WO2020075986A2 - 알코올 생성이 억제된 재조합 내산성 효모 및 이를 이용한 젖산의 제조방법 - Google Patents
알코올 생성이 억제된 재조합 내산성 효모 및 이를 이용한 젖산의 제조방법 Download PDFInfo
- Publication number
- WO2020075986A2 WO2020075986A2 PCT/KR2019/012326 KR2019012326W WO2020075986A2 WO 2020075986 A2 WO2020075986 A2 WO 2020075986A2 KR 2019012326 W KR2019012326 W KR 2019012326W WO 2020075986 A2 WO2020075986 A2 WO 2020075986A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- gene
- strain
- seq
- lactic acid
- acid
- Prior art date
Links
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 170
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 116
- 239000004310 lactic acid Substances 0.000 title claims abstract description 85
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 85
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 65
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 46
- 239000002253 acid Substances 0.000 title claims abstract description 29
- 108090000623 proteins and genes Proteins 0.000 claims description 157
- 108010011939 Pyruvate Decarboxylase Proteins 0.000 claims description 49
- 230000000694 effects Effects 0.000 claims description 33
- 102000004169 proteins and genes Human genes 0.000 claims description 15
- 102000007698 Alcohol dehydrogenase Human genes 0.000 claims description 14
- 108010021809 Alcohol dehydrogenase Proteins 0.000 claims description 14
- 108700023483 L-lactate dehydrogenases Proteins 0.000 claims description 10
- 230000001105 regulatory effect Effects 0.000 claims description 10
- 102000003855 L-lactate dehydrogenase Human genes 0.000 claims description 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 8
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 5
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 4
- 238000012217 deletion Methods 0.000 claims description 4
- 230000037430 deletion Effects 0.000 claims description 4
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 abstract description 11
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 abstract description 6
- 238000006243 chemical reaction Methods 0.000 abstract description 6
- 230000037361 pathway Effects 0.000 abstract 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 41
- 238000000034 method Methods 0.000 description 28
- 238000000855 fermentation Methods 0.000 description 21
- 230000004151 fermentation Effects 0.000 description 21
- 108020004414 DNA Proteins 0.000 description 19
- 101150104734 ldh gene Proteins 0.000 description 18
- 239000013598 vector Substances 0.000 description 18
- 101150034686 PDC gene Proteins 0.000 description 17
- 239000002609 medium Substances 0.000 description 17
- 239000000243 solution Substances 0.000 description 17
- 230000012010 growth Effects 0.000 description 15
- 230000014509 gene expression Effects 0.000 description 14
- 230000001965 increasing effect Effects 0.000 description 14
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 13
- 239000008103 glucose Substances 0.000 description 13
- 102000004190 Enzymes Human genes 0.000 description 11
- 108090000790 Enzymes Proteins 0.000 description 11
- 230000008569 process Effects 0.000 description 10
- 229940076788 pyruvate Drugs 0.000 description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 8
- 150000003839 salts Chemical class 0.000 description 8
- 108700028369 Alleles Proteins 0.000 description 7
- JJTUDXZGHPGLLC-UHFFFAOYSA-N lactide Chemical compound CC1OC(=O)C(C)OC1=O JJTUDXZGHPGLLC-UHFFFAOYSA-N 0.000 description 7
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 6
- 238000010353 genetic engineering Methods 0.000 description 6
- 244000005700 microbiome Species 0.000 description 6
- 229920000642 polymer Polymers 0.000 description 6
- 108020005345 3' Untranslated Regions Proteins 0.000 description 5
- 101150050255 PDC1 gene Proteins 0.000 description 5
- 239000001888 Peptone Substances 0.000 description 5
- 108010080698 Peptones Proteins 0.000 description 5
- 229940041514 candida albicans extract Drugs 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 230000007935 neutral effect Effects 0.000 description 5
- 235000019319 peptone Nutrition 0.000 description 5
- 108091008146 restriction endonucleases Proteins 0.000 description 5
- 239000012138 yeast extract Substances 0.000 description 5
- 101100351264 Candida albicans (strain SC5314 / ATCC MYA-2876) PDC11 gene Proteins 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 4
- 230000003115 biocidal effect Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 230000003472 neutralizing effect Effects 0.000 description 4
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 108020003589 5' Untranslated Regions Proteins 0.000 description 3
- 108091026890 Coding region Proteins 0.000 description 3
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 101100082596 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) PDC5 gene Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 101150067366 adh gene Proteins 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 235000010216 calcium carbonate Nutrition 0.000 description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 230000002503 metabolic effect Effects 0.000 description 3
- 238000006386 neutralization reaction Methods 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 239000013600 plasmid vector Substances 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000012552 review Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000002194 synthesizing effect Effects 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 241000186660 Lactobacillus Species 0.000 description 2
- 240000006024 Lactobacillus plantarum Species 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 229910052925 anhydrite Inorganic materials 0.000 description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 239000004621 biodegradable polymer Substances 0.000 description 2
- 229920002988 biodegradable polymer Polymers 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000034659 glycolysis Effects 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 239000013067 intermediate product Substances 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 229940039696 lactobacillus Drugs 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000005057 refrigeration Methods 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 229960002363 thiamine pyrophosphate Drugs 0.000 description 2
- 235000008170 thiamine pyrophosphate Nutrition 0.000 description 2
- 239000011678 thiamine pyrophosphate Substances 0.000 description 2
- YXVCLPJQTZXJLH-UHFFFAOYSA-N thiamine(1+) diphosphate chloride Chemical compound [Cl-].CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N YXVCLPJQTZXJLH-UHFFFAOYSA-N 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000003260 vortexing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- 238000010146 3D printing Methods 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108090000489 Carboxy-Lyases Proteins 0.000 description 1
- 102000004031 Carboxy-Lyases Human genes 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 208000009889 Herpes Simplex Diseases 0.000 description 1
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 1
- 101100536883 Legionella pneumophila subsp. pneumophila (strain Philadelphia 1 / ATCC 33152 / DSM 7513) thi5 gene Proteins 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- 229920000426 Microplastic Polymers 0.000 description 1
- 101150110652 PDC2 gene Proteins 0.000 description 1
- 208000034530 PLAA-associated neurodevelopmental disease Diseases 0.000 description 1
- 101150116328 Pcd gene Proteins 0.000 description 1
- 101100152887 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) THI3 gene Proteins 0.000 description 1
- 101100240664 Schizosaccharomyces pombe (strain 972 / ATCC 24843) nmt1 gene Proteins 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- IKHGUXGNUITLKF-XPULMUKRSA-N acetaldehyde Chemical compound [14CH]([14CH3])=O IKHGUXGNUITLKF-XPULMUKRSA-N 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000010363 gene targeting Methods 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- -1 lactic acid) Chemical class 0.000 description 1
- 229940072205 lactobacillus plantarum Drugs 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229950006238 nadide Drugs 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 235000006180 nutrition needs Nutrition 0.000 description 1
- 238000005580 one pot reaction Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 150000003138 primary alcohols Chemical class 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002250 progressing effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000007142 ring opening reaction Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 238000007039 two-step reaction Methods 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 239000007222 ypd medium 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
-
- 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
- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/165—Yeast isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/56—Lactic acid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01001—Alcohol dehydrogenase (1.1.1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01027—L-Lactate dehydrogenase (1.1.1.27)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
- C12Y401/01001—Pyruvate decarboxylase (4.1.1.1)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Definitions
- the present invention relates to a method for producing lactic acid using an acid-resistant yeast in which ethanol production is suppressed, more specifically, lactic acid production ability is imparted, conversion of pyruvate to acetaldehyde is suppressed, and as a result, acid resistance is suppressed. It relates to a method for producing yeast and lactic acid using the same.
