CN116904382B - 联产二羟基丙酮和波色因的宿主细胞及方法 - Google Patents
联产二羟基丙酮和波色因的宿主细胞及方法 Download PDFInfo
- Publication number
- CN116904382B CN116904382B CN202311008580.4A CN202311008580A CN116904382B CN 116904382 B CN116904382 B CN 116904382B CN 202311008580 A CN202311008580 A CN 202311008580A CN 116904382 B CN116904382 B CN 116904382B
- Authority
- CN
- China
- Prior art keywords
- host cell
- dihydroxyacetone
- gene
- seq
- glycerol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- RXKJFZQQPQGTFL-UHFFFAOYSA-N dihydroxyacetone Chemical compound OCC(=O)CO RXKJFZQQPQGTFL-UHFFFAOYSA-N 0.000 title claims abstract description 68
- 229940120503 dihydroxyacetone Drugs 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 36
- 101000892220 Geobacillus thermodenitrificans (strain NG80-2) Long-chain-alcohol dehydrogenase 1 Proteins 0.000 claims abstract description 14
- 101150033131 sthA gene Proteins 0.000 claims abstract description 7
- NIFJEQGFAKTKFF-KVPKETBZSA-N 1-[(2s,3r,4s,5r)-3,4,5-trihydroxyoxan-2-yl]propan-2-one Chemical compound CC(=O)C[C@@H]1OC[C@@H](O)[C@H](O)[C@H]1O NIFJEQGFAKTKFF-KVPKETBZSA-N 0.000 claims abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 6
- 101710191924 NADPH-dependent methylglyoxal reductase GRE2 Proteins 0.000 claims abstract description 5
- 239000005740 Boscalid Substances 0.000 claims abstract description 4
- WYEMLYFITZORAB-UHFFFAOYSA-N boscalid Chemical compound C1=CC(Cl)=CC=C1C1=CC=CC=C1NC(=O)C1=CC=CN=C1Cl WYEMLYFITZORAB-UHFFFAOYSA-N 0.000 claims abstract description 4
- 229940118790 boscalid Drugs 0.000 claims abstract description 4
- 241000588724 Escherichia coli Species 0.000 claims description 10
- 108010050375 Glucose 1-Dehydrogenase Proteins 0.000 claims description 10
- 108010000445 Glycerate dehydrogenase Proteins 0.000 claims description 10
- 108010029645 galactitol 2-dehydrogenase Proteins 0.000 claims description 10
- 101150000475 pntAB gene Proteins 0.000 claims description 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 8
- 239000013612 plasmid Substances 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 101100069419 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) GRE2 gene Proteins 0.000 claims description 3
- 230000001404 mediated effect Effects 0.000 claims description 3
- 108091033409 CRISPR Proteins 0.000 claims description 2
- 238000010354 CRISPR gene editing Methods 0.000 claims description 2
- 101150054144 GRE2 gene Proteins 0.000 claims description 2
- 101150019455 gdh gene Proteins 0.000 claims description 2
- 230000002401 inhibitory effect Effects 0.000 claims description 2
