CN116926156A - Preparation method of 2-carbonyl-containing polysubstituted butyric acid, D-pantothenic acid and panthenol - Google Patents
Preparation method of 2-carbonyl-containing polysubstituted butyric acid, D-pantothenic acid and panthenol Download PDFInfo
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- CN116926156A CN116926156A CN202210383075.7A CN202210383075A CN116926156A CN 116926156 A CN116926156 A CN 116926156A CN 202210383075 A CN202210383075 A CN 202210383075A CN 116926156 A CN116926156 A CN 116926156A
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- Prior art keywords
- hydroxy
- acid
- carbonyl
- dimethyl
- polysubstituted
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- GHOKWGTUZJEAQD-UHFFFAOYSA-N pantothenic acid Chemical compound OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 title claims abstract description 67
- 150000004652 butanoic acids Chemical class 0.000 title claims abstract description 38
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- SNPLKNRPJHDVJA-ZETCQYMHSA-N D-panthenol Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCCO SNPLKNRPJHDVJA-ZETCQYMHSA-N 0.000 title claims abstract description 16
- 229940101267 panthenol Drugs 0.000 title claims abstract description 16
- 235000020957 pantothenol Nutrition 0.000 title claims abstract description 16
- 239000011619 pantothenol Substances 0.000 title claims abstract description 16
- 238000000855 fermentation Methods 0.000 claims abstract description 45
- 230000004151 fermentation Effects 0.000 claims abstract description 45
- 108090000854 Oxidoreductases Proteins 0.000 claims abstract description 39
- 239000000758 substrate Substances 0.000 claims abstract description 33
- 102000004316 Oxidoreductases Human genes 0.000 claims abstract description 32
- 108090000790 Enzymes Proteins 0.000 claims abstract description 18
- 102000004190 Enzymes Human genes 0.000 claims abstract description 17
- 230000009471 action Effects 0.000 claims abstract description 11
- 238000010353 genetic engineering Methods 0.000 claims abstract description 4
- 238000006555 catalytic reaction Methods 0.000 claims abstract description 3
- 244000005700 microbiome Species 0.000 claims description 36
- 108090000623 proteins and genes Proteins 0.000 claims description 36
- 238000004519 manufacturing process Methods 0.000 claims description 29
- -1 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid Chemical compound 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 28
- 241000588724 Escherichia coli Species 0.000 claims description 25
- UCMIRNVEIXFBKS-UHFFFAOYSA-N beta-alanine Chemical compound NCCC(O)=O UCMIRNVEIXFBKS-UHFFFAOYSA-N 0.000 claims description 24
- OTOIIPJYVQJATP-BYPYZUCNSA-N (R)-pantoic acid Chemical compound OCC(C)(C)[C@@H](O)C(O)=O OTOIIPJYVQJATP-BYPYZUCNSA-N 0.000 claims description 22
- 101150058595 MDH gene Proteins 0.000 claims description 17
- 239000013598 vector Substances 0.000 claims description 16
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 12
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 12
- 229940000635 beta-alanine Drugs 0.000 claims description 12
- 239000013613 expression plasmid Substances 0.000 claims description 12
- 239000012634 fragment Substances 0.000 claims description 12
- 238000003259 recombinant expression Methods 0.000 claims description 12
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 claims description 11
- 230000035772 mutation Effects 0.000 claims description 10
- 101150028586 panE gene Proteins 0.000 claims description 10
- 239000013612 plasmid Substances 0.000 claims description 9
- 230000008569 process Effects 0.000 claims description 9
- 239000002253 acid Substances 0.000 claims description 7
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound OS(O)(=O)=O.O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 claims description 7
- 229960002064 kanamycin sulfate Drugs 0.000 claims description 7
- OTOIIPJYVQJATP-SCSAIBSYSA-N (2s)-2,4-dihydroxy-3,3-dimethylbutanoic acid Chemical compound OCC(C)(C)[C@H](O)C(O)=O OTOIIPJYVQJATP-SCSAIBSYSA-N 0.000 claims description 6
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims description 6
- 241000194107 Bacillus megaterium Species 0.000 claims description 6
- 244000063299 Bacillus subtilis Species 0.000 claims description 6
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 6
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 6
- AMIMRNSIRUDHCM-UHFFFAOYSA-N Isopropylaldehyde Chemical compound CC(C)C=O AMIMRNSIRUDHCM-UHFFFAOYSA-N 0.000 claims description 6
- 230000014509 gene expression Effects 0.000 claims description 6
- 239000013600 plasmid vector Substances 0.000 claims description 6
- 239000002994 raw material Substances 0.000 claims description 6
- 238000012408 PCR amplification Methods 0.000 claims description 5
- 230000002018 overexpression Effects 0.000 claims description 5
- 238000012163 sequencing technique Methods 0.000 claims description 5
- 230000001131 transforming effect Effects 0.000 claims description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
- 230000000694 effects Effects 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 3
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 3
- 241000186216 Corynebacterium Species 0.000 claims description 3
- 241000235646 Cyberlindnera jadinii Species 0.000 claims description 3
- 241000235058 Komagataella pastoris Species 0.000 claims description 3
- 241000187747 Streptomyces Species 0.000 claims description 3
- 229940006015 4-hydroxybutyric acid Drugs 0.000 claims description 2
- 102000005751 Alcohol Oxidoreductases Human genes 0.000 claims description 2
- 108010031132 Alcohol Oxidoreductases Proteins 0.000 claims description 2
- 101100456369 Aquifex aeolicus (strain VF5) mdh1 gene Proteins 0.000 claims description 2
- 101100518918 Bacillus subtilis (strain 168) panE gene Proteins 0.000 claims description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- 241000660147 Escherichia coli str. K-12 substr. MG1655 Species 0.000 claims description 2
- 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 2