- PLA Polylacic Acid
- PLA is a biodegradable polymer made by converting lactic acid into lactide and ring-opening and polymerizing it, and its raw material, lactic acid, is produced through fermentation.
- PLA can be widely used in disposable food containers, and has the strength that can be used alone or in the form of a composition or copolymer as a variety of industrial plastics including the automobile industry.
- it is a representative polymer that is also used in 3D printing in recent years. It is an eco-friendly polymer that generates less harmful gases and less odor when using 3D printers.
- biodegradable polymers are promising polymers that can solve the reality that environmental destruction is accelerating with waste plastics and micro plastics, which have become global problems in recent years, and advanced countries are pushing for expansion, and in order to produce PLA at a lower cost, Efforts are being made to improve the productivity of the monomer lactic acid.
- the traditional lactic acid production process is produced using lactic acid bacteria, and various types of Ca salts / Mg salts or neutralizing agents such as ammonia are used to prevent the growth of lactic acid produced by lactic acid bacteria or to stop the growth of bacteria.
- pH a neutral pH of 6 to 8
- fermentation proceeds.
- microorganisms are separated.
- sulfuric acid is added to convert lactate to lactic acid, and Ca salt is removed in the form of CaSO4. This process produces a larger amount of by-product CaSO4 than lactic acid and degrades process economy.
- lactic acid has L- and D-type optical isomers. Even in the case of lactic acid bacteria mainly producing L-type, about 5 to 10% of D-type are often produced together, and in the case of strains mainly producing D-type, D and L-type and D and ethanol There are many groups of microorganisms that have many varieties, such as being in the form of producing (Ellen I. Garvie, Microbiological Reviews , 106-139, 1980).
- D-form was mainly used for medical / drug delivery, but when applied to PLA, the crystallization rate by D-type lactide increased, and a phenomenon in which thermal properties were improved was found. Also, pure L-type polymer and pure water When stereocomplex PLA is structurally formed according to the processing conditions in which D-type polymer is mixed, the crystallinity caused by D-type is increased and the physical properties of the PLA are enhanced by increasing the heat resistance of existing PLA and PE / PP. Research and commercialization of methods are progressing rapidly and the field of application of PLA is expanding.
- PLA is a process in which lactic acid is produced through fermentation and then converted to lactide through a purification process.
- a process of converting lactic acid into a hydrogenated form is necessary, and since the pH for normal neutral fermentation is 6-7, it is converted to acidic pH using a large amount of sulfuric acid.
- a large amount of neutral salts are generated, and the economic efficiency is lowered due to the low value of the neutral salts along with the process investment cost for removing these neutral salts.
- acid-resistant yeast When lactic acid is produced using acid-grown yeast (hereinafter referred to as acid-resistant yeast), the fermentation process is simplified because it is not necessary to maintain the medium at a pH of 6 to 7 using a neutralizing agent during fermentation, and also removes a trailing neutralizing agent. No purification process is required. In addition, since yeast makes many components necessary for metabolism by itself, it can be cultured even in a medium having a relatively low nutritional level compared to bacteria, especially Lactobacillus, and thus a large number of post-purification processes can be omitted, thereby significantly reducing production cost.
- acid-resistant yeast which can reduce the cost of the process, must be able to finish fermentation with a pH of the fermentation broth below pKa value without using a neutralizing agent or in a minimal amount, and the meaning of commercial application only when the three major indicators of fermentation achieve similar levels to lactic acid bacteria There is.
- yeast when fermenting glucose, yeast metabolizes ethanol as a main product, and rarely produces lactic acid.
- a yeast strain having excellent acid resistance was selected, and the selected strain was made to have lactic acid production capacity by genetic engineering method.
- all ethanol-producing strains were selected.
- the metabolic circuit of lactic acid production consists of a one-step reaction in pyruvate, which is generated by the lactate dehydrogenase enzyme and then released out of the cell by active / diffusion through transport.
- pyruvate which is generated by the lactate dehydrogenase enzyme and then released out of the cell by active / diffusion through transport.
- the yeast proceeds in a two-step reaction that converts from pyruvate to ethanol, and a method of removing the PDC gene converting pyruvate to acetaldehyde and introducing LDH has been attempted.
- the present inventors tried to improve the ability of lactic acid production in acid-resistant yeast, and as a result of diligent efforts, the gene encoding lactate dehydrogenase was deleted while deleting the alcohol dehydrogenase enzyme from the acid-resistant yeast, thereby improving the ability to produce lactic acid.
- a gene encoding pyruvate decarboxylase was deleted, a recombinant strain in which a gene encoding lactate dehydrogenase was additionally introduced was prepared, and lactic acid was prepared using the recombinant strain.
- Another object of the present invention is to provide a method for producing lactic acid using the recombinant acid-resistant yeast.
- Another object of the present invention is to provide a gene having pyruvate decarboxylase activity derived from the acid-resistant yeast.
- the present invention is a gene encoding a pyruvate decarboxylase in the acid-resistant yeast YBC strain (KCTC13508BP) is deleted or weakened, and a lactate dehydrogenase-encoding gene is introduced. It provides a recombinant strain having the ability to produce.
- the present invention also, (a) culturing the recombinant strain to produce lactic acid; And (b) obtaining the produced lactic acid.
- the present invention also provides a gene encoding a protein having a pyruvate decarboxylase activity and having an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4.
- the present invention also provides a protein having an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4, which has pyruvate decarboxylase activity.
- the present invention also provides a captive motor of the g3002 gene having the nucleotide sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6.
- Figure 1 shows an example of a cassette (cassette) structure for removing the target gene from the YBC strain, (a) and (b) for the cassette to introduce LDH while removing the ORF of the target gene targeting g4423 It shows the case where two types of selection markers are used, and (c) shows an example of a cassette for removing the target gene.
- Figure 2 shows the results of confirming the PDC activity of the recombinant strains ⁇ g460, ⁇ g3002-1, ⁇ g3002-2 and ⁇ g6004 strains in which the PDC gene candidate is knocked out from the YBC strain.