- 230000017105 transposition Effects 0.000 claims description 2
- 241000672609 Escherichia coli BL21 Species 0.000 claims 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 abstract description 15
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-O 0.000 abstract description 11
- 108090000854 Oxidoreductases Proteins 0.000 abstract description 7
- 239000011521 glass Substances 0.000 abstract description 6
- 102000004190 Enzymes Human genes 0.000 abstract description 5
- 108090000790 Enzymes Proteins 0.000 abstract description 5
- 241000894006 Bacteria Species 0.000 abstract description 3
- 239000000758 substrate Substances 0.000 abstract description 3
- 238000009776 industrial production Methods 0.000 abstract description 2
- 238000004064 recycling Methods 0.000 abstract description 2
- 230000002194 synthesizing effect Effects 0.000 abstract description 2
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH 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](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 abstract 1
- 108010031318 Vitronectin Proteins 0.000 description 16
- 102100035140 Vitronectin Human genes 0.000 description 16
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 11
- CSCPPACGZOOCGX-UHFFFAOYSA-N acetone Substances CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 7
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 6
- 102000004316 Oxidoreductases Human genes 0.000 description 6
- 229930027917 kanamycin Natural products 0.000 description 6
- 229960000318 kanamycin Drugs 0.000 description 6
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound 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 SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 6
- 229930182823 kanamycin A Natural products 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 210000003491 skin Anatomy 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 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 5
- 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 4
- 229960000723 ampicillin Drugs 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000008929 regeneration Effects 0.000 description 4
- 238000011069 regeneration method Methods 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 3
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- BULLHNJGPPOUOX-UHFFFAOYSA-N chloroacetone Chemical compound CC(=O)CCl BULLHNJGPPOUOX-UHFFFAOYSA-N 0.000 description 3
- 239000005515 coenzyme Substances 0.000 description 3