- 102220493797 HLA class II histocompatibility antigen, DRB1 beta chain_D86S_mutation Human genes 0.000 claims description 2
- 101000962359 Homo sapiens NACHT, LRR and PYD domains-containing protein 10 Proteins 0.000 claims description 2
- 102220479935 Leucine-rich repeat-containing protein 26_R81A_mutation Human genes 0.000 claims description 2
- 101150074741 MDH1 gene Proteins 0.000 claims description 2
- 101150019925 MDH2 gene Proteins 0.000 claims description 2
- 101100384788 Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440) comC gene Proteins 0.000 claims description 2
- 101100020705 Mycoplasma gallisepticum (strain R(low / passage 15 / clone 2)) ldh gene Proteins 0.000 claims description 2
- 102100039260 NACHT, LRR and PYD domains-containing protein 10 Human genes 0.000 claims description 2
- WUGQZFFCHPXWKQ-UHFFFAOYSA-N Propanolamine Chemical compound NCCCO WUGQZFFCHPXWKQ-UHFFFAOYSA-N 0.000 claims description 2
- 101100290490 Rattus norvegicus Mdh1 gene Proteins 0.000 claims description 2
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 claims description 2
- 101150065293 apbA gene Proteins 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 238000009833 condensation Methods 0.000 claims description 2
- 230000005494 condensation Effects 0.000 claims description 2
- HHLFWLYXYJOTON-UHFFFAOYSA-N glyoxylic acid Chemical compound OC(=O)C=O HHLFWLYXYJOTON-UHFFFAOYSA-N 0.000 claims description 2
- 230000010354 integration Effects 0.000 claims description 2
- 101150025049 leuB gene Proteins 0.000 claims description 2
- WQVJUBFKFCDYDQ-BBWFWOEESA-N leubethanol Natural products C1=C(C)C=C2[C@H]([C@H](CCC=C(C)C)C)CC[C@@H](C)C2=C1O WQVJUBFKFCDYDQ-BBWFWOEESA-N 0.000 claims description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 2
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 2
- 102220093344 rs749571694 Human genes 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 claims description 2
- 229960003495 thiamine Drugs 0.000 claims description 2
- 235000019157 thiamine Nutrition 0.000 claims description 2
- 239000011721 thiamine Substances 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 8
- 238000009776 industrial production Methods 0.000 abstract description 4
- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 description 16
- 239000002609 medium Substances 0.000 description 12
- 235000019161 pantothenic acid Nutrition 0.000 description 9
- 239000011713 pantothenic acid Substances 0.000 description 9
- 229940055726 pantothenic acid Drugs 0.000 description 7
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 6
- 231100000331 toxic Toxicity 0.000 description 5
- 230000002588 toxic effect Effects 0.000 description 5
- SERHXTVXHNVDKA-BYPYZUCNSA-N (R)-pantolactone Chemical compound CC1(C)COC(=O)[C@@H]1O SERHXTVXHNVDKA-BYPYZUCNSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- SGWFGVQCRDTUQN-UHFFFAOYSA-N (2-prop-2-ynoyloxy-3-prop-2-ynoylsulfanylpropyl) prop-2-ynoate Chemical compound C#CC(=O)OCC(OC(=O)C#C)CSC(=O)C#C SGWFGVQCRDTUQN-UHFFFAOYSA-N 0.000 description 3
- XFXPMWWXUTWYJX-UHFFFAOYSA-N Cyanide Chemical compound N#[C-] XFXPMWWXUTWYJX-UHFFFAOYSA-N 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 102000003960 Ligases Human genes 0.000 description 3
- 108090000364 Ligases Proteins 0.000 description 3
- 235000010216 calcium carbonate Nutrition 0.000 description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 238000012994 industrial processing Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- RGJOEKWQDUBAIZ-IBOSZNHHSA-N CoASH Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCS)O[C@H]1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-IBOSZNHHSA-N 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 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 2
- 239000003054 catalyst Substances 0.000 description 2
- 230000019522 cellular metabolic process Effects 0.000 description 2
- RGJOEKWQDUBAIZ-UHFFFAOYSA-N coenzime A Natural products OC1C(OP(O)(O)=O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-UHFFFAOYSA-N 0.000 description 2
- 239000005516 coenzyme A Substances 0.000 description 2
- 229940093530 coenzyme a Drugs 0.000 description 2
- KDTSHFARGAKYJN-UHFFFAOYSA-N dephosphocoenzyme A Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 KDTSHFARGAKYJN-UHFFFAOYSA-N 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- LELOWRISYMNNSU-UHFFFAOYSA-N hydrogen cyanide Chemical compound N#C LELOWRISYMNNSU-UHFFFAOYSA-N 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000000543 intermediate Substances 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000013028 medium composition Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- OTOIIPJYVQJATP-UHFFFAOYSA-N pantoic acid Chemical compound OCC(C)(C)C(O)C(O)=O OTOIIPJYVQJATP-UHFFFAOYSA-N 0.000 description 2
- SERHXTVXHNVDKA-UHFFFAOYSA-N pantolactone Chemical compound CC1(C)COC(=O)C1O SERHXTVXHNVDKA-UHFFFAOYSA-N 0.000 description 2
- 229940115458 pantolactone Drugs 0.000 description 2
- SIEVQTNTRMBCHO-UHFFFAOYSA-N pantolactone Natural products CC1(C)OC(=O)CC1O SIEVQTNTRMBCHO-UHFFFAOYSA-N 0.000 description 2
- 229940014662 pantothenate Drugs 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- SERHXTVXHNVDKA-SCSAIBSYSA-N (3s)-3-hydroxy-4,4-dimethyloxolan-2-one Chemical compound CC1(C)COC(=O)[C@H]1O SERHXTVXHNVDKA-SCSAIBSYSA-N 0.000 description 1
- PKAUICCNAWQPAU-UHFFFAOYSA-N 2-(4-chloro-2-methylphenoxy)acetic acid;n-methylmethanamine Chemical compound CNC.CC1=CC(Cl)=CC=C1OCC(O)=O PKAUICCNAWQPAU-UHFFFAOYSA-N 0.000 description 1
- 108700037654 Acyl carrier protein (ACP) Proteins 0.000 description 1
- 102000048456 Acyl carrier protein (ACP) Human genes 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 201000004384 Alopecia Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000534000 Berula erecta Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010040954 Skin wrinkling Diseases 0.000 description 1