- Figure 3 is a comparison of the growth, ethanol production yield, sugar consumption rate and ethanol productivity of the recombinant strains ⁇ g3002-1, ⁇ g3002-2 in which the PDC gene candidate is knocked out in the YBC strain.
- Figure 4 shows the growth curve (A) and ethanol production capacity (B) of the recombinant strains ⁇ g460, ⁇ g3002-2 and ⁇ g6004 strains in which the PDC gene candidate is knocked out from the YBC strain.
- Figure 5 shows the lactic acid production yield (A), ethanol production yield (B) and lactic acid productivity (C) of the recombinant strains YBC1, YBC2 and YBC3 under pH 3 Flask culture conditions.
- Figure 6 shows the lactic acid production yield (A), ethanol production yield (B) and lactic acid productivity (C) of the recombinant strains YBC1, YBC2 and YBC3 under pH 4 Flask culture conditions.
- Figure 7 shows the glucose consumption (A) and lactic acid production capacity (B) of the recombinant strains YBC1 and YBC2 in the fermenter.
- Figure 8 shows the glucose consumption and lactic acid production capacity of the recombinant strain YBC2 after optimizing the culture conditions in the fermenter.
- Acid-resistant yeast rapidly consumes sugar even at an acidic pH and exhibits a high growth rate, and has the characteristic of converting the consumed sugar to metabolites under fermentation conditions.
- yeasts having these characteristics were selected through several yeast libraries, and the selected strains showed high growth and sugar consumption rate even under conditions of lactic acid concentration of 40 g / L to 80 g / L.
- the selected strains were subjected to metabolic circuit regulation using genetic engineering.
- a recombinant strain was prepared by additionally deleting the PDC gene and additionally introducing a gene encoding lactate dehydrogenase, thereby minimizing the toxicity of the intermediate product while minimizing the cytotoxicity of Cytosolic Acetyl-CoA
- a strain was developed without further affecting the supply of ethanol, while further reducing the ethanol production capacity and further improving the milk production capacity.
- the gene encoding pyruvate decarboxylase is deleted or weakened in the acid-resistant yeast YBC strain (KCTC13508BP), and the ability to generate lactic acid is introduced with a gene encoding lactate dehydrogenase. It relates to a recombinant strain having.
- the gene encoding the pyruvate decarboxylase may be characterized as a g3002 gene.
- the g3002 gene which is the gene having the greatest decrease in PDC activity when deleted in the YBC strain, was selected as the main PDC gene (main PDC gene).
- the g3002 gene is a gene of a very unique structure in which ORFs exist in two different places in the genome of the YBC strain, a gene in the scaffold 27 position and the scaffold 72 position in the genome sequence. , And the ORFs before and after on the genome are different genes and are separate independent genes.
- the two g3002 genes have a homology of 98.46% gene level to each other, and the promoter portion of the front end of the two genes has very different sequences, and it is estimated that expression is regulated by different mechanisms. It was estimated that one of the two genes would act as the primary PDC gene.
- the g3002 gene (hereinafter referred to as g3002-1 gene) located at 72 scaffold of the YBC1 strain is removed, and the recombinant strain YBC2 introducing the LDH gene and the g3002 gene located at 27 scaffold again from the recombinant strain YBC2 (hereinafter g3002- 2) (referred to as gene 2), while removing the LDH gene to prepare a recombinant strain YBC3, and culturing the recombinant strains, it was confirmed that lactic acid and ethanol production capacity and lactic acid productivity is improved.
- the g3002 gene may be characterized by being a gene at the scaffold 27 (g3002-2) position and the scaffold 72 (g3002-1) position on the genomic sequence of the YBC strain (KCTC13508BP), It can be characterized in that the g3002-1 gene at the scaffold 72 position is deleted or weakened.
- the recombinant strain may be characterized in that only one or both of the g3002-1 gene at the scaffold 72 position and the g3002-2 gene at the scaffold 27 position is deleted.
- the g3002-1 gene may be characterized in that it is a gene encoding the amino acid sequence represented by SEQ ID NO: 3, the g3002-2 gene is a gene encoding the amino acid sequence represented by SEQ ID NO: 4 It can be characterized by.
- the gene encoding the lactate dehydrogenase may be introduced by substituting the g3002 gene and being regulated by the promoter of the g3002 gene.
- the sequence of the promoter region of each of g3002-1 and g3002-2 is shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
- the gene encoding the lactate dehydrogenase is preferably L. helveticus- derived LDH gene, R. oryzae- derived LDH gene or L. plantarum- derived LDH gene, and more preferably L. plantarum- derived It is preferred that it is an LDH gene.
- the recombinant strain may be characterized in that the gene encoding the alcohol dehydrogenase (ADH gene) is further deleted, and the gene encoding the alcohol dehydrogenase is the g4423 gene. It can be characterized as.
- the recombinant strain may be characterized in that the LDH gene is additionally introduced in place of the ADH gene.
- the ability to produce ethanol may be reduced compared to the YBC strain (KCTC13508BP), which is the parent strain, by deletion or attenuation of the g3002 gene.
- the present invention in another aspect, (a) culturing the recombinant strain to produce lactic acid; And (b) obtaining the produced lactic acid.
- the present invention relates to a gene encoding a protein having pyruvate decarboxylase activity and having 90% homology with the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.
- the present invention relates to a gene encoding a protein having a pyruvate decarboxylase activity and having an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4.
- the gene may be characterized by having a nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.
- the present invention relates to a protein having pyruvate decarboxylase activity and having an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4.
- the present invention relates to a captive motor of the g3002 gene having the nucleotide sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6.
- the term 'acid-resistant yeast' means that at a pH of less than the pKa value of the organic acid, when the medium contains 1M or more organic acid (particularly lactic acid), at least 10% bio It is defined as yeast that can maintain a mass consumption rate (such as sugar consumption rate) or a specific growth rate of at least 10%. More specifically, in the present invention, 'acid resistant yeast' is a yeast capable of maintaining a biomass consumption rate of at least 10% (such as sugar consumption rate) or a specific growth rate of at least 10% at pH 2 to 4, compared to a case of pH 5 or higher. define.
- the recombinant yeast according to the present invention can be prepared by inserting the gene on the chromosome of the host yeast or introducing a vector containing the gene into the host yeast according to a conventional method.
- the host yeast a host cell having high DNA introduction efficiency and high expression efficiency of the introduced DNA is commonly used, and in one embodiment of the present invention, acid-resistant yeast is used, but is not limited thereto, and the target DNA is sufficiently Any yeast that can be expressed may be used.
- the recombinant yeast can be prepared according to any transformation method.
- Transformation refers to a phenomenon in which DNA is introduced into a host to cause DNA to be cloned as a factor of a chromosome or by chromosomal integration, thereby introducing an external DNA into a cell and causing an artificial genetic change.
- the conversion method includes an electroporation method, a lithium acetate-PEG method, and the like.
