US20210380996A1 - Microorganisms engineered to use unconventional sources of nitrogen - Google Patents
Microorganisms engineered to use unconventional sources of nitrogen Download PDFInfo
- Publication number
- US20210380996A1 US20210380996A1 US17/349,559 US202117349559A US2021380996A1 US 20210380996 A1 US20210380996 A1 US 20210380996A1 US 202117349559 A US202117349559 A US 202117349559A US 2021380996 A1 US2021380996 A1 US 2021380996A1
- Authority
- US
- United States
- Prior art keywords
- nitrogen
- gene
- certain embodiments
- cell
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 title abstract description 90
- 229910052757 nitrogen Inorganic materials 0.000 title abstract description 45
- 244000005700 microbiome Species 0.000 title description 12
- 238000000034 method Methods 0.000 claims abstract description 81
- XZMCDFZZKTWFGF-UHFFFAOYSA-N Cyanamide Chemical compound NC#N XZMCDFZZKTWFGF-UHFFFAOYSA-N 0.000 claims abstract description 34
- 238000000855 fermentation Methods 0.000 claims abstract description 6
- 230000004151 fermentation Effects 0.000 claims abstract description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 63
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 57
- 150000007523 nucleic acids Chemical class 0.000 claims description 56
- 108020004707 nucleic acids Proteins 0.000 claims description 51
- 102000039446 nucleic acids Human genes 0.000 claims description 51
- 239000000758 substrate Substances 0.000 claims description 43
- 239000004202 carbamide Substances 0.000 claims description 31
- 108030005585 Cyanamide hydratases Proteins 0.000 claims description 13
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 8
- 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 claims description 7
- 239000003242 anti bacterial agent Substances 0.000 claims description 7
- 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 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 6
- 230000003115 biocidal effect Effects 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 5
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 claims description 4
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 4
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 4
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 239000004310 lactic acid Substances 0.000 claims description 4
- 235000014655 lactic acid Nutrition 0.000 claims description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 239000008121 dextrose Substances 0.000 claims description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 2
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 2
- 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 claims description 2
- 229930091371 Fructose Natural products 0.000 claims 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 claims description 2
- 239000005715 Fructose Substances 0.000 claims description 2
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 claims description 2
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 2
- 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 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims description 2
- 235000011054 acetic acid Nutrition 0.000 claims description 2
- 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 claims description 2
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 claims description 2
- 235000019253 formic acid Nutrition 0.000 claims description 2
- 235000021588 free fatty acids Nutrition 0.000 claims description 2
- 229930182830 galactose Natural products 0.000 claims description 2
- 239000008101 lactose Substances 0.000 claims description 2
- 239000012978 lignocellulosic material Substances 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- 229920000877 Melamine resin Polymers 0.000 abstract description 55
- JDSHMPZPIAZGSV-UHFFFAOYSA-N melamine Chemical compound NC1=NC(N)=NC(N)=N1 JDSHMPZPIAZGSV-UHFFFAOYSA-N 0.000 abstract description 54
- -1 commodities Chemical class 0.000 abstract description 30
- 240000004808 Saccharomyces cerevisiae Species 0.000 abstract description 14
- 150000001875 compounds Chemical class 0.000 abstract description 6
- 241000894006 Bacteria Species 0.000 abstract description 5
- 239000003814 drug Substances 0.000 abstract description 2
- 239000012847 fine chemical Substances 0.000 abstract description 2
- 238000012776 robust process Methods 0.000 abstract 1
- 108090000623 proteins and genes Proteins 0.000 description 137
- 210000004027 cell Anatomy 0.000 description 66
- 239000013598 vector Substances 0.000 description 62
- 230000014509 gene expression Effects 0.000 description 43
- 239000002609 medium Substances 0.000 description 38
- 239000000047 product Substances 0.000 description 37
- 239000013612 plasmid Substances 0.000 description 33
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 31
- 101150023090 CAH gene Proteins 0.000 description 31
- 230000012010 growth Effects 0.000 description 29
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 28
- 241000588724 Escherichia coli Species 0.000 description 27
- 102000004190 Enzymes Human genes 0.000 description 26
- 108090000790 Enzymes Proteins 0.000 description 26
- 108020004414 DNA Proteins 0.000 description 23
- 108091028043 Nucleic acid sequence Proteins 0.000 description 23
- MASBWURJQFFLOO-UHFFFAOYSA-N ammeline Chemical compound NC1=NC(N)=NC(O)=N1 MASBWURJQFFLOO-UHFFFAOYSA-N 0.000 description 20
- OHJMTUPIZMNBFR-UHFFFAOYSA-N biuret Chemical compound NC(=O)NC(N)=O OHJMTUPIZMNBFR-UHFFFAOYSA-N 0.000 description 18
- 230000009466 transformation Effects 0.000 description 18
- 102000004169 proteins and genes Human genes 0.000 description 16
- XFNJVJPLKCPIBV-UHFFFAOYSA-N trimethylenediamine Chemical compound NCCCN XFNJVJPLKCPIBV-UHFFFAOYSA-N 0.000 description 16
- 235000018102 proteins Nutrition 0.000 description 15
- 238000013518 transcription Methods 0.000 description 15
- 230000035897 transcription Effects 0.000 description 15
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 14
- 230000003287 optical effect Effects 0.000 description 14
- 235000019270 ammonium chloride Nutrition 0.000 description 13
- 238000002744 homologous recombination Methods 0.000 description 13
- 230000006801 homologous recombination Effects 0.000 description 13
- 241000217808 Pseudomonas sp. ADP Species 0.000 description 12
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 12
- 125000000217 alkyl group Chemical group 0.000 description 12
- ZFSLODLOARCGLH-UHFFFAOYSA-N isocyanuric acid Chemical compound OC1=NC(O)=NC(O)=N1 ZFSLODLOARCGLH-UHFFFAOYSA-N 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 101150016414 DUR1,2 gene Proteins 0.000 description 11
- 108010012029 Guanine Deaminase Proteins 0.000 description 11
- 102000013587 Guanine deaminase Human genes 0.000 description 11
- 241000187562 Rhodococcus sp. Species 0.000 description 11
- 101150041447 guaD gene Proteins 0.000 description 11
- 230000037361 pathway Effects 0.000 description 11
- 101100438200 Pseudomonas sp. (strain ADP) atzD gene Proteins 0.000 description 10
- YSKUZVBSHIWEFK-UHFFFAOYSA-N ammelide Chemical compound NC1=NC(O)=NC(O)=N1 YSKUZVBSHIWEFK-UHFFFAOYSA-N 0.000 description 10
- 229910021529 ammonia Inorganic materials 0.000 description 10
- 108700023205 Allophanate hydrolases Proteins 0.000 description 9
- 108091026890 Coding region Proteins 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 241000235015 Yarrowia lipolytica Species 0.000 description 9
- 239000013604 expression vector Substances 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 230000001105 regulatory effect Effects 0.000 description 9
- 101150090745 trzD gene Proteins 0.000 description 9
- VILCJCGEZXAXTO-UHFFFAOYSA-N 2,2,2-tetramine Chemical compound NCCNCCNCCN VILCJCGEZXAXTO-UHFFFAOYSA-N 0.000 description 8
- 108010067937 Cyanuric acid amidohydrolase Proteins 0.000 description 8
- 239000007993 MOPS buffer Substances 0.000 description 8
- 108700019146 Transgenes Proteins 0.000 description 8
- 108010046334 Urease Proteins 0.000 description 8
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 8
- 230000001939 inductive effect Effects 0.000 description 8
- 229910000069 nitrogen hydride Inorganic materials 0.000 description 8
- MVXMNHYVCLMLDD-UHFFFAOYSA-N 4-methoxynaphthalene-1-carbaldehyde Chemical compound C1=CC=C2C(OC)=CC=C(C=O)C2=C1 MVXMNHYVCLMLDD-UHFFFAOYSA-N 0.000 description 7
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 7
- 108090001042 Hydro-Lyases Proteins 0.000 description 7
- 102000004867 Hydro-Lyases Human genes 0.000 description 7
- 102000004157 Hydrolases Human genes 0.000 description 7
- 108090000604 Hydrolases Proteins 0.000 description 7
- IMUDHTPIFIBORV-UHFFFAOYSA-N aminoethylpiperazine Chemical compound NCCN1CCNCC1 IMUDHTPIFIBORV-UHFFFAOYSA-N 0.000 description 7
- 229960000723 ampicillin Drugs 0.000 description 7
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 7
- STIAPHVBRDNOAJ-UHFFFAOYSA-N carbamimidoylazanium;carbonate Chemical compound NC(N)=N.NC(N)=N.OC(O)=O STIAPHVBRDNOAJ-UHFFFAOYSA-N 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- QGBSISYHAICWAH-UHFFFAOYSA-N dicyandiamide Chemical compound NC(N)=NC#N QGBSISYHAICWAH-UHFFFAOYSA-N 0.000 description 7
- 230000010076 replication Effects 0.000 description 7
- 241000894007 species Species 0.000 description 7
- 230000009261 transgenic effect Effects 0.000 description 7
- 108010010296 urea carboxylase (hydrolyzing) Proteins 0.000 description 7
- JYEUMXHLPRZUAT-UHFFFAOYSA-N 1,2,3-triazine Chemical compound C1=CN=NN=C1 JYEUMXHLPRZUAT-UHFFFAOYSA-N 0.000 description 6
- 108030006155 Biuret amidohydrolases Proteins 0.000 description 6
- RPNUMPOLZDHAAY-UHFFFAOYSA-N Diethylenetriamine Chemical compound NCCNCCN RPNUMPOLZDHAAY-UHFFFAOYSA-N 0.000 description 6
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 6
- 101100100932 Gordonia rubripertincta trzA gene Proteins 0.000 description 6
- 241000589774 Pseudomonas sp. Species 0.000 description 6
- 101100010928 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) tuf gene Proteins 0.000 description 6
- 101100176922 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) GUD1 gene Proteins 0.000 description 6
- 101150001810 TEAD1 gene Proteins 0.000 description 6
- 101150074253 TEF1 gene Proteins 0.000 description 6
- 102100029898 Transcriptional enhancer factor TEF-1 Human genes 0.000 description 6
- 241000798866 Yarrowia lipolytica CLIB122 Species 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 230000008901 benefit Effects 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- 108010052165 melamine deaminase Proteins 0.000 description 6
- 101150076017 triA gene Proteins 0.000 description 6
- 108020004705 Codon Proteins 0.000 description 5
- 241001646716 Escherichia coli K-12 Species 0.000 description 5
- 108091030071 RNAI Proteins 0.000 description 5
- WERYXYBDKMZEQL-UHFFFAOYSA-N butane-1,4-diol Chemical compound OCCCCO WERYXYBDKMZEQL-UHFFFAOYSA-N 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000009368 gene silencing by RNA Effects 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 108020004999 messenger RNA Proteins 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- 238000013519 translation Methods 0.000 description 5
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical group CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 4
- 101100218332 Pseudomonas sp. (strain ADP) atzC gene Proteins 0.000 description 4
- 101100437106 Pseudomonas sp. (strain ADP) atzE gene Proteins 0.000 description 4
- 101100437107 Pseudomonas sp. (strain ADP) atzF gene Proteins 0.000 description 4
- 229940088710 antibiotic agent Drugs 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 239000006143 cell culture medium Substances 0.000 description 4
- 210000000170 cell membrane Anatomy 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 230000010354 integration Effects 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 230000003204 osmotic effect Effects 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 230000008685 targeting Effects 0.000 description 4
- 239000007222 ypd medium Substances 0.000 description 4
- 241001103808 Albifimbria verrucaria Species 0.000 description 3
- 241000274790 Bradyrhizobium diazoefficiens USDA 110 Species 0.000 description 3
- 108700010070 Codon Usage Proteins 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- 230000005526 G1 to G0 transition Effects 0.000 description 3
- 241000235648 Pichia Species 0.000 description 3
- 241000589194 Rhizobium leguminosarum Species 0.000 description 3
- 241000093677 Williamsia sp. Species 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 125000004432 carbon atom Chemical group C* 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 230000002759 chromosomal effect Effects 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 230000001332 colony forming effect Effects 0.000 description 3
- 239000000356 contaminant Substances 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000010363 gene targeting Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 125000004433 nitrogen atom Chemical group N* 0.000 description 3
- 108091027963 non-coding RNA Proteins 0.000 description 3
- 102000042567 non-coding RNA Human genes 0.000 description 3
- HGASFNYMVGEKTF-UHFFFAOYSA-N octan-1-ol;hydrate Chemical compound O.CCCCCCCCO HGASFNYMVGEKTF-UHFFFAOYSA-N 0.000 description 3
- 238000005192 partition Methods 0.000 description 3
- 230000008488 polyadenylation Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- 230000002103 transcriptional effect Effects 0.000 description 3
- DNIAPMSPPWPWGF-VKHMYHEASA-N (+)-propylene glycol Chemical compound C[C@H](O)CO DNIAPMSPPWPWGF-VKHMYHEASA-N 0.000 description 2
- MEKDPHXPVMKCON-UHFFFAOYSA-N *.C.CC Chemical compound *.C.CC MEKDPHXPVMKCON-UHFFFAOYSA-N 0.000 description 2
- RHUYHJGZWVXEHW-UHFFFAOYSA-N 1,1-Dimethyhydrazine Chemical compound CN(C)N RHUYHJGZWVXEHW-UHFFFAOYSA-N 0.000 description 2
- YPFDHNVEDLHUCE-UHFFFAOYSA-N 1,3-propanediol Substances OCCCO YPFDHNVEDLHUCE-UHFFFAOYSA-N 0.000 description 2
- JAHNSTQSQJOJLO-UHFFFAOYSA-N 2-(3-fluorophenyl)-1h-imidazole Chemical compound FC1=CC=CC(C=2NC=CN=2)=C1 JAHNSTQSQJOJLO-UHFFFAOYSA-N 0.000 description 2
- 108020005345 3' Untranslated Regions Proteins 0.000 description 2
- 241001600126 Acidovorax citrulli Species 0.000 description 2
- 241000956177 Aspergillus kawachii IFO 4308 Species 0.000 description 2
- 241000479753 Aspergillus niger ATCC 1015 Species 0.000 description 2
- 241001370055 Aspergillus niger CBS 513.88 Species 0.000 description 2
- 241000075729 Aspergillus oryzae 3.042 Species 0.000 description 2
- 101100434663 Bacillus subtilis (strain 168) fbaA gene Proteins 0.000 description 2
- 101100351264 Candida albicans (strain SC5314 / ATCC MYA-2876) PDC11 gene Proteins 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 2
- ROSDSFDQCJNGOL-UHFFFAOYSA-N Dimethylamine Chemical compound CNC ROSDSFDQCJNGOL-UHFFFAOYSA-N 0.000 description 2
- 241000588722 Escherichia Species 0.000 description 2
- 241001522878 Escherichia coli B Species 0.000 description 2
- 241000660147 Escherichia coli str. K-12 substr. MG1655 Species 0.000 description 2
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 description 2
- 101150095274 FBA1 gene Proteins 0.000 description 2
- 241000333045 Fusarium graminearum PH-1 Species 0.000 description 2
- 241000527531 Fusarium oxysporum Fo5176 Species 0.000 description 2
- 101150081655 GPM1 gene Proteins 0.000 description 2
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 2
- 229910009891 LiAc Inorganic materials 0.000 description 2
- 239000006137 Luria-Bertani broth Substances 0.000 description 2
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 description 2
- SQMUXTYWFMYPKY-UHFFFAOYSA-N N#CC([Y])[Y] Chemical compound N#CC([Y])[Y] SQMUXTYWFMYPKY-UHFFFAOYSA-N 0.000 description 2
- NERMPXFJCIZSJW-UHFFFAOYSA-N O=C([Y])CC(=O)[Y] Chemical compound O=C([Y])CC(=O)[Y] NERMPXFJCIZSJW-UHFFFAOYSA-N 0.000 description 2
- 101150050255 PDC1 gene Proteins 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 241000235070 Saccharomyces Species 0.000 description 2
- 240000008223 Saccharomyces cerevisiae FostersB Species 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 2
- 101150050575 URA3 gene Proteins 0.000 description 2
- 108091023045 Untranslated Region Proteins 0.000 description 2
- 241000235013 Yarrowia Species 0.000 description 2
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 2
- 230000006652 catabolic pathway Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 101150084612 gpmA gene Proteins 0.000 description 2
- 239000003966 growth inhibitor Substances 0.000 description 2
- 125000001072 heteroaryl group Chemical group 0.000 description 2
- VKYKSIONXSXAKP-UHFFFAOYSA-N hexamethylenetetramine Chemical compound C1N(C2)CN3CN1CN2C3 VKYKSIONXSXAKP-UHFFFAOYSA-N 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 239000000543 intermediate Substances 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- ZXEKIIBDNHEJCQ-UHFFFAOYSA-N isobutanol Chemical compound CC(C)CO ZXEKIIBDNHEJCQ-UHFFFAOYSA-N 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- LVHBHZANLOWSRM-UHFFFAOYSA-N methylenebutanedioic acid Natural products OC(=O)CC(=C)C(O)=O LVHBHZANLOWSRM-UHFFFAOYSA-N 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 230000009057 passive transport Effects 0.000 description 2
- 229920005862 polyol Polymers 0.000 description 2
- 150000003077 polyols Chemical class 0.000 description 2
- 229920000166 polytrimethylene carbonate Polymers 0.000 description 2
- 230000003362 replicative effect Effects 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 description 2
- 235000011152 sodium sulphate Nutrition 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- LMBFAGIMSUYTBN-MPZNNTNKSA-N teixobactin Chemical compound C([C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H](CCC(N)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H]1C(N[C@@H](C)C(=O)N[C@@H](C[C@@H]2NC(=N)NC2)C(=O)N[C@H](C(=O)O[C@H]1C)[C@@H](C)CC)=O)NC)C1=CC=CC=C1 LMBFAGIMSUYTBN-MPZNNTNKSA-N 0.000 description 2
- 150000003505 terpenes Chemical class 0.000 description 2
- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea Chemical compound NC(N)=S UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 2
- 108700026220 vif Genes Proteins 0.000 description 2
- FZENGILVLUJGJX-NSCUHMNNSA-N (E)-acetaldehyde oxime Chemical compound C\C=N\O FZENGILVLUJGJX-NSCUHMNNSA-N 0.000 description 1
- CUNNCKOPAWXYDX-SFECMWDFSA-N (NZ)-N-[(2Z)-2-hydroxyiminocyclohexylidene]hydroxylamine Chemical compound O\N=C/1\CCCC\C\1=N\O CUNNCKOPAWXYDX-SFECMWDFSA-N 0.000 description 1
- ULUZGMIUTMRARO-UHFFFAOYSA-N (carbamoylamino)urea Chemical compound NC(=O)NNC(N)=O ULUZGMIUTMRARO-UHFFFAOYSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- DIIIISSCIXVANO-UHFFFAOYSA-N 1,2-Dimethylhydrazine Chemical compound CNNC DIIIISSCIXVANO-UHFFFAOYSA-N 0.000 description 1
- JGGFDEJXWLAQKR-UHFFFAOYSA-N 1,2-diaminoguanidine Chemical compound NNC(N)=NN JGGFDEJXWLAQKR-UHFFFAOYSA-N 0.000 description 1
- JIHQDMXYYFUGFV-UHFFFAOYSA-N 1,3,5-triazine Chemical compound C1=NC=NC=N1 JIHQDMXYYFUGFV-UHFFFAOYSA-N 0.000 description 1
- VSNHCAURESNICA-NJFSPNSNSA-N 1-oxidanylurea Chemical compound N[14C](=O)NO VSNHCAURESNICA-NJFSPNSNSA-N 0.000 description 1
- QWENRTYMTSOGBR-UHFFFAOYSA-N 1H-1,2,3-Triazole Chemical compound C=1C=NNN=1 QWENRTYMTSOGBR-UHFFFAOYSA-N 0.000 description 1
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 description 1
- ULRPISSMEBPJLN-UHFFFAOYSA-N 2h-tetrazol-5-amine Chemical compound NC1=NN=NN1 ULRPISSMEBPJLN-UHFFFAOYSA-N 0.000 description 1
- VIHRIIARIFUQLC-UHFFFAOYSA-N 3-hydrazinylpropanenitrile Chemical compound NNCCC#N VIHRIIARIFUQLC-UHFFFAOYSA-N 0.000 description 1
- NUKYPUAOHBNCPY-UHFFFAOYSA-N 4-aminopyridine Chemical compound NC1=CC=NC=C1 NUKYPUAOHBNCPY-UHFFFAOYSA-N 0.000 description 1
- NSPMIYGKQJPBQR-UHFFFAOYSA-N 4H-1,2,4-triazole Chemical compound C=1N=CNN=1 NSPMIYGKQJPBQR-UHFFFAOYSA-N 0.000 description 1
- WBZFUFAFFUEMEI-UHFFFAOYSA-M Acesulfame k Chemical compound [K+].CC1=CC(=O)[N-]S(=O)(=O)O1 WBZFUFAFFUEMEI-UHFFFAOYSA-M 0.000 description 1
- 241000726119 Acidovorax Species 0.000 description 1
- 241000589291 Acinetobacter Species 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 241001370313 Alepes vari Species 0.000 description 1
- 108090000531 Amidohydrolases Proteins 0.000 description 1
- 102000004092 Amidohydrolases Human genes 0.000 description 1
- 241000186063 Arthrobacter Species 0.000 description 1
- 108010011485 Aspartame Proteins 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 239000004156 Azodicarbonamide Substances 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000186146 Brevibacterium Species 0.000 description 1
- YQLLUYPCXXOWJH-UHFFFAOYSA-M C.C=C(N)NC(=O)[O-].CC(=O)NC(N)=O.CC1=NC(N)=NC(N)=N1.CC1=NC(N)=NC(N)=N1.CC1=NC(O)=NC(N)=N1.CC1=NC(O)=NC(O)=N1.[H]N(C)C#N Chemical compound C.C=C(N)NC(=O)[O-].CC(=O)NC(N)=O.CC1=NC(N)=NC(N)=N1.CC1=NC(N)=NC(N)=N1.CC1=NC(O)=NC(N)=N1.CC1=NC(O)=NC(O)=N1.[H]N(C)C#N YQLLUYPCXXOWJH-UHFFFAOYSA-M 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- QDHHCQZDFGDHMP-UHFFFAOYSA-N Chloramine Chemical compound ClN QDHHCQZDFGDHMP-UHFFFAOYSA-N 0.000 description 1
- 241001112696 Clostridia Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 241001509401 Gordonia rubripertincta Species 0.000 description 1
- 101100246753 Halobacterium salinarum (strain ATCC 700922 / JCM 11081 / NRC-1) pyrF gene Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- 241000235649 Kluyveromyces Species 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108010083922 Myrothecium verrucaria cyanamide hydratase Proteins 0.000 description 1
- FXHOOIRPVKKKFG-UHFFFAOYSA-N N,N-Dimethylacetamide Chemical compound CN(C)C(C)=O FXHOOIRPVKKKFG-UHFFFAOYSA-N 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 241001112159 Ogataea Species 0.000 description 1
- 241001099341 Ogataea polymorpha Species 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 208000001388 Opportunistic Infections Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 229920002562 Polyethylene Glycol 3350 Polymers 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 241000192142 Proteobacteria Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- NYTOUQBROMCLBJ-UHFFFAOYSA-N Tetranitromethane Chemical compound [O-][N+](=O)C([N+]([O-])=O)([N+]([O-])=O)[N+]([O-])=O NYTOUQBROMCLBJ-UHFFFAOYSA-N 0.000 description 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 1
- 241000223259 Trichoderma Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- NRAUADCLPJTGSF-ZPGVOIKOSA-N [(2r,3s,4r,5r,6r)-6-[[(3as,7r,7as)-7-hydroxy-4-oxo-1,3a,5,6,7,7a-hexahydroimidazo[4,5-c]pyridin-2-yl]amino]-5-[[(3s)-3,6-diaminohexanoyl]amino]-4-hydroxy-2-(hydroxymethyl)oxan-3-yl] carbamate Chemical compound NCCC[C@H](N)CC(=O)N[C@@H]1[C@@H](O)[C@H](OC(N)=O)[C@@H](CO)O[C@H]1\N=C/1N[C@H](C(=O)NC[C@H]2O)[C@@H]2N\1 NRAUADCLPJTGSF-ZPGVOIKOSA-N 0.000 description 1
