CN115747181A - Rare earth dependent alcohol dehydrogenase mutant and application thereof - Google Patents
Rare earth dependent alcohol dehydrogenase mutant and application thereof Download PDFInfo
- Publication number
- CN115747181A CN115747181A CN202211395902.0A CN202211395902A CN115747181A CN 115747181 A CN115747181 A CN 115747181A CN 202211395902 A CN202211395902 A CN 202211395902A CN 115747181 A CN115747181 A CN 115747181A
- Authority
- CN
- China
- Prior art keywords
- alcohol dehydrogenase
- rare earth
- dependent alcohol
- mutant
- hydroxymethyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102000007698 Alcohol dehydrogenase Human genes 0.000 title claims abstract description 49
- 108010021809 Alcohol dehydrogenase Proteins 0.000 title claims abstract description 49
- 229910052761 rare earth metal Inorganic materials 0.000 title claims abstract description 44
- 230000001419 dependent effect Effects 0.000 title claims abstract description 42
- 150000002910 rare earth metals Chemical class 0.000 title claims abstract description 41
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 57
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 44
- NOEGNKMFWQHSLB-UHFFFAOYSA-N 5-hydroxymethylfurfural Chemical compound OCC1=CC=C(C=O)O1 NOEGNKMFWQHSLB-UHFFFAOYSA-N 0.000 claims abstract description 18
- RJGBSYZFOCAGQY-UHFFFAOYSA-N hydroxymethylfurfural Natural products COC1=CC=C(C=O)O1 RJGBSYZFOCAGQY-UHFFFAOYSA-N 0.000 claims abstract description 18
- 241000894006 Bacteria Species 0.000 claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims description 25
- PCSKKIUURRTAEM-UHFFFAOYSA-N 5-hydroxymethyl-2-furoic acid Chemical compound OCC1=CC=C(C(O)=O)O1 PCSKKIUURRTAEM-UHFFFAOYSA-N 0.000 claims description 24
- 150000001413 amino acids Chemical group 0.000 claims description 12
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 claims description 11
- 238000007254 oxidation reaction Methods 0.000 claims description 9
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 claims description 8
- 235000001014 amino acid Nutrition 0.000 claims description 8
- 229940024606 amino acid Drugs 0.000 claims description 8
- 241000589776 Pseudomonas putida Species 0.000 claims description 7
- IKHGUXGNUITLKF-XPULMUKRSA-N acetaldehyde Chemical compound [14CH]([14CH3])=O IKHGUXGNUITLKF-XPULMUKRSA-N 0.000 claims description 6
- 230000035772 mutation Effects 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 4
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 4
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 4
- 229960001230 asparagine Drugs 0.000 claims description 4
- 235000009582 asparagine Nutrition 0.000 claims description 4
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 4
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 4
- 239000013604 expression vector Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 abstract description 35
- 108090000790 Enzymes Proteins 0.000 abstract description 35
- 230000000694 effects Effects 0.000 abstract description 25
- CHTHALBTIRVDBM-UHFFFAOYSA-N furan-2,5-dicarboxylic acid Chemical compound OC(=O)C1=CC=C(C(O)=O)O1 CHTHALBTIRVDBM-UHFFFAOYSA-N 0.000 abstract description 10
- 238000013461 design Methods 0.000 abstract description 8
- 238000000034 method Methods 0.000 abstract description 8
- 238000013135 deep learning Methods 0.000 abstract description 5
- 230000006870 function Effects 0.000 abstract description 5
- 238000012216 screening Methods 0.000 abstract description 5
- 150000001875 compounds Chemical class 0.000 abstract description 3
- 102000004169 proteins and genes Human genes 0.000 description 13
- 235000018102 proteins Nutrition 0.000 description 8
- MMXZSJMASHPLLR-UHFFFAOYSA-N pyrroloquinoline quinone Chemical compound C12=C(C(O)=O)C=C(C(O)=O)N=C2C(=O)C(=O)C2=C1NC(C(=O)O)=C2 MMXZSJMASHPLLR-UHFFFAOYSA-N 0.000 description 8
- 230000003647 oxidation Effects 0.000 description 7
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 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 5