- any commonly known genetic manipulation method can be used, and examples thereof include retrovirus vector, adenovirus vector, adeno-associated virus vector, and herpes simplex.
- Rex virus vectors, poxvirus vectors, lentiviral vectors, and non-viral vectors are used.
- Vector means a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host.
- the vector can be a plasmid, phage particle, or simply a potential genomic insert.
- the vector When transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Plasmids are currently the most commonly used form of vector, and linearized DNA is also the form commonly used for genomic integration of yeast.
- Typical plasmid vectors include (a) a replication initiation point for efficient replication to include a plasmid vector per host cell, (b) an antibiotic resistance gene or a nutritional demand marker allowing selection of host cells transformed with the plasmid vector. It has a structure that includes a restriction enzyme cleavage site that allows insertion of a gene (auxotrophic marker gene) and (c) a foreign DNA fragment. Even if a suitable restriction enzyme cleavage site does not exist, a vector and foreign DNA can be easily ligated using a synthetic oligonucleotide adapter or linker according to a conventional method (Gibson assembly) ), If necessary, a method of synthesizing and using the entire desired sequence is also commonly used.
- the gene is "operably linked" when placed in a functional relationship with other nucleic acid sequences.
- This can be a gene and regulatory sequence (s) linked in a manner that enables gene expression when an appropriate molecule (eg, a transcriptional activation protein) is coupled to the regulatory sequence (s).
- DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide;
- a promoter or enhancer is operably linked to a coding sequence when it affects transcription of the sequence;
- a ribosome binding site is operably linked to a coding sequence when it affects transcription of the sequence;
- the ribosome binding site is operably linked to the coding sequence when arranged to facilitate translation.
- operably linked means that the linked DNA sequences are in contact, and in the case of a secretory leader, they are in contact and are present in the reading frame. However, the enhancer does not need to be in contact. Linking these sequences is accomplished by ligation (linking) at convenient restriction enzyme sites. If such a site does not exist, a synthetic oligonucleotide adapter or linker according to a conventional method is used.
- the carbon source may be characterized by at least one selected from the group consisting of glucose, xylose, arabinose, sucrose, fructose, cellulose, galactose, glucose oligomer, and glycerol, but is not limited thereto.
- the culture may be performed in a condition that prevents microorganisms, such as E. coli, etc. from acting anymore (eg, impossible to produce metabolites).
- the culture may be characterized in that the pH is 1.0 to 6.5, preferably pH 1.0 to 6.0, more preferably 2.6 to 4.0, but is not limited thereto.
- Example 1 Confirming the expression rate of the gene encoding the pyruvate decarboxylase (PDC) in the YBC strain to identify the main expression gene
- the present inventors have selected a group of strains having acid resistance through tests on various yeast strains (Korean Patent Publication No. 2017-0025315).
- yeast fungus lactic acid was added to the medium at the beginning of the culture to check the growth and sugar consumption rate of microorganisms, and the strain having the best acid resistance was selected, and deposited as KCTC13508BP to the Korea Research Institute of Bioscience and Biotechnology Resource Center. have.
- KCTC13508BP is a strain similar to S. cerevisiae , has a diploid gene (Diploid), and has Crab-tree positive characteristics.
- G4136 Excluded as a gene attached to other PDC candidate genes.
- G5237 is 250 bp, so it is not possible to make the proper size of PDC.
- G460, g2550, g3002 and g6004 tentatively determined as primary PDC candidates.
- the g3002 gene was the closest to PDC1 at the annotation and the similarity was compared based on this.
- the g460 gene, the g3002 gene and the g6004 gene showed the highest similarity to the PDC1 gene of S. cerevisiae .
- genetic manipulation was performed to delete the target gene from the genome of the YBC strain.
- Example 2 Confirming the effect of lowering ethanol production by removing the target PDC gene from the YBC strain
- a recombinant strain knocking out the target PCD gene of the YBC strain identified in Example 1 was prepared, and the effect of PDC gene removal on the growth of the strain was confirmed.
- the ORF of each gene is removed and a gene cassette similar to Fig. 1 (c) with 5 'and 3' UTR and antibiotic markers is prepared to make Donor DNA It was used as.
- the 5'-UTR and 3'-UTR of the g460 gene are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively, and the 5'-UTR of the g3002 gene is shown in SEQ ID NO: 5 and SEQ ID NO: 6, and the sequence of 3'-UTR is shown. It is shown in No. 9 and SEQ ID No. 10.
- the 5'-UTR and 3'-UTR of the g6004 gene are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.
- g460 ORF inside-fwd CCAGACAATTGGTTGATATCACC (SEQ ID NO: 13)
- g6004 ORF inside-rev CATATCTTCGGACAGCTTAC (SEQ ID NO: 19)
- PDC activity was measured using the obtained ⁇ g460, ⁇ g3002 and ⁇ g6004 strains. PDC activity of the target strain was measured based on a well-known literature method (TC Hoppner, HW Doelle, European Journal of Applied Microbiology and Biotechnology , 17: 152-157, 1983).
- the solution required for activity measurement was prepared as follows.
- TPP Thiamine pyrophosphate
- Protein enzyme solution was prepared as follows.
- yeast cells are recovered by centrifugation.
- Vortexing was performed 5 times in total for 30 seconds, and the crushing solution was stored in ice for 1 minute between each vortexing to maintain a low temperature.
- Solution 1 was prepared prior to the PDC assay and maintained at room temperature and mixed with the following composition.
- PDC Activity Unit The definition of PDC Activity Unit is as follows.
- PDC activity of 1 unit is defined as an enzyme activity capable of oxidizing 1 ⁇ mol of NADH in 1 minute.
- the light path length is 0.6cm (96well plate, 0.2ml volume).
- PDC activity is defined as Unit / mg protein, and is calculated by dividing the measured total protein concentration.
- Unit / mg protein (Unit / ml enzyme) / (mg protein / ml enzyme)
- the obtained ⁇ g3002-1 strain and ⁇ g3002-2 strain were cultured at 150 ml in YP medium having a glucose concentration of 40 g / L and cultured at 30 ° C. and 200 rpm.
- the obtained ⁇ g460, ⁇ g3002-2 and ⁇ g6004 strains were cultured at 150 ml in YP medium with a glucose concentration of 40 g / L and cultured at 30 ° C. and 200 rpm.
- the g3002 gene is the main PDC (main PDC) gene in the YBC strain by synthesizing the results of cultivation and enzyme activity analysis of the strain in which each gene is deleted, and in particular, it was confirmed that the g3002-1 gene plays the main role. .
- Example 3 Production of lactic acid using recombinant strain in which PDC gene is removed and LDH is introduced
- the g3002-1 gene is the main PDC gene, but when the lactate dehydrogenase gene (LDH gene) is introduced for lactic acid production, the expression intensity of LDH is derived from the promoter at the front of the gene. LDH gene was introduced while removing the ORF of the target gene, so the effect on LDH expression was confirmed.