- 229960004998 acesulfame potassium Drugs 0.000 description 1
- 235000010358 acesulfame potassium Nutrition 0.000 description 1
- 239000000619 acesulfame-K Substances 0.000 description 1
- PXAJQJMDEXJWFB-UHFFFAOYSA-N acetone oxime Chemical compound CC(C)=NO PXAJQJMDEXJWFB-UHFFFAOYSA-N 0.000 description 1
- 230000009056 active transport Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- DFNYGALUNNFWKJ-UHFFFAOYSA-N aminoacetonitrile Chemical compound NCC#N DFNYGALUNNFWKJ-UHFFFAOYSA-N 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- IAOZJIPTCAWIRG-QWRGUYRKSA-N aspartame Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)OC)CC1=CC=CC=C1 IAOZJIPTCAWIRG-QWRGUYRKSA-N 0.000 description 1
- 239000000605 aspartame Substances 0.000 description 1
- 235000010357 aspartame Nutrition 0.000 description 1
- 229960003438 aspartame Drugs 0.000 description 1
- 238000000429 assembly Methods 0.000 description 1
- 230000000712 assembly Effects 0.000 description 1
- 101150109560 atzC gene Proteins 0.000 description 1
- 101150050997 atzD gene Proteins 0.000 description 1
- 101150107491 atzE gene Proteins 0.000 description 1
- 101150056434 atzF gene Proteins 0.000 description 1
- XOZUGNYVDXMRKW-AATRIKPKSA-N azodicarbonamide Chemical compound NC(=O)\N=N\C(N)=O XOZUGNYVDXMRKW-AATRIKPKSA-N 0.000 description 1
- 235000019399 azodicarbonamide Nutrition 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- DMSMPAJRVJJAGA-UHFFFAOYSA-N benzo[d]isothiazol-3-one Chemical compound C1=CC=C2C(=O)NSC2=C1 DMSMPAJRVJJAGA-UHFFFAOYSA-N 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- XEVRDFDBXJMZFG-UHFFFAOYSA-N carbonyl dihydrazine Chemical compound NNC(=O)NN XEVRDFDBXJMZFG-UHFFFAOYSA-N 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 238000012411 cloning technique Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- SOROIESOUPGGFO-UHFFFAOYSA-N diazolidinylurea Chemical compound OCNC(=O)N(CO)C1N(CO)C(=O)N(CO)C1=O SOROIESOUPGGFO-UHFFFAOYSA-N 0.000 description 1
- 229960001083 diazolidinylurea Drugs 0.000 description 1
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 1
- 229940043237 diethanolamine Drugs 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- WHRIKZCFRVTHJH-UHFFFAOYSA-N ethylhydrazine Chemical compound CCNN WHRIKZCFRVTHJH-UHFFFAOYSA-N 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 229960004979 fampridine Drugs 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000012224 gene deletion Methods 0.000 description 1
- 230000004545 gene duplication Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000012248 genetic selection Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000004312 hexamethylene tetramine Substances 0.000 description 1
- 235000010299 hexamethylene tetramine Nutrition 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- ZCTXEAQXZGPWFG-UHFFFAOYSA-N imidurea Chemical compound O=C1NC(=O)N(CO)C1NC(=O)NCNC(=O)NC1C(=O)NC(=O)N1CO ZCTXEAQXZGPWFG-UHFFFAOYSA-N 0.000 description 1
- 101150030475 impact gene Proteins 0.000 description 1
- 230000003116 impacting effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- ZLTPDFXIESTBQG-UHFFFAOYSA-N isothiazole Chemical compound C=1C=NSC=1 ZLTPDFXIESTBQG-UHFFFAOYSA-N 0.000 description 1
- MGIYRDNGCNKGJU-UHFFFAOYSA-N isothiazolinone Chemical compound O=C1C=CSN1 MGIYRDNGCNKGJU-UHFFFAOYSA-N 0.000 description 1
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- WRIRWRKPLXCTFD-UHFFFAOYSA-N malonamide Chemical compound NC(=O)CC(N)=O WRIRWRKPLXCTFD-UHFFFAOYSA-N 0.000 description 1
- CUONGYYJJVDODC-UHFFFAOYSA-N malononitrile Chemical compound N#CCC#N CUONGYYJJVDODC-UHFFFAOYSA-N 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- CXKWCBBOMKCUKX-UHFFFAOYSA-M methylene blue Chemical compound [Cl-].C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 CXKWCBBOMKCUKX-UHFFFAOYSA-M 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 238000009343 monoculture Methods 0.000 description 1
- 238000012267 monoculture fermentation Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- IONSZLINWCGRRI-UHFFFAOYSA-N n'-hydroxymethanimidamide Chemical compound NC=NO IONSZLINWCGRRI-UHFFFAOYSA-N 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- 229910017464 nitrogen compound Inorganic materials 0.000 description 1
- 150000002830 nitrogen compounds Chemical class 0.000 description 1
- LYGJENNIWJXYER-UHFFFAOYSA-N nitromethane Chemical compound C[N+]([O-])=O LYGJENNIWJXYER-UHFFFAOYSA-N 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 210000002706 plastid Anatomy 0.000 description 1
- 229920001223 polyethylene glycol 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
- 230000019525 primary metabolic process Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000008263 repair mechanism Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 239000007320 rich medium Substances 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- HBMJWWWQQXIZIP-UHFFFAOYSA-N silicon carbide Chemical compound [Si+]#[C-] HBMJWWWQQXIZIP-UHFFFAOYSA-N 0.000 description 1
- 229910010271 silicon carbide Inorganic materials 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- VGTPCRGMBIAPIM-UHFFFAOYSA-M sodium thiocyanate Chemical compound [Na+].[S-]C#N VGTPCRGMBIAPIM-UHFFFAOYSA-M 0.000 description 1
- IXBPPZBJIFNGJJ-UHFFFAOYSA-N sodium;cyanoiminomethylideneazanide Chemical compound [Na+].N#C[N-]C#N IXBPPZBJIFNGJJ-UHFFFAOYSA-N 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- IAHFWCOBPZCAEA-UHFFFAOYSA-N succinonitrile Chemical compound N#CCCC#N IAHFWCOBPZCAEA-UHFFFAOYSA-N 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- FAGUFWYHJQFNRV-UHFFFAOYSA-N tetraethylenepentamine Chemical compound NCCNCCNCCNCCN FAGUFWYHJQFNRV-UHFFFAOYSA-N 0.000 description 1
- 150000003536 tetrazoles Chemical class 0.000 description 1
- YUKQRDCYNOVPGJ-UHFFFAOYSA-N thioacetamide Chemical compound CC(N)=S YUKQRDCYNOVPGJ-UHFFFAOYSA-N 0.000 description 1
- DLFVBJFMPXGRIB-UHFFFAOYSA-N thioacetamide Natural products CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- LZGVDNRJCGPNDS-UHFFFAOYSA-N trinitromethane Chemical compound [O-][N+](=O)C([N+]([O-])=O)[N+]([O-])=O LZGVDNRJCGPNDS-UHFFFAOYSA-N 0.000 description 1
- 101150037367 trzA gene Proteins 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
-
- 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)
-
- 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
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y308/00—Hydrolases acting on halide bonds (3.8)
- C12Y308/01—Hydrolases acting on halide bonds (3.8) in C-halide substances (3.8.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/01—Hydro-lyases (4.2.1)
- C12Y402/01069—Cyanamide hydratase (4.2.1.69)
Definitions
- cell culture media is typically formulated to provide all nutrients necessary for the growth of a host cell line, with particular emphasis on meeting the cell line's requirements for carbon, nitrogen, phosphorus, sulfur, and other major nutrients.
- Some cell lines require additional components, including amino acids, trace minerals and metals, and complex growth factors. The presence of these nutrients provides a suitable growth environment for the organism of choice and, unfortunately, for any potential contaminating organisms. In this environment the production organism is required to compete directly with any contaminant organism in the cell culture.
- the invention relates to a genetically engineered organism, wherein the genetically engineered organism has been transformed by a nucleic acid molecule comprising any one or more of the sequences disclosed herein.
- the invention relates to a genetically engineered organism, wherein the genetically engineered organism has been transformed by a nucleic acid molecule; the nucleic acid molecule comprises a non-native gene; and the non-native gene encodes for a non-native enzyme selected from the group consisting of allophanate hydrolase, biuret amidohydrolase, cyanuric acid amidohydrolase, guanine deaminase, melamine deaminase, isopropylammelide isopropylaminohydrolase, cyanamide hydratase, urease, and urea carboxylase.
- a non-native enzyme selected from the group consisting of allophanate hydrolase, biuret amidohydrolase, cyanuric acid amidohydrolase, guanine deaminase, melamine deaminase, isopropylammelide isopropylaminohydrolase, cyanamide hydratase,
- the invention relates to a method, comprising the step of
- the substrate comprises a nitrogen-containing fraction and a non-nitrogen-containing fraction
- the nitrogen-containing fraction comprises, in an amount from about 10% by weight to about 100% by weight, a nitrogen-containing compound of any one of Formulas I-III, or a salt thereof;
- the genetically engineered organism converts the substrate to a product
- the invention relates to a method, comprising the step of
- the substrate comprises a nitrogen-containing fraction and a non-nitrogen-containing fraction
- the nitrogen-containing fraction comprises, in an amount from about 10% by weight to about 100% by weight, a nitrogen-containing compound selected from the group consisting of triazine, urea, melamine, cyanamide, 2-cyanoguanidine, ammeline, guanidine carbonate, ethylenediamine, ammelide, biuret, diethylenetriamine, triethylenetetramine, 1,3-diaminopropane, calcium cyanamide, cyanuric acid, aminoethylpiperazine, piperazine, and allophante; and
- a nitrogen-containing compound selected from the group consisting of triazine, urea, melamine, cyanamide, 2-cyanoguanidine, ammeline, guanidine carbonate, ethylenediamine, ammelide, biuret, diethylenetriamine, triethylenetetramine, 1,3-diaminopropane, calcium cyanamide, cyanuric acid, aminoethylpiperazine, piperazine, and allophante;
- the genetically engineered organism converts the substrate to a product.
- the invention relates to a method, comprising the step of
- the substrate comprises a nitrogen-containing fraction and a non-nitrogen-containing fraction
- the nitrogen containing fraction consists essentially of a nitrogen-containing compound selected from the group consisting of triazine, urea, melamine, cyanamide, 2-cyanoguanidine, ammeline, guanidine carbonate, ethylenediamine, ammelide, biuret, diethylenetriamine, triethylenetetramine, 1,3-diaminopropane, calcium cyanamide, cyanuric acid, aminoethylpiperazine, piperazine, and allophante; and
- the genetically engineered organism converts the substrate to a product.
- the invention relates to a method comprising the step of
- the substrate consists of a nitrogen-containing fraction and a non-nitrogen-containing fraction
- the nitrogen containing fraction consists of a nitrogen-containing compound selected from the group consisting of triazine, urea, melamine, cyanamide, 2-cyanoguanidine, ammeline, guanidine carbonate, ethylenediamine, ammelide, biuret, diethylenetriamine, triethylenetetramine, 1,3-diaminopropane, calcium cyanamide, cyanuric acid, aminoethylpiperazine, piperazine, and allophante; and
- the genetically engineered organism converts the substrate to a product.
- the invention relates to a product made by any one of the aforementioned methods.
- the invention relates to a recombinant vector comprising a gene operably linked to a promoter, wherein the gene encodes an enzyme; and the enzyme is allophanate hydrolase, biuret amidohydrolase, cyanuric acid amidohydrolase, guanine deaminase, melamine deaminase, isopropylammelide isopropylaminohydrolase, cyanamide hydratase, urease, or urea carboxylase.
- FIG. 1 depicts a schematic representation of the melamine degradation pathway.
- 1 Melamine deaminase (tzrA) (EC 3.5.4.-); 2—Ammeline deaminase (guanine deaminase) (EC 3.5.4.3); 3—N-isopropylammelide isopropylamino (Ammelide) hydrolyase (EC 3.5.99.4); 4—Cyanuric acid hydrolyase (EC 3.5.2.15); 4a—Carboxybiuret decarboxylase, spontaneous reaction; 5—Biuret amidohydrolase (EC 3.5.1.84); 6—Allophanate hydrolyase (EC 3.5.1.54).
- tzrA Melamine deaminase
- 2 Ammeline deaminase (guanine deaminase) (EC 3.5.4.3); 3—N-isopropylammelide isopropylamino (Ammelide)
- Nitrogen can be assimilated (as NH 3 ) by the action of the complete pathway acting on melamine, liberating 6 mol NH 3 per mol melamine, or via a subset of enzymes acting on pathway intermediates (e.g., steps 4, 4a, 5, and 6 acting on cyanuric acid releasing 3 mol NH 3 per mol cyanuric acid).
- FIG. 2 tabulates exemplary compounds capable of delivering nitrogen that could be accessed by an engineered organism.
- FIG. 3 tabulates DNA and protein sequences encoding the melamine degradation pathway.
- FIG. 4 depicts a schematic representation of the cyanamide assimilation pathway. After conversion of cyanamide to urea by cyanamide hydratase (EC 4.2.1.69), urea can be degraded either via urease (EC 3.5.1.5) or by urea carboxylase (EC 6.3.4.6) and allophante hydrolyase (EC 3.5.1.54).
- FIGS. 5-10 depict various plasmids of the invention.
- FIG. 11 tabulates the concentrations of the components in the MOPS medium used in Example 9.
- FIG. 12 depicts the growth progress of NS88 and NS91 (control) in media containing various concentrations of ammonium ion or melamine
- FIG. 13 depicts the growth progress of NS90 and NS91 (control) in media containing various concentrations of ammonium ion or biuret.
- FIG. 14 depicts images, taken after 48 h, of cultures grown in MOPS media with different nitrogen sources. From left to right: NS88 with 10 mM melamine; NS91 with 10 mM melamine; NS90 with 10 mM biuret (replicate 1); NS90 with 10 mM biuret (replicate 2); and NS91 with 10 mM biuret.
- FIG. 15 depicts a plasmid of the invention.
- FIG. 16 depicts a plasmid of the invention.
- FIG. 17 depicts the growth progress of NS100 (control) and NS101 in media containing no nitrogen source, urea, or cyanamide.
- FIG. 18 depicts the population fraction of NS100 (control) and NS101 in a urea-containing medium.
- FIG. 19 depicts the population fraction of NS100 (control) and NS101 in a cyanamide-containing medium.
- FIG. 20 depicts the growth progress of NS100 (control) and NS101 in media containing no nitrogen source, or media containing cyanamide.
- FIG. 21 depicts the growth of an organism of the invention in the presence of an antibiotic on various nitrogen-containing media (see FIG. 33 for composition of SC amino acid media).
- FIG. 22 tabulates the optical density at 600 nm after growth of four organisms of the invention on various media.
- FIG. 23 tabulates the optical density at 600 nm after growth of three organisms of the invention on various media.
- FIG. 26 depicts the growth of various organisms of the invention on 0.5 mM NH 4 Cl.
- the organisms described in FIGS. 26-28 for example NS120, NS91, NS107, and NS123, are E. coli strains derived from E. coli K12, E. coli B, E. coli Crooks, and E. coli MG1655 and are intended to show the breadth of the invention across various strains of E. coli.
- FIG. 27 depicts the growth of various organisms of the invention on a medium containing no nitrogen.
- FIG. 28 depicts the growth of various organisms of the invention on a medium containing 0.5 mM melamine
- FIG. 29 tabulates a summary of various plasmids of the invention.
- FIG. 30 tabulates a summary of various organisms of the invention.
- FIG. 31 tabulates the components and molar concentrations of each component in a MOPS defined medium, which is used, for example, with E. coli.
- FIG. 32 tabulates the components and weight concentrations of each component in a YNB medium, which is used, for example, with S. cerevisiae.
- FIG. 33 tabulates the components and weight concentrations of each component in a SC amino acid medium.
- the invention relates to a genetically engineered host organism, wherein the genetically engineered host organism has a non-native ability to obtain a growth-limiting nutrient from a complex substrate; and the complex substrate could not have been metabolized or used as a nutrient by the native host organism.
- the non-native ability will provide the organism with a significant competitive advantage, and provide a major barrier to the success of contaminants in a fermentation.
- the genetically engineered host organism is a bacterium, a yeast, a fungus, a mammalian cell, or an insect cell.
- the genetically engineered host organism is a bacterium or a yeast.
- the invention relates to a method of using the above-mentioned genetically engineered host organism, comprising contacting the genetically engineered host organism with a modified cell culture medium. In certain embodiments, the invention relates to a method of using the above-mentioned genetically engineered host organism, comprising contacting the genetically engineered host organism with a modified cell culture medium, wherein the genetically engineered host organism converts the cell culture medium to a product. In certain embodiments, using this approach provides a unique and targeted manner to promote the growth of the desired genetically engineered host organism. In certain embodiments, the above-mentioned methods minimize the growth of contaminant organisms, provide a valuable competitive advantage, and allow management of production of a range of valuable products.
- the inventive methods decrease or eliminate the need for use of prophylactic antibiotics in large scale yeast cultures. Avoiding unnecessary antibiotics is an important benefit due to emerging environmental considerations and societal pressures. Additionally, in certain embodiments, the technique can be applied to bacterial systems in which antibiotics may not be added.
- the genetically engineered host organism is a yeast; and the product is ethanol, isobutanol, lactic acid, an isoprenoid, a lipid, and enzyme product, or a high value specialty chemical.
- the genetically engineered host organism is a bacterium
- butanol is butanol, ethanol, isopropanol, 1,3-propanediol (PDO), 1,4-butanediol (BDO), succinic acid, itaconic acid, an enzyme product, a polyol, a protein product, or a high value specialty chemical.
- PDO 1,3-propanediol
- BDO 1,4-butanediol
- succinic acid 1, itaconic acid
- an enzyme product a polyol, a protein product, or a high value specialty chemical.
- the inventive technology is applicable in the production of one or more commodities, fine chemicals, and pharmaceuticals.
- Dry weight and “dry cell weight” mean weight determined in the relative absence of water. For example, reference to oleaginous cells as comprising a specified percentage of a particular component by dry weight means that the percentage is calculated based on the weight of the cell after substantially all water has been removed.
- Exogenous gene is a nucleic acid that codes for the expression of an RNA and/or protein that has been introduced into a cell (e.g., by transformation/transfection), and is also referred to as a “transgene.”
- a cell comprising an exogenous gene may be referred to as a recombinant cell, into which additional exogenous gene(s) may be introduced.
- the exogenous gene may be from a different species (and so heterologous), or from the same species (and so homologous), relative to the cell being transformed.
- an exogenous gene can include a homologous gene that occupies a different location in the genome of the cell or is under different control, relative to the endogenous copy of the gene.
- An exogenous gene may be present in more than one copy in the cell.
- An exogenous gene may be maintained in a cell as an insertion into the genome (nuclear or plastid) or as an episomal molecule.
- “Expression vector” or “expression construct” or “plasmid” or “recombinant DNA construct” is a vehicle for introducing a nucleic acid into a host cell.
- the nucleic acid can be one that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription and/or translation of a particular nucleic acid.
- the expression vector can be part of a plasmid, virus, or nucleic acid fragment, or other suitable vehicle.
- the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
- “Inducible promoter” is a promoter that mediates transcription of an operably linked gene in response to a particular stimulus.
- “In operable linkage” is a functional linkage between two nucleic acid sequences, such a control sequence (typically a promoter) and the linked sequence (typically a sequence that encodes a protein, also called a coding sequence).
- a promoter is in operable linkage with an exogenous gene if it can mediate transcription of the gene.
- Lysate is a solution containing the contents of lysed cells.
- “Lysis” is the breakage of the plasma membrane and optionally the cell wall of a biological organism sufficient to release at least some intracellular content, often by mechanical, viral or osmotic mechanisms that compromise its integrity.
- “Lysing” is disrupting the cellular membrane and optionally the cell wall of a biological organism or cell sufficient to release at least some intracellular content.
- Osmotic shock is the rupture of cells in a solution following a sudden reduction in osmotic pressure. Osmotic shock is sometimes induced to release cellular components of such cells into a solution.
- Plasmid refers to an extra chromosomal element often carrying genes that are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules.
- Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell.
- Transformation cassette refers to a specific vector containing a foreign gene and having elements, in addition to the foreign gene, that facilitate transformation of a particular host cell.
- Expression cassette refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.
- Promoter is a nucleic acid control sequence that directs transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
- Recombinant is a cell, nucleic acid, protein, or vector, which has been modified due to the introduction of an exogenous nucleic acid or the alteration of a native nucleic acid.
- recombinant cells can express genes that are not found within the native (non-recombinant) form of the cell or express native genes differently than those genes are expressed by a non-recombinant cell.
- Recombinant cells can, without limitation, include recombinant nucleic acids that encode for a gene product or for suppression elements such as mutations, knockouts, antisense, interfering RNA (RNAi) or dsRNA that reduce the levels of active gene product in a cell.
- RNAi interfering RNA
- a “recombinant nucleic acid” is a nucleic acid originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases, ligases, exonucleases, and endonucleases, or otherwise is in a form not normally found in nature.
- Recombinant nucleic acids may be produced, for example, to place two or more nucleic acids in operable linkage.
- an isolated nucleic acid or an expression vector formed in vitro by ligating DNA molecules that are not normally joined in nature are both considered recombinant for the purposes of this invention.
