- pdh pyruvate dehydrogenase
- accDABC acetyl-CoA carboxylase
- fabD malonyl-CoA: ACP transferase
- lcfA, yhfL, yhfT acyl-CoA synthetases.
- the present invention describes bacterial strains of the species B. subtilis, preferably strains CECT 7968, CECT 7969 and NCIMB 42026, capable of producing biodiesel (FAEE) from glycerin as a carbon source.
- Said bacterial strains of the species B. subtilis are transformed with an expression vector that expresses the plasmid pNAKA62 (in strains CECT 7968 and CECT 7969) or pNAKA143 (in strain NCIMB 42026), which carry the heterologous synthetic mutated genes: pdc (SEQ ID NO: 2 or SEQ ID NO: 18 or variants thereof) and adhB (SEQ ID NO: 4 or SEQ ID NO: 19 or variants thereof) originating from Z.
- pdc SEQ ID NO: 2 or SEQ ID NO: 18 or variants thereof
- adhB SEQ ID NO: 4 or SEQ ID NO: 19 or variants thereof
- 'tesA SEQ ID NO: 6 or SEQ ID NO: 17 or SEQ ID NOs: 20-21 or variants thereof
- atfl SEQ ID NO: 8 or SEQ ID NO: 22 or variants thereof
- said promoter is the Pspac promoter, inducible by isopropyl-D-thiogalactoside (IPTG).
- IPTG isopropyl-D-thiogalactoside
- the bacteria CECT 7968 CECT 7969 and NCIMB 42026 described in the present invention, over-express at least one of the genes encoding the enzymes ACC (EC 6.4.1.2) and Acil CoA synthetase (EC 6.2.1.3), thus enhancing the synthesis of biodiesel from glycerol, since these genes are expressed in very little (ACC) or zero amount (Acil CoA synthetase) in the exponential phase of bacterial growth.
- the bacterial strains CECT 7968, CECT 7969 and NCIMB 42026 show an increase in the synthesis of fatty acids and ethanol thanks to the transformation of the same with the expression vector pDR67 that carries the plasmid pNAKA62 or the plasmid pNAKA143, both capable of expressing,
- the four heterologous synthetic mutated genes SEQ ID NO: 2 or SEQ ID NO: 18 or variants thereof, SEQ ID NO: 4 or SEQ ID NO: 19 or variants thereof, SEQ ID NO: 6 or SEQ ID NO: 17 or SEQ ID NOs: 20-21 or variants thereof and SEQ ID NO: 8 or SEQ ID NO: 22, or variants thereof, and also, alternatively, said strains, by transformation thereof with a vector that carries a plasmid capable of over-expressing at least one of the genes of the ACC enzyme complex (EC 6.4.1.2) and / or acyl CoA synthetase (EC 6.2.1.3), increase the production
- the genes shown in Figure 2 within squares are heterologous synthetic mutated genes, which belong to species other than B. subtilis and, which have been obtained synthetically, optimizing the use of specific codons for this organism.
- the genes that appear included in an ellipse are the genes that, alternatively, can be overexpressed in said bacteria, by specific promoters, such as, for example, promoters inducible by IPTG (Pspac or Pspachy) and / or strong and constitutive promoters (Pacp), to increase and enhance the production of biodiesel.
- promoters facilitate and increase the production of biodiesel thanks to the increase in the expression of the genes of the ACC complexes (EC 6.4.1.2) and acyl-CoA synthetase (EC 6.2.1.3), during the exponential phase of the growth of B. subtilis , since its expression is low, in the case of the accDABC complex, or zero, in the case of the acyl-CoA synthetases complex.
- the increase in the synthesis and concentration of fatty acids produced by the modification of the metabolic pathways of the strains of the invention is a consequence of the expression of the heterologous synthetic mutated gene that codes for the enzyme ' tesA (SEQ ID NO: 6 or SEQ ID NO: 17 or SEQ ID NOs: 20-21 or variants thereof) originating in E. coli.
- Said enzyme catalyzes the hydrolysis of the thioester bond between acyl groups and the acyl transporter protein (ACP).
- ACP acyl transporter protein
- the strains of the present invention express the mutated catalytic domain of E. coli thioesterase ' tesA (SEQ ID NO: 6 or SEQ ID NO: 17 or variants thereof), by optimizing the use of specific codons for B. subtilis bacteria.
- sequences coding for restriction enzyme sites were added at the ends of SEQ ID NO: 6 and SEQ ID NO: 17 specific, as well as the ribosome binding sequence, giving rise to SEQ ID NO: 20 and 21, respectively.