- LDH gene lactate dehydrogenase gene
- the target strain was a strain (YBC1) that introduced the LDH gene while removing the primary alcohol dehydrogenase (ADH) gene in the existing wild type rather than the wild type strain.
- Candidate genes of the LDH gene to be introduced into the YBC strain were selected through literature (N. Ishida et. Al., Appl. Environ. Micobiol ., 1964-1970, 2005; M. Sauer et al., Biotechnology and Genetic Engineering Reviews , 27: 1, 229-256, 2010). Finally, the LDH gene derived from L. plantarum represented by SEQ ID NO: 4 was selected and introduced.
- the YBC1 strain is a strain that removes the main ADH gene g4423 gene of the YBC strain and introduces the LDH gene of SEQ ID NO: 28 derived from Lactobacillus plantarum at the position g4423, based on the information of g4423 and their UTR.
- FIGS. 1 (a) and 1 (b) Gene cassettes of FIGS. 1 (a) and 1 (b) with the ORF of the gene removed and 5 ′ and 3 ′ UTR and antibiotic markers were prepared and used as Donor DNA.
- the corresponding 5 'UTR for each allele of g4423 is shown in SEQ ID NO: 29 and SEQ ID NO: 30, and the 3' UTR is shown in SEQ ID NO: 31 and SEQ ID NO: 32.
- cloning method using restriction enzyme, Gibson assembly, and method using gene synthesis were used for the production of donor DNA. After synthesizing and introducing LDH of SEQ ID NO: 28 into the ORF site of g4423, Donor DNA was prepared and introduced into YBC to produce recombinant strain YBC1.
- the g3002 gene is a gene of a very unique structure in which two ORFs are present in the genome of the YBC strain, and in the genomic sequencing results, the gene is located at the scaffold 27 and scaffold 72 positions.
- the two g3002 genes have a homology of 98.46% gene level to each other, but the promoter portion of the front end of the two genes has very different sequences, and it is estimated that the expression is regulated by different mechanisms. It was estimated that one of the two genes would act as the primary PDC gene.
- inoculation OD was 0.5
- medium was YP medium (20g / L Peptone, 10g / L Yeast extract) using Glucose 6%
- Glucose 9% and CaCO3 3% were used for the recombinant strains having an inoculation OD value of 2 and a medium of YP medium (20 g / L Peptone, 10 g / L Yeast extract), and cultured at 30 ° C and 150 rpm with 100 ml of Flask culture. .
- the notable fact in this example is for an increase in lactic acid yield compared to a decrease in ethanol yield.
- the ethanol yield was reduced from 0.093 g / g to 0.075 g / g, 0.018 g / g.
- the decrease in yields of other by-products such as glycerol and acetate was reflected as an increase in lactic acid yield.
- g3002 confirmed a large increase in lactic acid production capacity (0.59 g / L-> 0.67 g / L without pH adjustment), and the expression of LDH with a decrease in PDC activity. It was found that there was an increase in yield and performance.
- YBC1 strain is Hestrin and Schramme medium (Glucose 120g / L, Peptone 5g / L, Yeast extract 5g / L, citric acid 1.15 g / L, K2HPO4 2.7g / L, MgSO4 ⁇ 7H2O 1g / L), 1L volume
- Glucose 120g / L Peptone 5g / L
- Yeast extract 5g / L citric acid 1.15 g / L
- K2HPO4 2.7g / L citric acid 1.15 g / L
- K2HPO4 2.7g / L citric acid 1.15 g / L
- K2HPO4 2.7g / L citric acid 1.15 g / L
- K2HPO4 2.7g / L citric acid 1.15 g / L
- K2HPO4 2.7g / L citric acid 1.15 g / L
- the YBC2 strain was cultured in a 1 L volume using a Hestrin and Schramme medium at a sugar concentration of 120 g / L in a fermenter, the culture temperature was 30 ° C, and air was supplied at 0.1 vvm to 3.6% of CaCO3 and adjusted to pH 4 400 The rpm level was maintained.
- the cultivation optimization was mainly carried out mainly on the condition change for the initial OD and oxygen supply rate, and the two best results were shown in FIG. 8.
- the medium was cultured at a sugar concentration of 120 g / L in a fermenter in a 1 L volume in YP medium (20 g / L Peptone, 10 g / L Yeast extract) for the YBC2 strain.
- Incubation temperature is 30 °C and supplying air at 0.025 ⁇ 0.05 vvm, dividing 3.6% of CaCO 3 into 3 times and adding it to fermentation time 5 hours, 13 hours and 23 hours, adjusted to pH 4 and maintaining the level of 300 ⁇ 400 rpm Did.
- the medium was cultured at a sugar concentration of 120 g / L in a fermenter in a 1 L volume in YP medium (20 g / L Peptone, 10 g / L Yeast extract) for the YBC2 strain.
- the incubation temperature was 30 ° C, and while supplying air at 0.05 vvm, 3.6% of CaCO3 was added to adjust the pH to 4 to maintain 400 rpm.
- Recombinant acid-resistant yeast according to the present invention inhibits ethanol production, increases the pyruvate pool in cells, strongly expresses LDH enzyme, and can produce lactic acid with high yield.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Botany (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
Description
Claims (12)
- 내산성 효모 YBC 균주(KCTC13508BP)에서 피루베이트 디카르복실라아제를 코딩하는 유전자가 결실 또는 약화되고, 락테이트 디하이드로게나아제를 코딩하는 유전자가 도입되어 있는 젖산 생성능을 가지는 재조합 균주.
- 제1항에 있어서, 상기 피루베이트 디카르복실라아제를 코딩하는 유전자는 g3002 유전자인 것을 특징으로 하는 재조합 균주.
- 제2항에 있어서, 상기 g3002 유전자는 서열번호 1 또는 서열번호 2의 염기서열을 가지는 것을 특징으로 하는 재조합 균주.
- 제1항에 있어서, 알코올 디하이드로게나아제를 코딩하는 유전자가 추가로 결실되어 있는 것을 특징으로 하는 재조합 균주.
- 제4항에 있어서, 상기 알코올 디하이드로게나아제를 코딩하는 유전자는 g4423 유전자인 것을 특징으로 하는 재조합 균주.
- 제1항에 있어서, 상기 락테이트 디하이드로게나아제를 코딩하는 유전자는 g3002 유전자와 치환하여 도입되어 g3002 유전자의 프로모터에 의하여 조절되는 것을 특징으로 하는 재조합 균주.
- 제1항에 있어서, 상기 g3002 유전자의 결실 또는 약화에 의하여 모균주인 YBC 균주(KCTC13508BP) 보다 에탄올 생성능이 감소되는 것을 특징으로 하는 재조합 균주.
- 다음을 단계를 포함하는 젖산의 제조방법;(a) 제1항 내지 제7항 중 어느 한 항의 재조합 균주를 배양하여 젖산을 생성시키는 단계; 및(b) 상기 생성된 젖산을 수득하는 단계.