- FBWADIKARMIWNM-UHFFFAOYSA-N N-3,5-dichloro-4-hydroxyphenyl-1,4-benzoquinone imine Chemical compound C1=C(Cl)C(O)=C(Cl)C=C1N=C1C=CC(=O)C=C1 FBWADIKARMIWNM-UHFFFAOYSA-N 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000003197 catalytic effect Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000004090 dissolution Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- VDJKJPMLWJWQIH-UHFFFAOYSA-M 5-ethylphenazin-5-ium;ethyl sulfate Chemical compound CCOS([O-])(=O)=O.C1=CC=C2[N+](CC)=C(C=CC=C3)C3=NC2=C1 VDJKJPMLWJWQIH-UHFFFAOYSA-M 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- KKEYFWRCBNTPAC-UHFFFAOYSA-N Terephthalic acid Chemical compound OC(=O)C1=CC=C(C(O)=O)C=C1 KKEYFWRCBNTPAC-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000009510 drug design Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000002741 site-directed mutagenesis Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000006860 carbon metabolism Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 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 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000013473 artificial intelligence Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000013406 biomanufacturing process Methods 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000010801 machine learning Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920002635 polyurethane Polymers 0.000 description 1
- 239000004814 polyurethane Substances 0.000 description 1
- LHBNLZDGIPPZLL-UHFFFAOYSA-K praseodymium(iii) chloride Chemical compound Cl[Pr](Cl)Cl LHBNLZDGIPPZLL-UHFFFAOYSA-K 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- -1 rare earth ion Chemical class 0.000 description 1
- 238000009790 rate-determining step (RDS) Methods 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 229930195727 α-lactose Natural products 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- 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.)
-
- 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
- C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
-
- 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
-
- 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
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a rare earth dependent alcohol dehydrogenase mutant and application thereof, and relates to the technical field of enzyme engineering. The invention adopts a method of combining a MutComute design tool based on deep learning and a FoldX tool based on an energy function to determine the site to be mutated of the rare earth-dependent alcohol dehydrogenase PedH, and obtains mutants with the activity of improving the activity of methanol, ethanol or 5-hydroxymethylfurfural by 130-260% by screening, wherein the stability of the two mutants is improved simultaneously. The invention has important significance for constructing artificial methylotrophic bacteria, preparing important compounds such as FDCA and the like by green organisms and solving the problem that the high stability and the high activity of the enzyme can not be considered at the same time.
Description
Technical Field
The invention relates to the technical field of enzyme engineering, in particular to a rare earth dependent alcohol dehydrogenase mutant and application thereof.
Background
To obtain enzymes with superior performance, humans are continually exploring the development of methods for engineering proteins, such as primary rational design, directed evolution, and semi-rational design. In recent years, with the great improvement of computer computing capability and the rapid development of structure biology, computational biology and artificial intelligence technologies, computer-aided protein design has received great attention and has become a new important direction for protein engineering. Protein computational designs based on structural modeling and energy calculations (e.g., rosetta, foldX, etc.) and machine learning (e.g., MLDE, mutcomute, etc.) have achieved considerable performance.
The oxidation of alcohols to ketones or aldehydes is an important reaction in organic synthesis. Compared with the chemical method, the enzymatic oxidation of alcohol is carried out under mild conditions, does not use any toxic reagent, and has higher catalytic specificity and selectivity and fewer byproducts, so the enzymatic oxidation of alcohol is always the first choice for the green oxidation of alcohol. Alcohol Dehydrogenases (ADHs) are customary oxidases. In 2012, the first rare earth-dependent alcohol dehydrogenase XoxF was discovered and reported to show higher catalytic activity compared to calcium-dependent alcohol dehydrogenase that also uses PQQ as a coenzyme. Different from common metal ions, the rare earth elements have unique 4 f-valence electronic structures, so that the rare earth elements have excellent optical, electric, magnetic and catalytic properties. The characteristics of rare earth ion electron transition, spin coupling, orbital hybridization and the like can provide more possibilities for enzyme engineering.