- any specific sequence of restriction enzymes other than those used in the present invention can be used for the same purpose.
- the bacterial strains described in the present invention can over-express the ACC enzyme complex and in turn, to enhance and increase the yield in Biodiesel synthesis can also overexpress the acyl-CoA ligase enzyme complex.
- Both complexes are overexpressed by transforming them with expression vectors that carry plasmids under the control of specific strong and constitutive promoters, such as the Pacp-accDABC promoter for overexpression of the acyl CoA carboxylase (ACC) or inducible promoters, such as the Pspac or Pspachy promoter, inducible in the presence of IPTG.
- the elongation cycle of fatty acids in B. subtilis is carried out by fatty acid synthetase (FAS).
- FAS fatty acid synthetase
- the FabF isoform of the fatty acid synthetase catalyzes the condensation of malonyl-ACP with acyl-ACP to produce elongation of the fatty acid
- the FabG isoform reduces the keto acid formed
- the FabZ isoform catalyzes the dehydration of the hydroxy acid
- the FabI isoform (and eventually FabL) reduces the double bond formed, generating a new acyl-ACP with two more carbons than at the beginning of the cycle.
- the FAS enzyme complex described in Figure 2, can be overexpressed in the B.
- subtilis strains of the present invention to increase the yield on the synthesis of biodiesel from glycerin as a carbon source.
- over-expression can be carried out by different strategies.
- One such strategy involves the elimination of the global repressor of fatty acid synthesis, FapR (fapR), which implies an FAS activity, in B. subtilis 5 times greater than in strains that had said repressor (fapR + ) (Schujman , GE et al. 2003).
- FapR the global repressor of fatty acid synthesis
- FapR + fatty acid synthesis
- Another strategy that can be used is the over-expression of the ACC enzyme complex (EC6.4.1.2). Said complex catalyzes the synthesis of malonyl-CoA from acetyl-CoA.
- strains of the present invention can, in order to increase the yield in biodiesel synthesis, over-express the genes comprising said ACC enzyme complex (accD, accA, accB and accC).
- accDABC a synthetic operon, which comprises the four genes together, while in the untransformed wild bacteria, the accDA and accBC operons are found separately.
- the synthetic accDABC operon obtained was included in the plasmid pGES468 under the control of a promoter that, as mentioned above, can be strong and constitutive, such as the Yacp-accDABC promoter, or inducible such as Pspac- accDABC, in the presence of, for example, IPTG, although any known promoter can be used gone in the state of the art.
- the heterologous mutated gene ' tesA (SEQ ID NO: 6 or SEQ ID NO: 17 or SEQ ID Nos: 20- 21 or variants thereof).
- the increase in the synthesis of ethanol, produced by the strains described in the present invention has been obtained by expressing the heterologous synthetic mutated genes encoding the pdc enzymes (SEQ ID NO: 2 or SEQ ID NO: 18 or variants of them) and adhB (SEQ ID NO: 4 or SEQ ID NO: 19 or variants thereof) of Z. mobilis.
- acetyl-CoA From acetyl-CoA, said enzymes, pdc and adhB, generate acetaldehyde and subsequently ethanol.
- the pdc and adhB genes of the bacterial species Z. mobilis have been successfully used to transform Gram-negative bacteria, such as E. coli (Kalscheuer, R., et al, 2006).
- the strains described in the present invention are able to esterify the synthesized ethanol together with the acyl moieties attached to the acyl-CoA resulting in the production of biodiesel from glycerin, obtained as a byproduct of the biodiesel manufacturing process in biorefineries, as a carbon source (see Figure 2).
- the microorganisms used in the present invention were deposited in the Spanish Type Culture Collection (CECT) located in the Research Building of the University of Valencia, Burjassot Campus, Burjassot 46100 (Valencia, Spain), with deposit number:
- the other microorganism used in the present invention has been deposited on 10
- NCIMB National Collections of Industrial, Food and Marine Bacteria
- NCIMB National Collections of Industrial, Food and Marine Bacteria
- thrC lacI-Pspachy-yhfL amyE:: Pspac-pdc-adh-atfl-'tesA.
- One of the objects of the present invention relates to a gene construct comprising at least the genes selected from SEQ ID NO: 2 or SEQ ID NO: 18, SEQ ID NO: 4 or SEQ ID NO: 19, SEQ ID NO: 6 or SEQ ID NO: 17 or SEQ ID NO: 20 or SEQ ID NO: 21 and SEQ ID NO: 8 or SEQ ID NO: 22, or variants thereof, together with a promoter.