- 피루베이트 디카르복실라아제 활성을 가지고, 서열번호 3 또는 서열번호 4로 표시되는 아미노산 서열을 가지는 단백질을 코딩하는 유전자.
- 제9항에 있어서, 서열번호 1 또는 서열번호 2로 표시되는 염기서열을 가지는 것을 특징으로 하는 유전자.
- 피루베이트 디카르복실라아제 활성을 가지고, 서열번호 3 또는 서열번호 4로 표시되는 아미노산 서열을 가지는 단백질.
- 서열번호 5 또는 서열번호 6으로 표시되는 염기서열을 가지는 g3002 유전자 프로모터.
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201980065667.0A CN112789353B (zh) | 2018-10-08 | 2019-09-23 | 抑制乙醇产生的重组耐酸酵母以及使用其制备乳酸的方法 |
JP2021520201A JP7530891B2 (ja) | 2018-10-08 | 2019-09-23 | アルコール生成が抑制された組換え耐酸性酵母及びこれを用いた乳酸の製造方法 |
US17/276,306 US12084665B2 (en) | 2018-10-08 | 2019-09-23 | Recombinant acid-resistant yeast in which alcohol production is inhibited and method for producing lactic acid by using same |
EP19872165.6A EP3865577A4 (en) | 2018-10-08 | 2019-09-23 | RECOMBINANT ACID RESISTANT YEAST WITH INHIBITING ALCOHOL PRODUCTION AND PROCESS FOR THE MANUFACTURE OF LACTIC ACID USING THE SAME |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020180119721A KR20200040017A (ko) | 2018-10-08 | 2018-10-08 | 알코올 생성이 억제된 재조합 내산성 효모 및 이를 이용한 젖산의 제조방법 |
KR10-2018-0119721 | 2018-10-08 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2020075986A2 true WO2020075986A2 (ko) | 2020-04-16 |
WO2020075986A3 WO2020075986A3 (ko) | 2020-05-28 |
Family
ID=70164968
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/KR2019/012326 WO2020075986A2 (ko) | 2018-10-08 | 2019-09-23 | 알코올 생성이 억제된 재조합 내산성 효모 및 이를 이용한 젖산의 제조방법 |
Country Status (6)
Country | Link |
---|---|
US (1) | US12084665B2 (ko) |
EP (1) | EP3865577A4 (ko) |
JP (1) | JP7530891B2 (ko) |
KR (1) | KR20200040017A (ko) |
CN (1) | CN112789353B (ko) |
WO (1) | WO2020075986A2 (ko) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3929282A3 (en) * | 2020-06-24 | 2022-03-16 | SK Innovation Co., Ltd. | Recombinant acid-resistant yeast having improved lactic-acid-producing ability |
EP4008771A1 (en) * | 2020-11-12 | 2022-06-08 | SK Innovation Co., Ltd. | Synthetic promoter based on gene from acid-resistant yeast |
US11655478B2 (en) | 2018-04-17 | 2023-05-23 | Sk Innovation Co., Ltd. | Promoter derived from organic acid-resistant yeast and method for expression of target gene by using same |
US11680270B2 (en) | 2019-10-08 | 2023-06-20 | Sk Innovation Co., Ltd. | Recombinant acid-resistant yeast with inhibited lactate metabolism and alcohol production and method of producing lactic acid using the same |
Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2001204464A (ja) | 2000-01-27 | 2001-07-31 | Toyota Motor Corp | 乳酸の製造方法 |
US7141410B2 (en) | 2000-11-22 | 2006-11-28 | Natureworks Llc | Methods and materials for the production of organic products in cells of Candida species |
JP4095889B2 (ja) | 2002-12-13 | 2008-06-04 | トヨタ自動車株式会社 | 高光学純度な乳酸の製造方法 |
US7534597B2 (en) | 2002-05-30 | 2009-05-19 | Cargill, Inc. | Methods and materials for the production of L-lactic acid in yeast |
JP4692173B2 (ja) | 2005-09-13 | 2011-06-01 | 東レ株式会社 | D−乳酸デヒドロゲナーゼ活性を有するポリペプチド、これをコードする遺伝子およびd−乳酸の製造方法 |
US9353388B2 (en) | 2013-02-05 | 2016-05-31 | Samsung Electronics Co., Ltd. | Microorganism over-expressing lactic acid transporter gene and having inhibitory pathway of lactic acid degradation, and method of producing lactic acid using the microorganism |
KR101686900B1 (ko) | 2014-06-20 | 2016-12-16 | 한국생명공학연구원 | 신규한 피키아 쿠드리압즈비 ng7 균주 및 이의 용도 |
KR20170025315A (ko) | 2014-08-29 | 2017-03-08 | 에스케이이노베이션 주식회사 | 3-hp를 생산할 수 있는 재조합 효모 및 이를 이용한 3-hp의 제조방법 |
KR20180044508A (ko) | 2016-10-24 | 2018-05-03 | (주) 한호기술 | 접이식 휠체어 |
KR20180044509A (ko) | 2016-10-24 | 2018-05-03 | 와토스코리아 주식회사 | 앵글밸브 |
Family Cites Families (31)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IT1294728B1 (it) | 1997-09-12 | 1999-04-12 | Biopolo S C A R L | Ceppi di lievito per la riproduzione di acido lattico |
JP4460876B2 (ja) | 2003-11-07 | 2010-05-12 | 株式会社豊田中央研究所 | 有機酸存在下におけるプロモーター及びその利用 |
US20050112737A1 (en) | 2003-11-20 | 2005-05-26 | A. E. Staley Manufacturing Co. | Lactic acid producing yeast |
JP4700395B2 (ja) | 2005-04-13 | 2011-06-15 | 株式会社豊田中央研究所 | 酸性条件下で使用するためのプロモーター及びその利用 |
EP1960516B1 (en) | 2005-11-23 | 2012-12-26 | Cargill, Incorporated | Lactic acid-producing yeast cells having nonfunctional l-or d-lactate: ferricytochrome c oxidoreductase gene |
CA2645361A1 (en) | 2006-03-13 | 2007-09-20 | Cargill Inc. | Yeast cells having disrupted pathway from dihydroxyacetone phosphate to glycerol |
EP2060632A1 (en) | 2007-10-29 | 2009-05-20 | Technische Universität Berlin | Method of modifying a yeast cell for the production of ethanol |
JP2010075171A (ja) * | 2008-08-25 | 2010-04-08 | Kirin Holdings Co Ltd | キャンディダ・ユティリスによる高効率乳酸製造法 |
IN2012DN01521A (ko) * | 2009-08-21 | 2015-06-05 | Asahi Glass Co Ltd | |
WO2011038364A1 (en) | 2009-09-27 | 2011-03-31 | Opx Biotechnologies, Inc. | Method for producing 3-hydroxypropionic acid and other products |
JP2012061006A (ja) * | 2011-12-22 | 2012-03-29 | Toyota Motor Corp | 耐酸性微生物を用いた有機酸及びアルコールの製造方法 |
WO2014003439A1 (ko) | 2012-06-26 | 2014-01-03 | 한국생명공학연구원 | 에탄올 생산 경로가 봉쇄된 클루이베로마이세스 막시아누스 균주 및 이의 용도 |
KR102144998B1 (ko) | 2013-08-30 | 2020-08-14 | 삼성전자주식회사 | 효모에 내산성을 부여하는 폴리펩티드, 그를 코딩하는 폴리뉴클레오티드, 그 양이 증가되어 있는 효모 세포, 상기 효모 세포를 이용한 산물의 생산 방법 및 내산성 효모 세포를 생산하는 방법 |
US9617569B2 (en) | 2013-11-15 | 2017-04-11 | Samsung Electronics Co., Ltd. | Genetically engineered yeast cell producing lactate including acetaldehyde dehydrogenase, method of producing yeast cell, and method of producing lactate using the same |
KR20150064802A (ko) | 2013-12-03 | 2015-06-12 | 삼성전자주식회사 | 글리세롤-3-포스페이트 데히드로게나제가 불활성화되고 글리세르알데히드-3-포스페이트 데히드로게나제가 활성화된 효모 세포 및 그를 이용한 락테이트를 생산하는 방법 |
KR102163724B1 (ko) | 2014-02-13 | 2020-10-08 | 삼성전자주식회사 | 내산성을 갖는 효모 세포 및 이의 용도 |
KR101577134B1 (ko) * | 2014-05-09 | 2015-12-14 | 씨제이제일제당 (주) | 젖산 생산이 향상된 미생물 및 이를 이용하여 젖산을 생산하는 방법 |
KR102277898B1 (ko) | 2014-07-03 | 2021-07-15 | 삼성전자주식회사 | 산물 생산능이 향상된 효모 및 그를 이용한 산물을 생산하는 방법 |
KR20160012561A (ko) | 2014-07-24 | 2016-02-03 | 삼성전자주식회사 | Erg5의 활성이 증가되도록 유전적으로 조작된, 내산성을 갖는 효모 세포 및 그를 이용하여 락테이트를 생산하는 방법 |
KR102227975B1 (ko) | 2014-07-24 | 2021-03-15 | 삼성전자주식회사 | 방사선 감수성 보완 키나아제의 활성이 증가되도록 유전적으로 조작된, 내산성을 갖는 효모 세포 및 그를 이용하여 락테이트를 생산하는 방법 |
EP3205721B1 (en) * | 2014-10-10 | 2019-08-28 | JMTC Enzyme Corporation | Transformant and method for producing same, and method for producing lactic acid |
KR102303832B1 (ko) | 2015-05-12 | 2021-09-17 | 삼성전자주식회사 | 내산성을 갖는 효모 세포, 상기 효모 세포를 제조하는 방법 및 이의 용도 |
KR101704212B1 (ko) * | 2015-06-12 | 2017-02-08 | 씨제이제일제당 (주) | 젖산을 생산하는 미생물 및 이를 이용한 젖산 제조 방법 |
KR20170008151A (ko) | 2015-07-13 | 2017-01-23 | 에스케이이노베이션 주식회사 | 메틸말로닐-CoA 리덕테이즈 코딩 유전자를 함유하는 미생물 변이체 및 이의 용도 |
KR101759673B1 (ko) | 2015-12-28 | 2017-07-31 | 서울대학교산학협력단 | 생장 속도가 증대된 유전적으로 조작된 효모 세포 및 그를 사용하여 목적 물질을 생산하는 방법 |
KR101965364B1 (ko) | 2016-08-03 | 2019-04-03 | 한국생명공학연구원 | Upc2를 발현하는 재조합 균주 및 이의 용도 |
KR102140596B1 (ko) | 2018-04-17 | 2020-08-04 | 에스케이이노베이션 주식회사 | 유기산 내성 효모 유래 신규 프로모터 및 이를 이용한 목적유전자의 발현방법 |
KR102140597B1 (ko) | 2018-04-17 | 2020-08-03 | 에스케이이노베이션 주식회사 | 에탄올 생산 경로가 억제된 내산성 효모 및 이를 이용한 젖산의 제조방법 |
KR20210041903A (ko) | 2019-10-08 | 2021-04-16 | 에스케이이노베이션 주식회사 | 락테이트 대사 및 알코올 생성이 억제된 재조합 내산성 효모 및 이를 이용한 젖산의 제조방법 |
KR20210128742A (ko) | 2020-04-17 | 2021-10-27 | 에스케이이노베이션 주식회사 | 글리세롤 생성이 억제된 재조합 내산성 효모 및 이를 이용한 젖산의 제조방법 |
KR20210158676A (ko) | 2020-06-24 | 2021-12-31 | 에스케이이노베이션 주식회사 | 젖산 생산능이 증가된 재조합 내산성 효모 |
-
2018
- 2018-10-08 KR KR1020180119721A patent/KR20200040017A/ko not_active Application Discontinuation
-
2019
- 2019-09-23 CN CN201980065667.0A patent/CN112789353B/zh active Active
- 2019-09-23 EP EP19872165.6A patent/EP3865577A4/en active Pending
- 2019-09-23 WO PCT/KR2019/012326 patent/WO2020075986A2/ko active Application Filing
- 2019-09-23 JP JP2021520201A patent/JP7530891B2/ja active Active
- 2019-09-23 US US17/276,306 patent/US12084665B2/en active Active
Patent Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2001204464A (ja) | 2000-01-27 | 2001-07-31 | Toyota Motor Corp | 乳酸の製造方法 |
US7141410B2 (en) | 2000-11-22 | 2006-11-28 | Natureworks Llc | Methods and materials for the production of organic products in cells of Candida species |
US7534597B2 (en) | 2002-05-30 | 2009-05-19 | Cargill, Inc. | Methods and materials for the production of L-lactic acid in yeast |
JP4095889B2 (ja) | 2002-12-13 | 2008-06-04 | トヨタ自動車株式会社 | 高光学純度な乳酸の製造方法 |
JP4692173B2 (ja) | 2005-09-13 | 2011-06-01 | 東レ株式会社 | D−乳酸デヒドロゲナーゼ活性を有するポリペプチド、これをコードする遺伝子およびd−乳酸の製造方法 |
US9353388B2 (en) | 2013-02-05 | 2016-05-31 | Samsung Electronics Co., Ltd. | Microorganism over-expressing lactic acid transporter gene and having inhibitory pathway of lactic acid degradation, and method of producing lactic acid using the microorganism |
KR101686900B1 (ko) | 2014-06-20 | 2016-12-16 | 한국생명공학연구원 | 신규한 피키아 쿠드리압즈비 ng7 균주 및 이의 용도 |
KR20170025315A (ko) | 2014-08-29 | 2017-03-08 | 에스케이이노베이션 주식회사 | 3-hp를 생산할 수 있는 재조합 효모 및 이를 이용한 3-hp의 제조방법 |