- a recombinant nucleic acid Once a recombinant nucleic acid is made and introduced into a host cell or organism, it may replicate using the in vivo cellular machinery of the host cell; however, such nucleic acids, once produced recombinantly, although subsequently replicated intracellularly, are still considered recombinant for purposes of this invention.
- a “recombinant protein” is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid.
- Sonication is a process of disrupting biological materials, such as a cell, by use of sound wave energy.
- Transformation refers to the transfer of a nucleic acid fragment into a host organism or the genome of a host organism, resulting in genetically stable inheritance.
- Host organisms containing the transformed nucleic acid fragments are referred to as “recombinant”, “transgenic” or “transformed” organisms.
- isolated polynucleotides of the present invention can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell.
- Such a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell.
- expression vectors include, for example, one or more cloned genes under the transcriptional control of 5′ and 3′ regulatory sequences and a selectable marker.
- Such vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or location-specific expression), a transcription initiation start site, a ribosome binding site, a transcription termination site, and/or a polyadenylation signal.
- a microorganism is genetically modified to improve or provide de novo growth characteristics on a variety of feedstock materials.
- Genes and gene products may be introduced into microbial host cells.
- Suitable host cells for expression of the genes and nucleic acid molecules are microbial hosts that can be found broadly within the fungal or bacterial families and which grow over a wide range of temperature, pH values, and solvent tolerances.
- suitable host strains include but are not limited to fungal or yeast species, such as Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, Kluyveromyces , or bacterial species, such as member of the proteobacteria and actinomycetes as well as the specific genera Acinetobacter, Arthrobacter, Brevibacterium, Acidovorax, Bacillus, Clostridia, Streptomyces, Escherichia, Salmonella, Pseudomonas , and Cornyebacterium.
- fungal or yeast species such as Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, Kluyveromyces , or bacterial species, such as member of the proteobacteria and actinomycetes as well as the specific genera Acinetobacter, Arthrobacter, Brevibacterium, Acidovorax, Bacillus, Clostridia, Streptomyces, Escherich
- E. coli is well suited to use as the host microorganism in the invention fermentative processes.
- Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct chimeric genes to produce the any one of the gene products of the instant sequences. These chimeric genes could then be introduced into appropriate microorganisms via transformation techniques to provide high-level expression of the enzymes.
- a gene encoding an enzyme can be cloned in a suitable plasmid, and the aforementioned starting parent strain as a host can be transformed with the resulting plasmid.
- This approach can increase the copy number of each of the genes encoding the enzymes and, as a result, the activities of these enzymes can be increased.
- the plasmid is not particularly limited so long as it can autonomously replicate in the microorganism.
- Vectors or cassettes useful for the transformation of suitable host cells are well known in the art.
- the vector or cassette contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration.
- Suitable vectors comprise a region 5′ of the gene harboring transcriptional initiation controls and a region 3′ of the DNA fragment which controls transcriptional termination. It is most preferred when both control regions are derived from genes homologous to the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host.
- Promoters, cDNAs, and 3′UTRs, as well as other elements of the vectors can be generated through cloning techniques using fragments isolated from native sources (see, for example, Molecular Cloning: A Laboratory Manual, Sambrook et al. (3d edition, 2001, Cold Spring Harbor Press; and U.S. Pat. No. 4,683,202 (incorporated by reference)). Alternatively, elements can be generated synthetically using known methods (see, for example, Gene. 1995 Oct. 16; 164(1):49-53).
- Homologous recombination is the ability of complementary DNA sequences to align and exchange regions of homology.
- Transgenic DNA (“donor”) containing sequences homologous to the genomic sequences being targeted (“template”) is introduced into the organism and then undergoes recombination into the genome at the site of the corresponding genomic homologous sequences.
- homologous recombination is a precise gene targeting event, hence, most transgenic lines generated with the same targeting sequence will be essentially identical in terms of phenotype, necessitating the screening of far fewer transformation events.
- homologous recombination also targets gene insertion events into the host chromosome, potentially resulting in excellent genetic stability, even in the absence of genetic selection. Because different chromosomal loci will likely impact gene expression, even from heterologous promoters/UTRs, homologous recombination can be a method of querying loci in an unfamiliar genome environment and to assess the impact of these environments on gene expression.
- a particularly useful genetic engineering approach using homologous recombination is to co-opt specific host regulatory elements such as promoters/UTRs to drive heterologous gene expression in a highly specific fashion.
- homologous recombination is a precise gene targeting event, it can be used to precisely modify any nucleotide(s) within a gene or region of interest, so long as sufficient flanking regions have been identified. Therefore, homologous recombination can be used as a means to modify regulatory sequences impacting gene expression of RNA and/or proteins. It can also be used to modify protein coding regions in an effort to modify enzyme activities such as substrate specificity, affinities and Km, and thus affecting the desired change in metabolism of the host cell. Homologous recombination provides a powerful means to manipulate the host genome resulting in gene targeting, gene conversion, gene deletion, gene duplication, gene inversion and exchanging gene expression regulatory elements such as promoters, enhancers and 3′UTRs.
- Homologous recombination can be achieved by using targeting constructs containing pieces of endogenous sequences to “target” the gene or region of interest within the endogenous host cell genome.
- targeting sequences can either be located 5′ of the gene or region of interest, 3′ of the gene/region of interest or even flank the gene/region of interest.
- Such targeting constructs can be transformed into the host cell either as a supercoiled plasmid DNA with additional vector backbone, a PCR product with no vector backbone, or as a linearized molecule.
- Other methods of increasing recombination efficiency include using PCR to generate transforming transgenic DNA containing linear ends homologous to the genomic sequences being targeted.
- Vectors for transformation of microorganisms in accordance with the present invention can be prepared by known techniques familiar to those skilled in the art in view of the disclosure herein.
- a vector typically contains one or more genes, in which each gene codes for the expression of a desired product (the gene product) and is operably linked to one or more control sequences that regulate gene expression or target the gene product to a particular location in the recombinant cell.
- Subsection 1 describes control sequences typically contained on vectors as well as novel control sequences provided by the present invention.
- Subsection 2 describes genes typically contained in vectors as well as novel codon optimization methods and genes prepared using them provided by the invention.
- Control sequences are nucleic acids that regulate the expression of a coding sequence or direct a gene product to a particular location in or outside a cell.
- Control sequences that regulate expression include, for example, promoters that regulate transcription of a coding sequence and terminators that terminate transcription of a coding sequence.
- Another control sequence is a 3′ untranslated sequence located at the end of a coding sequence that encodes a polyadenylation signal.
- Control sequences that direct gene products to particular locations include those that encode signal peptides, which direct the protein to which they are attached to a particular location in or outside the cell.
- an exemplary vector design for expression of an exogenous gene in a microbe contains a coding sequence for a desired gene product (for example, a selectable marker, or an enzyme) in operable linkage with a promoter active in microalgae.
- a desired gene product for example, a selectable marker, or an enzyme
- the coding sequence can be transformed into the cells such that it becomes operably linked to an endogenous promoter at the point of vector integration.
- the promoter used to express an exogenous gene can be the promoter naturally linked to that gene or can be a heterologous promoter.
- a promoter can generally be characterized as either constitutive or inducible. Constitutive promoters are generally active or function to drive expression at all times (or at certain times in the cell life cycle) at the same level. Inducible promoters, conversely, are active (or rendered inactive) or are significantly up- or down-regulated only in response to a stimulus. Both types of promoters find application in the methods of the invention. Inducible promoters useful in the invention include those that mediate transcription of an operably linked gene in response to a stimulus, such as an exogenously provided small molecule, temperature (heat or cold), lack of nitrogen in culture media, etc. Suitable promoters can activate transcription of an essentially silent gene or upregulate, preferably substantially, transcription of an operably linked gene that is transcribed at a low level.
- termination region control sequence is optional, and if employed, then the choice is be primarily one of convenience, as the termination region is relatively interchangeable.
- the termination region may be native to the transcriptional initiation region (the promoter), may be native to the DNA sequence of interest, or may be obtainable from another source. See, for example, Chen and Orozco, Nucleic Acids Res. (1988) 16:8411.
- a gene typically includes a promoter, coding sequence, and termination control sequences.
- a gene When assembled by recombinant DNA technology, a gene may be termed an expression cassette and may be flanked by restriction sites for convenient insertion into a vector that is used to introduce the recombinant gene into a host cell.
- the expression cassette can be flanked by DNA sequences from the genome or other nucleic acid target to facilitate stable integration of the expression cassette into the genome by homologous recombination.
- the vector and its expression cassette may remain unintegrated (e.g., an episome), in which case, the vector typically includes an origin of replication, which is capable of providing for replication of the heterologous vector DNA.
- a common gene present on a vector is a gene that codes for a protein, the expression of which allows the recombinant cell containing the protein to be differentiated from cells that do not express the protein.
- a gene, and its corresponding gene product is called a selectable marker or selection marker. Any of a wide variety of selectable markers can be employed in a transgene construct useful for transforming the organisms of the invention.
- transgenes For optimal expression of a recombinant protein, it is beneficial to employ coding sequences that produce mRNA with codons optimally used by the host cell to be transformed.
- proper expression of transgenes can require that the codon usage of the transgene matches the specific codon bias of the organism in which the transgene is being expressed.
- the precise mechanisms underlying this effect are many, but include the proper balancing of available aminoacylated tRNA pools with proteins being synthesized in the cell, coupled with more efficient translation of the transgenic messenger RNA (mRNA) when this need is met.
- mRNA transgenic messenger RNA
- a genetically engineered microorganism may comprise and express more than one exogenous gene.
- One or more genes can be expressed using an inducible promoter, which allows the relative timing of expression of these genes to be controlled. Expression of the two or more exogenous genes may be under control of the same inducible promoter or under control of different inducible promoters. In the latter situation, expression of a first exogenous gene can be induced for a first period of time (during which expression of a second exogenous gene may or may not be induced) and expression of a second or further exogenous gene can be induced for a second period of time (during which expression of a first exogenous gene may or may not be induced).
- Provided herein are vectors and methods for engineering microbes to grow on non-traditional growth media.
- Cells can be transformed by any suitable technique including, e.g., biolistics, electroporation, glass bead transformation and silicon carbide whisker transformation. Any convenient technique for introducing a transgene into a microorganism can be employed in the present invention. Transformation can be achieved by, for example, the method of D. M. Morrison (Methods in Enzymology 68, 326 (1979)), the method by increasing permeability of recipient cells for DNA with calcium chloride (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)), or the like.
- transgenes in oleaginous yeast e.g., Yarrowia lipolytica
- yeast e.g., Yarrowia lipolytica
- Examples of expression of exogenous genes in bacteria such as E. coli are well known; see, for example, Molecular Cloning: A Laboratory Manual, Sambrook et al. (3d edition, 2001, Cold Spring Harbor Press).
- an exemplary vector design for expression of a gene in a microorganism contains a gene encoding an enzyme in operable linkage with a promoter active in the microorganism.
- the gene can be transformed into the cells such that it becomes operably linked to an endogenous promoter at the point of vector integration.
- the vector can also contain a second gene that encodes a protein.
- one or both gene(s) is/are followed by a 3′ untranslated sequence containing a polyadenylation signal.
- Expression cassettes encoding the two genes can be physically linked in the vector or on separate vectors. Co-transformation of microbes can also be used, in which distinct vector molecules are simultaneously used to transform cells (see, for example, Protist 2004 December; 155(4):381-93). The transformed cells can be optionally selected based upon the ability to grow in the presence of the antibiotic or other selectable marker under conditions in which cells lacking the resistance cassette would not grow.
- the invention relates to use of an atypical nitrogen-containing feedstock comprising, consisting essentially of, or consisting of a nitrogen-containing compound of any one of Formulas I-III.
- a non-genetically engineered organism i.e., a native organism, could not metabolize (i.e., use as a source of nitrogen) the nitrogen-containing compounds in the feedstock.
- the invention relates to any one of the aforementioned nitrogen-containing feedstocks, wherein the nitrogen-containing compound is a compound of formula I or a salt thereof:
- R is —OH, —CO 2 H, —NO 2 , —CN, substituted or unsubstituted amino, or substituted or unsubstituted alkyl;
- n 0, 1, 2, 3, 4, or 5.
- the invention relates to any one of the aforementioned nitrogen-containing feedstocks, wherein the nitrogen-containing compound is a compound of formula II or a salt thereof:
- X is —NH—, —N(alkyl)-, —O—, —C(R 1 ) 2 —, —S—, or absent;
- Y is —H, —NH 2 , —N(H)(alkyl), —N(alkyl) 2 , —CO 2 H, —CN, or substituted or unsubstituted alkyl;
- R 1 is —H, —OH, —CO 2 H, —NO 2 , —CN, substituted or unsubstituted amino, or substituted or unsubstituted alkyl.
- the invention relates to any one of the aforementioned nitrogen-containing feedstocks, wherein the nitrogen-containing compound is a compound of formula III or a salt thereof:
- Y is —H, —NH 2 , —N(H)(alkyl), —N(alkyl) 2 , —CO 2 H, —CN, or substituted or unsubstituted alkyl.
- the invention relates to any one of the aforementioned nitrogen-containing feedstocks, wherein the nitrogen-containing compound is selected from the group consisting of:
- the invention relates to any one of the aforementioned nitrogen-containing feedstocks, wherein the nitrogen-containing compound is selected from the group consisting of Hydrazine, 5-Aminotetrazole, Tetrazole, Melamine, Cyanamide, 2-Cyanoguanidine, Sodium azide, Carbohydrazide, 1,2,3-Triazole, 1,2,4-Triazole, 1,3-Diaminoguanidine HCl, Ammeline, 1,3,5-triazine, Aminoacetonitrile, Cyanoethylhydrazine, Azodicarbonamide, Biurea, Formamidoxime, 1,2-Dimethylhydrazine, 1,1-Dimethylhydrazine, ethylhydrazine, Ethylenediamine, Sodium dicyanamide, Guanidine carbonate, Methylamine, Ammelide, Hydroxylamine, Malononitrile, Biuret, Diethyltriamine, Hexamethylenetetra
- the invention relates to an isolated nucleic acid molecule, wherein the nucleic acid molecule encodes an enzyme that provides the organism with the ability to assimilate a nitrogen source that otherwise would not have been accessible to the native organism; and the enzyme is allophanate hydrolase, biuret amidohydrolase, cyanuric acid amidohydrolase, guanine deaminase, ammeline hydrolase, ammelide hydrolyase, melamine deaminase, isopropylammelide isopropylaminohydrolase, cyanamide hydratase, urease, or urea carboxylase.
- the enzyme is allophanate hydrolase, biuret amidohydrolase, cyanuric acid amidohydrolase, guanine deaminase, ammeline hydrolase, ammelide hydrolyase, melamine deaminase, isopropylammelide isopropylaminohydrolase, cyanamide hydrat
- the invention relates to an isolated nucleic acid molecule, wherein the nucleic acid molecule is selected from the group consisting of trzE from Rhodococcus sp. strain Mel, trzE from Rhizobium leguminosarum , trzC MEL, trzC 12227, cah from Fusarium oxysporum Fo5176, cah from F. pseudograminaearum CS3096, cah from Gibberella zeae PH-1, cah from Aspergillus kawachii IFO 4308, cah from A. niger CBS 513.88, cah from A. niger ATCC 1015, cah from A.
- oryzae 3.042 cah from S. cerevisiae FostersB, atzF from Pseudomonas sp. strain ADP, DUR1,2 from S. cerevisiae , YALI0E 07271g from Y. lipolytica CLIB122, atzE from Pseudomonas sp. strain ADP, atzD from Pseudomonas sp. strain ADP, trzD from Pseudomonas sp. strain NRRLB-12227, atzD from Rhodococcus sp. Mel, trzD from Rhodococcus sp. Mel, guaD from E.
- the invention relates to an isolated nucleic acid molecule comprising any one of the sequences disclosed herein. In certain embodiments, the invention relates to an isolated nucleic acid molecule having at least 85% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to an isolated nucleic acid molecule having at least 90% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to an isolated nucleic acid molecule having at least 95% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to an isolated nucleic acid molecule having at least 99% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to an isolated nucleic acid molecule having any one of the sequences disclosed herein.
- a recombinant vector comprising any one of the aformentioned nucleic acid molecules operably linked to a promoter.
- the invention relates to a recombinant vector comprising any one of the sequences disclosed herein. In certain embodiments, the invention relates to a recombinant vector having at least 85% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to a recombinant vector having at least 90% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to a recombinant vector having at least 95% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to a recombinant vector having at least 99% sequence homology with any one of the sequences disclosed herein.
- the invention relates to a genetically engineered organism, wherein the genetically engineered organism has been transformed by a nucleic acid molecule or a recombinant vector comprising any one of the sequences disclosed herein.
- the nucleic acid molecule or recombinant vector has at least 85% sequence homology with any one of the sequences disclosed herein.
- the nucleic acid molecule or recombinant vector has at least 90% sequence homology with any one of the sequences disclosed herein.
- the nucleic acid molecule or recombinant vector has at least 95% sequence homology with any one of the sequences disclosed herein.
- the nucleic acid molecule or recombinant vector has at least 99% sequence homology with any one of the sequences disclosed herein.
- the invention relates to a genetically engineered organism, wherein the genetically engineered organism has been transformed by a nucleic acid molecule or a recombinant vector having any one of the sequences disclosed herein.
- the invention relates to a genetically engineered organism, wherein the genetically engineered organism has been transformed by a nucleic acid molecule; the nucleic acid molecule comprises a non-native gene; and the non-native gene encodes for a non-native enzyme selected from the group consisting of allophanate hydrolase, biuret amidohydrolase, cyanuric acid amidohydrolase, guanine deaminase, ammeline hydrolase, ammelide hydrolyase, melamine deaminase, and isopropylammelide isopropylaminohydrolase, cyanamide hydratase, urease, or urea carboxylase.
- a non-native enzyme selected from the group consisting of allophanate hydrolase, biuret amidohydrolase, cyanuric acid amidohydrolase, guanine deaminase, ammeline hydrolase, ammelide hydrolyase, melamine deamina
- the invention relates to any one of the aforementioned genetically engineered organisms, wherein the non-native gene is selected from the group consisting of atzF, DUR1,2 YALI0E 07271g, atzE, atzD, trzC, trzD, trzE, atzD, guaD, blr3880, GUD1/Y DL238C, YAL10E2 5740p, trzA, triA, atzC, and cah.
- the non-native gene is selected from the group consisting of atzF, DUR1,2 YALI0E 07271g, atzE, atzD, trzC, trzD, trzE, atzD, guaD, blr3880, GUD1/Y DL238C, YAL10E2 5740p, trzA, triA, atzC, and cah.
- the invention relates to any one of the aforementioned genetically engineered organisms, wherein the non-native gene is selected from the group consisting of atzF, DUR1,2 YALI0E 07271g, atzE, atzD, trzD, atzD, guaD, blr3880, GUD1/Y DL238C, YAL10E2 5740p, trzA, triA, atzC, and cah.
- the non-native gene is selected from the group consisting of atzF, DUR1,2 YALI0E 07271g, atzE, atzD, trzD, atzD, guaD, blr3880, GUD1/Y DL238C, YAL10E2 5740p, trzA, triA, atzC, and cah.
- the non-native gene can each be obtained from chromosomal DNA of any one of the aforementioned microorganisms by isolating a DNA fragment complementing auxotrophy of a variant strain lacking the enzymatic activity.
- the nucleotide sequence of these gene of the organism has already been elucidated (Biochemistry, Vol. 22, pp. 5243-5249, 1983; J. Biochem. Vol. 95, pp. 909-916, 1984; Gene, Vol. 27, pp. 193-199, 1984; Microbiology, Vol. 140, pp. 1817-1828, 1994; Mol. Gene Genet. Vol.
- the genes can be obtained by PCR using primers synthesized based on each of the elucidated nucleotide sequences, and the chromosome DNA as a template.
- the invention relates to any one of the aforementioned genetically engineered organisms, wherein the non-native gene is selected from the group consisting of trzE from Rhodococcus sp. strain Mel, trzE from Rhizobium leguminosarum , trzC MEL, trzC 12227, cah from Fusarium oxysporum Fo5176, cah from F. pseudograminaearum CS3096, cah from Gibberella zeae PH-1, cah from Aspergillus kawachii IFO 4308, cah from A. niger CBS 513.88, cah from A. niger ATCC 1015, cah from A.
- oryzae 3.042 cah from S. cerevisiae FostersB, atzF from Pseudomonas sp. strain ADP, DUR1,2 from S. cerevisiae , YALI0E 07271g from Y. lipolytica CLIB122, atzE from Pseudomonas sp. strain ADP, atzD from Pseudomonas sp. strain ADP, trzD from Pseudomonas sp. strain NRRLB-12227, atzD from Rhodococcus sp. Mel, trzD from Rhodococcus sp. Mel, guaD from E.
- the invention relates to any one of the aforementioned genetically engineered organisms, wherein the genetically engineered organism is a species of the genus Yarrowia, Saccharomyces, Ogataea, Pichia , or Escherichia.
- the invention relates to any one of the aforementioned genetically engineered organisms, wherein the genetically engineered organism is selected from the group consisting of Yarrowia lipolytica, Saccharomyces cerevisiae, Ogataea polymorpha, Pichia pastoris , and Escherichia coli.
- the genetically engineered organism is not Rhodococcus sp. Strain Mel.
- the invention relates to a method, comprising the step of
- the substrate comprises a nitrogen-containing fraction and a non-nitrogen-containing fraction
- the nitrogen-containing fraction comprises, in an amount from about 10% by weight to about 100% by weight, a nitrogen-containing compound of any one of Formulas I-III;
- the genetically engineered organism converts the substrate to a product.
- the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have a low molecular weight. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have a molecular weight between about 30 Da and about 800 Da. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have a molecular weight between about 40 Da and about 600 Da.
- the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have a molecular weight of about 40 Da, about 50 Da, about 60 Da, about 70 Da, about 80 Da, about 90 Da, about 100 Da, about 110 Da, about 120 Da, about 130 Da, about 140 Da, about 150 Da, about 160 Da, about 170 Da, about 180 Da, about 190 Da, about 200 Da, about 220 Da, about 240 Da, about 260 Da, about 280 Da, about 300 Da, about 320 Da, about 340 Da, about 360 Da, about 380 Da, about 400 Da, about 420 Da, about 440 Da, about 460 Da, about 480 Da, about 500 Da, about 520 Da, about 540 Da, bout 560 Da, about 580 Da, or about 600 Da.
- the nitrogen-containing compounds have a molecular weight of about 40 Da, about 50 Da, about 60 Da, about 70 Da, about 80 Da, about 90 Da, about 100 Da, about 110 Da, about 120 Da, about 130 Da, about 140 Da,
- the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have less than 12 carbon atoms. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have less than 8 carbon atoms. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have 1, 2, 3, 4, 5, 6, or 7 carbon atoms.
- the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nitrogen atoms.
- the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have 0, 1, 2, 3, 4, 5, 6, 7, or 8 oxygen atoms.