A rare-earth dependent alcohol dehydrogenase PedH from Pseudomonas putida can realize heterologous soluble expression in Escherichia coli, and is convenient for enzyme engineering modification. There are studies showing that PedH-expressing Pseudomonas putida can grow on ethanol as the sole carbon source. In addition, pedH can catalyze methanol to generate formaldehyde, and the oxidation of methanol is a key rate-limiting step of carbon metabolism and is an indispensable step in natural methylotrophic bacteria and artificial methylotrophic bacteria. With the rapid growth of the world population and the rapid development of the industry, resource shortage and environmental pollution problems have become great challenges for human beings, and green bio-manufacturing represented by one-carbon metabolism is receiving increasing attention. Methanol, methane and other monocarbon compounds are ideal raw materials for green biological production of bulk chemicals due to the advantages of wide raw material source, low price, high reducing power and the like.
PedH is capable of catalyzing the formation of 5-hydroxymethyl-2-furancarboxylic acid (HFCA) from 5-Hydroxymethylfurfural (HMF) and further oxidation to 5-carboxaldehyde-2-carboxylic acid (FFCA). FFCA is a precursor to 2,5-furandicarboxylic acid (FDCA). FDCA is an important bio-based platform compound, has the potential to be used as a substitute of terephthalic acid (PTA) for synthesizing renewable polyethylene-2, 5-furandicarboxylate (PEF), and is widely applied to the production of various bio-based high molecular polymers, such as polyamide, polyester, polyurethane, and the like.
However, like most alcohol dehydrogenases, pedH suffers from problems of low activity and stability during catalytic oxidation, which limits its industrial application. Therefore, the method of combining the MutComute design tool based on deep learning and the FoldX tool based on the energy function is used for determining the site to be mutated of the rare earth-dependent alcohol dehydrogenase PedH, and screening to obtain the mutant with improved stability and/or improved activity on methanol, ethanol and 5-hydroxymethylfurfural. Can promote the development of carbon metabolism, improve the problems of resource shortage and environmental pollution, and promote the research of green biological manufacturing of important compounds such as FDCA and the like.
Disclosure of Invention
In order to solve the problem that the activity and the stability of the original rare earth-dependent alcohol dehydrogenase PedH derived from Pseudomonas putida are low, the invention provides a rare earth-dependent alcohol dehydrogenase PedH mutant and application thereof.
The invention uses a deep learning-based MutCompute online design tool to perform on rare earth-dependent alcohol dehydrogenase PedH (amino acid sequence is shown as SEQ ID NO:)Shown as IDNO.1 and shown as SEQ ID NO. 2) to obtain a series of predicted mutants, and calculating the binding free energy change delta G of each predicted mutant by using a FoldX tool based on an energy function. The first 10 mutants with the highest MutComute prediction score and the 10 mutants with the lowest Δ Δ G calculated by FoldX were selected for site-directed mutagenesis. Culturing, expressing and purifying the 20 mutants, determining the specific enzyme activities of the mutants on methanol, ethanol and 5-hydroxymethylfurfural by using an enzyme labeling instrument, and determining the dissolution temperature T of each mutant by using a protein stability analyzer m Finally obtaining the mutant of the rare earth dependent alcohol dehydrogenase PedH with improved activity and/or stability by combining mutation.
The mutant is Q207N, H486Y, Q207N/H486Y. Wherein Q207N represents: the amino acid at the 207 th position is mutated from glutamine to asparagine; H486Y represents: the 486 th amino acid is mutated from histidine into tyrosine; Q207N/H486Y represents: the amino acid at position 207 was mutated from glutamine to asparagine and the amino acid at position 486 was mutated from histidine to tyrosine.