- 206010042496 Sunburn Diseases 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 231100000360 alopecia Toxicity 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- SNPLKNRPJHDVJA-UHFFFAOYSA-N dl-panthenol Chemical compound OCC(C)(C)C(O)C(=O)NCCCO SNPLKNRPJHDVJA-UHFFFAOYSA-N 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 230000003628 erosive effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000003779 hair growth Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000013048 microbiological method Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 150000002948 pantothenic acids Chemical class 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 229940046001 vitamin b complex Drugs 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
Classifications
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- 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
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/002—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by oxidation/reduction reactions
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/02—Preparation of carboxylic acid amides from carboxylic acids or from esters, anhydrides, or halides thereof by reaction with ammonia or amines
-
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
- C12N15/1031—Mutagenizing nucleic acids mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
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- 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
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- 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)
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- 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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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- 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
- C12P7/42—Hydroxy-carboxylic acids
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- 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)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03015—(S)-2-Hydroxy-acid oxidase (1.1.3.15)
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Abstract
The invention relates to the technical field of genetic engineering and fermentation engineering, and particularly discloses a preparation method of polysubstituted butyric acid containing 2-carbonyl, which takes polysubstituted butyric acid containing (S) -2-hydroxy as a substrate, and adds (S) -2-hydroxy oxidase to carry out enzyme catalytic reaction, wherein the substrate is converted into polysubstituted butyric acid containing 2-carbonyl under the action of the (S) -2-hydroxy oxidase. The invention further discloses a preparation method of D-pantothenic acid and panthenol. The invention is safe and environment-friendly, has high conversion rate and yield, and is suitable for industrial production.
Description
Technical Field
The invention relates to the technical field of genetic engineering and fermentation engineering, in particular to a preparation method of 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid, D-pantothenic acid and panthenol.
Background
D-pantothenic acid is a member of the vitamin B complex. In cells, pantothenic acid is used primarily for biosynthesis of coenzyme A (CoA) and Acyl Carrier Protein (ACP), which are of great importance for cellular metabolism and which are involved in more than 100 different intermediate reactions in cellular metabolism. The panthenol is used as one of pantothenic acid derivatives, and is mainly used for preparing hair care products and topical cosmetics, and can be used for preventing and treating small wrinkles, inflammation, sunburn and erosion, preventing alopecia, and promoting hair growth. Calcium D-pantothenate is a commercially extremely important product form of pantothenic acid, and has been widely used in the pharmaceutical, feed and food industries.
At present, D-pantothenic acid is mostly synthesized by adopting a pure chemical synthesis method, wherein isobutyraldehyde, hydrocyanic acid and formaldehyde are condensed to obtain a racemate of 2-hydroxy-3, 3-dimethyl-4-hydroxybutyric acid, and the racemate is condensed with beta-alanine and then subjected to chiral resolution to obtain D-pantothenic acid. Because the synthesis by a pure chemical method needs to use highly toxic cyanide and involves a high-pressure operation environment, and a toxic chiral catalyst is needed in the chiral resolution step. To overcome these drawbacks, microbial synthesis methods have been the subject of research in recent years. The existing industrial production method is to produce a precursor by a chemical method and then carry out chiral resolution by a microbiological method or an enzymatic method. The resolution process is generally to hydrolyze and separate a substrate with a certain configuration, reform an S-configuration into a racemate, and repeat the steps to finally obtain a single R-configuration. The time consumption is longer, the operation is tedious, the yield is not high, and the industrial processing production is not facilitated.
Polysubstituted butyric acids containing 2-carbonyl groups, such as 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid, are extremely important intermediates for the production of pantothenic acid and panthenol, and also oxidation products of pantoic acid. Therefore, in actual production, 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid also has extremely high economic value, however, in actual production, 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid also has the problems that the yield is not high, the industrial production is not suitable, and the 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid cannot be fully supplied to meet the production needs.
Therefore, the invention aims to develop a brand-new preparation method of polysubstituted butyric acid containing 2-carbonyl, D-pantothenic acid and panthenol so as to better meet the actual production needs.
Disclosure of Invention
One of the technical problems to be solved by the invention is to provide a preparation method of polysubstituted butyric acid containing 2-carbonyl, which is safe and environment-friendly, has higher yield and is more suitable for industrial processing production.