- 239000002537 cosmetic Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 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 3
- 239000000463 material Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- KOGFZZYPPGQZFZ-QVAPDBTGSA-N (2s,3r,4s,5r)-2-(2-hydroxypropyl)oxane-3,4,5-triol Chemical compound CC(O)C[C@@H]1OC[C@@H](O)[C@H](O)[C@H]1O KOGFZZYPPGQZFZ-QVAPDBTGSA-N 0.000 description 1
- MTZYZFYTYOADPI-UHFFFAOYSA-N 3-(oxan-2-yl)propan-1-ol Chemical compound OCCCC1CCCCO1 MTZYZFYTYOADPI-UHFFFAOYSA-N 0.000 description 1
- 108010031132 Alcohol Oxidoreductases Proteins 0.000 description 1
- 102000005751 Alcohol Oxidoreductases Human genes 0.000 description 1
- 235000018185 Betula X alpestris Nutrition 0.000 description 1
- 235000018212 Betula X uliginosa Nutrition 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- 241000218657 Picea Species 0.000 description 1
- 108050007763 Soluble pyridine nucleotide transhydrogenases Proteins 0.000 description 1
- -1 aldehyde ketones Chemical class 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000007036 catalytic synthesis reaction Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000004177 elastic tissue Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000000434 stratum corneum Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 235000019605 sweet taste sensations Nutrition 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
-
- 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
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
- C12P7/28—Acetone-containing products
-
- 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)
- C12Y101/01006—Glycerol dehydrogenase (1.1.1.6)
-
- 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)
- C12Y101/01283—Methylglyoxal reductase (NADPH-dependent) (1.1.1.283)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
- C12Y106/01—Oxidoreductases acting on NADH or NADPH (1.6) with NAD+ or NADP+ as acceptor (1.6.1)
- C12Y106/01001—NAD(P)+ transhydrogenase (B-specific) (1.6.1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
- C12Y106/01—Oxidoreductases acting on NADH or NADPH (1.6) with NAD+ or NADP+ as acceptor (1.6.1)
- C12Y106/01002—NAD(P)+ Transhydrogenase (AB-specific) (1.6.1.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种联产二羟基丙酮和波色因的宿主细胞，其中所述宿主细胞进行了如下工程改造：引入了编码NADPH依赖性甲基乙二醛还原酶GRE2的基因，以及编码甘油脱氢酶GDH的基因。还公开了联产二羟基丙酮和波色因的方法。本发明的宿主菌共表达甘油脱氢酶、波色因还原酶和辅因子转化酶，并敲除sthA基因，从而实现辅因子NADPH和NAD+的循环利用。以甘油和1‑C‑(β‑D‑吡喃木糖基)‑丙酮为底物，通过控制甘油的量，最终达到同时高产二羟基丙酮和波色因的目的。这种方法合成玻色因和二羟基丙酮具有产量高、效率快、成本低和安全性好等优势，适用于玻色因和二羟基丙酮的工业化生产。
Description
技术领域
本发明涉及一种联产二羟基丙酮和波色因的宿主细胞及方法，涉及生物合成技术领域。
背景技术