The specific technical scheme is as follows:
the invention provides a rare earth dependent alcohol dehydrogenase mutant which is obtained by mutating wild type alcohol dehydrogenase from Pseudomonas putida (Pseudomonas putida), wherein the amino acid sequence of the wild type alcohol dehydrogenase is shown as SEQ ID NO.1, and the specific mutation is (1) and/or (2):
(1) The amino acid at the 207 th position is mutated from glutamine to asparagine;
(2) The amino acid at position 486 was mutated from histidine to tyrosine.
The invention also provides application of the rare earth dependent alcohol dehydrogenase mutant in catalyzing methanol to generate formaldehyde, catalyzing ethanol to generate acetaldehyde, or catalyzing 5-hydroxymethylfurfural to generate 5-hydroxymethyl-2-furancarboxylic acid.
The invention also provides a gene for coding the rare earth dependent alcohol dehydrogenase mutant.
Preferably, the nucleotide sequence of the gene is shown as SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO. 9.
The invention also provides application of the gene in catalyzing methanol to generate formaldehyde, catalyzing ethanol to generate acetaldehyde, or catalyzing 5-hydroxymethyl furfural to generate 5-hydroxymethyl-2-furancarboxylic acid.
The invention also provides an expression vector containing the gene. The original expression vector of the recombinant vector used in the present invention was pET28a.
The invention also provides a genetic engineering bacterium for expressing the rare earth dependent alcohol dehydrogenase mutant. The host cell of the genetically engineered bacterium used in the present invention is e.coli BL21 (DE 3).
The invention also provides application of the genetically engineered bacterium in catalyzing methanol to generate formaldehyde, catalyzing ethanol to generate acetaldehyde, or catalyzing 5-hydroxymethylfurfural to generate 5-hydroxymethyl-2-furancarboxylic acid.
The invention also provides a preparation method of formaldehyde, acetaldehyde or 5-hydroxymethyl-2-furancarboxylic acid, which is characterized in that the rare earth dependent alcohol dehydrogenase mutant is used for catalyzing methanol, ethanol or 5-hydroxymethyl furfural to perform oxidation reaction to prepare formaldehyde, acetaldehyde or 5-hydroxymethyl-2-furancarboxylic acid.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the invention, based on the rare earth dependent alcohol dehydrogenase, a MutCompute online design tool based on deep learning and a FoldX tool based on an energy function are used for predicting a to-be-mutated site, and a mutant with improved activity and/or stability is obtained by screening, so that the problem that high stability and high activity cannot be considered at the same time is solved. Wherein, the specific enzyme activities of the mutant Q207N, the mutant H486Y and the combined mutant Q207N/H486Y to ethanol are respectively 2.4 times, 2.6 times and 3.6 times of the original enzyme PedH, the specific enzyme activities to methanol are respectively 2.3 times, 2.8 times and 3.6 times of the original enzyme PedH, and the specific enzyme activities to 5-hydroxymethylfurfural are respectively 2.3 times, 2.6 times and 2.8 times of the original enzyme PedH. Wherein, the mutant H486Y and the combined mutant Q207N/H486Y have improved stability while improving the specific enzyme activity, and the Tm value of the dissolution temperature is respectively improved by 2.3 ℃ and 2.2 ℃ compared with the original enzyme PedH.
(2) The rational design method combining the MutComute design tool and the FoldX tool can rapidly obtain the rare earth-dependent alcohol dehydrogenase mutant with high stability and high activity by screening a small mutant library.
Drawings
FIG. 1 is a SDS-PAGE graph of expression purification of the PedH protein; m from left to right in order: maker,1: ultrafiltrate, 2: eluent, 3: flow-through, 4: and (6) supernatant fluid.
FIG. 2 is a diagram showing the activity ratios of the wild type PedH and the mutants Q207N, H486Y and Q207N/H486Y on three substrates.
FIG. 3 shows T of wild type PedH and Q207N, H486Y, Q207N/H486Y mutants m A map of values.