The second technical problem to be solved by the invention is to provide a preparation method of D-pantothenic acid, which is safe and environment-friendly, has higher yield and is more suitable for industrial processing production.
The technical problem solved by the invention is also to provide a novel preparation method of panthenol.
A preparation method of polysubstituted butyric acid containing 2-carbonyl takes polysubstituted butyric acid containing (S) -2-hydroxy as a substrate, and (S) -2-hydroxy oxidase is added for enzyme catalytic reaction, and the substrate is converted into polysubstituted butyric acid containing 2-carbonyl under the action of the (S) -2-hydroxy oxidase.
Further, the (S) -2-hydroxy-containing polysubstituted butyric acid is S-2-hydroxy-3, 3-dimethyl-4-hydroxybutyric acid, and the 2-carbonyl-containing polysubstituted butyric acid is 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid.
Further, the (S) -2-hydroxy-containing polysubstituted butyric acid is S-2-hydroxy-3, 3-dimethyl-4-aldehyde butyric acid, and the 2-carbonyl-containing polysubstituted butyric acid is 2-carbonyl-3, 3-dimethyl-4-aldehyde butyric acid.
Further, the (S) -2-hydroxy-containing polysubstituted butyric acid is taken as a substrate, recombinant microorganism containing (S) -2-hydroxy oxidase coding genes is added for fermentation culture, and in the fermentation process, the (S) -2-hydroxy-containing polysubstituted butyric acid is converted into the 2-carbonyl-containing polysubstituted butyric acid under the action of (S) -2-hydroxy oxidase generated by over-expression of the recombinant microorganism.
Further, the (S) -2-hydroxyl oxidase coding gene comprises one or more of Mdh, mdh1, mdh2, leuB and PMDH 2.
Further, the (S) -2-hydroxy oxidase gene causes the overexpressed enzyme to increase the activity on the substrate by mutation or evolution.
Further, the (S) -2-hydroxyl oxidase gene comprises a mutant mdh gene mutated at the position of I12V, R81A, M Q, D86S, L149A, G179D, A G, and the nucleotide sequence of the mutant mdh gene is shown as SEQ ID NO. 1.
Further, the preparation method of the mutant mdh gene comprises the following steps: designing a mutation primer according to the locus of a mutated base, amplifying a plurality of gene fragments with different mutations by using the escherichia coli MG1655 genome as a template through PCR, and integrating the plurality of gene fragments to obtain the mdh gene with the mutation.
Further, the S-2-hydroxy-3, 3-dimethyl-4-aldehyde butyric acid is prepared from glyoxylate and isobutyraldehyde serving as raw materials.
A process for preparing D-pantothenic acid, wherein the above-mentioned 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid is reacted with (R) -carbonyl reductase to obtain (R) -2-hydroxy-3, 3-dimethyl-4-hydroxybutyric acid, and the (R) -2-hydroxy-3, 3-dimethyl-4-hydroxybutyric acid is reacted with beta-alanine to obtain D-pantothenic acid.
A process for preparing D-pantothenic acid, wherein 2-carbonyl-3, 3-dimethyl-4-aldehyde butyric acid as described above is converted into 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid by carbonyl reductase, and then into (R) -2-hydroxy-3, 3-dimethyl-4-hydroxybutyric acid, and D-pantothenic acid is obtained by condensing (R) -2-hydroxy-3, 3-dimethyl-4-hydroxybutyric acid with beta-alanine.
Further, taking (S) -2-hydroxy-3, 3-dimethyl-4-hydroxybutyric acid and beta-alanine as raw materials, adding recombinant microorganisms containing (S) -2-hydroxy oxidase coding genes, (R) -carbonyl reductase coding genes and condensed enzyme genes for fermentation, and converting the (S) -2-hydroxy-3, 3-dimethyl-4-hydroxybutyric acid into 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid under the action of (S) -2-hydroxy oxidase overexpressed by the recombinant microorganisms in the fermentation process; 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid is converted to (R) -2-hydroxy-3, 3-dimethyl-4-hydroxybutyric acid under the action of a recombinant microorganism overexpressed (R) -carbonyl reductase; (R) -2-hydroxy-3, 3-dimethyl-4-hydroxybutyric acid and beta-alanine are subjected to the action of recombinant microorganism overexpressed condensed enzyme to obtain D-pantothenic acid.
Further, the (R) -carbonyl reductase can convert ketone organic matters into alcohol organic matters, and the encoding genes of the (R) -carbonyl reductase comprise one or more of panE, PAN5, apbA and ylbQ; the condensation enzyme coding gene comprises one or more of panC and cmk.
Further, in the fermentation process, the fermentation temperature is 20-90 ℃ and the pH is 6.0-8.0.
Further, the fermentation medium adopted in the fermentation comprises the following raw materials: 11-13 g/L of M9 salt, 1-3 g/L of magnesium sulfate, 0.1-0.4 g/L of calcium chloride, 0.01-0.02 mg/mL of thiamine, 30-50 g/L of D-glucose, 2-6 g/L of yeast powder and 40-60 mu g/mL of kanamycin sulfate.
Further, it includes constructing the recombinant microorganism by genetic engineering methods including plasmid expression or genomic integration.
Further, the recombinant microorganism is constructed by a plasmid expression method, wherein the construction method comprises the following steps: obtaining a target gene through PCR amplification, connecting the obtained target gene to a plasmid vector, converting the plasmid vector into competent cells, and sequencing to obtain a recombinant expression plasmid vector; and (3) transforming the recombinant expression plasmid vector into a microorganism to obtain the recombinant microorganism.