近年来，随着人们的物质生活越来越丰富，对美的追求也成为日常消费的主流。因此，美妆护肤市场在整个市场中占据了越来越重要的地位。二羟基丙酮和玻色因是美妆市场的两大重要原材料，其生物酶法的合成技术具有效率高、成本低、无毒无害、经济价值高、产品质量高等一系列的优点，因此具有重要的市场价值。
二羟基丙酮(dihydroxyacetone，DHA)是一种有机化合物，无色至浅黄色的液体，具有甜味。在美容行业中，二羟基丙酮被广泛地用于制作自然的晒黑效果，因为它能够与皮肤表面的角质层反应，从而使皮肤看起来更显棕褐色。此外，它还被用于食品、医药和化学制品中。甘油脱氢酶是一种酶，它可以催化甘油的氧化反应。在这个反应中，甘油被氧化为二羟基丙酮以及NADH(还原型辅酶)。通过这个反应，甘油脱氢酶可以合成二羟基丙酮。需要注意的是，甘油脱氢酶合成二羟基丙酮的反应需要有NAD+参与，而NAD+是一种辅酶，需要从其他代谢过程中得到。因此，甘油脱氢酶合成二羟基丙酮的速率和NAD+的浓度有关。
玻色因(Pro-Xylane)，又名羟丙基四氢吡喃醇，常用于制备护肤品。它是一种天然多糖，主要来源于植物，如桦树、橡树和云杉等。玻色因可以促进胶原蛋白和弹性纤维的生成，从而改善皮肤的弹性和紧致度，同时还有助于提高皮肤的保湿能力。市面上的玻色因主要来源于植物提取或者化学合成，这些方法成本高、纯度低、操作复杂，且化学合成会导致非理想产物和分离困难等问题。目前玻色因的生物合成主要基于醛酮还原酶催化1-C-(β-D-吡喃木糖基)-丙酮生成，但是这种方法对辅酶的依赖性极强，且成本极高，限制了玻色因的产量和生产成本。
发明内容
本发明公开了一种联产二羟基丙酮和波色因的宿主细胞，其中宿主细胞进行了如下工程改造：引入了编码NADPH依赖性甲基乙二醛还原酶GRE2的基因，以及编码甘油脱氢酶GDH的基因。
优选的，所述宿主细胞还进行了如下工程改造：过表达pntAB基因。
优选的，所述宿主细胞还进行了如下工程改造：抑制sthA基因的表达。
优选的，所述NADPH依赖性甲基乙二醛还原酶GRE2的氨基酸序列如SEQ ID NO:1所示。
优选的，所述甘油脱氢酶GDH的氨基酸序列如SEQ ID NO:3所示。
优选的，pntAB基因的序列如SEQ ID NO:5所示。
优选的，通过CRISPR转座和crRNA的方式敲除sthA基因。
优选的，所述宿主细胞为大肠杆菌，尤其是大肠杆菌BL21、Rossetta、W3110或MG1655。
本发明还公开了一种质粒，构建有启动子介导的GRE2基因、GDH基因以及pntAB基因。质粒载体可以是Pet32a载体，启动子为T7-LacO启动子。
一种联产二羟基丙酮和波色因的方法，其特征在于其步骤包括：
(1)构建上述的宿主细胞；
(2)挑取宿主细胞单克隆，在培养基中进行培养；
(3)将1-C-(β-D-吡喃木糖基)-丙酮和甘油加入培养基中，继续培养，获得二羟基丙酮和波色因。
其中1-C-(β-D-吡喃木糖基)-丙酮可以使用木糖和氯丙酮聚合获得。
本发明构建了联产二羟基丙酮和波色因的宿主菌。在宿主菌中共表达甘油脱氢酶、波色因还原酶和辅因子转化酶，并敲除sthA基因，从而实现辅因子NADPH和NAD+的循环利用。以甘油和1-C-(β-D-吡喃木糖基)-丙酮为底物，通过控制甘油的量，最终达到同时高产二羟基丙酮和波色因的目的。这种方法合成玻色因和二羟基丙酮具有产量高、效率快、成本低和安全性好等优势，适用于玻色因和二羟基丙酮的工业化生产。
附图说明
图1玻色因还原酶Gre2催化模型。
图2 1-C-(β-D-吡喃木糖基)-丙酮合成模型。
图3玻色因还原酶Gre2催化生成玻色因结果。
图4甘油脱氢酶GDH催化模型。
图5Gre2-GDH催化生成玻色因和二羟基丙酮结果。
图6NAD+和NADPH再生体系模型。
图7NAD+和NADPH再生体系模型在Gre2-GDH联产玻色因和二羟基丙酮中的模型。
图8Donor序列的模型。
图9crRNA序列的模型。
具体实施方式
以下结合附图，通过实施例进一步说明本发明，但不作为对本发明的限制。以下提供了本发明实施方案中所使用的具体材料及其来源。但是，应当理解的是，这些仅仅是示例性的，并不意图限制本发明，与如下试剂和仪器的类型、型号、品质、性质或功能相同或相似的材料均可以用于实施本发明。下述实施例中所使用的实验方法如无特别说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
为了实现玻色因的合成，我们在大肠杆菌中引入了羰基还原酶AKR，来自毕赤酵母的NADPH依赖性甲基乙二醛还原酶GRE2广泛应用在各类醛酮类化合物的羰基还原上，因此我们尝试使用Gre2来实现1-C-(β-D-吡喃木糖基)-丙酮到玻色因的催化(见图1)。
玻色因还原酶Gre2的氨基酸序列见SEQ ID NO:1，经大肠杆菌密码子优化后得到SEQ ID NO:2。序列合成于广州艾基生物，并构建到pET28a上。构建好的质粒转染到BL21(DE3)中，涂布在含50mg/L卡那霉素的LB培养基上，37℃过夜培养挑取单克隆，于含50mg/L卡那霉素的LB液体培养基中培养至OD600值达到0.6，加入终浓度为0.05、0.1、0.2、0.5和1mM的IPTG，28℃诱导过夜。
1-C-(β-D-吡喃木糖基)-丙酮使用木糖和氯丙酮进行合成(见图2)。饱和木糖的DMF水溶液(含1M氢氧化钠)中加入1/10体积氯丙酮，在90℃反应过夜。1-C-(β-D-吡喃木糖基)-丙酮产量大概在88％。
将获得的1-C-(β-D-吡喃木糖基)-丙酮按照10％溶于PBS溶液中，加入到上述获得的菌株中，30℃反应12h，HPLC测定玻色因含量。结果见图3，玻色因还原酶Gre2能够将1-C-(β-D-吡喃木糖基)-丙酮还原成玻色因。尽管玻色因还原酶Gre2的表达量逐渐升高，玻色因的产量上升并不明显。我们推测在全细胞催化合成玻色因的过程中，NADPH的浓度不够导致了玻色因产量升高不明显。
实施例2