Detailed Description
Reagents used in upstream genetic engineering: coli BL21 (DE 3), plasmid pET28a, etc. used in the examples of the present invention were purchased from Novagen corporation; the gene synthesis, primer synthesis and sequence sequencing of the PedH are completed by the Ongko bioengineering GmbH.
Reagents used for the catalytic reaction: methanol, ethanol, tris and hydrochloric acid were purchased from national chemical group, inc.; PQQ (pyrroloquinoline quinone) was purchased from shishiei (shanghai) chemical industry development limited; praseodymium chloride and 5-hydroxymethylfurfural were obtained from the Aladdin reagent (Shanghai) Inc.; DCPIP (2, 6-dichlorophenolindophenol) was purchased from Shanghai Maxin Biotechnology, inc.; PES (phenazine ethosulfate) was purchased from Shanghai Yi En chemical technology, inc.
Definition of enzyme activity unit (U): the amount of enzyme required for consuming 1. Mu.M DCPIP per minute in the reaction system.
Example 1
And (3) constructing an original rare earth-dependent alcohol dehydrogenase recombinant strain.
Entrusted with Strobiological engineering, inc. to synthesize the gene sequence of original rare earth-dependent alcohol dehydrogenase (the amino acid sequence is shown in SEQ ID NO. 1), the sequence is shown in SEQ ID NO.2, and the gene sequence is inserted between the enzyme cutting sites BamHI and Hind III of the vector pET28a to obtain the recombinant plasmid pET28a-PedH. Transforming the recombinant plasmid into E.co, i BL21 (DE 3) competent cells by adopting a heat shock method, and obtaining the original rare earth-dependent alcohol dehydrogenase recombinant strain after sequencing verification is correct.
Example 2
Designing the site to be mutated of the rare earth dependent alcohol dehydrogenase.
High performance mutants of PedH were predicted using the deep learning based MutComute on-line design tool (https:// mutcomute. Com /). Specifically, after logging in a website to register a user, a PDB number (6 zcw) of the PedH is input, and the Predict is clicked to obtain a series of predicted mutants. The binding free energy change Δ Δ G for each predicted mutant was then calculated using the FoldX tool based on an energy function. 2 potential mutation sites are obtained by screening according to the MutComute prediction score and the delta G value calculated by FoldX.
Example 3
And (3) construction of the rare earth dependent alcohol dehydrogenase mutant.
Primers were designed according to the mutants obtained in example 2, and PedH was subjected to site-directed mutagenesis.
The specific method comprises the following steps:
1. whole plasmid PCR
Using pET28a-PedH plasmid as template, designing the upstream and downstream primers (Table 1) covering the mutation site to perform whole plasmid PCR.
TABLE 1 primers used for site-directed mutagenesis construction
| Primer name | Primer sequence (5 'to 3') |
| Q207N-F | TAATGCATATAATCCGGAAAATGGTGAACTGC |
| Q207N-R | CCGGATTATATGCATTAATTTTACCAACAACACCAAATTCA |
| H486Y-F | GAAGTTTGGCGTTATAAAAATTATGCACCGCTGTGG |
| H486Y-R | TTATAACGCCAAACTTCTTTACCGCTAACCGG |
PCR amplification System:
25. Mu.L of DNA polymerase was added,
1 μ L of upstream primer (10 μ M),
1 μ L of downstream primer (10 μ M),
template (5 ng/. Mu.L) 1. Mu.L,
ddH 2 O 22μL。
PCR amplification conditions:
1) Pre-denaturation: 3min at 98 ℃;
2) Denaturation: 10s at 98 ℃; annealing: 15s at 60 ℃; extension: 1min at 72 ℃; the cycle is 33 times in total;
3) And (3) post-extension: 5min at 72 ℃;
4) Storing at 4 deg.C.