Further, the microorganism includes one or more of E.coli, bacillus, corynebacterium, yeast or Streptomyces.
Further, the microorganism includes one or more of Escherichia coli (Escherichia coli), bacillus subtilis (Bacillus subtilis), bacillus megaterium (Bacillus megaterium), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), corynebacterium glutamicum (Corynebacterium glutamicum), saccharomyces cerevisiae (Saccharomyces cerevisiae), candida utilis (Candida lutea) or Pichia pastoris.
A process for preparing panthenol, R-pantoic acid is also generated in the course of preparing D-pantothenic acid, and the obtained R-pantoic acid is reacted with beta-alaninol to obtain panthenol.
The technical problems solved by the invention are realized by adopting the following technical scheme:
the beneficial effects are that: the preparation method of the polysubstituted butyric acid containing 2-carbonyl can be used for producing a large amount of polysubstituted butyric acid containing 2-carbonyl, such as 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid, high-pressure operation is not needed in the production process, and highly toxic cyanide is not needed in the synthesis process, so that the preparation method is simple to operate, safe and environment-friendly and high in yield.
The preparation method of D-pantothenic acid provided by the invention has the advantages that high-pressure operation is not needed in the preparation process, extremely toxic cyanide is not needed in the synthesis process, toxic chiral catalyst is not needed, the operation is simple, safety and environmental friendliness are realized, and the problems of difficult separation, substrate loss, complicated steps and the like caused by chiral resolution are avoided by adopting a direct fermentation method, so that the preparation method is more suitable for industrial batch production.
The preparation method of panthenol disclosed by the invention is safe and environment-friendly, has high conversion rate and yield, and is suitable for industrial production.
Detailed Description
In order that the manner in which the invention is attained, as well as the features and advantages thereof, will be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
As used herein and unless otherwise indicated, the term "pantoic acid" is 2, 4-dihydroxy-3, 3-dimethylbutyric acid. The term "pantothenic acid" refers to 2, 4-dihydroxy-3, 3-, dimethylbutyryl-3-alanine. The term "panthenol" refers to 2, 4-dihydroxy-N- (3-hydroxypropyl) -3, 3-dimethylbutyramide.
The term "amplification" refers to an increase in intracellular activity of one or more enzymes in a microorganism encoded by an appropriate DNA, for example, by increasing the gene copy number, using a strong promoter or using a gene encoding an appropriate enzyme having high activity, and selectively binding these methods.
The term "wild" refers to an object that may be found in nature.
Unless specifically stated otherwise, the terms "first," "second," and the like do not denote any order or importance, but rather the terms first, second, etc. are used to distinguish one object from another.
As used herein and unless otherwise indicated, the term "about" refers to a measurable value such as an amount, time period, or the like, and is meant to include a variation of ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%, from a given value, provided that such variation is suitable for practicing the disclosed method.
As described above, one of the purposes of the present invention is to provide a process for preparing 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid and D-pantothenic acid, which is safe and environment-friendly and has high yield.
Some embodiments of the present invention disclose a process for preparing 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid and D-pantothenic acid by adding (R, S) -2-hydroxy-3, 3-dimethyl-4-aldehydebutyric acid to a medium as a fermentation substrate and fermenting the fermentation substrate by a microorganism.
According to some embodiments of the invention, wherein when the fermentation substrate does not contain β -alanine, more R-pantoic acid is obtained by bacterial or fungal fermentation and less D-pantothenic acid.
According to some embodiments of the present invention, wherein (R, S) -2-hydroxy-3, 3-dimethyl-4-aldehydebutyric acid and beta-alanine are added simultaneously to a culture medium as fermentation substrates for fermentation, R-pantoic acid and D-pantothenic acid can be obtained simultaneously, and the amount of D-pantothenic acid produced is significantly increased.
The invention has the following concept and mechanism:
subsequent production of panthenol after the production of R-pantoic acid can be achieved by either chemical or biological fermentation methods as reported previously.
According to some embodiments of the invention, wherein the bacteria or fungi include, but are not limited to, wild or genetically engineered E.coli, bacillus, yeast, corynebacterium or Streptomyces. Such as Escherichia coli (Escherichia coli), bacillus subtilis (Bacillus subtilis), bacillus megaterium (Bacillus megaterium), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), corynebacterium glutamicum (Corynebacterium glutamicum), saccharomyces cerevisiae (Saccharomyces cerevisiae), candida utilis (Candida lutea) or Pichia pastoris.
In the test process, the invention adopts a DNA polymerase Phanta Max Super-Fidelity DNA Polymerase and non-ligase dependent single fragment rapid cloning kitII One Step Cloning Kit。
LB medium composition: 10g/L of tryptone, 5g/L of yeast powder, 10g/L of sodium chloride and 1.5% of agar powder are added into a solid culture medium.
The antibiotic concentration is: kanamycin sulfate 50. Mu.g/mL.
Method for detecting R-pantoic acid and D-pantothenic acid: pantothenate was quantitatively detected using HPLC-RID equipped with a Berle chromatographic column (1250140 Aminex HPX-87H Column 300x 7.8mm).
The recombinant microorganism can be realized by means of plasmid expression or gene recombination. The construction is schematically carried out by a plasmid expression method, and the construction method comprises the following steps: obtaining the (S) -2-hydroxyl oxidase coding gene or (R) -carbonyl reductase coding gene or condensed enzyme coding gene through PCR amplification, connecting the obtained gene to a plasmid vector, converting the plasmid vector into competent cells, and sequencing to obtain a recombinant expression plasmid vector; and (3) transforming the recombinant expression plasmid vector into a microorganism to obtain the recombinant microorganism.
The mutation primers were designed based on the genomic sequence of E.coli MG1655 published by NCBI:
mdh_1_F：
GGAAGATCTATGAAAGTCGCAGTCCTCGGCGCTGCTGGCGGTGTCGGCCAGGCGCTTGC
mdh_1_R：
GTTAACGTTAAACAGGTCGGAACGAGACTGACCCGGTTTAGCCGCTACGCCTGCAGAG
mdh_2_F：CCGGGTCAGTCTCGTTCCGACCTGTTTAACGTTAAC
mdh_2_R：GGAACGAATGATATCTGCCGTGGTAACGCCGAACAG
mdh_3_F：GGCGTTACCACGGCAGATATCATTCGTTCC
mdh_3_R：CGGCAGAATGGTAACATCAGAGTGACCGCC
mdh_4_F：GGTCACTCTGATGTTACCATTCTGCCG
mdh_4_R：GCCCATAGACAGGGTACCAGACCCGCCACCGGCCTTCGC
mdh_5_F:GGTGGCGGGTCTGGTACCCTGTCTATGGGC
mdh_5_R:CCCAAGCTTTTACTTATTAACGAACTCTTCGCC
mdh_ori_F:GGAAGATCTATGAAAGTCGCAGTCCTCGGCGCTGCTGGCGGT
mdh_ori_R:CAAGCTTTTACTTATTAACGAACTCTTCGCC
five mutated gene fragments are obtained respectively by taking an MG1655 genome as a template through PCR amplification, the five gene fragments are integrated to obtain a mutated mdh gene with seven mutation sites, the nucleotide sequence of the gene is shown as SEQ ID NO. 1, the gene is connected to a pze vector containing an IPTG inducible promoter by a non-ligase dependent single-fragment rapid cloning kit, then the gene is transformed to BW25113 competent cells, a kanamycin sulfate resistant plate is coated for overnight culture, positive clones are picked for sequencing verification, and the correct recombinant vector is named pze-mdh-m7 respectively.
As a control, a wild type mdh gene fragment is obtained by PCR amplification by taking an MG1655 genome as a template, the nucleotide sequence of the wild type mdh gene fragment is shown as SEQ ID NO. 2, the wild type mdh gene fragment is connected to a pze vector containing an IPTG inducible promoter by a non-ligase dependent single-fragment rapid cloning kit, then the vector is transformed into BW25113 competent cells, a kanamycin sulfate resistant plate is coated for overnight culture, sequencing verification is carried out on positive clones, and the correct recombinant vector is named as pze-mdh-ori respectively.
Example 1
Recombinant expression plasmid vector pze-mdh-m7 was transformed into E.coli BW25113 to obtain recombinant microorganism, and single colony of the recombinant microorganism was inoculated into 2mL of LB liquid medium containing 50. Mu.g/mL kanamycin sulfate, and cultured overnight (about 14 h) at 37℃and 220 rpm. A100 mL Erlenmeyer flask containing 10mL of fermentation medium (the composition of the fermentation medium was as described above) was transferred to the flask at an initial OD of 0.05, and the flask was cultured at 30℃and 220 rpm. 10ml of fermentation broth was added to each flask, and 0.5g CaCO3 was added to adjust the pH of the broth. After 12h of incubation, the broth was collected and when OD600 = 5 was detected, 10 μl of 1M TPTG solution was added for induction, while 1ml of substrate containing and 11mg was added for fermentation.
The overexpressed (S) -2-hydroxy oxidase oxidizes (S) -2-hydroxy-3, 3-dimethyl-4-hydroxybutyric acid in the substrate to 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid.
The fermentation medium used in this example is as described in example 1.
TABLE 1 fermentation Medium composition Table
The initial substrate concentration was 1g/L and the remaining substrate concentration was measured by HPLC.
In the implementation process of the embodiment, D, L pantolactone is used as a substrate for some parallel components, or the hydrolyzed pantolactone is used as a substrate, and the difference of results is small, so that the pantolactone can be directly used for production in actual production, and the production is more convenient and quick.
Example 2
As a control for example 1, the recombinant expression plasmid vector pze-mdh-ori was transformed into E.coli BW25113 to obtain a recombinant microorganism, and a single colony of the recombinant microorganism was inoculated into 2mL of LB liquid medium containing 50. Mu.g/mL kanamycin sulfate and cultured overnight at 37℃and 220rpm (about 14 hours). A100 mL Erlenmeyer flask containing 10mL of fermentation medium (the composition of the fermentation medium was as described above) was transferred to the flask at an initial OD of 0.05, and the flask was cultured at 30℃and 220 rpm. Each fermentation bottle was charged with 0.5g CaCO3 to adjust the pH of the fermentation broth. After 12h of incubation, the broth was collected and when OD600 = 5 was detected, 10 μl of 1M TPTG solution was added for induction, while 1ml of substrate containing 11mg was added for fermentation. The initial concentration of substrate was 1g/L.
The overexpressed (S) -2-hydroxy oxidase oxidizes (S) -2-hydroxy-3, 3-dimethyl-4-hydroxybutyric acid in the substrate to 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid.
The fermentation medium used in this example is as described in example 1.
Example 1 and example 2 substrate consumption was measured at 12h and 24h fermentation, two parallel groups were set for each example, and the results of the measurements are shown in Table 2. Compared with the wild type mdh gene, the enzyme produced by over-expression of the mutant mdh gene can promote the conversion of the substrate more effectively and improve the conversion rate.