KR20180044508A (ko) | 2016-10-24 | 2018-05-03 | (주) 한호기술 | 접이식 휠체어 |
KR20180044509A (ko) | 2016-10-24 | 2018-05-03 | 와토스코리아 주식회사 | 앵글밸브 |
Non-Patent Citations (5)
Title |
---|
ELLEN I. GARVIE, MICROBIOLOGICAL REVIEWS, 1980, pages 106 - 139 |
MICHAEL SAUER ET AL., BIOTECHNOLOGY AND GENETIC ENGINEERING REVIEWS, vol. 27, no. 1, 2010, pages 229 - 256 |
N. ISHIDA, APPL. ENVIRON. MICOBIOL., 2005, pages 1964 - 1970 |
See also references of EP3865577A4 |
T. C. HOPPNERH. W. DOELLE, EUROPEAN JOURNAL OF APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 17, 1983, pages 152 - 157 |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11655478B2 (en) | 2018-04-17 | 2023-05-23 | Sk Innovation Co., Ltd. | Promoter derived from organic acid-resistant yeast and method for expression of target gene by using same |
US11680270B2 (en) | 2019-10-08 | 2023-06-20 | Sk Innovation Co., Ltd. | Recombinant acid-resistant yeast with inhibited lactate metabolism and alcohol production and method of producing lactic acid using the same |
EP3929282A3 (en) * | 2020-06-24 | 2022-03-16 | SK Innovation Co., Ltd. | Recombinant acid-resistant yeast having improved lactic-acid-producing ability |
US11898173B2 (en) | 2020-06-24 | 2024-02-13 | Sk Innovation Co., Ltd. | Recombinant acid-resistant yeast having improved lactic-acid-producing ability |
EP4008771A1 (en) * | 2020-11-12 | 2022-06-08 | SK Innovation Co., Ltd. | Synthetic promoter based on gene from acid-resistant yeast |
US11788095B2 (en) | 2020-11-12 | 2023-10-17 | Sk Innovation Co., Ltd. | Synthetic promoter based on gene from acid-resistant yeast |
Also Published As
Publication number | Publication date |
---|---|
CN112789353A (zh) | 2021-05-11 |
WO2020075986A3 (ko) | 2020-05-28 |
CN112789353B (zh) | 2024-08-16 |
EP3865577A4 (en) | 2022-08-17 |
KR20200040017A (ko) | 2020-04-17 |
EP3865577A2 (en) | 2021-08-18 |
JP7530891B2 (ja) | 2024-08-08 |
US20220056459A1 (en) | 2022-02-24 |
JP2022504813A (ja) | 2022-01-13 |
US12084665B2 (en) | 2024-09-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2020075986A2 (ko) | 알코올 생성이 억제된 재조합 내산성 효모 및 이를 이용한 젖산의 제조방법 | |
WO2019203436A1 (ko) | 에탄올 생산 경로가 억제된 내산성 효모 및 이를 이용한 젖산의 제조방법 | |
WO2019203435A1 (ko) | 유기산 내성 효모 유래 신규 프로모터 및 이를 이용한 목적유전자의 발현방법 | |
WO2019027267A2 (ko) | Atp 포스포리보실 전이효소 변이체 및 이를 이용한 l-히스티딘 생산방법 | |
WO2013162274A1 (ko) | 신규한 d형 젖산 생산균주 및 그의 용도 | |
WO2018124440A2 (ko) | 신규한 이소프로필말레이트 신타제 변이체 및 이를 이용한 l-류신의 생산 방법 | |
WO2013095071A2 (ko) | L-라이신 생산능을 갖는 미생물을 이용하여 l-라이신을 생산하는 방법 | |
WO2016024771A1 (ko) | O-포스포세린 생산 미생물 및 이를 이용한 o-포스포세린 또는 l-시스테인 생산 방법 | |
WO2013105802A2 (ko) | 자일로즈 이용능이 부여된 코리네박테리움 속 미생물 및 이를 이용한 l-라이신의 생산방법 | |
US11680270B2 (en) | Recombinant acid-resistant yeast with inhibited lactate metabolism and alcohol production and method of producing lactic acid using the same | |
US20210324346A1 (en) | Recombinant acid-resistant yeast with suppressed glycerol production and method of producing lactic acid using the same | |
WO2016200207A1 (ko) | 젖산을 생산하는 미생물 및 이를 이용한 젖산 제조 방법 | |
WO2014003439A1 (ko) | 에탄올 생산 경로가 봉쇄된 클루이베로마이세스 막시아누스 균주 및 이의 용도 | |
WO2015170914A1 (ko) | 젖산 생산이 향상된 미생물 및 이를 이용하여 젖산을 생산하는 방법 | |
WO2022163933A1 (ko) | 신규한 abc 트랜스포터 atp-결합 단백질 변이체 및 이를 이용한 l-글루탐산 생산 방법 | |
WO2010104224A1 (ko) | 글리세롤 산화대사경로를 차단시킨 재조합 균주를 이용한 1、3-프로판디올의 제조방법 | |
WO2022154190A1 (ko) | 신규한 포스포노아세테이트 하이드롤라제 변이체 및 이를 이용한 xmp 또는 gmp 생산 방법 | |
WO2022154191A1 (ko) | 신규한 2,5-다이케토-d-글루콘산 리덕타제 변이체 및 이를 이용한 xmp 또는 gmp 생산 방법 | |
WO2022225320A1 (ko) | 신규한 포스포글리세린산 디하이드로게나제 변이체 및 이를 이용한 xmp 또는 gmp 생산 방법 | |
WO2022004953A1 (ko) | 아세토인 생산능을 갖는 유전적으로 조작된 효모 및 이를 이용한 아세토인 생산방법 | |
WO2020116941A2 (ko) | 디카르복시산 생산을 위한 미생물 및 이를 이용한 디카르복시산 생산방법 | |
WO2020085556A1 (ko) | 재조합 코리네박테리움 글루타미쿰 균주 및 이를 이용한 카다베린의 생산방법 | |
WO2022215800A1 (ko) | 신규한 분지쇄아미노산 투과효소 변이체 및 이를 이용한 l-발린 생산 방법 | |
WO2015046978A1 (ko) | 2,3-부탄디올의 생성능이 증강된 재조합 미생물 및 이를 이용한 2,3-부탄디올의 생산 방법 | |
WO2020180132A1 (ko) | D-글루타메이트 영양요구성 대장균 및 이를 이용한 목적 물질 생산 방법 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 19872165 Country of ref document: EP Kind code of ref document: A2 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2101001985 Country of ref document: TH |
|
ENP | Entry into the national phase |
Ref document number: 2021520201 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2019872165 Country of ref document: EP Effective date: 20210510 |