2. Transformation and validation
Directly transforming the PCR product into E.coli BL21 (DE 3) competent cells by adopting a heat shock method, and obtaining rare earth-dependent alcohol dehydrogenase mutant strains after sequencing verification, wherein the mutant strains are named as a mutant strain Q207N (the nucleotide sequence for coding the rare earth-dependent alcohol dehydrogenase mutant is shown as SEQ ID No. 7), a mutant strain H486Y (the nucleotide sequence for coding the rare earth-dependent alcohol dehydrogenase mutant is shown as SEQ ID No. 8) and a mutant strain Q207N/H486Y (the nucleotide sequence for coding the rare earth-dependent alcohol dehydrogenase mutant is shown as SEQ ID No. 9).
Example 4
Strain culture and protein expression and purification.
Single colonies of the recombinant bacteria (original rare earth-dependent alcohol dehydrogenase recombinant bacteria and rare earth-dependent alcohol dehydrogenase mutant strains) with the correct sequencing in example 3 were picked up in 5mL of LB liquid medium (containing 50. Mu.g/mL of kanamycin), and cultured with shaking at 37 ℃ for 12 hours. The resulting mixture was inoculated into 50mL of LB liquid medium containing 50. Mu.g/mL kanamycin and 10g/L α -lactose in an amount of 2% and cultured with shaking at 30 ℃ for 12 hours.
After the culture was completed, the bacterial solution was centrifuged at 4000 Xg and 4 ℃ for 15min, and the supernatant was discarded to collect the cells. After the cells were washed with 100mM Tris-HCl buffer solution, pH 8.0, they were resuspended in Tris-HCl buffer solution and placed in an ice-water bath for ultrasonication until they were clarified. The cell-breaking solution was centrifuged at 8000 Xg at 4 ℃ for 20min, the supernatant was subjected to affinity chromatography using a nickel column, impurities were washed with a washing buffer (100 mM Tris-HCl buffer, 150mM NaCl,50mM imidazole, pH 8.0), the target protein was eluted with an elution buffer (100 mM Tris-HCl buffer, 150mM NaCl,250mM imidazole, pH 8.0), and the separated target protein was subjected to desalting concentration in an ultrafiltration tube to obtain a pure target protein, which was then subjected to SDS-PAGE detection, and the results are shown in FIG. 1. A distinct protein band between 55kDa and 70kDa is present in lane 1 (ultrafiltrate), lane 2 (eluate) and lane 4 (supernatant), which coincides with the theoretical protein molecular weight of PedH (63.0 kDa), indicating successful purification of PedH.
Example 5
And (3) measuring the enzyme activity of the rare earth dependent alcohol dehydrogenase.
And quantitatively analyzing the amount of DCPIP consumed in the reaction system by using an enzyme reader, and calculating to obtain the specific enzyme activities of the original rare earth-dependent alcohol dehydrogenase and the rare earth-dependent alcohol dehydrogenase mutant on the substrates of methanol, ethanol and 5-hydroxymethylfurfural. The reaction system contained appropriate amounts of enzyme solution, 1.5. Mu.M PQQ, 1.5. Mu.M MPrCl 3 15mM substrate, 200. Mu.L total. The reaction medium was Tris-HCl buffer (100mM, pH 8.0) containing 1mM PES and 150. Mu.M DCPIP. The specific enzyme activity determination result is shown in figure 2, and the specific enzyme activities of the mutant Q207N, the H486Y and the combined mutant Q207N/H486Y on three substrates are obviously improved compared with the original enzyme PedH. The specific enzyme activities of the original enzyme PedH to ethanol, methanol and 5-hydroxymethylfurfural are 0.433U/mg, 0.088U/mg and 0.053U/mg respectively. The specific enzyme activities of the mutants Q207N, H486Y and the combined mutant Q207N/H486Y to ethanol are respectively 2.4 times, 2.6 times and 3.6 times of the original enzyme PedH, and the specific enzyme activities to methanol are respectively the original enzyme PedH of 2.3, 2.8 and 3.6 times, and specific enzyme activity to 5-hydroxymethyl furfural of 2.3, 2.6 and 2.8 times of original enzyme PedH.