If the microorganism itself contains a reductase gene expressing the reduction of 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid to D-pantolactone, the reductase gene reducing 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid to D-pantolactone is knocked out. Otherwise, the substrate is directly converted to pantothenic acid or other products after consumption, and accumulation of 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid cannot be achieved.
TABLE 2 results of detection of substrate consumption
Example 3
This example is different from example 1 in that a recombinant microorganism was obtained by transforming a recombinant expression plasmid vector pze-mdh-m7 into E.coli BW25113 with the panE gene knocked out. panE is a reductase gene contained in E.coli BW25113 itself which reduces 2-carbonyl-3, 3-dimethyl-4-hydroxybutyrate to D-pantolactone.
Example 4
This example is different from example 2 in that a recombinant microorganism was obtained by transforming a recombinant expression plasmid vector pze-mdh-m7 into E.coli BW25113 with the panE gene knocked out. panE is a reductase gene contained in E.coli BW25113 itself which reduces 2-carbonyl-3, 3-dimethyl-4-hydroxybutyrate to D-pantolactone.
Example 3 and example 4 substrate consumption was measured at 24h fermentation, two parallel groups were set for each example, and the results of the measurements are shown in Table 3. It is known that the enzyme produced by over-expression can effectively promote the conversion of the substrate, realize the accumulation of the product and effectively improve the yield and conversion rate of the product.
Table 3 2 Table of the results of detection of carbonyl-3, 3-dimethyl-4-hydroxybutyric acid
Example 5
Example 5 production of pantothenic acid by fermentation with recombinant E.coli.
The recombinant expression plasmid vector pze-mdh-M7 is transformed into recombinant escherichia coli WL148, and the background of the escherichia coli WL148 is WL148 (M1-93_panC (E.coli), cm_M1-93_panE (E.coli), dilvA, dtdcB, dldhA, dsad: FRT-M1-93-yihU, dadhE:: FRT-trc-panC (C.Glutam), dfrd:: FRT-M1-93-yqhE). Because the recombinant escherichia coli WL148 contains the (R) -carbonyl reductase encoding gene panE and the condensed enzyme encoding gene panC, no recombinant plasmid containing the (R) -carbonyl reductase encoding gene panE and the condensed enzyme encoding gene panC is required to be additionally constructed.
After Plasmid extraction using Plasmid Mini Kit I, recombinant E.coli WL148 was transformed and cultured overnight with ampicillin resistant plates, and positive clones were picked and inoculated with 2mL of LB liquid medium containing 50. Mu.g/mL of ampicillin, and cultured overnight at 37℃and 220rpm (about 14 h). A100 mL Erlenmeyer flask containing 10mL of fermentation medium was transferred at an initial OD of 0.05, and incubated at 30℃and 220 rpm. Each fermentation bottle was charged with 0.5g CaCO3 to adjust the pH of the fermentation broth. After 12h of incubation, the broth was collected and when OD600 = 5 was detected, 10. Mu.L of 1M TPTG solution was added for induction, and substrate was added to give an initial concentration of 10 g/L2-hydroxy-3, 3-dimethyl-4-aldehydebutyric acid and an initial concentration of 5.8g/L beta-alanine, and after 30h of incubation, the broth was collected and assayed for the concentration of 2-hydroxy-3, 3-dimethyl-4-aldehydebutyric acid and pantothenic acid (see Table below). The fermentation result shows that the reductase generated by the overexpression of the mutant mdh gene can obviously improve the conversion rate of the substrate.
In this example, recombinant E.coli WL148, which was not transformed with the recombinant expression plasmid, was used as a control. The results are shown in Table 4.
TABLE 4 pantothenate assay results Table
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
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Claims (20)
1. A preparation method of polysubstituted butyric acid containing 2-carbonyl is characterized in that polysubstituted butyric acid containing (S) -2-hydroxy is taken as a substrate, and (S) -2-hydroxy oxidase is added for enzyme catalytic reaction, and the substrate is converted into polysubstituted butyric acid containing 2-carbonyl under the action of the (S) -2-hydroxy oxidase.
2. The method for producing 2-carbonyl group-containing polysubstituted butyric acid according to claim 1, wherein said (S) -2-hydroxy group-containing polysubstituted butyric acid is S-2-hydroxy-3, 3-dimethyl-4-hydroxybutyric acid, and said 2-carbonyl group-containing polysubstituted butyric acid is 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid.
3. The method for producing 2-carbonyl group-containing polysubstituted butyric acid according to claim 1, wherein the (S) -2-hydroxy group-containing polysubstituted butyric acid is S-2-hydroxy-3, 3-dimethyl-4-aldehydebutyric acid, and the 2-carbonyl group-containing polysubstituted butyric acid is 2-carbonyl-3, 3-dimethyl-4 aldehydebutyric acid.
4. The method for producing 2-carbonyl group-containing polysubstituted butyric acid according to claim 1, wherein (S) -2-hydroxy group-containing polysubstituted butyric acid is used as a substrate, a recombinant microorganism containing a gene encoding (S) -2-hydroxy oxidase is added for fermentation culture, and in the fermentation process, the (S) -2-hydroxy group-containing polysubstituted butyric acid is converted into 2-carbonyl group-containing polysubstituted butyric acid under the action of (S) -2-hydroxy oxidase produced by overexpression of the recombinant microorganism.