- the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have an octanol-water partition coefficient (log P) less than about 5. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have an octanol-water partition coefficient (log P) from about ⁇ 0.5 to about 5. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have an octanol-water partition coefficient (log P) of about ⁇ 0.5, about 0, about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, or about 4.5.
- the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds are soluble in water at about 20° C. at a concentration of between about 0.01 g/L to about 1000 g/L. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds are soluble in water at about 20° C.
- g/L at a concentration of about 0.01 g/L, about 0.05 g/L, about 0.1 g/L, about 0.5 g/L, about 1 g/L, about 5 g/L, about 10 g/L, about 15 g/L, about 20 g/L, about 25 g/L, about 30 g/L, about 35 g/L, about 40 g/L, about 45 g/L, about 50 g/L, about 55 g/L, about 60 g/L, about 65 g/L, about 70 g/L, about 75 g/L, about 80 g/L, about 85 g/L, about 90 g/L, about 95 g/L, or about 100 g/L.
- the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds move through the cell membrane by passive transport.
- Passive transport includes diffusion, facilitated diffusion, and filtration.
- the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds move through the cell membrane by active transport, such as, for example, via an ATP-Binding Cassette (ABC) transporter or other known transmembrane transporter.
- active transport such as, for example, via an ATP-Binding Cassette (ABC) transporter or other known transmembrane transporter.
- ABSC ATP-Binding Cassette
- the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds are transported through the cell membrane.
- the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds are substantially non-biocidal.
- the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds are substantially biodegradable.
- the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing fraction comprises the nitrogen-containing compound in about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% by weight.
- the invention relates to a method, comprising the step of
- the substrate comprises a nitrogen-containing fraction and a non-nitrogen-containing fraction
- the genetically engineered organism converts the substrate to a product.
- the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing fraction comprises the nitrogen-containing compound in about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% by weight.
- the invention relates to a method, comprising the step of
- the substrate comprises a nitrogen-containing fraction and a non-nitrogen-containing fraction
- the nitrogen containing fraction consists essentially of a nitrogen-containing compound selected from the group consisting of triazine, urea, melamine, cyanamide, 2-cyanoguanidine, ammeline, guanidine carbonate, ethylenediamine, ammelide, biuret, diethylenetriamine, triethylenetetramine, 1,3-diaminopropane, calcium cyanamide, cyanuric acid, aminoethylpiperazine, piperazine, and allophante; and
- the genetically engineered organism converts the substrate to a product.
- the invention relates to a method, comprising the step of
- the substrate consists of a nitrogen-containing fraction and a non-nitrogen-containing fraction
- the nitrogen containing fraction consists of a nitrogen-containing compound selected from the group consisting of triazine, urea, melamine, cyanamide, 2-cyanoguanidine, ammeline, guanidine carbonate, ethylenediamine, ammelide, biuret, diethylenetriamine, triethylenetetramine, 1,3-diaminopropane, calcium cyanamide, cyanuric acid, aminoethylpiperazine, piperazine, and allophante; and the genetically engineered organism converts the substrate to a product.
- the invention relates to any one of the aforementioned methods, wherein the genetically engineered organism sequesters the product.
- the invention relates to any one of the aforementioned methods, wherein a plurality of genetically engineered organisms is used.
- the invention relates to any one of the aforementioned methods, wherein the substrate does not comprise an antibiotic.
- the invention relates to any one of the aforementioned methods, wherein the substrate does not comprise ammonium sulfate.
- the invention relates to any one of the aforementioned methods, wherein the substrate does not comprise urea.
- the invention relates to any one of the aforementioned methods, wherein a non-genetically engineered organism, i.e., a native organism, could not metabolize (i.e., use as a source of nitrogen) the nitrogen-containing compound.
- a non-genetically engineered organism i.e., a native organism
- the genetically engineered organism is not Rhodococcus sp. Strain Mel.
- the invention relates to any one of the aforementioned methods, wherein the substrate comprises lignocellulosic material, glucose, xylose, sucrose, acetic acid, formic acid, lactic acid, butyric acid, a free fatty acid, dextrose, glycerol, fructose, lactose, galactose, mannose, rhamnose, or arabinose, or a combination thereof.
- the substrate comprises lignocellulosic material, glucose, xylose, sucrose, acetic acid, formic acid, lactic acid, butyric acid, a free fatty acid, dextrose, glycerol, fructose, lactose, galactose, mannose, rhamnose, or arabinose, or a combination thereof.
- the invention relates to any one of the aforementioned methods, wherein the pH of the substrate is from about 2.5 to about 10.
- the invention relates to any one of the aforementioned methods, wherein the genetically engineered organism is contacted with the substrate at a temperature of from about 15° C. to about 80° C.
- the invention relates to any one of the aforementioned methods, wherein the genetically engineered organism is contacted with the substrate over a time period of from about 6 h to about 10 d.
- the invention relates to any one of the aforementioned methods, wherein the genetically engineered organism is contacted with the substrate in a fermentor.
- the invention relates to any one of the aforementioned methods, wherein the genetically engineered organism is contacted with the substrate in an industrial-size fermentor.
- the invention relates to any one of the aforementioned methods, wherein a plurality of genetically engineered organisms is contacted with a plurality of substrates in a plurality of fermentors, wherein the plurality of fermentors are arranged in parallel.
- the invention relates to any one of the aforementioned methods, wherein the product is ethanol, isopropanol, lactic acid, an isoprenoid, a lipid, a high-value specialty chemical, butanol, 1,3-propanediol, 1,4-butanediol, succinic acid, an expressed protein product, an enzyme product, a polyol, a pharmaceutical product, itaconic acid, or a high value specialty chemical.
- the invention relates to a product made by any one of the aforementioned methods.
- the oleaginous yeast Yarrowia lipolytica may be engineered to convert melamine into ammonia.
- Melamine C 3 N 6 H 6
- Yarrowia is a highly nitrogenous compound that can only be degraded by a very limited number of organisms including Rhodococcus sp. Strain Mel. Incorporating the pathway for melamine degradation into Yarrowia , accompanied with a modification in the media composition to use melamine as the predominant nitrogen source, will generate a more robust industrial production solution applicable to a number of applications.
- the advantage confirmed by this modification is significant enough to provide advantage in multiple applications including situations where the core technology may be significant genetic burden on the organism.
- Genes from FIG. 3 will be cloned into a host strain such as Yarrowia lipolytica, Saccharomyces cerevisiae , or Escherichia coli .
- Enzymes native to the host organism such as allophante hydrolase or guanine deaminase may be overexpressed with a heterologous promoter. Functional expression will be assayed by enzymatic activity and the ability to confer nitrogen limited growth on the appropriate pathway intermediate.
- strains able to degrade melamine will be selected for improved utilization of the pathway via melamine limited continuous culturing or other selective methods. Similar strategies can be devised for nitrogen compounds listed in FIG. 2 .
- Vector pNC10 contains an E. coli pMB1 origin of replication and ampicillin resistance gene, a S. cerevisiae 2 ⁇ m origin of replication and URA3 gene, and a multiple cloning site containing the 8-bp recognition sequences for PacI, PmeI, and AscI.
- DNA of interest is inserted in the multiple cloning site via yeast mediated homologous recombination (YML) cloning. (Shanks et al. 2006; Shanks et al. 2009). Briefly, target DNA sequences are amplified by PCR using primers with 20-40 bp overhang homology to adjacent DNA segments in the final vector.
- pNC10 or another suitable base vector is then restriction digested, creating a linearized plasmid.
- PCR products and linear plasmid are transformed in S. cerevisiae , and the native S. cerevisiae gap repair mechanism assembles an intact plasmid based on homology overhangs.
- the complete vector can then be isolated from S. cerevisiae via a DNA extraction protocol and used to transform E. coli .
- Concentrated vector can then be recovered from E. coli via DNA plasmid mini-prep or other suitable standard molecular biology protocols. See FIG. 5 .
- Melamine assimilation genes can be expressed in S. cerevisiae by construction of a vector using the yeast mediated ligation described above.
- Expression vectors consist of an S. cerevisiae functional promoter, a gene encoding an enzyme of the melamine assimilation pathway, and an S. cerevisiae functional terminator. Assemblies of the promoter-gene-terminator motif can be incorporated into a single strain, either on a replicating plasmid or integrated into a chromosome. Possible promoters and terminators are listed below, see also Sun et al. 2012. A representative plasmid, expressing the trzA melamine hydratase under control of the Y. lipolytica TEF1 promoter and terminator is shown below.
- Plasmid AJS35 is an example of the melamine dehydratase trzA transcribed via the Y. lipolytica TEF1 promoter and terminator. See FIG. 6 .
- Strains NS98 and NS99 are industrial S. cerevisiae strains carrying plasmids pNC96 (hyg R , and a codon optimized trzE from Rhodococcus sp. MEL and pNC97 (hyg R , and a codon optimized trzE from Rhizobium leguminosarum ), respectively.
- Strain NS100 is the same industrial S. cerevisiae stain carrying plasmid pNC67 (hyg R , nat R ) which serves as a control strain.
- Strains NS98, NS99, and NS100 were grown in defined YNB medium with 10 mM urea and 100 ⁇ g/mL hygromycin to stationary phase aerobically at 30° C. 1/1000 v/v inoculations were then made into the same defined medium with either 10 mM urea, 10 mM biuret, or no additional nitrogen and grown under the same conditions. Optical density was measured after 72 hours, as shown in FIG. 23 . Strains NS98 and NS99 were able to grow to an optical density approximately double that of NS100 in medium containing biuret, and also approximately double that with medium with no nitrogen supply. This shows that S. cerevisiae strains expressing trzE genes are advantaged in their utilization of biuret.
- GPM1 terminator gtctgaagaatgaatgatttgaTGATTTCTTTTTCCCTCCATTTTTCTTAC TGAATATATCAATGATATAGACTTGTATAGTTTATTATTTCAAATTAAGTA GCTATATATAGTCAAGATAACGTTTGTTTGACACGATTACATTATTCGTCG ACATCTTTTTTCAGCCTGTCGTGGTAGCAATTTGAGGAGTATTATTAATTG AATAGGTTCATTTTGCGCTCGCATAAACAGTTTTCGTCAGGGACAGTATGT TGGAATGAGTGGTAATTAATGGTGACATGACATGTTATAGCAATAACCTTG ATGTTTACATCGTAGTTTAATGTACACCCCGCGAATTCGTTCAAGTAggag tgcaccaattgcaagggaa S .
- FBA1 terminator gttaattcaaattaattgatatAGTTTTTTAATGAGTATTGAATCTGTTTA GAAATAATGGAATATTATTTTTATTTATTTATTTATATTATTGGTCGGCTC TTTTCTTCTGAAGGTCAATGACAAAATGATATGAAGGAAATAATGATTTCT AAAATTTTACAACGTAAGATATTTTTACAaaagcctagctcatctt Y .
- PDC1 terminator acaagctaagttgactgctgctACCAACGCTAAGCAATAAGCGATTTAATC TCTAATTATTAGTTAAAGTTTTATAAGCATTTTTATGTAACGAAAAATAAA TTGGTTCATATTATTACTGCACTGTCACTTACCATGGAAAGACCAGACAAG AAGTTGCCGACACGACAGTCTGTTGAattggcttaagtctgggtccgctt S .
- CYC1 terminator caggccccttttcctttgtcgaTATCATGTAATTAGTTATGTCACGCTTAC ATTCACGCCCTCCTCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGAC AACCTGAAGTCTAGGTCCCTATTTATTTTTTTTAATAGTTATGTTAGTATT AAGAACGTTATTTATATTTCAAATTTTTCTTTTTTCTGTACAAACGCGT GTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACG CTCGAAGGCTTTAATTTGC
- Melamine assimilation genes can be expressed in E. coli by construction of a vector using the yeast mediated ligation described above.
- Expression vectors consist of an E. coli functional promoter, a gene encoding an enzyme of the melamine assimilation pathway, and an E. coli functional terminator.
- several genes can be expressed from a single promoter as part of a gene operon; in this case inter-gene linker sequences are placed between genes. Sequences that can act as promoters, terminators, and linkers are listed below, as well as two representative E.
- AJS67 expressing genes for degradation of melamine to cyanuric acid with release of 3 NH 3 per melamine
- AJS68 expressing genes for degradation of cyanuric acid to NH 3 and CO 2 with release of 3 NH 3 per cyanuric acid
- S. cerevisiae has the native ability to convert urea to NH 3 and CO 2 via the actions of urea carboxylase and allophante hydrolase, encoded in the fusion gene DUR1,2. Therefore, functional expression of cyanamide hydrolase is sufficient to convert cyanamide to NH 3 .
- a representative cyanamide hydratase expression vector is shown below, with Y. lipolytica TEF1 promoter and terminator and a S. cerevisiae codon-optimized cyanamide hydratase (cah) from Myrothecium verrucaria. See FIG. 9 .
- Example 6 The gene expression methods described in Example 6 can also be used in example 8. Unlike S. cerevisiae , most E. coli strains are unable to utilize urea as a nitrogen source, so these additional conversion steps must also be engineered. Either a urea carboxylase/allophante hydrolase system or a urease enzyme with appropriate accessory enzymes must be expressed in addition to a cyanamide hydrolase. Urease can be found in some E. coli isolates (Collins and Falkow 1990) or heterologously expressed (Cussac et al. 1992). Alternatively, the DUR1,2 genes from S. cerevisiae could be expressed, as shown below in plasmid AJS70, along with a cyanamide hydratase. See FIG. 10 .
- E. coli strains containing partial or complete melamine utilization pathways were constructed, as shown in FIGS. 29 and 30 .
- Vector and strain construction was as described in example 6. All vectors contain the ampicillin resistance gene, and 100 ug/mL ampicillin was added to all culture medium. These strains were grown in MOPS defined medium with different nitrogen sources.
- NS93 triA, native guaD selected for improved ammeline utilization (steps 1, 2)
- NS103 triA, guaD, trzC (steps 1, 2, 3)
- NS109 triA, guaD, trzC, trzD 12227, trzE, DUR1,2 (steps 1-6)
- NS110 triA, guaD, trzC, atzD ADP, trzE, DUR1,2 (steps 1-6)
- FIG. 12 shows the growth progress of NS88 and NS91 (control) in media containing various concentrations of ammonium chloride or melamine NS88 grown on 1 mM melamine reaches an optical density comparable to that of the equivalent use of 2 mM ammonium chloride, suggesting that 2 mM ammonia are liberated from melamine by triA and the natively encoded guaD genes.
- the control strain NS91 does not grow with melamine as nitrogen source.
- FIG. 13 shows the growth progress of NS90 and NS91 (control) in media containing various concentrations of ammonium chloride or biuret.
- NS90 grown on 1 mM biuret reaches an optical density comparable to that of the equivalent use of 3 mM ammonium chloride, suggesting that 3 mM ammonia are liberated from biuret by trzE and the DUR1,2.
- the control strain NS91 does not grow with biuret as nitrogen source.
- FIG. 24 shows the growth progress of NS91, NS103, NS109, and NS110 in medium containing 0.25 mM melamine as sole nitrogen source. An average of all four strains grown on different ammonium chloride concentrations from 0 to 1.5 mM is also shown as a standard curve for growth with limiting nitrogen. NS91 grown on melamine is similar to the 0 mM ammonium chloride control.
- NS103 grown on 0.25 mM melamine is similar to 1-0.75 mM ammonium chloride, suggesting it is approximately utilizating the predicted 3 mM ammonia per 1 mM melamine
- Strains NS109 and NS110 grown on 0.25 mM melamine are similar to 1.5-1.25 mM ammonium chloride, suggesting it is approximately utilizating the predicted 6 mM ammonia per 1 mM melamine
- FIG. 25 shows the growth progress of NS91, NS103, NS109, and NS110 in medium containing 0.25 mM ammeline as sole nitrogen source.
- An average of all four strains grown on different ammonium chloride concentrations from 0 to 1.5 mM is also shown as a standard curve for growth with limiting nitrogen.
- NS91 grown on ammeline is similar to the 0 mM ammonium chloride control.
- NS103 grown on 0.25 mM ammeline is similar to 0.5 mM ammonium chloride, suggesting it is approximately utilizating the predicted 2 mM ammonia per 1 mM ammeline.
- Strains NS109 and NS110 grown on 0.25 mM ammeline are similar to 1.25-1.0 mM ammonium chloride, suggesting it is approximately utilizating the predicted 5 mM ammonia per 1 mM ammeline.
- FIGS. 26, 27, and 28 show E. coli strains derived from E. coli K12, E. coli MG1655, E. coli B, and E. coli Crooks (C) containing either pNC121 with the complete melamine utilization pathway, or pNC53, a control vector. See FIGS. 29 and 30 for strain details. All the strains containing pNC121 are able to grow on 0.5 mM melamine as sole nitrogen source ( FIG. 28 ). This indicates that the melamine utilization pathway is broadly applicable to E. coli strains that are commonly utilized for biotechnology applications.
- Strains can also be selected for improved utilization of melamine derived nitrogen sources, in one example NS88 was passaged for 11 serial transfers in MOPS defined medium with 0.5 mM ammeline as sole nitrogen source. After the final passage, single colonies were isolated, and one was designated as NS93. NS93 and NS91 were grown overnight in medium with 0.5 mM ammonium chloride as sole nitrogen source, and then inoculated in medium with 0.5 mM ammeline as sole nitrogen source. NS91 exhibited a maximum growth rate of 0.024 hr ⁇ 1 on ammeline, while NS93 exhibited a maximum growth rate of 0.087 hr ⁇ 1 .
- Precultures were grown in LB media with 100 mg/L ampicillin, 0.1 mL were directly inoculated into 5 mL MOPS media with 100 mg/L ampicillin and the indicated nitrogen source. Grown at 37° C. in a drum roller at 30 rpm. See FIG. 14 .
- NS100 industrial S. cerevisiae strain with pNC67 (hyg R , nat R )
- NS101 industrial S. cerevisiae strain with pNC93 (hyg R , cah)
- NS111 S. cerevisiae NRRL Y-2223 with pNC93 (hyg R , cah)
- NS112 S. cerevisiae NRRL Y-2223 with pNC67 (hyg R , nat R )
- Optical density of NS100 and NS101 grown in defined medium with different nitrogen sources were grown overnight in YPD medium, washed once in an equal volume of sterile water, and inoculated at 3.33% v/v.
- Strain NS101 is able to grow to an optical density with cyanamide comparable to that with urea, while NS100 grows to an optical density comparable to that with no nitrogen present in the medium.
- Data are averages of 3 replicate wells in a 96 well plate; 150 ⁇ L per well.
- YNB medium contained 20 g/L glucose, 1.7 g/L YNB base medium without amino acids or ammonium sulfate, 5 g/L sodium sulfate, 100 ⁇ g/mL hygromycin, and either 10 mM urea, 10 mM cyanamide, or no nitrogen source. Inoculation was with 5 ⁇ L of culture pregrown for 24 hrs in the same medium with urea as nitrogen source. See FIG. 17 .
- strains NS100, NS101, NS111, and NS112 were grown in defined YNB medium with 10 mM urea and 100 ⁇ g/mL hygromycin to stationary phase aerobically at 30° C. 1/1000 v/v inoculations were then made into the same defined medium with either 10 mM urea, 10 mM cyanamide, or no additional nitrogen and grown under the same conditions. Optical density was measured after 72 hours, as shown in FIG. 22 . Strains NS101 and NS111, two different S.
- cerevisiae strains carrying the cah gene were able to grow to an optical density comparable to that with urea; however, NS100 and NS112 only were able to grow to an optical density equal to or lower than in media with no nitrogen source. This shows that multiple S. cerevisiae strains are able to utilize cyanamide in the presence of the cah gene.
- Strains NS100 (hyg R , nat R ) and NS101 (hyg R , cah) were grown in defined medium with 100 ⁇ g/mL hygromycin with urea as nitrogen source, and then both inoculated into defined medium containing either 10 mM urea or 10 mM cyanamide as nitrogen source. Upon growth to stationary phase, 1/100 v/v serial transfers were made to fresh medium with the same composition. The culture population was monitored via counting the number of hyg R , nat R colony forming units and subtracting from the number of hyg R colony forming units. See FIG. 18 and FIG. 19 for one experiment in defined minimal medium. A second experiment is shown in FIG. 21 .
- the second experiment included both defined minimal (YNB) and defined complex (YNB+SC amino acids) medium compositions.
- the defined YNB medium contained 20 g/L glucose, 1.7 g/L YNB base medium without amino acids or ammonium sulfate, 5 g/L sodium sulfate, and either 10 mM urea, 10 mM cyanamide, or no nitrogen source. Medium compositions are additionally given in FIGS. 32 and 33 . Growth occurred aerobically at 30° C. Colony forming units were counted by serial dilutions in YPD media with either 300 ⁇ g/mL hygromycin or 100 ⁇ g/mL nourseothricin, and are the average of 3 dilution counts. See FIG. 18 and FIG. 19 .
- Optical density of NS100 and NS101 grown in rich YPD medium with 100 ⁇ g/mL hydgromycin and with and without 10 mM cyanamide NS100 and NS101 were grown overnight in YNB medium, and inoculated at 3.33% v/v. NS101 experiences a shorter lag phase than NS100 in the presence of 10 mM cyanamide.
- cyanamide in addition to functioning as a sole source of nitrogen, can also act as a deterrent for microbial growth.
- Data are averages of 3 replicate wells in a 96 well plate; 150 ⁇ L per well. 30° C., YPD medium or YPD medium with 10 mM cyanamide. Inoculation was with 5 ⁇ L of culture pregrown for 24 hrs in the YNB medium with urea as nitrogen source.
- This assay measured the conversion rate of cyanamide to urea.
- cyanamide was hydrated to urea by cyanamide hydratase, which was detected in cell free extract of a S. cerevisiae strain expressing the cah gene and a control strain without cah.
- a commercial kit (Megazyme, Ireland) was used to detect urea via enzymatic conversion of urea to ammonia followed by NADPH linked conversion of ammonia and 2-oxoglutarate to NADP+, H 2 O, and glutamic acid.
- Cell free extracts were prepared by growing S. cerevisiae strains in 50 mL yeast extract, peptone, dextrose (YPD) medium with 300 ⁇ g/mL hygromycin to an optical density between 1-2. Cells were harvested by centrifugation, washed once in an equal volume of water, and re-suspended in Y-PER lysis buffer (Thermo Scientific, USA) following the manufacturer's instructions. After incubation at room temperature for 20 minutes, the lysate was centrifuged at 14,000 ⁇ g for 10 mM and the supernatant was recovered as the cell free extract. Total protein was measured by a Nanodrop spectrophotometer (Thermo Scientific, USA).
- Sequence 1 is the DNA sequence of the allophanate hydrolase atzF gene in
- Sequence 2 is the DNA sequence of allophanate hydrolase DUR1,2 gene in S. cerevisiae.
- Sequence 3 is the DNA sequence of allophanate hydrolase YALI0E07271g gene in Y. lipolytica CLIB122.
- Sequence 4 is the DNA sequence of the biuret amidohydrolase atzE gene in Pseudomonas sp. strain ADP.
- Sequence 5 is the DNA sequence of the cyanuric acid amidohydrolase atzD gene in Pseudomonas sp. strain ADP.
- Sequence 6 is the DNA sequence of the cyanuric acid amidohydrolase trzD gene in Pseudomonas sp. strain NRRLB-12227 (formerly Acidovorax citrulli ).
- Sequence 7 is the DNA sequence of the cyanuric acid amidohydrolase atzD trzD gene in Rhodococcus sp. Mel.
- Sequence 8 is the DNA sequence of the guanine deaminase guaD gene in E. coli K12 strain MG1566.
- Sequence 9 is the DNA sequence of the guanine deaminase blr3880 gene in Bradyrhizobium japonicum USDA 110.
- Sequence 10 is the DNA sequence of the guanine deaminase GUD1/YDL238C gene in S. cerevisiae.
- Sequence 11 is the DNA sequence of the guanine deaminase YALI0E25740p gene in Y. lipolytica CLIB122.
- Sequence 12 is the DNA sequence of the melamine deaminase trzA gene in Williamsia sp. NRRL B-15444R (formerly R. corallinus ).
- Sequence 13 is the DNA sequence of the melamine deaminase triA gene in Pseudomonas sp. strain NRRL B-12227 (formerly Acidovorax citrulli ).
- Sequence 14 is the DNA sequence of the isopropylammelide isopropylaminohydrolase atzC gene in Pseudomonas sp. strain ADP.
- Sequence 15 is the cDNA sequence of the Myrothecium verrucaria cyanamide hydratase (cah) gene.
- Sequences 16-21 are DNA sequences of the invention.
- Sequences 22-37 are the sequences of various cyanamide hydratase (cah) genes for use in the invention.
- Sequences 38 and 39 are the sequences of various trzC genes for use in the invention.
- Sequences 40 and 41 are the sequences of various trzE genes for use in the invention.
- Sequence 42 is the sequence of plasmid pNC10.
- Sequence 43 is the sequence of plasmid pNC53.
- Sequence 44 is the sequence of plasmid pNC67.
- Sequence 45 is the sequence of plasmid pNC85.
- Sequence 46 is the sequence of plasmid pNC86.
- Sequence 47 is the sequence of plasmid pNC87.
- Sequence 48 is the sequence of plasmid pNC93.
- Sequence 49 is the sequence of plasmid pNC96.
- Sequence 50 is the sequence of plasmid pNC97.
- Sequence 51 is the sequence of plasmid pNC101.
- Sequence 52 is the sequence of plasmid pNC120.
- Sequence 53 is the sequence of plasmid pNC121.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
- This application claims the benefit of priority to U.S. Provisional Patent Application Ser. Nos. 61/748,901, filed Jan. 4, 2013, and 61/782,351, filed Mar. 14, 2013; the contents of both of which are hereby incorporated by reference.
- In the fermentation industry, cell culture media is typically formulated to provide all nutrients necessary for the growth of a host cell line, with particular emphasis on meeting the cell line's requirements for carbon, nitrogen, phosphorus, sulfur, and other major nutrients. Some cell lines require additional components, including amino acids, trace minerals and metals, and complex growth factors. The presence of these nutrients provides a suitable growth environment for the organism of choice and, unfortunately, for any potential contaminating organisms. In this environment the production organism is required to compete directly with any contaminant organism in the cell culture.
- Even in robust hosts, the combination of opportunistic infections of the culture and the metabolic burden resulting from the demands of product manufacture is a major concern in monoculture operations. Industrial robustness is typically considered a multigenic trait specific to the host strain and thus difficult to engineer predictably into organisms late in the development process. Addition of selective growth inhibitors, such as bacterial antibiotics, is one method used to create a more robust fermentation environment for host organisms that are resistant to the growth inhibitor. However, antibiotic addition is often undesirable or unfeasible, and spontaneously resistant contaminations frequently result.
- Accordingly, there exists a need for rationally engineered traits that, when engineered into a host organism, create a robust monoculture fermentation environment.
- In certain embodiments, the invention relates to a genetically engineered organism, wherein the genetically engineered organism has been transformed by a nucleic acid molecule comprising any one or more of the sequences disclosed herein.
- In certain embodiments, the invention relates to a genetically engineered organism, wherein the genetically engineered organism has been transformed by a nucleic acid molecule; the nucleic acid molecule comprises a non-native gene; and the non-native gene encodes for a non-native enzyme selected from the group consisting of allophanate hydrolase, biuret amidohydrolase, cyanuric acid amidohydrolase, guanine deaminase, melamine deaminase, isopropylammelide isopropylaminohydrolase, cyanamide hydratase, urease, and urea carboxylase.
- In certain embodiments, the invention relates to a method, comprising the step of
- contacting any one of the aforementioned genetically engineered organisms with a substrate,
- wherein
- the substrate comprises a nitrogen-containing fraction and a non-nitrogen-containing fraction;
- the nitrogen-containing fraction comprises, in an amount from about 10% by weight to about 100% by weight, a nitrogen-containing compound of any one of Formulas I-III, or a salt thereof;
- a native organism of the same species as the genetically engineered organism could not metabolize (i.e., use as a source of nitrogen) the nitrogen-containing compound;
- the genetically engineered organism converts the substrate to a product; and
- the compound of formula I is
- wherein, independently for each occurrence,
-
- is a five-, six, nine-, or ten-membered aryl or heteroaryl group;
- R is —OH, —CO2H, —NO2, —CN, substituted or unsubstituted amino, or substituted or unsubstituted alkyl; and
- n is 0, 1, 2, 3, 4, or 5;
- the compound of formula II is
-
- wherein, independently for each occurrence,
- X is —NH—, —N(alkyl)-, —O—, —C(R1)2—, —S—, or absent;
- Y is —H, —NH2, —N(H)(alkyl), —N(alkyl)2, —CO2H, —CN, or substituted or unsubstituted alkyl; and
- R1 is —H, —OH, —CO2H, —NO2, —CN, substituted or unsubstituted amino, or substituted or unsubstituted alkyl; and
- the compound of formula III is
- wherein, independently for each occurrence,
-
- wherein, independently for each occurrence,
- Y is —H, —NH2, —N(H)(alkyl), —N(alkyl)2, —CO2H, —CN, or substituted or unsubstituted alkyl.
- wherein, independently for each occurrence,
- In certain embodiments, the invention relates to a method, comprising the step of
- contacting any one of the aforementioned genetically engineered organisms with a substrate,
- wherein
- the substrate comprises a nitrogen-containing fraction and a non-nitrogen-containing fraction;
- the nitrogen-containing fraction comprises, in an amount from about 10% by weight to about 100% by weight, a nitrogen-containing compound selected from the group consisting of triazine, urea, melamine, cyanamide, 2-cyanoguanidine, ammeline, guanidine carbonate, ethylenediamine, ammelide, biuret, diethylenetriamine, triethylenetetramine, 1,3-diaminopropane, calcium cyanamide, cyanuric acid, aminoethylpiperazine, piperazine, and allophante; and
- the genetically engineered organism converts the substrate to a product.
- In certain embodiments, the invention relates to a method, comprising the step of
- contacting any one of the aforementioned genetically engineered organisms with a substrate,
- wherein
- the substrate comprises a nitrogen-containing fraction and a non-nitrogen-containing fraction;
- the nitrogen containing fraction consists essentially of a nitrogen-containing compound selected from the group consisting of triazine, urea, melamine, cyanamide, 2-cyanoguanidine, ammeline, guanidine carbonate, ethylenediamine, ammelide, biuret, diethylenetriamine, triethylenetetramine, 1,3-diaminopropane, calcium cyanamide, cyanuric acid, aminoethylpiperazine, piperazine, and allophante; and
- the genetically engineered organism converts the substrate to a product.
- In certain embodiments, the invention relates to a method comprising the step of
- contacting any one of the aforementioned genetically engineered organisms with a substrate,
- wherein
- the substrate consists of a nitrogen-containing fraction and a non-nitrogen-containing fraction;
- the nitrogen containing fraction consists of a nitrogen-containing compound selected from the group consisting of triazine, urea, melamine, cyanamide, 2-cyanoguanidine, ammeline, guanidine carbonate, ethylenediamine, ammelide, biuret, diethylenetriamine, triethylenetetramine, 1,3-diaminopropane, calcium cyanamide, cyanuric acid, aminoethylpiperazine, piperazine, and allophante; and
- the genetically engineered organism converts the substrate to a product.
- In certain embodiments, the invention relates to a product made by any one of the aforementioned methods.
- In certain embodiments, the invention relates to a recombinant vector comprising a gene operably linked to a promoter, wherein the gene encodes an enzyme; and the enzyme is allophanate hydrolase, biuret amidohydrolase, cyanuric acid amidohydrolase, guanine deaminase, melamine deaminase, isopropylammelide isopropylaminohydrolase, cyanamide hydratase, urease, or urea carboxylase.
-
FIG. 1 depicts a schematic representation of the melamine degradation pathway. 1—Melamine deaminase (tzrA) (EC 3.5.4.-); 2—Ammeline deaminase (guanine deaminase) (EC 3.5.4.3); 3—N-isopropylammelide isopropylamino (Ammelide) hydrolyase (EC 3.5.99.4); 4—Cyanuric acid hydrolyase (EC 3.5.2.15); 4a—Carboxybiuret decarboxylase, spontaneous reaction; 5—Biuret amidohydrolase (EC 3.5.1.84); 6—Allophanate hydrolyase (EC 3.5.1.54). Nitrogen can be assimilated (as NH3) by the action of the complete pathway acting on melamine, liberating 6 mol NH3 per mol melamine, or via a subset of enzymes acting on pathway intermediates (e.g.,steps -
FIG. 2 tabulates exemplary compounds capable of delivering nitrogen that could be accessed by an engineered organism. -
FIG. 3 tabulates DNA and protein sequences encoding the melamine degradation pathway. -
FIG. 4 depicts a schematic representation of the cyanamide assimilation pathway. After conversion of cyanamide to urea by cyanamide hydratase (EC 4.2.1.69), urea can be degraded either via urease (EC 3.5.1.5) or by urea carboxylase (EC 6.3.4.6) and allophante hydrolyase (EC 3.5.1.54). -
FIGS. 5-10 depict various plasmids of the invention. -
FIG. 11 tabulates the concentrations of the components in the MOPS medium used in Example 9. -
FIG. 12 depicts the growth progress of NS88 and NS91 (control) in media containing various concentrations of ammonium ion or melamine -
FIG. 13 depicts the growth progress of NS90 and NS91 (control) in media containing various concentrations of ammonium ion or biuret. -
FIG. 14 depicts images, taken after 48 h, of cultures grown in MOPS media with different nitrogen sources. From left to right: NS88 with 10 mM melamine; NS91 with 10 mM melamine; NS90 with 10 mM biuret (replicate 1); NS90 with 10 mM biuret (replicate 2); and NS91 with 10 mM biuret. -
FIG. 15 depicts a plasmid of the invention. -
FIG. 16 depicts a plasmid of the invention. -
FIG. 17 depicts the growth progress of NS100 (control) and NS101 in media containing no nitrogen source, urea, or cyanamide. -
FIG. 18 depicts the population fraction of NS100 (control) and NS101 in a urea-containing medium. -
FIG. 19 depicts the population fraction of NS100 (control) and NS101 in a cyanamide-containing medium. -
FIG. 20 depicts the growth progress of NS100 (control) and NS101 in media containing no nitrogen source, or media containing cyanamide. -
FIG. 21 depicts the growth of an organism of the invention in the presence of an antibiotic on various nitrogen-containing media (seeFIG. 33 for composition of SC amino acid media). -
FIG. 22 tabulates the optical density at 600 nm after growth of four organisms of the invention on various media. -
FIG. 23 tabulates the optical density at 600 nm after growth of three organisms of the invention on various media. -
FIG. 24 depicts the growth of four organisms of the invention (NS91=control) on 0.25 mM melamine, as compared to the standard curves for a native organism on NH4Cl. Because melamine has six nitrogen atoms, organisms having the ability to utilize melamine should be approximately six times more efficient (see, for example, NS110 on 0.25 mM melamine, as compared to a native organism on 1.5 mM NH4Cl). -
FIG. 25 depicts the growth of four organisms of the invention (NS91=control) on 0.25 mM ammeline, as compared to the standard curves for a native organism on NH4Cl. Because ammeline has five nitrogen atoms, organisms having the ability to utilize melamine should be approximately five times more efficient (see, for example, NS110 on 0.25 mM ammeline, as compared to a native organism on 1.25 mM NH4Cl). -
FIG. 26 depicts the growth of various organisms of the invention on 0.5 mM NH4Cl. Importantly, the organisms described inFIGS. 26-28 , for example NS120, NS91, NS107, and NS123, are E. coli strains derived from E. coli K12, E. coli B, E. coli Crooks, and E. coli MG1655 and are intended to show the breadth of the invention across various strains of E. coli. -
FIG. 27 depicts the growth of various organisms of the invention on a medium containing no nitrogen. -
FIG. 28 depicts the growth of various organisms of the invention on a medium containing 0.5 mM melamine -
FIG. 29 tabulates a summary of various plasmids of the invention. -
FIG. 30 tabulates a summary of various organisms of the invention. -
FIG. 31 tabulates the components and molar concentrations of each component in a MOPS defined medium, which is used, for example, with E. coli. -
FIG. 32 tabulates the components and weight concentrations of each component in a YNB medium, which is used, for example, with S. cerevisiae. -
FIG. 33 tabulates the components and weight concentrations of each component in a SC amino acid medium. - In certain embodiments, the invention relates to a genetically engineered host organism, wherein the genetically engineered host organism has a non-native ability to obtain a growth-limiting nutrient from a complex substrate; and the complex substrate could not have been metabolized or used as a nutrient by the native host organism. In certain embodiments, the non-native ability will provide the organism with a significant competitive advantage, and provide a major barrier to the success of contaminants in a fermentation. In certain embodiments, the genetically engineered host organism is a bacterium, a yeast, a fungus, a mammalian cell, or an insect cell. In certain embodiments, the genetically engineered host organism is a bacterium or a yeast.
- In certain embodiments, the invention relates to a method of using the above-mentioned genetically engineered host organism, comprising contacting the genetically engineered host organism with a modified cell culture medium. In certain embodiments, the invention relates to a method of using the above-mentioned genetically engineered host organism, comprising contacting the genetically engineered host organism with a modified cell culture medium, wherein the genetically engineered host organism converts the cell culture medium to a product. In certain embodiments, using this approach provides a unique and targeted manner to promote the growth of the desired genetically engineered host organism. In certain embodiments, the above-mentioned methods minimize the growth of contaminant organisms, provide a valuable competitive advantage, and allow management of production of a range of valuable products.
- In certain embodiments, the inventive methods decrease or eliminate the need for use of prophylactic antibiotics in large scale yeast cultures. Avoiding unnecessary antibiotics is an important benefit due to emerging environmental considerations and societal pressures. Additionally, in certain embodiments, the technique can be applied to bacterial systems in which antibiotics may not be added.
- In certain embodiments, the genetically engineered host organism is a yeast; and the product is ethanol, isobutanol, lactic acid, an isoprenoid, a lipid, and enzyme product, or a high value specialty chemical.
- In certain embodiments, the genetically engineered host organism is a bacterium;
- and the product is butanol, ethanol, isopropanol, 1,3-propanediol (PDO), 1,4-butanediol (BDO), succinic acid, itaconic acid, an enzyme product, a polyol, a protein product, or a high value specialty chemical.
- In certain embodiments, the inventive technology is applicable in the production of one or more commodities, fine chemicals, and pharmaceuticals.
- “Dry weight” and “dry cell weight” mean weight determined in the relative absence of water. For example, reference to oleaginous cells as comprising a specified percentage of a particular component by dry weight means that the percentage is calculated based on the weight of the cell after substantially all water has been removed.
- “Exogenous gene” is a nucleic acid that codes for the expression of an RNA and/or protein that has been introduced into a cell (e.g., by transformation/transfection), and is also referred to as a “transgene.” A cell comprising an exogenous gene may be referred to as a recombinant cell, into which additional exogenous gene(s) may be introduced. The exogenous gene may be from a different species (and so heterologous), or from the same species (and so homologous), relative to the cell being transformed. Thus, an exogenous gene can include a homologous gene that occupies a different location in the genome of the cell or is under different control, relative to the endogenous copy of the gene. An exogenous gene may be present in more than one copy in the cell. An exogenous gene may be maintained in a cell as an insertion into the genome (nuclear or plastid) or as an episomal molecule.
- “Expression vector” or “expression construct” or “plasmid” or “recombinant DNA construct” is a vehicle for introducing a nucleic acid into a host cell. The nucleic acid can be one that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription and/or translation of a particular nucleic acid. The expression vector can be part of a plasmid, virus, or nucleic acid fragment, or other suitable vehicle. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
- “Inducible promoter” is a promoter that mediates transcription of an operably linked gene in response to a particular stimulus.
- “In operable linkage” is a functional linkage between two nucleic acid sequences, such a control sequence (typically a promoter) and the linked sequence (typically a sequence that encodes a protein, also called a coding sequence). A promoter is in operable linkage with an exogenous gene if it can mediate transcription of the gene.
- “Lysate” is a solution containing the contents of lysed cells.
- “Lysis” is the breakage of the plasma membrane and optionally the cell wall of a biological organism sufficient to release at least some intracellular content, often by mechanical, viral or osmotic mechanisms that compromise its integrity.
- “Lysing” is disrupting the cellular membrane and optionally the cell wall of a biological organism or cell sufficient to release at least some intracellular content.
- “Osmotic shock” is the rupture of cells in a solution following a sudden reduction in osmotic pressure. Osmotic shock is sometimes induced to release cellular components of such cells into a solution.
- The terms “plasmid”, “vector” and “cassette” refer to an extra chromosomal element often carrying genes that are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell. “Transformation cassette” refers to a specific vector containing a foreign gene and having elements, in addition to the foreign gene, that facilitate transformation of a particular host cell. “Expression cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.
- “Promoter” is a nucleic acid control sequence that directs transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
- “Recombinant” is a cell, nucleic acid, protein, or vector, which has been modified due to the introduction of an exogenous nucleic acid or the alteration of a native nucleic acid. Thus, e.g., recombinant cells can express genes that are not found within the native (non-recombinant) form of the cell or express native genes differently than those genes are expressed by a non-recombinant cell. Recombinant cells can, without limitation, include recombinant nucleic acids that encode for a gene product or for suppression elements such as mutations, knockouts, antisense, interfering RNA (RNAi) or dsRNA that reduce the levels of active gene product in a cell. A “recombinant nucleic acid” is a nucleic acid originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases, ligases, exonucleases, and endonucleases, or otherwise is in a form not normally found in nature. Recombinant nucleic acids may be produced, for example, to place two or more nucleic acids in operable linkage. Thus, an isolated nucleic acid or an expression vector formed in vitro by ligating DNA molecules that are not normally joined in nature, are both considered recombinant for the purposes of this invention. Once a recombinant nucleic acid is made and introduced into a host cell or organism, it may replicate using the in vivo cellular machinery of the host cell; however, such nucleic acids, once produced recombinantly, although subsequently replicated intracellularly, are still considered recombinant for purposes of this invention. Similarly, a “recombinant protein” is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid.
- “Sonication” is a process of disrupting biological materials, such as a cell, by use of sound wave energy.
- “Transformation” refers to the transfer of a nucleic acid fragment into a host organism or the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “recombinant”, “transgenic” or “transformed” organisms. Thus, isolated polynucleotides of the present invention can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell. Such a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. Typically, expression vectors include, for example, one or more cloned genes under the transcriptional control of 5′ and 3′ regulatory sequences and a selectable marker. Such vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or location-specific expression), a transcription initiation start site, a ribosome binding site, a transcription termination site, and/or a polyadenylation signal.
- A. Overview
- In certain embodiments of the invention, a microorganism is genetically modified to improve or provide de novo growth characteristics on a variety of feedstock materials.
- Genes and gene products may be introduced into microbial host cells. Suitable host cells for expression of the genes and nucleic acid molecules are microbial hosts that can be found broadly within the fungal or bacterial families and which grow over a wide range of temperature, pH values, and solvent tolerances. Examples of suitable host strains include but are not limited to fungal or yeast species, such as Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, Kluyveromyces, or bacterial species, such as member of the proteobacteria and actinomycetes as well as the specific genera Acinetobacter, Arthrobacter, Brevibacterium, Acidovorax, Bacillus, Clostridia, Streptomyces, Escherichia, Salmonella, Pseudomonas, and Cornyebacterium.
- E. coli is well suited to use as the host microorganism in the invention fermentative processes.
- Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct chimeric genes to produce the any one of the gene products of the instant sequences. These chimeric genes could then be introduced into appropriate microorganisms via transformation techniques to provide high-level expression of the enzymes.
- For example, a gene encoding an enzyme can be cloned in a suitable plasmid, and the aforementioned starting parent strain as a host can be transformed with the resulting plasmid. This approach can increase the copy number of each of the genes encoding the enzymes and, as a result, the activities of these enzymes can be increased. The plasmid is not particularly limited so long as it can autonomously replicate in the microorganism.
- Vectors or cassettes useful for the transformation of suitable host cells are well known in the art. Typically the vector or cassette contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a
region 5′ of the gene harboring transcriptional initiation controls and aregion 3′ of the DNA fragment which controls transcriptional termination. It is most preferred when both control regions are derived from genes homologous to the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host. - Promoters, cDNAs, and 3′UTRs, as well as other elements of the vectors, can be generated through cloning techniques using fragments isolated from native sources (see, for example, Molecular Cloning: A Laboratory Manual, Sambrook et al. (3d edition, 2001, Cold Spring Harbor Press; and U.S. Pat. No. 4,683,202 (incorporated by reference)). Alternatively, elements can be generated synthetically using known methods (see, for example, Gene. 1995 Oct. 16; 164(1):49-53).
- B. Homologous Recombination
- Homologous recombination is the ability of complementary DNA sequences to align and exchange regions of homology. Transgenic DNA (“donor”) containing sequences homologous to the genomic sequences being targeted (“template”) is introduced into the organism and then undergoes recombination into the genome at the site of the corresponding genomic homologous sequences.
- The ability to carry out homologous recombination in a host organism has many practical implications for what can be carried out at the molecular genetic level and is useful in the generation of an oleaginous microbe that can produced tailored oils. By its very nature homologous recombination is a precise gene targeting event, hence, most transgenic lines generated with the same targeting sequence will be essentially identical in terms of phenotype, necessitating the screening of far fewer transformation events. Homologous recombination also targets gene insertion events into the host chromosome, potentially resulting in excellent genetic stability, even in the absence of genetic selection. Because different chromosomal loci will likely impact gene expression, even from heterologous promoters/UTRs, homologous recombination can be a method of querying loci in an unfamiliar genome environment and to assess the impact of these environments on gene expression.
- A particularly useful genetic engineering approach using homologous recombination is to co-opt specific host regulatory elements such as promoters/UTRs to drive heterologous gene expression in a highly specific fashion.
- Because homologous recombination is a precise gene targeting event, it can be used to precisely modify any nucleotide(s) within a gene or region of interest, so long as sufficient flanking regions have been identified. Therefore, homologous recombination can be used as a means to modify regulatory sequences impacting gene expression of RNA and/or proteins. It can also be used to modify protein coding regions in an effort to modify enzyme activities such as substrate specificity, affinities and Km, and thus affecting the desired change in metabolism of the host cell. Homologous recombination provides a powerful means to manipulate the host genome resulting in gene targeting, gene conversion, gene deletion, gene duplication, gene inversion and exchanging gene expression regulatory elements such as promoters, enhancers and 3′UTRs.
- Homologous recombination can be achieved by using targeting constructs containing pieces of endogenous sequences to “target” the gene or region of interest within the endogenous host cell genome. Such targeting sequences can either be located 5′ of the gene or region of interest, 3′ of the gene/region of interest or even flank the gene/region of interest. Such targeting constructs can be transformed into the host cell either as a supercoiled plasmid DNA with additional vector backbone, a PCR product with no vector backbone, or as a linearized molecule. In some cases, it may be advantageous to first expose the homologous sequences within the transgenic DNA (donor DNA) with a restriction enzyme. This step can increase the recombination efficiency and decrease the occurrence of undesired events. Other methods of increasing recombination efficiency include using PCR to generate transforming transgenic DNA containing linear ends homologous to the genomic sequences being targeted.
- C. Vectors and Vector Components
- Vectors for transformation of microorganisms in accordance with the present invention can be prepared by known techniques familiar to those skilled in the art in view of the disclosure herein. A vector typically contains one or more genes, in which each gene codes for the expression of a desired product (the gene product) and is operably linked to one or more control sequences that regulate gene expression or target the gene product to a particular location in the recombinant cell.
- This subsection is divided into subsections.
Subsection 1 describes control sequences typically contained on vectors as well as novel control sequences provided by the present invention.Subsection 2 describes genes typically contained in vectors as well as novel codon optimization methods and genes prepared using them provided by the invention. - 1. Control Sequences
- Control sequences are nucleic acids that regulate the expression of a coding sequence or direct a gene product to a particular location in or outside a cell. Control sequences that regulate expression include, for example, promoters that regulate transcription of a coding sequence and terminators that terminate transcription of a coding sequence. Another control sequence is a 3′ untranslated sequence located at the end of a coding sequence that encodes a polyadenylation signal. Control sequences that direct gene products to particular locations include those that encode signal peptides, which direct the protein to which they are attached to a particular location in or outside the cell.
- Thus, an exemplary vector design for expression of an exogenous gene in a microbe contains a coding sequence for a desired gene product (for example, a selectable marker, or an enzyme) in operable linkage with a promoter active in microalgae. Alternatively, if the vector does not contain a promoter in operable linkage with the coding sequence of interest, the coding sequence can be transformed into the cells such that it becomes operably linked to an endogenous promoter at the point of vector integration.
- The promoter used to express an exogenous gene can be the promoter naturally linked to that gene or can be a heterologous promoter.
- A promoter can generally be characterized as either constitutive or inducible. Constitutive promoters are generally active or function to drive expression at all times (or at certain times in the cell life cycle) at the same level. Inducible promoters, conversely, are active (or rendered inactive) or are significantly up- or down-regulated only in response to a stimulus. Both types of promoters find application in the methods of the invention. Inducible promoters useful in the invention include those that mediate transcription of an operably linked gene in response to a stimulus, such as an exogenously provided small molecule, temperature (heat or cold), lack of nitrogen in culture media, etc. Suitable promoters can activate transcription of an essentially silent gene or upregulate, preferably substantially, transcription of an operably linked gene that is transcribed at a low level.
- Inclusion of termination region control sequence is optional, and if employed, then the choice is be primarily one of convenience, as the termination region is relatively interchangeable. The termination region may be native to the transcriptional initiation region (the promoter), may be native to the DNA sequence of interest, or may be obtainable from another source. See, for example, Chen and Orozco, Nucleic Acids Res. (1988) 16:8411.
- 2. Genes and Codon Optimization
- Typically, a gene includes a promoter, coding sequence, and termination control sequences. When assembled by recombinant DNA technology, a gene may be termed an expression cassette and may be flanked by restriction sites for convenient insertion into a vector that is used to introduce the recombinant gene into a host cell. The expression cassette can be flanked by DNA sequences from the genome or other nucleic acid target to facilitate stable integration of the expression cassette into the genome by homologous recombination. Alternatively, the vector and its expression cassette may remain unintegrated (e.g., an episome), in which case, the vector typically includes an origin of replication, which is capable of providing for replication of the heterologous vector DNA.
- A common gene present on a vector is a gene that codes for a protein, the expression of which allows the recombinant cell containing the protein to be differentiated from cells that do not express the protein. Such a gene, and its corresponding gene product, is called a selectable marker or selection marker. Any of a wide variety of selectable markers can be employed in a transgene construct useful for transforming the organisms of the invention.
- For optimal expression of a recombinant protein, it is beneficial to employ coding sequences that produce mRNA with codons optimally used by the host cell to be transformed. Thus, proper expression of transgenes can require that the codon usage of the transgene matches the specific codon bias of the organism in which the transgene is being expressed. The precise mechanisms underlying this effect are many, but include the proper balancing of available aminoacylated tRNA pools with proteins being synthesized in the cell, coupled with more efficient translation of the transgenic messenger RNA (mRNA) when this need is met. When codon usage in the transgene is not optimized, available tRNA pools are not sufficient to allow for efficient translation of the heterologous mRNA resulting in ribosomal stalling and termination and possible instability of the transgenic mRNA.
- D. Expression of Two or More Exogenous Genes
- Further, a genetically engineered microorganism may comprise and express more than one exogenous gene. One or more genes can be expressed using an inducible promoter, which allows the relative timing of expression of these genes to be controlled. Expression of the two or more exogenous genes may be under control of the same inducible promoter or under control of different inducible promoters. In the latter situation, expression of a first exogenous gene can be induced for a first period of time (during which expression of a second exogenous gene may or may not be induced) and expression of a second or further exogenous gene can be induced for a second period of time (during which expression of a first exogenous gene may or may not be induced). Provided herein are vectors and methods for engineering microbes to grow on non-traditional growth media.
- E. Transformation
- Cells can be transformed by any suitable technique including, e.g., biolistics, electroporation, glass bead transformation and silicon carbide whisker transformation. Any convenient technique for introducing a transgene into a microorganism can be employed in the present invention. Transformation can be achieved by, for example, the method of D. M. Morrison (Methods in Enzymology 68, 326 (1979)), the method by increasing permeability of recipient cells for DNA with calcium chloride (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)), or the like.
- Examples of expression of transgenes in oleaginous yeast (e.g., Yarrowia lipolytica) can be found in the literature (see, for example, Bordes et al., J Microbiol Methods, Jun. 27 (2007)). Examples of expression of exogenous genes in bacteria such as E. coli are well known; see, for example, Molecular Cloning: A Laboratory Manual, Sambrook et al. (3d edition, 2001, Cold Spring Harbor Press).
- Vectors for transformation of microorganisms in accordance with the present invention can be prepared by known techniques familiar to those skilled in the art. In one embodiment, an exemplary vector design for expression of a gene in a microorganism contains a gene encoding an enzyme in operable linkage with a promoter active in the microorganism. Alternatively, if the vector does not contain a promoter in operable linkage with the gene of interest, the gene can be transformed into the cells such that it becomes operably linked to an endogenous promoter at the point of vector integration. The vector can also contain a second gene that encodes a protein. Optionally, one or both gene(s) is/are followed by a 3′ untranslated sequence containing a polyadenylation signal. Expression cassettes encoding the two genes can be physically linked in the vector or on separate vectors. Co-transformation of microbes can also be used, in which distinct vector molecules are simultaneously used to transform cells (see, for example, Protist 2004 December; 155(4):381-93). The transformed cells can be optionally selected based upon the ability to grow in the presence of the antibiotic or other selectable marker under conditions in which cells lacking the resistance cassette would not grow.
- In certain embodiments, the invention relates to use of an atypical nitrogen-containing feedstock comprising, consisting essentially of, or consisting of a nitrogen-containing compound of any one of Formulas I-III. In certain embodiments, a non-genetically engineered organism, i.e., a native organism, could not metabolize (i.e., use as a source of nitrogen) the nitrogen-containing compounds in the feedstock.
- In certain embodiments, the invention relates to any one of the aforementioned nitrogen-containing feedstocks, wherein the nitrogen-containing compound is a compound of formula I or a salt thereof:
- wherein, independently for each occurrence,
-
- R is —OH, —CO2H, —NO2, —CN, substituted or unsubstituted amino, or substituted or unsubstituted alkyl; and
- n is 0, 1, 2, 3, 4, or 5.
- In certain embodiments, the invention relates to any one of the aforementioned nitrogen-containing feedstocks, wherein the nitrogen-containing compound is a compound of formula II or a salt thereof:
- wherein, independently for each occurrence,
- X is —NH—, —N(alkyl)-, —O—, —C(R1)2—, —S—, or absent;
- Y is —H, —NH2, —N(H)(alkyl), —N(alkyl)2, —CO2H, —CN, or substituted or unsubstituted alkyl; and
- R1 is —H, —OH, —CO2H, —NO2, —CN, substituted or unsubstituted amino, or substituted or unsubstituted alkyl.
- In certain embodiments, the invention relates to any one of the aforementioned nitrogen-containing feedstocks, wherein the nitrogen-containing compound is a compound of formula III or a salt thereof:
- wherein, independently for each occurrence,
- Y is —H, —NH2, —N(H)(alkyl), —N(alkyl)2, —CO2H, —CN, or substituted or unsubstituted alkyl.
- In certain embodiments, the invention relates to any one of the aforementioned nitrogen-containing feedstocks, wherein the nitrogen-containing compound is selected from the group consisting of:
- In certain embodiments, the invention relates to any one of the aforementioned nitrogen-containing feedstocks, wherein the nitrogen-containing compound is selected from the group consisting of Hydrazine, 5-Aminotetrazole, Tetrazole, Melamine, Cyanamide, 2-Cyanoguanidine, Sodium azide, Carbohydrazide, 1,2,3-Triazole, 1,2,4-Triazole, 1,3-Diaminoguanidine HCl, Ammeline, 1,3,5-triazine, Aminoacetonitrile, Cyanoethylhydrazine, Azodicarbonamide, Biurea, Formamidoxime, 1,2-Dimethylhydrazine, 1,1-Dimethylhydrazine, ethylhydrazine, Ethylenediamine, Sodium dicyanamide, Guanidine carbonate, Methylamine, Ammelide, Hydroxylamine, Malononitrile, Biuret, Diethyltriamine, Hexamethylenetetramine, Triethylenetetramine, 1,3-Diaminopropane, Triethylenetetramine, 1,3-Diaminopropane, Hydroxyurea, Tetraethylenepentamine, Thiourea, Succinonitrile, Calcium cyanamide, Cyanuric acid, Aminoethylpiperazine, Piperazine, Dimethylamine, Ethylamine, dalfampridine, Tetranitromethane, Imidazolidinyl urea, Trinitromethane, malonamide, Chloramine, Allophante, Trimethylamine, Nitromethane, Acetaldoxime, Diazolidinyl urea, 1,2-Cyclohexanedione dioxime, Acetone oxime, Thioacetamide, Sodium thiocyanate, Isothiazole, Thiazole, Dimethylacetamide, Isothiazolinone, Methylene blue, Diethanolamine, Aspartame, Benzisothiazolinone, and Acesulfame potassium.
- In certain embodiments, the invention relates to an isolated nucleic acid molecule, wherein the nucleic acid molecule encodes an enzyme that provides the organism with the ability to assimilate a nitrogen source that otherwise would not have been accessible to the native organism; and the enzyme is allophanate hydrolase, biuret amidohydrolase, cyanuric acid amidohydrolase, guanine deaminase, ammeline hydrolase, ammelide hydrolyase, melamine deaminase, isopropylammelide isopropylaminohydrolase, cyanamide hydratase, urease, or urea carboxylase.
- In certain embodiments, the invention relates to an isolated nucleic acid molecule, wherein the nucleic acid molecule is selected from the group consisting of trzE from Rhodococcus sp. strain Mel, trzE from Rhizobium leguminosarum, trzC MEL,
trzC 12227, cah from Fusarium oxysporum Fo5176, cah from F. pseudograminaearum CS3096, cah from Gibberella zeae PH-1, cah from Aspergillus kawachii IFO 4308, cah from A. niger CBS 513.88, cah from A. niger ATCC 1015, cah from A. oryzae 3.042, cah from S. cerevisiae FostersB, atzF from Pseudomonas sp. strain ADP, DUR1,2 from S. cerevisiae, YALI0E 07271g from Y. lipolytica CLIB122, atzE from Pseudomonas sp. strain ADP, atzD from Pseudomonas sp. strain ADP, trzD from Pseudomonas sp. strain NRRLB-12227, atzD from Rhodococcus sp. Mel, trzD from Rhodococcus sp. Mel, guaD from E. coli K12 strain MG1566, blr3880 from Bradyrhizobium japonicum USDA 110, GUD1/Y DL238C from S. cerevisiae, YAL10E2 5740p from Y. lipolytica CLIB122, trzA from Williamsia sp. NRRL B-15444R, triA from Pseudomonas sp. strain NRRL B-12227, atzC from Pseudomonas sp. strain ADP, and cah from Myrothecium verrucaria. - In certain embodiments, the invention relates to an isolated nucleic acid molecule comprising any one of the sequences disclosed herein. In certain embodiments, the invention relates to an isolated nucleic acid molecule having at least 85% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to an isolated nucleic acid molecule having at least 90% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to an isolated nucleic acid molecule having at least 95% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to an isolated nucleic acid molecule having at least 99% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to an isolated nucleic acid molecule having any one of the sequences disclosed herein.
- A recombinant vector comprising any one of the aformentioned nucleic acid molecules operably linked to a promoter.
- In certain embodiments, the invention relates to a recombinant vector comprising any one of the sequences disclosed herein. In certain embodiments, the invention relates to a recombinant vector having at least 85% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to a recombinant vector having at least 90% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to a recombinant vector having at least 95% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to a recombinant vector having at least 99% sequence homology with any one of the sequences disclosed herein.
- In certain embodiments, the invention relates to a genetically engineered organism, wherein the genetically engineered organism has been transformed by a nucleic acid molecule or a recombinant vector comprising any one of the sequences disclosed herein. In certain embodiments, the nucleic acid molecule or recombinant vector has at least 85% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the nucleic acid molecule or recombinant vector has at least 90% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the nucleic acid molecule or recombinant vector has at least 95% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the nucleic acid molecule or recombinant vector has at least 99% sequence homology with any one of the sequences disclosed herein. In certain embodiments, the invention relates to a genetically engineered organism, wherein the genetically engineered organism has been transformed by a nucleic acid molecule or a recombinant vector having any one of the sequences disclosed herein.
- In certain embodiments, the invention relates to a genetically engineered organism, wherein the genetically engineered organism has been transformed by a nucleic acid molecule; the nucleic acid molecule comprises a non-native gene; and the non-native gene encodes for a non-native enzyme selected from the group consisting of allophanate hydrolase, biuret amidohydrolase, cyanuric acid amidohydrolase, guanine deaminase, ammeline hydrolase, ammelide hydrolyase, melamine deaminase, and isopropylammelide isopropylaminohydrolase, cyanamide hydratase, urease, or urea carboxylase.
- In certain embodiments, the invention relates to any one of the aforementioned genetically engineered organisms, wherein the non-native gene is selected from the group consisting of atzF, DUR1,2 YALI0E 07271g, atzE, atzD, trzC, trzD, trzE, atzD, guaD, blr3880, GUD1/Y DL238C, YAL10E2 5740p, trzA, triA, atzC, and cah. In certain embodiments, the invention relates to any one of the aforementioned genetically engineered organisms, wherein the non-native gene is selected from the group consisting of atzF, DUR1,2 YALI0E 07271g, atzE, atzD, trzD, atzD, guaD, blr3880, GUD1/Y DL238C, YAL10E2 5740p, trzA, triA, atzC, and cah. Any organism may be used as a source of the non-native gene, as long as the organisms has the desired enzymatic activity The non-native gene can each be obtained from chromosomal DNA of any one of the aforementioned microorganisms by isolating a DNA fragment complementing auxotrophy of a variant strain lacking the enzymatic activity. Alternatively, if the nucleotide sequence of these gene of the organism has already been elucidated (Biochemistry, Vol. 22, pp. 5243-5249, 1983; J. Biochem. Vol. 95, pp. 909-916, 1984; Gene, Vol. 27, pp. 193-199, 1984; Microbiology, Vol. 140, pp. 1817-1828, 1994; Mol. Gene Genet. Vol. 218, pp. 330-339, 1989; and Molecular Microbiology, Vol. 6, pp. 317-326, 1992), the genes can be obtained by PCR using primers synthesized based on each of the elucidated nucleotide sequences, and the chromosome DNA as a template.
- In certain embodiments, the invention relates to any one of the aforementioned genetically engineered organisms, wherein the non-native gene is selected from the group consisting of trzE from Rhodococcus sp. strain Mel, trzE from Rhizobium leguminosarum, trzC MEL,
trzC 12227, cah from Fusarium oxysporum Fo5176, cah from F. pseudograminaearum CS3096, cah from Gibberella zeae PH-1, cah from Aspergillus kawachii IFO 4308, cah from A. niger CBS 513.88, cah from A. niger ATCC 1015, cah from A. oryzae 3.042, cah from S. cerevisiae FostersB, atzF from Pseudomonas sp. strain ADP, DUR1,2 from S. cerevisiae, YALI0E 07271g from Y. lipolytica CLIB122, atzE from Pseudomonas sp. strain ADP, atzD from Pseudomonas sp. strain ADP, trzD from Pseudomonas sp. strain NRRLB-12227, atzD from Rhodococcus sp. Mel, trzD from Rhodococcus sp. Mel, guaD from E. coli K12 strain MG1566, blr3880 from Bradyrhizobium japonicum USDA 110, GUD1/Y DL238C from S. cerevisiae, YAL10E2 5740p from Y. lipolytica CLIB122, trzA from Williamsia sp. NRRL B-15444R, triA from Pseudomonas sp. strain NRRL B-12227, atzC from Pseudomonas sp. strain ADP, and cah from Myrothecium verrucaria. - In certain embodiments, the invention relates to any one of the aforementioned genetically engineered organisms, wherein the genetically engineered organism is a species of the genus Yarrowia, Saccharomyces, Ogataea, Pichia, or Escherichia.
- In certain embodiments, the invention relates to any one of the aforementioned genetically engineered organisms, wherein the genetically engineered organism is selected from the group consisting of Yarrowia lipolytica, Saccharomyces cerevisiae, Ogataea polymorpha, Pichia pastoris, and Escherichia coli.
- In certain embodiments, the genetically engineered organism is not Rhodococcus sp. Strain Mel.
- In certain embodiments, the invention relates to a method, comprising the step of
- contacting any one of the aforementioned genetically engineered organisms with a substrate,
- wherein
- the substrate comprises a nitrogen-containing fraction and a non-nitrogen-containing fraction;
- the nitrogen-containing fraction comprises, in an amount from about 10% by weight to about 100% by weight, a nitrogen-containing compound of any one of Formulas I-III;
- a native organism of the same species as the genetically engineered organism could not metabolize (i.e., use as a source of nitrogen) the nitrogen-containing compound; and
- the genetically engineered organism converts the substrate to a product.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have a low molecular weight. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have a molecular weight between about 30 Da and about 800 Da. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have a molecular weight between about 40 Da and about 600 Da. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have a molecular weight of about 40 Da, about 50 Da, about 60 Da, about 70 Da, about 80 Da, about 90 Da, about 100 Da, about 110 Da, about 120 Da, about 130 Da, about 140 Da, about 150 Da, about 160 Da, about 170 Da, about 180 Da, about 190 Da, about 200 Da, about 220 Da, about 240 Da, about 260 Da, about 280 Da, about 300 Da, about 320 Da, about 340 Da, about 360 Da, about 380 Da, about 400 Da, about 420 Da, about 440 Da, about 460 Da, about 480 Da, about 500 Da, about 520 Da, about 540 Da, bout 560 Da, about 580 Da, or about 600 Da.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have less than 12 carbon atoms. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have less than 8 carbon atoms. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have 1, 2, 3, 4, 5, 6, or 7 carbon atoms.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nitrogen atoms.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have 0, 1, 2, 3, 4, 5, 6, 7, or 8 oxygen atoms.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have an octanol-water partition coefficient (log P) less than about 5. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have an octanol-water partition coefficient (log P) from about −0.5 to about 5. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds have an octanol-water partition coefficient (log P) of about −0.5, about 0, about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, or about 4.5.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds are soluble in water at about 20° C. at a concentration of between about 0.01 g/L to about 1000 g/L. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds are soluble in water at about 20° C. at a concentration of about 0.01 g/L, about 0.05 g/L, about 0.1 g/L, about 0.5 g/L, about 1 g/L, about 5 g/L, about 10 g/L, about 15 g/L, about 20 g/L, about 25 g/L, about 30 g/L, about 35 g/L, about 40 g/L, about 45 g/L, about 50 g/L, about 55 g/L, about 60 g/L, about 65 g/L, about 70 g/L, about 75 g/L, about 80 g/L, about 85 g/L, about 90 g/L, about 95 g/L, or about 100 g/L.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds move through the cell membrane by passive transport. Passive transport includes diffusion, facilitated diffusion, and filtration.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds move through the cell membrane by active transport, such as, for example, via an ATP-Binding Cassette (ABC) transporter or other known transmembrane transporter.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds are transported through the cell membrane.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds are substantially non-biocidal.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing compounds are substantially biodegradable.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing fraction comprises the nitrogen-containing compound in about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% by weight.
- In certain embodiments, the invention relates to a method, comprising the step of
- contacting any one of the aforementioned genetically engineered organisms with a substrate,
- wherein
- the substrate comprises a nitrogen-containing fraction and a non-nitrogen-containing fraction;
-
- the nitrogen-containing fraction comprises, in an amount from about 10% by weight to about 100% by weight, a nitrogen-containing compound selected from the group consisting of triazine, urea, melamine, cyanamide, 2-cyanoguanidine, ammeline, guanidine carbonate, ethylenediamine, ammelide, biuret, diethylenetriamine, triethylenetetramine, 1,3-diaminopropane, calcium cyanamide, cyanuric acid, aminoethylpiperazine, piperazine, and allophante; and
- the genetically engineered organism converts the substrate to a product.′
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the nitrogen-containing fraction comprises the nitrogen-containing compound in about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% by weight.