Example 6
T of rare earth-dependent alcohol dehydrogenases m And (4) measuring the value.
The enzyme solution concentrations of the original rare earth-dependent alcohol dehydrogenase and the rare earth-dependent alcohol dehydrogenase mutant are uniformly adjusted to 10 μ M, and added into a high-precision quartz glass capillary tube set, and T is measured by using a protein stability analyzer (Prometheus NT. Plex) m The values, determined as shown in FIG. 3, can be seen in the dissolution temperature T of mutant Q207N with wild type PedH m The values are not obviously different, but the stability of the mutant H486Y and the combined mutant Q207N/H486Y is improved, and the dissolution temperature T is improved m The values were increased by 2.3 ℃ and 2.2 ℃ respectively compared with the wild type PedH (63.7 ℃).
Claims (9)
1. A rare earth dependent alcohol dehydrogenase mutant is characterized by being obtained by mutating wild type alcohol dehydrogenase from Pseudomonas putida (Pseudomonas putida), wherein the amino acid sequence of the wild type alcohol dehydrogenase is shown as SEQ ID NO.1, and the specific mutation is (1) and/or (2):
(1) The amino acid at the 207 th site is mutated from glutamine to asparagine;
(2) The amino acid at position 486 was mutated from histidine to tyrosine.
2. The use of the rare earth-dependent alcohol dehydrogenase mutant of claim 1 in catalyzing methanol to formaldehyde, ethanol to acetaldehyde, or 5-hydroxymethylfurfural to 5-hydroxymethyl-2-furancarboxylic acid.
3. A gene encoding the rare earth-dependent alcohol dehydrogenase mutant according to claim 1.
4. The gene of claim 3, wherein the nucleotide sequence of the gene is as shown in SEQ ID No.7, SEQ ID No.8 or SEQ ID No. 9.
5. The use of the gene of claim 3 in catalyzing methanol to formaldehyde, ethanol to acetaldehyde, or 5-hydroxymethylfurfural to 5-hydroxymethyl-2-furancarboxylic acid.
6. An expression vector comprising the gene of claim 3.
7. A genetically engineered bacterium expressing the rare earth-dependent alcohol dehydrogenase mutant of claim 1.
8. The use of the genetically engineered bacterium of claim 7 in catalyzing methanol to formaldehyde, catalyzing ethanol to acetaldehyde, or catalyzing 5-hydroxymethylfurfural to 5-hydroxymethyl-2-furancarboxylic acid.
9. A preparation method of formaldehyde, acetaldehyde or 5-hydroxymethyl-2-furancarboxylic acid, which is characterized in that the rare earth-dependent alcohol dehydrogenase mutant of claim 1 is used for catalyzing methanol, ethanol or 5-hydroxymethyl furfural to perform oxidation reaction to prepare formaldehyde, acetaldehyde or 5-hydroxymethyl-2-furancarboxylic acid.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211395902.0A CN115747181A (en) | 2022-11-04 | 2022-11-04 | Rare earth dependent alcohol dehydrogenase mutant and application thereof |
| PCT/CN2022/138388 WO2024092967A1 (en) | 2022-11-04 | 2022-12-12 | Rare earth-dependent alcohol dehydrogenase mutant and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211395902.0A CN115747181A (en) | 2022-11-04 | 2022-11-04 | Rare earth dependent alcohol dehydrogenase mutant and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115747181A true CN115747181A (en) | 2023-03-07 |
Family
ID=85368417
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211395902.0A Pending CN115747181A (en) | 2022-11-04 | 2022-11-04 | Rare earth dependent alcohol dehydrogenase mutant and application thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN115747181A (en) |
| WO (1) | WO2024092967A1 (en) |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140377817A1 (en) * | 2013-06-25 | 2014-12-25 | Ut-Battelle, Llc | Heat stable, fe dependent alcohol dehydrogenase for aldehyde detoxification |
| EP3052635A4 (en) * | 2013-10-04 | 2017-06-21 | Genomatica, Inc. | Alcohol dehydrogenase variants |
| EP3222712A1 (en) * | 2016-03-22 | 2017-09-27 | Universität zu Köln | Alcohol dehydrogenase from pichia pastoris and use thereof |
| CN109402076A (en) * | 2018-10-30 | 2019-03-01 | 江南大学 | A kind of Alcohol dehydrogenase mutant and its application in regenerating coenzyme |
-
2022
- 2022-11-04 CN CN202211395902.0A patent/CN115747181A/en active Pending
- 2022-12-12 WO PCT/CN2022/138388 patent/WO2024092967A1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| WO2024092967A1 (en) | 2024-05-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108118041B (en) | Phospholipase D mutant, recombinant genetic engineering bacterium, and preparation methods and applications thereof | |
| KR19990087330A (en) | Cloning and Expression of the Gene Encoding Secondary Alcohol Dehydrogenase of Thermoaerobacter Ethanolicus 39E and Biochemical Characterization of the Enzyme | |
| Inoue et al. | Type 1 Cu structure of blue nitrite reductase from Alcaligenes xylosoxidans GIFU 1051 at 2.05 Å resolution: comparison of blue and green nitrite reductases | |
| Obradors et al. | Site‐directed mutagenesis studies of the metal‐binding center of the iron‐dependent propanediol oxidoreductase from Escherichia coli | |
| Aro-Kärkkäinen et al. | L-arabinose/D-galactose 1-dehydrogenase of Rhizobium leguminosarum bv. trifolii characterised and applied for bioconversion of L-arabinose to L-arabonate with Saccharomyces cerevisiae | |
| CN113249349B (en) | Mutant alcohol dehydrogenase, recombinant vector, preparation method and application thereof | |
| CN116240187A (en) | Prolyl hydroxylase alpha 1 subunit mutant, coding gene and application thereof in catalyzing hydroxylation of proline | |
| CN115747181A (en) | Rare earth dependent alcohol dehydrogenase mutant and application thereof | |
| CN113088501A (en) | Glutamic acid dehydrogenase mutant for producing L-glufosinate-ammonium and L-glufosinate-ammonium production method | |
| CN111004794B (en) | Subtilisin E mutant with improved thermal stability and application thereof | |
| CN117384873A (en) | Glycerol phosphate oxidase mutant and screening method, preparation method and application thereof | |
| CN113151210B (en) | Peroxidase mutant with high specific enzyme activity and application thereof | |
| CN116376855A (en) | Rare earth-dependent thermostable alcohol dehydrogenase mutant and application thereof | |
| CN115927228B (en) | Mutant and construction method and application thereof | |
| CN114525266B (en) | Phospholipase D mutant from Antarctic bacteria and application thereof | |
| CN114480342B (en) | Mutant PET hydrolase, recombinant vector, recombinant engineering bacterium and application thereof | |
| CN114621944B (en) | Arginine deiminase mutant with improved enzyme activity | |
| CN117535265A (en) | Recombinant 9 DEG N DNA polymerase and application thereof in DNA information storage | |
| CN116445455B (en) | Heat-resistant alkali-resistant xylanase mutant and application thereof | |
| CN109735511B (en) | Preparation method of xanthine oxidase for clinical detection | |
| CN118516320A (en) | D-lactate dehydrogenase mutant, preparation method, encoding DNA and D-lactate production method and application thereof | |
| CN117165543A (en) | Alcohol dehydrogenase mutant derived from escherichia coli and application thereof | |
| CN117165544A (en) | Alcohol dehydrogenase mutant and application thereof in furfuryl alcohol synthesis | |
| CN116574709A (en) | DNA polymerase BstX mutant and construction method and application thereof | |
| CN118272339A (en) | Protein, preparation method and application thereof, biological material and application thereof |
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 |