5. The method for producing 2-carbonyl group-containing polysubstituted butyric acid according to claim 1, wherein the (S) -2-hydroxyoxidase encoding gene comprises one or more of Mdh, mdh1, mdh2, leuB and PMDH 2.
6. The method for producing 2-carbonyl group-containing polysubstituted butyric acid according to claim 1, wherein the (S) -2-hydroxyoxidase gene increases the activity of the overexpressed enzyme on the substrate by mutation or evolution.
7. The method for preparing 2-carbonyl group-containing polysubstituted butyric acid according to claim 6, wherein the (S) -2-hydroxy oxidase gene comprises a mutant mdh gene mutated at position I12V, R81A, M85Q, D86S, L149A, G179D, A G, and the nucleotide sequence of the mutant mdh gene is shown in SEQ ID NO. 1.
8. The method for preparing polysubstituted butyric acid containing 2-carbonyl group according to claim 7, wherein the preparation method of mutant mdh gene comprises the following steps: designing a mutation primer according to the locus of a mutated base, amplifying a plurality of gene fragments with different mutations by using the escherichia coli MG1655 genome as a template through PCR, and integrating the plurality of gene fragments to obtain the mdh gene with the mutation.
9. The method for producing 2-carbonyl group-containing polysubstituted butyric acid according to claim 3, wherein said S-2-hydroxy-3, 3-dimethyl-4-aldehydebutyric acid is produced by using glyoxylate and isobutyraldehyde as raw materials.
10. A process for preparing D-pantothenic acid, wherein the 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid of claim 2 is reacted with (R) -carbonyl reductase to give (R) -2-hydroxy-3, 3-dimethyl-4-hydroxybutyric acid, and the (R) -2-hydroxy-3, 3-dimethyl-4-hydroxybutyric acid is reacted with beta-alanine to give D-pantothenic acid.
11. A process for preparing D-pantothenic acid, wherein 2-carbonyl-3, 3-dimethyl-4-aldehyde butyric acid according to claim 3 is converted into 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid by carbonyl reductase, and then into (R) -2-hydroxy-3, 3-dimethyl-4-hydroxybutyric acid, and D-pantothenic acid is obtained by condensing (R) -2-hydroxy-3, 3-dimethyl-4-hydroxybutyric acid with beta-alanine.
12. The process for producing D-pantothenic acid according to claim 10, wherein (S) -2-hydroxy-3, 3-dimethyl-4-hydroxybutyric acid and beta-alanine are used as raw materials, a recombinant microorganism containing a gene encoding (S) -2-hydroxy oxidase, a gene encoding (R) -carbonyl reductase and a gene encoding a condensed enzyme is added for fermentation, and in the fermentation process, (S) -2-hydroxy-3, 3-dimethyl-4-hydroxybutyric acid is converted into 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid under the action of (S) -2-hydroxy oxidase overexpressed by the recombinant microorganism; 2-carbonyl-3, 3-dimethyl-4-hydroxybutyric acid is converted to (R) -2-hydroxy-3, 3-dimethyl-4-hydroxybutyric acid under the action of a recombinant microorganism overexpressed (R) -carbonyl reductase; (R) -2-hydroxy-3, 3-dimethyl-4-hydroxybutyric acid and beta-alanine are subjected to the action of recombinant microorganism overexpressed condensed enzyme to obtain D-pantothenic acid.
13. The process for preparing D-pantothenic acid according to claim 10, wherein said (R) -carbonyl reductase is capable of converting a ketone organic compound into an alcohol organic compound, and wherein said gene encoding (R) -carbonyl reductase comprises one or more of panE, PAN5, apbA and ylbQ; the condensation enzyme coding gene comprises one or more of panC and cmk.
14. The method according to claim 4 or 12, wherein the fermentation temperature is 20-90 ℃ and the pH is 6.0-8.0 during fermentation.
15. The method according to claim 4 or 12, characterized in that the fermentation medium used in the fermentation comprises the following raw materials: 11-13 g/L of M9 salt, 1-3 g/L of magnesium sulfate, 0.1-0.4 g/L of calcium chloride, 0.01-0.02 mg/mL of thiamine, 30-50 g/L of D-glucose, 2-6 g/L of yeast powder and 40-60 mu g/mL of kanamycin sulfate.
16. The method of claim 4 or 12, comprising constructing the recombinant microorganism by genetic engineering methods comprising plasmid expression or genomic integration.
17. The method according to claim 4 or 12, wherein the recombinant microorganism is constructed by means of plasmid expression by: obtaining a target gene through PCR amplification, connecting the obtained target gene to a plasmid vector, converting the plasmid vector into competent cells, and sequencing to obtain a recombinant expression plasmid vector; and (3) transforming the recombinant expression plasmid vector into a microorganism to obtain the recombinant microorganism.
18. The method of claim 17, wherein the microorganism comprises one or more of escherichia coli, bacillus, corynebacterium, yeast, or streptomyces.
19. The method of claim 17, wherein the microorganism comprises one or more of Escherichia coli (Escherichia coli), bacillus subtilis (Bacillus subtilis), bacillus megaterium (Bacillus megaterium), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), corynebacterium glutamicum (Corynebacterium glutamicum), saccharomyces cerevisiae (Saccharomyces cerevisiae), candida utilis (Candida) or Pichia pastoris.
20. A process for the preparation of panthenol, characterized in that the process according to any one of claims 10 to 13, wherein R-pantoic acid is also produced during the preparation, and the obtained R-pantoic acid is reacted with β -alaninol to obtain panthenol.
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