为了提升玻色因的产量，同时生产二羟基丙酮。我们在大肠杆菌中构建了甘油脱氢酶体系，催化甘油产二羟基丙酮，同时生产NADH(见图4)。我们使用的是大肠杆菌中NADH依赖性的甘油脱氢酶GDH，氨基酸序列见SEQ ID NO:3，经大肠杆菌密码子优化后得到SEQID NO:4。序列合成于广州艾基生物，并构建到pET32a上。构建好的质粒转染到实施例1中BL21(DE3)中，涂布在含50mg/L卡那霉素和氨苄霉素的LB培养基上，37℃过夜培养挑取单克隆，于含50mg/L卡那霉素和氨苄霉素的LB液体培养基中培养至OD600值达到0.6，加入终浓度为0.2mM的IPTG，28℃诱导过夜。将获得的1-C-(β-D-吡喃木糖基)-丙酮和甘油按照10％溶于PBS溶液中，加入到上述获得的菌株中，30℃反应12h，HPLC测定玻色因含量。结果见图5，加入甘油脱氢酶后，甘油可以被催化成二羟基丙酮，玻色因的产量有明显升高。
实施例3
为了提升玻色因和二羟基丙酮的产量。我们在大肠杆菌中构建了NAD+和NADPH再生的体系。首先，通过过表达pntAB(核苷酸序列见SEQ ID NO:5)来消耗NADH再生NADPH，同时消耗NADP+再生NAD+，同时来敲除可溶型吡啶核苷酸转氢酶sthA基因来抑制NADPH转化成NADH，以及抑制NAD+转化成NADP+(见图6)。这样可以在大肠杆菌体内提高NAD+和NADPH的浓度，来实现玻色因和二羟基丙酮底物浓度的提升(见图7)。为了解决这一问题，我们将T7-LacO启动子介导的GRE2、GDH、pntAB构建到Pet32a载体上(见图8)。同时在PSL1142载体(addgene)上插入crRNA-sthA和crRNA-IS168(见图9)(方法参考文献Zhang Y,Sun X,WangQ,Xu J,Dong F,Yang S,Yang J,Zhang Z,Qian Y,Chen J,Zhang J,Liu Y,Tao R,JiangY,Yang J,Yang S.Multicopy Chromosomal Integration Using CRISPR-AssociatedTransposases.ACS Synth Biol.2020Aug 21；9(8):1998-2008.doi:10.1021/acssynbio.0c00073.Epub 2020Jul 2.Erratumin:ACS Synth Biol.2020Oct 16；9(10):2860.PMID:32551502.)。将两个质粒转化到Rosetta(DE3)菌株中，涂布在含50mg/L卡那霉素和氨苄霉素的的LB培养基上，37℃过夜培养挑取单克隆，于含50mg/L卡那霉素和氨苄霉素的LB液体培养基中培养至OD600值达到0.6，加入终浓度为0.2mM的IPTG，28℃诱导过夜。菌株离心后，将获得的1-C-(β-D-吡喃木糖基)-丙酮和甘油按照10％V/V溶于PBS溶液中，加入到上述获得的菌株沉淀中，30℃反应12h，HPLC测定玻色因含量。插入NAD+和NADPH再生体系后，二羟基丙酮和玻色因的产量分别达到37.2g/L和55.1g/L。
Claims (7)
1.一种联产二羟基丙酮和波色因的宿主细胞，其中所述宿主细胞进行了如下工程改造：引入了编码NADPH依赖性甲基乙二醛还原酶GRE2的基因，以及编码甘油脱氢酶GDH的基因，其中GRE2的氨基酸序列如SEQ ID NO:1所示，GDH的氨基酸序列如SEQ ID NO:3所示，所述宿主细胞为大肠杆菌。
2.根据权利要求1所述的宿主细胞，其特征在于所述宿主细胞还进行了如下工程改造：过表达pntAB基因，其中pntAB基因的序列如SEQ ID NO:5所示。
3.根据权利要求2所述的宿主细胞，其特征在于所述宿主细胞还进行了如下工程改造：抑制sthA基因的表达。
4.根据权利要求3所述的宿主细胞，其特征在于通过CRISPR转座和crRNA的方式敲除sthA基因。
5.根据权利要求1-4中任一项所述的宿主细胞，其特征在于所述宿主细胞为大肠杆菌BL21、Rossetta、W3110或MG1655。
6.一种质粒，其特征在于构建有启动子介导的GRE2基因、GDH基因以及pntAB基因，其中GRE2的氨基酸序列如SEQ ID NO:1所示，GDH的氨基酸序列如SEQ ID NO:3所示，pntAB基因的序列如SEQ ID NO:5所示。
7.一种联产二羟基丙酮和波色因的方法，其特征在于其步骤包括：
（1）构建权利要求1-5中任一项所述的宿主细胞；
（2）挑取宿主细胞单克隆，在培养基中进行培养；
（3）将1-C-(β-D-吡喃木糖基)-丙酮和甘油加入培养基中，继续培养，获得二羟基丙酮和波色因。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2023108354446 | 2023-07-10 | ||
| CN202310835444 | 2023-07-10 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116904382A CN116904382A (zh) | 2023-10-20 |
| CN116904382B true CN116904382B (zh) | 2024-03-08 |
Family
ID=88367976
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311008580.4A Active CN116904382B (zh) | 2023-07-10 | 2023-08-11 | 联产二羟基丙酮和波色因的宿主细胞及方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116904382B (zh) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106350476A (zh) * | 2016-08-31 | 2017-01-25 | 中国科学院青岛生物能源与过程研究所 | 联产异戊二烯和1,3‑丙二醇的基因工程菌及构建方法和应用 |