- In certain embodiments, the invention relates to a method, comprising the step of
- contacting any one of the aforementioned genetically engineered organisms with a substrate,
- wherein
- the substrate comprises a nitrogen-containing fraction and a non-nitrogen-containing fraction;
- the nitrogen containing fraction consists essentially of a nitrogen-containing compound selected from the group consisting of triazine, urea, melamine, cyanamide, 2-cyanoguanidine, ammeline, guanidine carbonate, ethylenediamine, ammelide, biuret, diethylenetriamine, triethylenetetramine, 1,3-diaminopropane, calcium cyanamide, cyanuric acid, aminoethylpiperazine, piperazine, and allophante; and
- the genetically engineered organism converts the substrate to a product.
- In certain embodiments, the invention relates to a method, comprising the step of
- contacting any one of the aforementioned genetically engineered organisms with a substrate,
- wherein
- the substrate consists of a nitrogen-containing fraction and a non-nitrogen-containing fraction;
- the nitrogen containing fraction consists of a nitrogen-containing compound selected from the group consisting of triazine, urea, melamine, cyanamide, 2-cyanoguanidine, ammeline, guanidine carbonate, ethylenediamine, ammelide, biuret, diethylenetriamine, triethylenetetramine, 1,3-diaminopropane, calcium cyanamide, cyanuric acid, aminoethylpiperazine, piperazine, and allophante; and the genetically engineered organism converts the substrate to a product.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the genetically engineered organism sequesters the product.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein a plurality of genetically engineered organisms is used.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the substrate does not comprise an antibiotic.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the substrate does not comprise ammonium sulfate.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the substrate does not comprise urea.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein a non-genetically engineered organism, i.e., a native organism, could not metabolize (i.e., use as a source of nitrogen) the nitrogen-containing compound. In certain embodiments, the genetically engineered organism is not Rhodococcus sp. Strain Mel.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the substrate comprises lignocellulosic material, glucose, xylose, sucrose, acetic acid, formic acid, lactic acid, butyric acid, a free fatty acid, dextrose, glycerol, fructose, lactose, galactose, mannose, rhamnose, or arabinose, or a combination thereof.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the pH of the substrate is from about 2.5 to about 10.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the genetically engineered organism is contacted with the substrate at a temperature of from about 15° C. to about 80° C.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the genetically engineered organism is contacted with the substrate over a time period of from about 6 h to about 10 d.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the genetically engineered organism is contacted with the substrate in a fermentor.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the genetically engineered organism is contacted with the substrate in an industrial-size fermentor.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein a plurality of genetically engineered organisms is contacted with a plurality of substrates in a plurality of fermentors, wherein the plurality of fermentors are arranged in parallel.
- In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the product is ethanol, isopropanol, lactic acid, an isoprenoid, a lipid, a high-value specialty chemical, butanol, 1,3-propanediol, 1,4-butanediol, succinic acid, an expressed protein product, an enzyme product, a polyol, a pharmaceutical product, itaconic acid, or a high value specialty chemical.
- In certain embodiments, the invention relates to a product made by any one of the aforementioned methods.
- The following examples are provided to illustrate the invention. It will be understood, however, that the specific details given in each example have been selected for purpose of illustration and are not to be construed as limiting the scope of the invention. Generally, the experiments were conducted under similar conditions unless noted.
- The oleaginous yeast Yarrowia lipolytica may be engineered to convert melamine into ammonia. Melamine (C3N6H6) is a highly nitrogenous compound that can only be degraded by a very limited number of organisms including Rhodococcus sp. Strain Mel. Incorporating the pathway for melamine degradation into Yarrowia, accompanied with a modification in the media composition to use melamine as the predominant nitrogen source, will generate a more robust industrial production solution applicable to a number of applications. The advantage confirmed by this modification is significant enough to provide advantage in multiple applications including situations where the core technology may be significant genetic burden on the organism.
- Genes from
FIG. 3 , or suitable homologs, will be cloned into a host strain such as Yarrowia lipolytica, Saccharomyces cerevisiae, or Escherichia coli. Enzymes native to the host organism, such as allophante hydrolase or guanine deaminase may be overexpressed with a heterologous promoter. Functional expression will be assayed by enzymatic activity and the ability to confer nitrogen limited growth on the appropriate pathway intermediate. Ultimately, strains able to degrade melamine will be selected for improved utilization of the pathway via melamine limited continuous culturing or other selective methods. Similar strategies can be devised for nitrogen compounds listed inFIG. 2 . - Vector pNC10 contains an E. coli pMB1 origin of replication and ampicillin resistance gene, a
S. cerevisiae 2 μm origin of replication and URA3 gene, and a multiple cloning site containing the 8-bp recognition sequences for PacI, PmeI, and AscI. DNA of interest is inserted in the multiple cloning site via yeast mediated homologous recombination (YML) cloning. (Shanks et al. 2006; Shanks et al. 2009). Briefly, target DNA sequences are amplified by PCR using primers with 20-40 bp overhang homology to adjacent DNA segments in the final vector. pNC10 or another suitable base vector is then restriction digested, creating a linearized plasmid. PCR products and linear plasmid are transformed in S. cerevisiae, and the native S. cerevisiae gap repair mechanism assembles an intact plasmid based on homology overhangs. - The complete vector can then be isolated from S. cerevisiae via a DNA extraction protocol and used to transform E. coli. Concentrated vector can then be recovered from E. coli via DNA plasmid mini-prep or other suitable standard molecular biology protocols. See
FIG. 5 . - Grow overnight a 5 mL culture of a S. cerevisiae ura3 auxotroph strain in YPD at 30 C.
- Transfer 1.5 mL of overnight culture to 50 mL fresh YPD (OD˜0.3) and shake at 200 rpm, 30° C. in a flask. Allow to grow for approx. 4-5 hrs to an OD of 1.0.
- Centrifuge cells at >5,000 rpm for 1 mM, resuspend in 50 mL sterile water and repeat.
- Add 1 mL of 100 mM Lithium acetate to cell pellet and transfer cells to a 1.5 mL tube.
- Spin cells for 10 sec at >12,000 rpm, remove supernatant, and resuspend in 400-800 μL of 100 mM LiAc (each transformation uses 50 μL of this cell suspension).
- Prepare a transformation master mix of the following, per sample
-
X number of transformations + 1 50% PEG 3350 240 μL 1M LiAc 36 μL Salmon sperm DNA* (2 mg/mL) 50 μL *SS DNA should be first boiled for 10 min and rapidly cooled to 4° C. - Prepare one 1.5 mL tube for each transformation. Per tube, add: 5 μL of digested vector, 5 μL of each PCR insert (assuming a good PCR amplification, approx. 100-200 ng DNA), and water to bring the final volume to 34 μL. Add 326 μL master mix, and then 50 μL of cell suspension. Vortex tubes to completely mix contents.
- Incubate for 30 min at 30° C., then mix by inverting and place in 42° C. water bath for 30 min. (Note optimal time at 42° C. varies strain to strain).
- Spin down cells for 10 sec at >12,000 rpm, remove PEG mixture and resuspend in 1 mL sterile water. Spin down again, remove 800 μL, and use final 200 μL to resuspend and spread on SD-URA plates. Incubate at 30° C. for 2-4 days.
- Melamine assimilation genes, or a subset of them, can be expressed in S. cerevisiae by construction of a vector using the yeast mediated ligation described above. Expression vectors consist of an S. cerevisiae functional promoter, a gene encoding an enzyme of the melamine assimilation pathway, and an S. cerevisiae functional terminator. Assemblies of the promoter-gene-terminator motif can be incorporated into a single strain, either on a replicating plasmid or integrated into a chromosome. Possible promoters and terminators are listed below, see also Sun et al. 2012. A representative plasmid, expressing the trzA melamine hydratase under control of the Y. lipolytica TEF1 promoter and terminator is shown below.
- Plasmid AJS35 is an example of the melamine dehydratase trzA transcribed via the Y. lipolytica TEF1 promoter and terminator. See
FIG. 6 . - Strains NS98 and NS99 are industrial S. cerevisiae strains carrying plasmids pNC96 (hygR, and a codon optimized trzE from Rhodococcus sp. MEL and pNC97 (hygR, and a codon optimized trzE from Rhizobium leguminosarum), respectively. Strain NS100 is the same industrial S. cerevisiae stain carrying plasmid pNC67 (hygR, natR) which serves as a control strain.
- Strains NS98, NS99, and NS100 were grown in defined YNB medium with 10 mM urea and 100 μg/mL hygromycin to stationary phase aerobically at 30° C. 1/1000 v/v inoculations were then made into the same defined medium with either 10 mM urea, 10 mM biuret, or no additional nitrogen and grown under the same conditions. Optical density was measured after 72 hours, as shown in
FIG. 23 . Strains NS98 and NS99 were able to grow to an optical density approximately double that of NS100 in medium containing biuret, and also approximately double that with medium with no nitrogen supply. This shows that S. cerevisiae strains expressing trzE genes are advantaged in their utilization of biuret. - DNA that can be used as promoters for gene transcription in S. cerevisiae
-
S. cerevisiae TPI promoter aggaacccatcaggttggtggaaGATTACCCGTTCTAAGACTTTTCAGCTT CCTCTATTGATGTTACACCTGGACACCCCTTTTCTGGCATCCAGTTTTTAA TCTTCAGTGGCATGTGAGATTCTCCGAAATTAATTAAAGCAATCACACAAT TCTCTCGGATACCACCTCGGTTGAAACTGACAGGTGGTTTGTTACGCATGC TAATGCAAAGGAGCCTATATACCTTTGGCTCGGCTGCTGTAACAGGGAATA TAAAGGGCAGCATAATTTAGGAGTTTAGTGAACTTGCAACATTTACTATTT TCCCTTCTTACGTAAATATTTTTCTTTTTAATTCTAAATCAATCTTTTTCA ATTTTTTGTTTGTATTCTTTTCTTGCTTAAAtctataac tacaaaaaacacatacataaactaaaa S. cerevisiae GPM1 promoter ttgctacgcaggctgcacaattacACGAGAATGCTCCCGCCTAGGATTTAA GGCTAAGGGACGTGCAATGCAGACGACAGATCTAAATGACCGTGTCGGTGA AGTGTTCGCCAAACTTTTCGGTTAACACATGCAGTGATGCACGCGCGATGG TGCTAAGTTACATATATATATATATATATATATATATATATATATAGCCAT AGTGATGTCTAAGTAACCTTTATGGTATATTTCTTAATGTGGAAAGATACT AGCGCGCGCACCCACACACAAGCTTCGTCTTTTCTTGAAGAAAAGAGGAAG CTCGCTAAATGGGATTCCACTTTCCGTTCCCTGCCAGCTGATGGAAAAAGG TTAGTGGAACGATGAAGAATAAAAAGAGAGATCCACTGAGGTGAAATTTCA GCTGACAGCGAGTTTCATGATCGTGATGAACAATGGTAACGAGTTGTGGCT GTTGCCAGGGAGGGTGGTTCTCAACTTTTAATGTATGGCCAAATCGCTACT TGGGTTTGTTATATAACAAAGAAGAAATAATGAACTGATTCTCTTCCTCCT TCTTGTCCTTTCTTAATTCTGTTGTAATTACCTTCCTTTGTAATTTTTTTT GTAATTATTCTtcttaataatccaaacaaacacacatattacaata S. cerevisiae TDH3 promoter tgctgtaacccgtacatgcccaaaATAGGGGGCGGGTTACACAGAATATAT AACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATAT AATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCA CCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTA GCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCT CAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGA CCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTC TCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAA TTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGT AATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAG TCTTTTTTTTAGTTTTAAAACACCAAGAacttagtttcgaataaacacaca taaacaaacaaa S. cerevisiae FBA1 promoter gcaccgctggcttgaacaacaataCCAGCCTTCCAACTTCTGTAAATAACG GCGGTACGCCAGTGCCACCAGTACCGTTACCTTTCGGTATACCTCCTTTCC CCATGTTTCCAATGCCCTTCATGCCTCCAACGGCTACTATCACAAATCCTC ATCAAGCTGACGCAAGCCCTAAGAAATGAATAACAATACTGACAGTACTAA ATAATTGCCTACTTGGCTTCACATACGTTGCATACGTCGATATAGATAATA ATGATAATGACAGCAGGATTATCGTAATACGTAATAGTTGAAAATCTCAAA AATGTGTGGGTCATTACGTAAATAATGATAGGAATGGGATTCTTCTATTTT TCCTTTTTCCATTCTAGCAGCCGTCGGGAAAACGTGGCATCCTCTCTTTCG GGCTCAATTGGAGTCACGCTGCCGTGAGCATCCTCTCTTTCCATATCTAAC AACTGAGCACGTAACCAATGGAAAAGCATGAGCTTAGCGTTGCTCCAAAAA AGTATTGGATGGTTAATACCATTTGTCTGTTCTCTTCTGACTTTGACTCCT CAAAAAAAAAAAATCTACAATCAACAGATCGCTTCAATTACGCCCTCACAA AAACTTTTTTCCTTCTTCTTCGCCCACGTTAAATTTTATCCCTCATGTTGT CTAACGGATTTCTGCACTTGATTTATTATAAAAAGACAAAGACATAATACT TCTCTATCAATTTCAGTTATTGTTCTTCCTTGCGTTATTCTTCTGTTCTTC TTTTTCTTTTGTcatatataaccataaccaagtaatacatattcaaa Y. lipolytica TEF1 promoter tataaacggtattttcacaattgcACCCCAGCCAGACCGATAGCCGGTCGC AATCCGCCACCCACAACCGTCTACCTCCCACAGAACCCCGTCACTTCCACC CTTTTCCACCAGATCATATGTCCCAACTTGCCAAATTAAAACCGTGCGAAT TTTCAAAATAAACTTTGGCAAAGAGGCTGCAAAGGAGGGGCTGGTGAGGGC GTCTGGAAGTCGACCAGAGACCGGGTTGGCGGCGCATTTGTGTCCCAAAAA ACAGCCCCAATTGCCCCAATTGACCCCAAATTGACCCAGTAGCGGGCCCAA CCCCGGCGAGAGCCCCCTTCTCCCCACATATCAAACCTCCCCCGGTTCCCA CACTTGCCGTTAAGGGCGTAGGGTACTGCAGTCTGGAATCTACGCTTGTTC AGACTTTGTACTAGTTTCTTTGTCTGGCCATCCGGGTAACCCATGCCGGAC GCAAAATAGACTACTGAAAATTTTTTTGCTTTGTGGTTGGGACTTTAGCCA AGGGTATAAAAGACCACCGTCCCCGAATTACCTTTCCTCTTCTTTTCTCTC TCTCCTTGTCAACTCACACCCGAAATCGTtaagcatttccttctgagtata agaatcattcaaa S. cerevisiae PDC1 promoter gcataatattgtccgctgcccgttTTTCTGTTAGACGGTGTCTTGATCTAC TTGCTATCGTTCAACACCACCTTATTTTCTAACTATTTTTTTTTTAGCTCA TTTGAATCAGCTTATGGTGATGGCACATTTTTGCATAAACCTAGCTGTCCT CGTTGAACATAGGAAAAAAAAATATATAAACAAGGCTCTTTCACTCTCCTT GGAATCAGATTTGGGTTTGTTCCCTTTATTTTCATATTTCTTGTCATATTC TTTTCTCAATTATTATCTTCTACTCATAacctcacgcaaaataacacagtc aaatcaatcaaa S. cerevisiae TEF1 promoter CATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGG ACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAA TTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTT TGGAAAAGAAAAAAGAGACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGC AATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATT TTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTAATAAACGGTCTT CAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTT ACTTCTTGCTCATTAGAAAGAaagcatagcaatctaatctaagttttaatt acaaa
DNA sequences that can be used as terminators of gene transcription -
S. cerevisiae TPI terminator taagattaatataattatataaAAATATTATCTTCTTTTCTTTATATCTAG TGTTATGTAAAATAAATTGATGACTACGGAAAGCTTTTTTATATTGTTTCT TTTTCATTCTGAGCCACTTAAATTTCGTGAATGTTCTTGTAAGGGACGGTA GATTTACAAGTGATACAACAAAAAGCAAGGCGCTTTTTCTAATAAAAAGAA GAAAAGCATTTAACAATTGAACACCTCTATATCAACGAAGAATATTACTTT GTCTCTAAATCCTTGTAAAATGTGTACGATCTCTATATGGGTTACTCATAA gtgtaccgaagactgcattgaaag S. cerevisiae GPM1 terminator gtctgaagaatgaatgatttgaTGATTTCTTTTTCCCTCCATTTTTCTTAC TGAATATATCAATGATATAGACTTGTATAGTTTATTATTTCAAATTAAGTA GCTATATATAGTCAAGATAACGTTTGTTTGACACGATTACATTATTCGTCG ACATCTTTTTTCAGCCTGTCGTGGTAGCAATTTGAGGAGTATTATTAATTG AATAGGTTCATTTTGCGCTCGCATAAACAGTTTTCGTCAGGGACAGTATGT TGGAATGAGTGGTAATTAATGGTGACATGACATGTTATAGCAATAACCTTG ATGTTTACATCGTAGTTTAATGTACACCCCGCGAATTCGTTCAAGTAggag tgcaccaattgcaaagggaa S. cerevisiae TDH3 terminator gtgaatttactttaaatcttgcATTTAAATAAATTTTCTTTTTATAGCTTT ATGACTTAGTTTCAATTTATATACTATTTTAATGACATTTTCGATTCATTG ATTGAAAGCTTTGTGTTTTTTCTTGATGCGCTATTGCATTGTTCTTGTCTT TTTCGCCACATGTAATATCTGTAGTAGATACCTGATACATTGTGGATGCTG AGTGAAATTTTAGTTAATAATGGAGGCGCTCTTAATAATTTTGGGGATATT GGCTTTTTTTTTTAAAGTTTACAAATGAATTTTTTCCGCCAGGATAACGAT TCTGAAGTTACTCTTAGCGTTCCTATCGGTACAGCCATCAAATCATGCCTA TAAATCATGCCTATATTTGCGTGCAGTCAGTATCATCTACATGAAAAAAAC TCCCGCAATTTCTTATAGAATACGTTGAAAATTAAATGTACGCGCCAAGAT AAGATAACATATATCTAGATGCAGTAATATACACAGATTCCCGCGGA S. cerevisiae FBA1 terminator gttaattcaaattaattgatatAGTTTTTTAATGAGTATTGAATCTGTTTA GAAATAATGGAATATTATTTTTATTTATTTATTTATATTATTGGTCGGCTC TTTTCTTCTGAAGGTCAATGACAAAATGATATGAAGGAAATAATGATTTCT AAAATTTTACAACGTAAGATATTTTTACAaaagcctagctcatctt Y. lipolytica TEF1 terminator gctgcttgtacctagtgcaaccccagtttgttaaaAATTAGTAGTCAAAAA CTTCTGAGTTAGAAATTTGTGAGTGTAGTGAGATTGTAGAGTATCATGTGT GTCCGTAAGTGAAGTGTTATTGACTCTTAGTTAGTTTATCTAGTACTCGTT TAGTTGACACTGATCTAGTATTTTACGAGGCGTATGACTTTAGCCAAGTGT TGTACTTAGTCTTCTCTCCAAACATGAGAGGGCTCTGTCACTCAGTCGGCC TATGGGTGAGATGGCTTGGTGAGATCTTTCGATAGTCTCGTCAAGATGGTA GGATGATGGGGGAATACATTACTGCTCTCGTCAAGGAAACCACAATCAGAT CACACCATCCTCCATGGTAtccgatgactctcttctccacagt S. cerevisiae PDC1 terminator acaagctaagttgactgctgctACCAACGCTAAGCAATAAGCGATTTAATC TCTAATTATTAGTTAAAGTTTTATAAGCATTTTTATGTAACGAAAAATAAA TTGGTTCATATTATTACTGCACTGTCACTTACCATGGAAAGACCAGACAAG AAGTTGCCGACACGACAGTCTGTTGAattggcttaagtctgggtccgctt S. cerevisiae CYC1 terminator caggccccttttcctttgtcgaTATCATGTAATTAGTTATGTCACGCTTAC ATTCACGCCCTCCTCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGAC AACCTGAAGTCTAGGTCCCTATTTATTTTTTTTAATAGTTATGTTAGTATT AAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAAACGCGT GTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACG CTCGAAGGCTTTAATTTGC - Melamine assimilation genes, or a subset of them, can be expressed in E. coli by construction of a vector using the yeast mediated ligation described above. Expression vectors consist of an E. coli functional promoter, a gene encoding an enzyme of the melamine assimilation pathway, and an E. coli functional terminator. Alternatively, several genes can be expressed from a single promoter as part of a gene operon; in this case inter-gene linker sequences are placed between genes. Sequences that can act as promoters, terminators, and linkers are listed below, as well as two representative E. coli expression plasmids, AJS67 (expressing genes for degradation of melamine to cyanuric acid with release of 3 NH3 per melamine) and AJS68 (expressing genes for degradation of cyanuric acid to NH3 and CO2 with release of 3 NH3 per cyanuric acid)
-
E. coli Ptach promoter agctggtgacaattaatcatcggctcgtataatgtgtggaattgaatcgat ataaggaggttaatca E. coli trpT′ terminator ctcaaaatatattttccctctatcttctcgttgcgcttaatttgactaatt ctcattagcgaggcgcgcctttccataggctccgcccc inter-gene operon linkers lacZ-lacY linker ggaaatccatt galT-galK linker ggaacgacc - See
FIG. 7 andFIG. 8 . - The gene expression methods described in example 5 can also be used in example 7. S. cerevisiae has the native ability to convert urea to NH3 and CO2 via the actions of urea carboxylase and allophante hydrolase, encoded in the fusion gene DUR1,2. Therefore, functional expression of cyanamide hydrolase is sufficient to convert cyanamide to NH3. A representative cyanamide hydratase expression vector is shown below, with Y. lipolytica TEF1 promoter and terminator and a S. cerevisiae codon-optimized cyanamide hydratase (cah) from Myrothecium verrucaria. See
FIG. 9 . - The gene expression methods described in Example 6 can also be used in example 8. Unlike S. cerevisiae, most E. coli strains are unable to utilize urea as a nitrogen source, so these additional conversion steps must also be engineered. Either a urea carboxylase/allophante hydrolase system or a urease enzyme with appropriate accessory enzymes must be expressed in addition to a cyanamide hydrolase. Urease can be found in some E. coli isolates (Collins and Falkow 1990) or heterologously expressed (Cussac et al. 1992). Alternatively, the DUR1,2 genes from S. cerevisiae could be expressed, as shown below in plasmid AJS70, along with a cyanamide hydratase. See
FIG. 10 . - Several E. coli strains containing partial or complete melamine utilization pathways were constructed, as shown in
FIGS. 29 and 30 . Vector and strain construction was as described in example 6. All vectors contain the ampicillin resistance gene, and 100 ug/mL ampicillin was added to all culture medium. These strains were grown in MOPS defined medium with different nitrogen sources. - E. coli strains and melamine utilization genes
- NS88—triA (step 1)
- NS89—trzA, guaD, trzC (
steps - NS90—trzD, trzE, DUR1,2 (
steps - NS91—none (control strain)
- NS93—triA, native guaD selected for improved ammeline utilization (
steps 1, 2) - NS103—triA, guaD, trzC (
steps - NS109—triA, guaD, trzC,
trzD 12227, trzE, DUR1,2 (steps 1-6) - NS110—triA, guaD, trzC, atzD ADP, trzE, DUR1,2 (steps 1-6)
-
FIG. 12 shows the growth progress of NS88 and NS91 (control) in media containing various concentrations of ammonium chloride or melamine NS88 grown on 1 mM melamine reaches an optical density comparable to that of the equivalent use of 2 mM ammonium chloride, suggesting that 2 mM ammonia are liberated from melamine by triA and the natively encoded guaD genes. The control strain NS91 does not grow with melamine as nitrogen source. -
FIG. 13 shows the growth progress of NS90 and NS91 (control) in media containing various concentrations of ammonium chloride or biuret. NS90 grown on 1 mM biuret reaches an optical density comparable to that of the equivalent use of 3 mM ammonium chloride, suggesting that 3 mM ammonia are liberated from biuret by trzE and the DUR1,2. The control strain NS91 does not grow with biuret as nitrogen source. -
FIG. 24 shows the growth progress of NS91, NS103, NS109, and NS110 in medium containing 0.25 mM melamine as sole nitrogen source. An average of all four strains grown on different ammonium chloride concentrations from 0 to 1.5 mM is also shown as a standard curve for growth with limiting nitrogen. NS91 grown on melamine is similar to the 0 mM ammonium chloride control. NS103 grown on 0.25 mM melamine is similar to 1-0.75 mM ammonium chloride, suggesting it is approximately utilizating the predicted 3 mM ammonia per 1 mM melamine Strains NS109 and NS110 grown on 0.25 mM melamine are similar to 1.5-1.25 mM ammonium chloride, suggesting it is approximately utilizating the predicted 6 mM ammonia per 1 mM melamine -
FIG. 25 shows the growth progress of NS91, NS103, NS109, and NS110 in medium containing 0.25 mM ammeline as sole nitrogen source. An average of all four strains grown on different ammonium chloride concentrations from 0 to 1.5 mM is also shown as a standard curve for growth with limiting nitrogen. NS91 grown on ammeline is similar to the 0 mM ammonium chloride control. NS103 grown on 0.25 mM ammeline is similar to 0.5 mM ammonium chloride, suggesting it is approximately utilizating the predicted 2 mM ammonia per 1 mM ammeline. Strains NS109 and NS110 grown on 0.25 mM ammeline are similar to 1.25-1.0 mM ammonium chloride, suggesting it is approximately utilizating the predicted 5 mM ammonia per 1 mM ammeline. -
FIGS. 26, 27, and 28 show E. coli strains derived from E. coli K12, E. coli MG1655, E. coli B, and E. coli Crooks (C) containing either pNC121 with the complete melamine utilization pathway, or pNC53, a control vector. SeeFIGS. 29 and 30 for strain details. All the strains containing pNC121 are able to grow on 0.5 mM melamine as sole nitrogen source (FIG. 28 ). This indicates that the melamine utilization pathway is broadly applicable to E. coli strains that are commonly utilized for biotechnology applications. - Strains can also be selected for improved utilization of melamine derived nitrogen sources, in one example NS88 was passaged for 11 serial transfers in MOPS defined medium with 0.5 mM ammeline as sole nitrogen source. After the final passage, single colonies were isolated, and one was designated as NS93. NS93 and NS91 were grown overnight in medium with 0.5 mM ammonium chloride as sole nitrogen source, and then inoculated in medium with 0.5 mM ammeline as sole nitrogen source. NS91 exhibited a maximum growth rate of 0.024 hr−1 on ammeline, while NS93 exhibited a maximum growth rate of 0.087 hr−1.
- Cultures grown aerobically at 37° C. with 100 mg/L ampicillin. Pre-cultures were grown in LB media with 100 mg/L ampicillin, washed once with an equal volume of MOPS media containing no nitrogen, and inoculated at 5% v/v of the final fermentation volume. The content of the MOPS medium is outlined in
FIG. 11 . - Precultures were grown in LB media with 100 mg/L ampicillin, 0.1 mL were directly inoculated into 5 mL MOPS media with 100 mg/L ampicillin and the indicated nitrogen source. Grown at 37° C. in a drum roller at 30 rpm. See
FIG. 14 . - NS100—industrial S. cerevisiae strain with pNC67 (hygR, natR)
- NS101—industrial S. cerevisiae strain with pNC93 (hygR, cah)
- NS111—S. cerevisiae NRRL Y-2223 with pNC93 (hygR, cah)
- NS112—S. cerevisiae NRRL Y-2223 with pNC67 (hygR, natR)
- See
FIG. 16 . - Optical density of NS100 and NS101 grown in defined medium with different nitrogen sources. NS100 and NS101 were grown overnight in YPD medium, washed once in an equal volume of sterile water, and inoculated at 3.33% v/v. Strain NS101 is able to grow to an optical density with cyanamide comparable to that with urea, while NS100 grows to an optical density comparable to that with no nitrogen present in the medium. Data are averages of 3 replicate wells in a 96 well plate; 150 μL per well. 30° C., YNB medium contained 20 g/L glucose, 1.7 g/L YNB base medium without amino acids or ammonium sulfate, 5 g/L sodium sulfate, 100 μg/mL hygromycin, and either 10 mM urea, 10 mM cyanamide, or no nitrogen source. Inoculation was with 5 μL of culture pregrown for 24 hrs in the same medium with urea as nitrogen source. See
FIG. 17 . - Additionally, strains NS100, NS101, NS111, and NS112 were grown in defined YNB medium with 10 mM urea and 100 μg/mL hygromycin to stationary phase aerobically at 30° C. 1/1000 v/v inoculations were then made into the same defined medium with either 10 mM urea, 10 mM cyanamide, or no additional nitrogen and grown under the same conditions. Optical density was measured after 72 hours, as shown in
FIG. 22 . Strains NS101 and NS111, two different S. cerevisiae strains carrying the cah gene, were able to grow to an optical density comparable to that with urea; however, NS100 and NS112 only were able to grow to an optical density equal to or lower than in media with no nitrogen source. This shows that multiple S. cerevisiae strains are able to utilize cyanamide in the presence of the cah gene. - Strains NS100 (hygR, natR) and NS101 (hygR, cah) were grown in defined medium with 100 μg/mL hygromycin with urea as nitrogen source, and then both inoculated into defined medium containing either 10 mM urea or 10 mM cyanamide as nitrogen source. Upon growth to stationary phase, 1/100 v/v serial transfers were made to fresh medium with the same composition. The culture population was monitored via counting the number of hygR, natR colony forming units and subtracting from the number of hygR colony forming units. See
FIG. 18 andFIG. 19 for one experiment in defined minimal medium. A second experiment is shown inFIG. 21 . The second experiment included both defined minimal (YNB) and defined complex (YNB+SC amino acids) medium compositions. The defined YNB medium contained 20 g/L glucose, 1.7 g/L YNB base medium without amino acids or ammonium sulfate, 5 g/L sodium sulfate, and either 10 mM urea, 10 mM cyanamide, or no nitrogen source. Medium compositions are additionally given inFIGS. 32 and 33 . Growth occurred aerobically at 30° C. Colony forming units were counted by serial dilutions in YPD media with either 300 μg/mL hygromycin or 100 μg/mL nourseothricin, and are the average of 3 dilution counts. SeeFIG. 18 andFIG. 19 . - Optical density of NS100 and NS101 grown in rich YPD medium with 100 μg/mL hydgromycin and with and without 10 mM cyanamide. NS100 and NS101 were grown overnight in YNB medium, and inoculated at 3.33% v/v. NS101 experiences a shorter lag phase than NS100 in the presence of 10 mM cyanamide. Thus, cyanamide, in addition to functioning as a sole source of nitrogen, can also act as a deterrent for microbial growth. Data are averages of 3 replicate wells in a 96 well plate; 150 μL per well. 30° C., YPD medium or YPD medium with 10 mM cyanamide. Inoculation was with 5 μL of culture pregrown for 24 hrs in the YNB medium with urea as nitrogen source.
- See
FIG. 20 . - This assay measured the conversion rate of cyanamide to urea. In the first step, cyanamide was hydrated to urea by cyanamide hydratase, which was detected in cell free extract of a S. cerevisiae strain expressing the cah gene and a control strain without cah. In the second step of the assay, a commercial kit (Megazyme, Ireland) was used to detect urea via enzymatic conversion of urea to ammonia followed by NADPH linked conversion of ammonia and 2-oxoglutarate to NADP+, H2O, and glutamic acid.
- Cell free extracts were prepared by growing S. cerevisiae strains in 50 mL yeast extract, peptone, dextrose (YPD) medium with 300 μg/mL hygromycin to an optical density between 1-2. Cells were harvested by centrifugation, washed once in an equal volume of water, and re-suspended in Y-PER lysis buffer (Thermo Scientific, USA) following the manufacturer's instructions. After incubation at room temperature for 20 minutes, the lysate was centrifuged at 14,000×g for 10 mM and the supernatant was recovered as the cell free extract. Total protein was measured by a Nanodrop spectrophotometer (Thermo Scientific, USA).
- Add together in a 100 μL volume:
- 10 μL of 50 mM NaPO4, pH 7.7;
- 10 μL of 200 mM cyanamide made fresh
- 5-20 μL cell free extract
- balance water (60 μL for 20 μL CFE)
- add 100 uL of above sample to 2.9 mL Megazyme urea/ammonia assay reagents and monitor at 340 nm.
-
Cyanamide hydratase activity μmol Standard Strain Genotype mg−1 min−1 Deviation NS100 hygR natR 0.019 0.001 N5101 hygR cah 0.073 0.002 -
Sequence 1 is the DNA sequence of the allophanate hydrolase atzF gene in - Pseudomonas sp. strain ADP.
-
Sequence 2 is the DNA sequence of allophanate hydrolase DUR1,2 gene in S. cerevisiae. -
Sequence 3 is the DNA sequence of allophanate hydrolase YALI0E07271g gene in Y. lipolytica CLIB122. -
Sequence 4 is the DNA sequence of the biuret amidohydrolase atzE gene in Pseudomonas sp. strain ADP. -
Sequence 5 is the DNA sequence of the cyanuric acid amidohydrolase atzD gene in Pseudomonas sp. strain ADP. -
Sequence 6 is the DNA sequence of the cyanuric acid amidohydrolase trzD gene in Pseudomonas sp. strain NRRLB-12227 (formerly Acidovorax citrulli). -
Sequence 7 is the DNA sequence of the cyanuric acid amidohydrolase atzD trzD gene in Rhodococcus sp. Mel. - Sequence 8 is the DNA sequence of the guanine deaminase guaD gene in E. coli K12 strain MG1566.
- Sequence 9 is the DNA sequence of the guanine deaminase blr3880 gene in Bradyrhizobium japonicum USDA 110.
-
Sequence 10 is the DNA sequence of the guanine deaminase GUD1/YDL238C gene in S. cerevisiae. - Sequence 11 is the DNA sequence of the guanine deaminase YALI0E25740p gene in Y. lipolytica CLIB122.
- Sequence 12 is the DNA sequence of the melamine deaminase trzA gene in Williamsia sp. NRRL B-15444R (formerly R. corallinus).
-
Sequence 13 is the DNA sequence of the melamine deaminase triA gene in Pseudomonas sp. strain NRRL B-12227 (formerly Acidovorax citrulli). -
Sequence 14 is the DNA sequence of the isopropylammelide isopropylaminohydrolase atzC gene in Pseudomonas sp. strain ADP. -
Sequence 15 is the cDNA sequence of the Myrothecium verrucaria cyanamide hydratase (cah) gene. - Sequences 16-21 are DNA sequences of the invention.
- Sequences 22-37 are the sequences of various cyanamide hydratase (cah) genes for use in the invention.
-
Sequences 38 and 39 are the sequences of various trzC genes for use in the invention. -
Sequences -
Sequence 42 is the sequence of plasmid pNC10. - Sequence 43 is the sequence of plasmid pNC53.
-
Sequence 44 is the sequence of plasmid pNC67. -
Sequence 45 is the sequence of plasmid pNC85. - Sequence 46 is the sequence of plasmid pNC86.
-
Sequence 47 is the sequence of plasmid pNC87. -
Sequence 48 is the sequence of plasmid pNC93. -
Sequence 49 is the sequence of plasmid pNC96. -
Sequence 50 is the sequence of plasmid pNC97. - Sequence 51 is the sequence of plasmid pNC101.
-
Sequence 52 is the sequence of plasmid pNC120. - Sequence 53 is the sequence of plasmid pNC121.
- All of the U.S. patents and U.S. published patent applications cited herein are hereby incorporated by reference.
- Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Claims (7)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/349,559 US20210380996A1 (en) | 2013-01-04 | 2021-06-16 | Microorganisms engineered to use unconventional sources of nitrogen |
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201361748901P | 2013-01-04 | 2013-01-04 | |
US201361782351P | 2013-03-14 | 2013-03-14 | |
PCT/US2014/010332 WO2014107660A2 (en) | 2013-01-04 | 2014-01-06 | Microorganisms engineered to use unconventional sources of nitrogen |
US201514759878A | 2015-07-08 | 2015-07-08 | |
US15/679,312 US10316323B2 (en) | 2013-01-04 | 2017-08-17 | Microorganisms engineered to use unconventional sources of nitrogen |
US16/392,322 US11041162B2 (en) | 2013-01-04 | 2019-04-23 | Microorganisms engineered to use unconventional sources of nitrogen |
US17/349,559 US20210380996A1 (en) | 2013-01-04 | 2021-06-16 | Microorganisms engineered to use unconventional sources of nitrogen |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/392,322 Continuation US11041162B2 (en) | 2013-01-04 | 2019-04-23 | Microorganisms engineered to use unconventional sources of nitrogen |
Publications (1)
Publication Number | Publication Date |
---|---|
US20210380996A1 true US20210380996A1 (en) | 2021-12-09 |
Family
ID=51062558
Family Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/759,878 Active 2034-02-24 US9765348B2 (en) | 2013-01-04 | 2014-01-06 | Microorganisms engineered to use unconventional sources of nitrogen |
US15/679,312 Active US10316323B2 (en) | 2013-01-04 | 2017-08-17 | Microorganisms engineered to use unconventional sources of nitrogen |
US16/392,322 Active US11041162B2 (en) | 2013-01-04 | 2019-04-23 | Microorganisms engineered to use unconventional sources of nitrogen |
US17/349,559 Pending US20210380996A1 (en) | 2013-01-04 | 2021-06-16 | Microorganisms engineered to use unconventional sources of nitrogen |
Family Applications Before (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/759,878 Active 2034-02-24 US9765348B2 (en) | 2013-01-04 | 2014-01-06 | Microorganisms engineered to use unconventional sources of nitrogen |
US15/679,312 Active US10316323B2 (en) | 2013-01-04 | 2017-08-17 | Microorganisms engineered to use unconventional sources of nitrogen |
US16/392,322 Active US11041162B2 (en) | 2013-01-04 | 2019-04-23 | Microorganisms engineered to use unconventional sources of nitrogen |
Country Status (6)
Country | Link |
---|---|
US (4) | US9765348B2 (en) |
EP (1) | EP2941481A4 (en) |
CN (1) | CN105264061A (en) |
AU (1) | AU2014203893B2 (en) |
CA (2) | CA2895838C (en) |
WO (1) | WO2014107660A2 (en) |
Families Citing this family (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2941481A4 (en) | 2013-01-04 | 2016-11-02 | Novogy Inc | Microorganisms engineered to use unconventional sources of nitrogen |
EP3129479A4 (en) * | 2014-04-08 | 2017-10-25 | Novogy Inc. | Selective advantage in fermentation |
EP3247200A4 (en) | 2015-01-21 | 2018-06-13 | Basf Se | Plants having increased tolerance to herbicides |
JP2018515121A (en) * | 2015-05-21 | 2018-06-14 | 天津科技大学 | One kind of polylactic acid monomer producing bacteria, its construction method and lactic acid production method |
CN105705630B (en) * | 2015-05-21 | 2018-06-12 | 天津科技大学 | A kind of polymer grade lactic acid monomer production bacterium and its construction method and lactic acid manufacturing technology |
US11208649B2 (en) | 2015-12-07 | 2021-12-28 | Zymergen Inc. | HTP genomic engineering platform |
US9988624B2 (en) | 2015-12-07 | 2018-06-05 | Zymergen Inc. | Microbial strain improvement by a HTP genomic engineering platform |
CA3030803A1 (en) * | 2016-07-15 | 2018-01-18 | Basf Se | Plants having increased tolerance to herbicides |
US10138489B2 (en) | 2016-10-20 | 2018-11-27 | Algenol Biotech LLC | Cyanobacterial strains capable of utilizing phosphite |
BR112020013929A2 (en) * | 2018-01-17 | 2020-12-01 | Basf Se | plants or parts of the plant, seed, plant cells, plant product, progeny or descending plant, methods for controlling weeds, for producing a plant and for producing a descending plant, nucleic acid molecule, expression cassette, vector, polypeptide, method to produce a plant product and plant product |
EP3891268A4 (en) * | 2018-12-04 | 2022-08-03 | Nanyang Technological University | Genetically engineered cyanobacteria for growth in unsterilized conditions using antibiotic-free selection |
US20220389465A1 (en) * | 2019-11-27 | 2022-12-08 | Regents Of The University Of Minnesota | Methods of reducing biuret in urea compositions |
US11812753B2 (en) | 2020-07-22 | 2023-11-14 | Winfield Solutions, Llc | Solvent compositions promoting plant growth |
CN113980820A (en) * | 2021-11-04 | 2022-01-28 | 锬酃藏虫草生物科技(深圳)有限公司 | Method for culturing cordycepin and adenosine contents in cordyceps militaris |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6096947A (en) * | 1997-01-14 | 2000-08-01 | Pioneer Hi-Bred International, Inc. | Methods for improving transformation efficiency |
UY24959A1 (en) * | 1997-04-18 | 1998-10-14 | Mogen Int Nv | CYANAMIDE HYDRATASE ENCODER GENE AS A SELECTION MARKER FOR PLANTS |
DE02771969T1 (en) * | 2001-11-06 | 2005-02-10 | The University Of British Columbia, Vancouver | MODULATION OF UREA REMOVAL IN WINE YEAST |
EP2276830A4 (en) | 2008-04-14 | 2013-03-20 | Univ British Columbia | Functional enhancement of yeast to minimize production of ethyl carbamate via modified transporter expression |
WO2009155917A2 (en) | 2008-06-26 | 2009-12-30 | Pp Energy Aps | Device for enabling access to a wind turbine |
US8637269B2 (en) * | 2009-07-10 | 2014-01-28 | Bioo Scientific Corporation | Method for the detection of melamine |
US8367389B2 (en) * | 2009-09-11 | 2013-02-05 | Regents Of The University Of Minnesota | Methods, compositions and devices utilizing structurally stable cyanuric acid hydrolase |
EP2941481A4 (en) | 2013-01-04 | 2016-11-02 | Novogy Inc | Microorganisms engineered to use unconventional sources of nitrogen |
-
2014
- 2014-01-06 EP EP14735413.8A patent/EP2941481A4/en not_active Withdrawn
- 2014-01-06 CA CA2895838A patent/CA2895838C/en active Active
- 2014-01-06 WO PCT/US2014/010332 patent/WO2014107660A2/en active Application Filing
- 2014-01-06 AU AU2014203893A patent/AU2014203893B2/en active Active
- 2014-01-06 CN CN201480012382.8A patent/CN105264061A/en active Pending
- 2014-01-06 CA CA3109035A patent/CA3109035A1/en not_active Abandoned
- 2014-01-06 US US14/759,878 patent/US9765348B2/en active Active
-
2017
- 2017-08-17 US US15/679,312 patent/US10316323B2/en active Active
-
2019
- 2019-04-23 US US16/392,322 patent/US11041162B2/en active Active
-
2021
- 2021-06-16 US US17/349,559 patent/US20210380996A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
US10316323B2 (en) | 2019-06-11 |
WO2014107660A2 (en) | 2014-07-10 |
WO2014107660A3 (en) | 2014-08-28 |
AU2014203893B2 (en) | 2017-11-02 |
US20160115492A1 (en) | 2016-04-28 |
CA2895838C (en) | 2021-03-30 |
US11041162B2 (en) | 2021-06-22 |
EP2941481A2 (en) | 2015-11-11 |
CN105264061A (en) | 2016-01-20 |
US20180051294A1 (en) | 2018-02-22 |
EP2941481A4 (en) | 2016-11-02 |
US20190316140A1 (en) | 2019-10-17 |
CA2895838A1 (en) | 2014-07-10 |
AU2014203893A1 (en) | 2015-07-09 |
CA3109035A1 (en) | 2014-07-10 |
US9765348B2 (en) | 2017-09-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20210380996A1 (en) | Microorganisms engineered to use unconventional sources of nitrogen | |
US11535829B2 (en) | Microorganisms engineered to use unconventional sources of phosphorous or sulfur | |
US20210155411A1 (en) | Yeast cells having disrupted pathway from dihydroxyacetone phosphate to glycerol | |
US11492647B2 (en) | Increasing lipid production in oleaginous yeast | |
US20200109377A1 (en) | Increasing Cellular Lipid Production by Increasing the Activity of Diacylglycerol Acyltransferase and Decreasing the Activity of Triacylglycerol Lipase | |
US10760105B2 (en) | Enhanced production of core lipids in oleaginous yeasts | |
AU2015243881B2 (en) | Selective advantage in fermentation | |
US10443047B2 (en) | Increasing cellular lipid production by increasing the activity of diacylglycerol acyltransferase and decreasing the activity of triacylglycerol lipase | |
EP3356550B1 (en) | A system for improved production titers in fermentations | |
US20210230573A1 (en) | Microorganisms and the production of fine chemicals | |
CN112680389A (en) | Method for improving tolerance and utilization rate of microorganisms to methanol |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
AS | Assignment |
Owner name: NOVOGY, INC., MASSACHUSETTS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SOUTH, COLIN R.;SHAW, ARTHUR J., IV;REEL/FRAME:066420/0412 Effective date: 20140702 Owner name: GINKGO BIOWORKS, INC., MASSACHUSETTS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:NOVOGY, INC.;REEL/FRAME:066420/0434 Effective date: 20201016 |