- the gene construct described in the present invention comprises at least the genes selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 17 and SEQ ID NO: 8, or variants thereof, together with a promoter.
- the gene construct described herein comprises at least the genes selected from SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 and SEQ ID NO: 22, or variants thereof, together with a promoter.
- the gene construct is characterized in that it further comprises at least one of the nucleotide sequences or variants thereof, which encode for at least one of the genes that form the Acyl CoA synthetase enzyme complexes (EC6 .2.1.3) and / or ACC (EC6.4.1.2) selected from: IcfA, yhfL, yhfl 1 , accD, accA, accB or accC and / or any combination thereof.
- the gene construct is characterized in that said genes belong to the bacterial species B. subtilis.
- the gene construct described in the present invention is characterized in that the promoter is inducible or constitutive.
- the inducible promoter is preferably the Pspac promoter or the Pspachy promoter, inducible by the addition in the culture medium of isopropyl-D-thiogalactoside (IPTG).
- the constitutive promoter is preferably the Pacp promoter. Any other promoter, either inducible or constitutive, known in the state of the art for the same purpose, can also be used.
- the gene construct described in the present invention is characterized in that it is selected from any of the following: pNAKA62, pNAKA 143, pNAKA52, pNAKA53 and / or combinations thereof.
- Another object described in the present invention relates to an expression vector characterized by comprising the sequences SEQ ID NO: 2 or SEQ ID NO: 18, SEQ ID NO: 4 or
- SEQ ID NO: 19 SEQ ID NO: 6 or SEQ ID NO: 17 or SEQ ID NO: 20 or SEQ ID NO: 21 and SEQ ID NO: 8 or SEQ ID NO: 22 or variants thereof.
- the expression vector described in the present invention comprises at least the genes selected from: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 17 and SEQ ID NO : 8 or variants thereof.
- the expression vector described in the present invention comprises at least the genes selected from SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 and SEQ ID NO : 22 or variants thereof.
- the expression vector is characterized in that it further comprises at least one of the nucleotide sequences or variants thereof, which code for at least one of the genes that form the Acil CoA enzyme complexes synthetase (EC6.2.1.3) and / or ACC (EC6.4.1.2) selected from: lcfA, yhfL, yhfT, accD, accA, accB or accC and / or any combination thereof.
- the expression vector is characterized by the nucleotide sequences or variants thereof, which code for at least one of the genes that form the Acyl CoA synthetase (EC6.2.1.3) and / or ACC ( EC6.4.1.2) (IcfA, yhfL, yhfT, accD, accA, accB or accC) belong to the bacterial species B. subtilis.
- the expression vector is characterized in that it comprises at least one of the gene constructs described in the present invention.
- the expression vector is characterized in that it consists of plasmid pNAKA62. In another preferred embodiment, the expression vector consists of plasmids pNAKA62 and pNAKA52. In another preferred embodiment, the expression vector consists of plasmids pNAKA62 and pNAKA53.
- the expression vector is characterized in that it consists of plasmid pNAKA143. In another preferred embodiment, the expression vector consists of plasmids pNAKA 143 and pNAKA53.
- the cell is preferably a bacterial cell.
- the cell is a bacterial cell of the genus Bacillus, preferably of the species B. subtilis.
- the host cell is selected from any of the following: CECT 7968 and / or CECT 7969 and / or NCIMB 42026. Another object described in the present invention relates to the use of the cell / s s
- Another object of the present invention relates to a biofuel production method comprising cultivating the bacterial strains described in the present invention in the presence of a carbon source, under suitable conditions.
- the method described in the present invention is characterized in that the carbon source is glycerin, preferably glycerin as a byproduct.
- the method of the invention is characterized in that it further comprises the isolation of the biofuel produced.
- the method of the invention is characterized in that the isolation of the biofuel is carried out by any method available in the art usually applied in the sector, preferably it is carried out by organic solvents, such as: hexane, ethyl acetate, etc.
- the method of the invention is characterized in that the biofuel obtained is composed of ethyl esters of branched and fully saturated fatty acids.
- the method of the invention is characterized in that the fatty acid ethyl esters are preferably: iso-C15 (13-methyltetradecanoic acid), anteiso-C15: 0 (12-methyltetradecanoic acid), n-C15: 0 ( n-pentadecanoic acid); iso-C16: 0 (14-methylpentadecanoic acid), n-C16: 0 (n-hexadecanoic acid), iso-C17: 0 (15-methylhexadecanoic acid), anteiso-C17: 0 (14-methylhexadecanoic acid), n-C17: 0 ( n-heptadecanoic acid), iso-C18: 0 (16-methylheptadecanoic acid) and n-C18: 0 (n-octadecanoic acid).
- the method of the invention is characterized in that the biofuel is biodie sel.
- the method of the invention is characterized in that it further comprises the addition of at least one additive.
- Another object of the present invention relates to a biofuel composition
- a biofuel composition comprising branched and fully saturated ethyl esters of fatty acids.
- the composition of the invention is characterized in that the fatty acid ethyl esters are formed by carbonated and fully saturated chains of the type: ⁇ SO-C15 (13-methyltetradecanoic acid), anteiso-C15: 0 (12-methyltetradecanoic acid ), n-C15: 0 (n-pentadecanoic); iso-C16: 0 (14-methylpentadecanoic), n-C16: 0 (n-hexadecanoic), iso-C17: 0 (15-methylhexadecanoic), anteiso-C17: 0 (14-methylhexadecanoic), n-C17: 0 ( n-heptadecanoic acid), iso-C18: 0 (16-methylheptadecanoic acid) and n-C18: 0 (n-octadecanoic acid).
- ⁇ SO-C15 13-methyltetradecanoic acid
- the composition of the invention is characterized in that it is a biodiesel composition and can also comprise at least one suitable additive.
- the examples detailed below are intended to illustrate the invention without limiting the scope thereof.
- Example 1. Characterization of the growth of B. subtilis in media containing glycerol recovered from different industrial processes, as a carbon source.
- subtilis JH642 bacteria could not be directly analyzed by absorbance measure, due to the turbidity generated by the impurities of commercial samples. For this reason, aliquots of the culture medium were obtained and centrifuged before proceeding to the absorbance measurement, and resuspended in fresh medium, adding the same centrifuged volume. Bacteria grown in AS reached, over time, growth values similar to those obtained for cultures of wild B. subtilis JH642 bacteria grown in glycerol, although the latency phase (period of time during which the bacterial inoculum adapts to the conditions of the medium on which it has been sown and made metabolic adjustment) was higher and the growth rate slower. On the other hand, the growth of wild B.
- subtilis JH642 bacteria in AF was lower and at a very low rate, so it was a less suitable source for the growth of wild B. subtilis bacteria. Therefore, by means of the present example, it is revealed that wild bacterial strains of the species B. subtilis are capable of growing in cultures with crude glycerin as the sole source of carbon.
- Example 2. Obtaining the CECT 7968, CECT 7969 and NCIMB 42026 strains of the species B. subtilis capable of producing biodiesel from glycerin as a carbon source.
- a disadvantage in the expression of heterologous genes in bacteria of the species B. subtilis is the different use of codons of said Gram positive bacterial species, with respect to the use of said codons by other Gram negative bacterial species. This is because of the different availability of tRNA for each codon in different organisms.
- adhB heterologous synthetic mutated genes SEQ ID NO: 4
- fc SEQ ID NO: 2
- tesA SEQ ID NO: 6 or SEQ ID NO: 17
- atfl SEQ ID NO: 8
- the frequency of codon use in a series of genes coding for ribosomal proteins (rplA, rplJ with access number to GenBank: D50303.1; rplC, rplD, rplB, rpsC with access number to the GenBank: U43929.1; rplF, rpsE with access to GenBank: L47971.1; rpsD with access to GenBank: S45404.1 and rpsG with access to GenBank: D64127.1), which They are highly expressed in B. subtilis.
- heterologous synthetic mutated sequences were obtained for each of the genes of interest, said sequences being SEQ ID NO: 2 or variants thereof for the pdc gene, SEQ ID NO: 4 or variants thereof for the adhB gene, SEQ ID NO: 8 or variants thereof for the atfl gene and SEQ ID Nos: 6 and 17 or variants thereof for the ' tes A. gene.
- sequences optimized for expression in B. subtilis are described in the present example, by the method described herein, but, any other sequence (s) of said genes can be used, obtained by the optimization method described herein. , for its stable and increased expression in B. subtilis, provided that said optimization of the origin gene sequence code for the same protein, that is, said optimization must be conservative, not alter at any time the amino acid sequence of the final protein.
- the restriction site sequences for the enzymes HindIII, BglII, BamHI, Xbal and Sal ⁇ have been used, and any other restriction enzyme known in the state of the art can be used for the same purpose.
- the optimized sequences of the four synthetic heterologous genes that, in addition, comprise at their ends, the sequences with the restriction sites for the enzymes and the rbs sequence are: SEQ ID NO: 18 or variants thereof for the pdc gene, SEQ ID NO: 19 or variants thereof for the adhB gene, SEQ. ID NOs. 20-21 or variants thereof for the ' tesA gene and SEQ ID NO: 22 or variants thereof for the atfl gene.
- the vectors obtained were subsequently used to transform competent E. coli DH5a bacteria to conserve and amplify the plasmids.
- heterologous synthetic mutated genes were subcloned into the expression vector pDR67 (Ireton K, 1993), obtaining, on the one hand, plasmid pNAKA62 (pDR67 (HindIII / BglII) + pdc-adh-atfl- 'tesA ), which contains the synthetic heterologous mutated sequences of the four genes of interest, pdc-adh-atfl- 'tesA (SEQ ID NO: 2-SEQ ID NO:
- plasmid pNAKA143 (pDR67 (HindIII / BglII) + pdc-adh-atfl- 'tesA), which contains the heterologous mutated sequences synthetic of the four genes of interest, pdc-adh-atfl- 'tesA (SEQ ID NO: 2-SEQ ID NO: 4-SEQ ID NO: 8-SEQ ID NO: 17, respectively).
- pNAKA62 and pNAKA143 the four genes are under the control of the Yspac promoter, inducible in the presence of isopropyl- ⁇ -
- D-thiogalactoside in the culture medium
- any other plasmid known in the state of the art can be used for the same purpose, under the control of an inducible or constitutive promoter, depending on the requirements of the process.
- said plasmids pNAKA62 and pNAKA143 contain the initial and final region of the amyE gene, flanking the operon and a resistance cassette for the chloramphenicol antibiotic.
- said strains LN72 and LN143 are capable of expressing the heterologous synthetic genes pdc (SEQ ID NO: 2), adh (SEQ ID NO: 4), atfl (SEQ ID NO: 8) and 'tesA (SEQ ID NO: 6) in the case of strain LN72 and heterologous synthetic genes pdc (SEQ ID NO: 2), adh (SEQ ID NO: 4), atfl (SEQ ID NO: 8) and 'tes A (SEQ ID NO: 17 ) in the case of strain LN145, thus being able to increase the synthesis of fatty acids and ethanol, in the presence of glycerin as a carbon source and produce biodiesel.
- acyl-CoA ligase complex EC6.2.1.3
- ACC complex EC6.4.1.2
- IcfA and yltfL genes were chosen. Briefly, said genes were amplified by PCR using as a template chromosomal DNA from the wild strain of B. subtilis JH642 and as primers those defined as SEQ ID NOs: 9-13.
- the plasmid pNAKA60 was constructed by subcloning the lacl gene, which codes for the Lac repressor, and the Vspac ⁇ promoter in the vector pDG1731 (Guérout-Fleury AM., 1996). Said plasmid pNAKA60 contains the initial and final region of the thrC gene, flanking Iacl-Pspac h and a resistance cassette for the spectinomycin antibiotic. Next, the IcfA and yltfL genes encoding acyl-CoA ligases were subcloned into said plasmid, generating plasmids pNAKA52 and pNAKA53, respectively.
- the CECT 7968, CECT 7969 and NCIMB 42026 strains therefore, contain the amyE and thrC locus with all the necessary genes for biodiesel biosynthesis under the control of the IPTG-inducible promoter, Pspac, although as mentioned above Throughout the present invention, any promoter, whether inducible or constitutive, known in the state of the art can be selected.
- AccDABC acetyl-CoA carboxylase
- plasmid pGES4608 contains the accDABC operon under control of the IPTG-inducible promoter, Yspac, although, as mentioned above, any promoter, whether inducible or constitutive, known in the state of The technique.
- the transformation of strains B. subtilis CECT 7968, CECT 7969 and NCIMB 42026 is carried out to increase the expression of the acetyl-CoA carboxylase enzyme complex and increase the yield of biodiesel synthesis by the strains described in the present invention.
- Example 3 Production of biodiesel using strains of B. subtilis CECT 7968, 7969 and NCIMB 42026.
- Strains of B. subtilis CECT 7968 and CECT 7969 described in the present invention were grown in 50 ml of SPI minimum culture medium formed by (NH 4 ) 2 S0 4 , 2.0 g / 1; KH 2 P0 4 , 14.0 g / 1; K 2 HP0 4 , 6.0 g / 1; Na Citrate, 1.0 g / 1; MgS0 4 7H 2 0, 0.2 g / 1.
- composition of the biofuel obtained showed the presence of ethyl esters of fatty acids or biodiesel of carbon chain and fully saturated, preferably formed by: iso-C15 (13-methyltetradecanoic acid), anteiso-C15: 0 (12 - methyltetradecanoic acid), n-C15: 0 (n-pentadecanoic acid); iso-C16: 0 (14-methylpentadecanoic), n-C16: 0 (n-hexadecanoic), iso-C17: 0 (15-methylhexadecanoic), anteiso-C17: 0 (14-methylhexadecanoic), n-C17: 0 ( n-heptadecanoic acid), iso-C18: 0 (16-methylheptadecanoic acid) and n-C18: 0 (n-octadecanoic acid).
- Karmakar A Karmakar S, Mukherjee S. Properties of various plants and animate feedstocks for biodiesel production. Bioresource Technology. 2010 Oct; 101 (2010) 7201-10.
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
BR112014003797A BR112014003797A8 (pt) | 2011-08-18 | 2012-08-17 | construção gênica, vetor de expressão, célula, uso da mesma, método de produção de biocombustíveis e composição de biocombustíveis |
MX2014001809A MX2014001809A (es) | 2011-08-18 | 2012-08-17 | Produccion de biodiesel a partir de glicerina. |
EP12824241.9A EP2746389A4 (en) | 2011-08-18 | 2012-08-17 | PRODUCTION OF BIODIESEL FROM GLYCERIN |
US14/239,228 US20150068108A1 (en) | 2011-08-18 | 2012-08-17 | Production of biodiesel from glycerine |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ES201131400A ES2396823B1 (es) | 2011-08-18 | 2011-08-18 | Producción de biodiesel a partir de glicerina. |
ESP201131400 | 2011-08-18 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2013024196A1 true WO2013024196A1 (es) | 2013-02-21 |
Family
ID=47711358
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/ES2012/070628 WO2013024196A1 (es) | 2011-08-18 | 2012-08-17 | Producción de biodiesel a partir de glicerina |
Country Status (8)
Country | Link |
---|---|
US (1) | US20150068108A1 (es) |
EP (1) | EP2746389A4 (es) |
AR (1) | AR087600A1 (es) |
BR (1) | BR112014003797A8 (es) |
ES (1) | ES2396823B1 (es) |
MX (1) | MX2014001809A (es) |
UY (1) | UY34269A (es) |
WO (1) | WO2013024196A1 (es) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105331647B (zh) * | 2015-07-03 | 2019-02-22 | 赣南师范大学 | 一种微生物体内合成脂肪醇乙酸酯的方法 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008119082A2 (en) * | 2007-03-28 | 2008-10-02 | Ls9, Inc. | Enhanced production of fatty acid derivatives |
WO2010130806A1 (en) * | 2009-05-14 | 2010-11-18 | Deinove | Recombinant bacteria and the uses thereof for producing ethanol |
WO2011038134A1 (en) | 2009-09-25 | 2011-03-31 | Ls9, Inc. | Production of fatty acid derivatives |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE102004052115A1 (de) * | 2004-10-26 | 2006-04-27 | Westfälische Wilhelms-Universität Münster | Mikroorganismen zur Herstellung von Fettsäureestern, Verfahren zu deren Herstellung und deren Verwendung |
CA2650773C (en) * | 2006-05-19 | 2014-12-02 | Jay D. Keasling | Microorganisms transformed with acetyl-coa carboxylase and wax synthase for production of fatty acid derivatives |
CA2759273C (en) * | 2009-04-27 | 2018-01-09 | Ls9, Inc. | Production of fatty acid esters |
CN102071228B (zh) * | 2009-11-25 | 2013-09-25 | 青岛生物能源与过程研究所 | 一种微生物体内合成生物柴油的方法 |
-
2011
- 2011-08-18 ES ES201131400A patent/ES2396823B1/es not_active Expired - Fee Related
-
2012
- 2012-08-17 BR BR112014003797A patent/BR112014003797A8/pt not_active Application Discontinuation
- 2012-08-17 EP EP12824241.9A patent/EP2746389A4/en not_active Withdrawn
- 2012-08-17 AR ARP120103050A patent/AR087600A1/es unknown
- 2012-08-17 US US14/239,228 patent/US20150068108A1/en not_active Abandoned
- 2012-08-17 WO PCT/ES2012/070628 patent/WO2013024196A1/es active Application Filing
- 2012-08-17 UY UY0001034269A patent/UY34269A/es not_active Application Discontinuation
- 2012-08-17 MX MX2014001809A patent/MX2014001809A/es unknown
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008119082A2 (en) * | 2007-03-28 | 2008-10-02 | Ls9, Inc. | Enhanced production of fatty acid derivatives |
WO2010130806A1 (en) * | 2009-05-14 | 2010-11-18 | Deinove | Recombinant bacteria and the uses thereof for producing ethanol |
WO2011038134A1 (en) | 2009-09-25 | 2011-03-31 | Ls9, Inc. | Production of fatty acid derivatives |
Non-Patent Citations (20)
Title |
---|
ATSUMI S; LIAO JC.: "Metabolic engineering for advanced biofuels production from Escherichia coli.", CURR OPIN BIOTECHNOL., vol. 19, no. 5, October 2008 (2008-10-01), pages 414 - 9 |
CHOI WJ.: "Glycerol-based biorefinery for fuels and chemicals.", RECENT PAT BIOTECHNOL, vol. 2, no. 3, 2008, pages 173 - 80 |
DA SILVA GP; MACK M, CONTIERO.: "Glycerol: a promising and abundant carbon source for industrial microbiology", J. BIOTECHNOL ADV, vol. 27, no. 1, 2009, pages 30 - 9 |
DAVIS, M.S.; SOLBIATI, J.; CRONAN, J.E., JR.: "Overproduction of acetyl-CoA carboxylase activity increases the rate offatty acid biosynthesis in Escherichia coli", J. BIOL. CHEM., vol. 275, 2000, pages 28593 - 28598 |
DUAN, Y. ET AL.: "De novo biosynthesis of biodiesel by Escherichia coli in optimized fed-batch cultivation. art e20265", PLOS ONE, vol. 6, no. 5, 2011, XP055026594 * |
GUÉROUT-FLEURY AM; FRANDSEN N; STRAGIER P.: "Plasmids for ectopic integration in Bacillus subtilis.", GENE, vol. 180, 1996, pages 57 - 61 |
HONG, W.K. ET AL.: "Enhanced production of ethanol from glycerol by engineered Hansenula polymorpha expressing pyruvate decarboxylase and aldehyde dehydrogenase genes from Zymomonas mobilis", BIOTECHNOL. LETT, vol. 32, no. 8, August 2010 (2010-08-01), pages 1077 - 1082, XP019813441 * |
IRETON K; RUDNER DZ; SIRANOSIAN KJ; GROSSMAN AD.: "Integration of multiple developmental signals in Bacillus subtilis through the SpoOA transcription factor.", GENES & DEV, vol. 7, 1993, pages 283 - 294 |
KALSCHEUER R; STOLTING T; STEINBUCHEL A.: "Microdiesel: Escherichia coli engineered for fuel production.", MICROBIOLOGY., vol. 152, September 2006 (2006-09-01), pages 2529 - 36 |
KALSCHEUER, R. ET AL.: "Microdiesel: Escherichia coli engineered for fuel production", MICROBIOLOGY, vol. 152, no. 9, 2006, pages 2529 - 2536, XP007903430 * |
KALSCHEUER, R.; T. STOLTING; A. STEINBUCHEL.: "Microdiesel: Escherichia coli engineered for fuel production", MICROBIOLOGY, vol. 152, 2006, pages 2529 - 36 |
KARMAKAR A; KARMAKAR S; MUKHERJEE S.: "Properties of various plants and animalsfeedstocks for biodiesel production.", BIORESOURCE TECHNOLOGY, vol. 101, October 2010 (2010-10-01), pages 7201 - 10 |
OH BR; SEO JW; HEO SY; HONG WK; LUO LH; JOE MH; PARK DH; KIM CH.: "Efficient production of ethanol from crude glycerol by a Klebsiella pneumoniae mutant strain", BIORESOUR TECHNOL., vol. 102, no. 4, February 2011 (2011-02-01), pages 3918 - 22 |
SCHUJMAN GE; ALTABE S; DE MENDOZA D.: "A malonyl-CoA-dependent switch in the bacterial response to a dysfunction of lipid metabolism.", MOLECULAR MICROBIOLOGY, vol. 68, no. 4, 2008, pages 987 - 996 |
See also references of EP2746389A4 |
SHAMS YAZDANI S; GONZALEZ R.: "Engineering Escherichia colifor the efficient conversion of glycerol to ethanol and co-products.", METAB ENG., vol. 10, no. 6, November 2008 (2008-11-01), pages 340 - 51 |
SHARP, P.M. ET AL.: "Codon usage patterns in Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster and Homo sapiens; a review of the considerable within-species diversity", NUCLEIC ACIDS RESEARCH, vol. 16, no. 17, 1988, pages 8207 - 8211, XP001122248 * |
SHIELDS, D.C. ET AL.: "Synonymous codon usage in Bacillus subtilis reflects both translational selection and mutational biases", NUCLEIC ACIDS RESEARCH, vol. 15, no. 19, 1987, pages 8023 - 8040, XP055139633 * |
STEEN, E.J. ET AL.: "Microbial production of fatty-acid-derived fuels and chemicals from plant biomass", NATURE, vol. 463, no. 7280, January 2010 (2010-01-01), pages 559 - 562, XP055011271 * |
WESTERS ET AL., BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1694, 2004, pages 299 - 310 |
Also Published As
Publication number | Publication date |
---|---|
US20150068108A1 (en) | 2015-03-12 |
MX2014001809A (es) | 2014-09-15 |
BR112014003797A2 (pt) | 2017-06-13 |
ES2396823A1 (es) | 2013-02-27 |
ES2396823B1 (es) | 2014-01-28 |
EP2746389A4 (en) | 2015-01-07 |
AR087600A1 (es) | 2014-04-03 |
EP2746389A1 (en) | 2014-06-25 |
BR112014003797A8 (pt) | 2017-06-20 |
UY34269A (es) | 2013-04-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA2876178C (en) | Recombinant microorganisms expressing a non-specific acyltransferase make biodiesel | |
Lennen et al. | A process for microbial hydrocarbon synthesis: overproduction of fatty acids in Escherichia coli and catalytic conversion to alkanes | |
ES2899431T3 (es) | Métodos y productos para la producción de ésteres de cera | |
ES2702621T3 (es) | Microorganismos recombinantes y sus usos | |
Cao et al. | Increasing unsaturated fatty acid contents in Escherichia coli by coexpression of three different genes | |
US10246726B2 (en) | Photosynthetic production of 3-hydroxybutyrate from carbon dioxide | |
CN102216463A (zh) | 微生物的乙醇生产 | |
KR100971792B1 (ko) | Butyryl-CoA를 중간체로 하여 부탄올을 생합성하는 능력을가지는 효모를 이용하여 부탄올을 제조하는 방법 | |
JP2015524283A (ja) | 脂肪酸および脂肪酸由来生成物の生成のため微生物および方法 | |
ES2647412T3 (es) | Variantes enzimáticas con propiedades de éster sintasa mejoradas | |
AU2014229307B2 (en) | Engineering microorganisms | |
US20140004580A1 (en) | Modified photosynthetic microorganisms for producing lipids | |
Lee et al. | Heterologous co-expression of accA, fabD, and thioesterase genes for improving long-chain fatty acid production in Pseudomonas aeruginosa and Escherichia coli | |
Yang et al. | Expression of phosphotransacetylase in Rhodosporidium toruloides leading to improved cell growth and lipid production | |
ES2908400T3 (es) | Variantes de polipéptidos de fusión relacionados con omega-hidroxilasa con propiedades mejoradas | |
CN101748069A (zh) | 一种重组蓝藻 | |
DK2465868T3 (en) | Enhancement of lipid production | |
WO2013024196A1 (es) | Producción de biodiesel a partir de glicerina | |
ES2633587T3 (es) | Producción de ácidos grasos por expresión heteróloga de agrupamientos de genes a partir de mixobacterias | |
JP2023027261A (ja) | 中鎖脂肪酸誘導体の生産のための改良された活性を有するチオエステラーゼ変種 | |
KR101402108B1 (ko) | 불포화 지방산을 생산하는 재조합 대장균, 및 이를 이용한 불포화 지방산의 제조방법 | |
KR101275090B1 (ko) | 지방산을 생산하는 재조합 대장균, 및 이를 이용한 지방산의 제조방법 | |
KR102116473B1 (ko) | 지방산을 생산하는 재조합 대장균 및 이를 이용한 바이오디젤의 제조방법 | |
KR101765219B1 (ko) | 불포화 지방산을 생산하는 재조합 대장균, 및 이를 이용한 불포화 지방산의 제조방법 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 12824241 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: MX/A/2014/001809 Country of ref document: MX |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2012824241 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 14239228 Country of ref document: US |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112014003797 Country of ref document: BR |
|
ENP | Entry into the national phase |
Ref document number: 112014003797 Country of ref document: BR Kind code of ref document: A2 Effective date: 20140218 |