| CN109312373A (zh) * | 2016-03-09 | 2019-02-05 | 布拉斯肯有限公司 | 用于共生产乙二醇和三碳化合物的微生物和方法 |
| CN116333907A (zh) * | 2022-07-13 | 2023-06-27 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | 一种芽孢杆菌及其制备玻色因的方法 |
-
2023
- 2023-08-11 CN CN202311008580.4A patent/CN116904382B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109312373A (zh) * | 2016-03-09 | 2019-02-05 | 布拉斯肯有限公司 | 用于共生产乙二醇和三碳化合物的微生物和方法 |
| CN106350476A (zh) * | 2016-08-31 | 2017-01-25 | 中国科学院青岛生物能源与过程研究所 | 联产异戊二烯和1,3‑丙二醇的基因工程菌及构建方法和应用 |
| CN116333907A (zh) * | 2022-07-13 | 2023-06-27 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | 一种芽孢杆菌及其制备玻色因的方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116904382A (zh) | 2023-10-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7044860B2 (ja) | 遺伝子工学菌 | |
| CA2529583C (en) | Oxidoreductase from pichia capsulata | |
| US20210254109A1 (en) | D-Glucaric Acid Producing Bacterium, and Method for Manufacturing D-Glucaric Acid | |
| Xu et al. | Biotechnological routes to pyruvate production | |
| WO2007142210A1 (ja) | 光学活性アルコールの製造方法 | |
| CN106868030B (zh) | 重组载体、含有其的工程菌及在产α-酮戊二酸的应用 | |
| Huwig et al. | Enzymatic synthesis of L-tagatose from galactitol with galactitol dehydrogenase from Rhodobacter sphaeroides D | |
| CN116904382B (zh) | 联产二羟基丙酮和波色因的宿主细胞及方法 | |
| CN110184288A (zh) | 没食子酸和原儿茶酸的制备方法及其反应催化剂的制备方法 | |
| CN108410831A (zh) | 酮酸还原酶、基因、工程菌及在合成手性芳香2-羟酸中的应用 | |
| WO2014003502A1 (ko) | 3-하이드록시프로피온산을 생산하는 재조합 미생물 및 이를 이용한 3-하이드록시프로피온산의 생산방법 | |
| CN107663517B (zh) | 一种l-乳酸催化反应体系及l-乳酸的制备方法 | |
| CN116376950A (zh) | 一种核酸构建体、细胞和四氢姜黄素的制备方法及应用 | |
| KR101919556B1 (ko) | 미생물 Acetobacterium woodii를 이용한 포르메이트(formate)의 생산 방법 | |
| Feng et al. | Whole-cell biotransformation for simultaneous synthesis of allitol and D-gluconic acid in recombinant Escherichia coli | |
| CN112126614B (zh) | 一种利用全细胞转化制备覆盆子酮的方法 | |
| CN110734936B (zh) | 一种多酶级联生产(r/s)-羟基蛋氨酸的方法 | |
| JP2008283917A (ja) | 乳酸の製造方法 | |
| Li et al. | Metabolic engineering of Escherichia coli for the production of glyoxylate from xylose | |
| Park et al. | Enantioselective bioconversion using Escherichia coli cells expressing Saccharomyces cerevisiae reductase and Bacillus subtilis glucose dehydrogenase | |
| KR20200121746A (ko) | 2,3-부탄디올 생산용 메탄자화균 형질전환체 | |
| US20220220490A1 (en) | A method for biologically producing sugar alcohol from agar | |
| KR102028161B1 (ko) | 형질전환 미생물을 이용한 2,3-부탄디올 생산방법 | |
| KR20200136242A (ko) | 포도당으로부터 글리세롤로의 전환률 향상을 위한 유도성 프로모터의 이용 | |
| JP2006503559A (ja) | Ｄ−マンニトールを製造するための方法並びに微生物 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |