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WO2022151611A1 - 细胞色素p450酶突变体及其应用 - Google Patents

细胞色素p450酶突变体及其应用 Download PDF

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WO2022151611A1
WO2022151611A1 PCT/CN2021/089667 CN2021089667W WO2022151611A1 WO 2022151611 A1 WO2022151611 A1 WO 2022151611A1 CN 2021089667 W CN2021089667 W CN 2021089667W WO 2022151611 A1 WO2022151611 A1 WO 2022151611A1
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pet
optionally substituted
group
preparation
enzyme
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PCT/CN2021/089667
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French (fr)
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洪浩
詹姆斯•盖吉
张娜
焦学成
马玉磊
曹珊
程逸冰
孙凯华
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凯莱英生命科学技术(天津)有限公司
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Priority to JP2023541086A priority Critical patent/JP7576179B2/ja
Priority to KR1020237027471A priority patent/KR20230141801A/ko
Priority to EP21918810.9A priority patent/EP4257679A4/en
Priority to US18/267,326 priority patent/US20240010997A1/en
Publication of WO2022151611A1 publication Critical patent/WO2022151611A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • C12N9/0081—Cholesterol monooxygenase (cytochrome P 450scc)(1.14.15.6)
    • 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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • 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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • 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
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/02—Oxidoreductases acting on a peroxide as acceptor (1.11) with H2O2 as acceptor, one oxygen atom of which is incorporated into the product (1.11.2)
    • C12Y111/02004—Fatty-acid peroxygenase (1.11.2.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/15—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced iron-sulfur protein as one donor, and incorporation of one atom of oxygen (1.14.15)
    • C12Y114/15006—Cholesterol monooxygenase (side-chain-cleaving) (1.14.15.6), i.e. cytochrome P450scc

Definitions

  • the present invention relates to the field of enzyme technology, in particular, to a cytochrome P450 enzyme mutant and its application.
  • Catalyzing the inverse Markov oxidation of olefinic raw materials can simplify the synthetic route of many important chemical raw materials.
  • the direct inverse Markov oxidation reaction of olefins to generate corresponding carbonyl compounds is a very important challenge in organic synthesis, which often requires the participation of efficient catalysts.
  • the use of noble metals as catalysts has problems such as low conversion efficiency and enantioselectivity, requiring multi-step catalysis, and three wastes [G.Dong,P.Teo,Z.K.Wickens,R.H.Grubbs,Primary alcohols from terminal olefins: Formal anti-Markovnikov hydration via triple relay catalysis. Science 333, 1609–1612 (2011)].
  • Cytochrome P450 monooxygenases are a family of heme-dependent enzymes that use oxygen as an oxidant to selectively activate C-H bonds under mild conditions, catalyzing a variety of synthesis that is difficult to achieve by traditional chemical methods. Reactions, including the oxidation of alkenes, have great potential for applications in fine chemistry and synthesis of drugs and their metabolites.
  • P450 (BM3) derived from Bacillus megaterium is a self-sufficient monooxygenase, that is, the redox protein chaperone involved in electron transfer is partially fused to a peptide chain of P450 oxidase. This fusion and recombination structure greatly improves the electron transfer efficiency and the electron coupling efficiency of the oxidation reaction.
  • BM3 is also one of the P450 enzymes with high catalytic efficiency.
  • the main purpose of the present invention is to provide a cytochrome P450 enzyme mutant and its application, so as to solve the problem that there is no P450 enzyme that can efficiently catalyze the anti-Markov oxidation reaction of olefin compounds in the prior art.
  • a cytochrome P450 enzyme mutant comprising: (a) a protein with one or more amino acid mutations in the sequence of SEQ ID NO: 1, And the protein has the anti-Markov oxidation activity of cytochrome P450 enzyme; or (b) is derived from Bacillus megaterium strain, and has an amino acid sequence with more than 80% homology with SEQ ID NO: 1, and has cytochrome P450 enzyme. Anti-Markov oxidation activity.
  • the mutant is a protein with any one or more of 1-14 amino acid mutations in the sequence of SEQ ID NO: 1, preferably 2-14, more preferably 3-14, further preferably 10-14 amino acid mutations , and the protein has the anti-Markov oxidation activity of cytochrome P450 enzymes.
  • the mutant is derived from Bacillus megaterium strain, and has 85% or more, preferably 90% or more, more preferably 95% or more, and more preferably 99% or more homology with SEQ ID NO: 1, and has cell homology Anti-Markov oxidation activity of pigment P450 enzyme; further preferably, the mutant has any one or more of 1 to 14 on the basis of SEQ ID NO: 1, preferably 2 to 14, more preferably 3 to 14, further preferably 10 to 14 amino acid mutations.
  • mutant is the following amino acid mutation on the basis of SEQ ID NO: 1:
  • a DNA molecule encoding any one of the above mutants is provided.
  • a recombinant plasmid is provided, wherein the above-mentioned DNA molecule is connected to the recombinant plasmid.
  • the recombinant plasmid is selected from any one of the following: pET-21b(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a (+), pET-14b, pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET -29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET- 40b(+),
  • a host cell is provided, the host cell containing any of the above-mentioned recombinant plasmids.
  • the host cell is a prokaryotic cell or a eukaryotic cell, preferably the eukaryotic cell is a yeast cell.
  • the host cell is a competent cell, preferably the competent cell is Escherichia coli BL21 cell or Escherichia coli W3110.
  • a method for preparing a carbonyl compound or an alcohol compound comprising: using any of the above cytochrome P450 enzyme mutants to catalyze olefin compounds Carry out a direct inverse Markov oxidation reaction to form carbonyl compounds and alcohol compounds wherein R represents an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group.
  • R represents an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group with 1-20 carbon atoms; preferably , R represents an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group with 1-10 carbon atoms; preferably, substituted Refers to being substituted by halogen atom, nitrogen atom, sulfur atom, hydroxyl, nitro, cyano, methoxy, ethoxy, carboxyl, carboxymethyl, carboxyethyl or methylenedioxy; preferably, olefins
  • the compound is a styrene compound substituted or unsubstituted at any position on the benzene ring, and the reaction is generate The inverse Markov oxidation
  • the wild enzyme is transformed into protein by means of directional evolution, the activity and selectivity of the enzyme are improved, and the P450 enzyme that can be used for industrial production is developed.
  • BM3 has the ability to catalyze the anti-Markov oxidation reaction of alkene compounds through enzyme screening.
  • its activity is low and the selectivity of anti-Markov oxidation is poor.
  • the inventors carried out protein modification of the wild enzyme by means of directed evolution, improved the activity and selectivity of the enzyme, and developed a P450 enzyme that can be used for industrial production.
  • the inventors of the present invention improve the P450 enzyme activity and the selectivity of anti-Markov oxidation derived from the wild-type strain of Bacillus megaterium through the method of directed evolution, and reduce the usage amount of the enzyme.
  • a mutation site was introduced into the wild-type P450 enzyme SEQ ID NO: 1 by means of whole plasmid PCR, the activity and selectivity of the mutant were detected, and the activity and selectivity (ie the proportion of aldehyde in the total product) were selected to improve mutants.
  • the anti-Markov oxidation reaction catalyzed by P450 enzyme is as follows:
  • the cytochrome P450 enzyme mutants provided by the present invention can catalyze alkene substrates to generate aldehydes, and the aldehydes can be further reduced to alcohols by coenzymes.
  • the wild-type P450 enzyme sequence SEQ ID NO: 1 derived from Bacillus megaterium is as follows:
  • site-directed mutagenesis refers to the introduction of desired changes (usually changes in a favorable direction) into the target DNA fragment (which can be a genome or a plasmid) by methods such as polymerase chain reaction (PCR), including base additions, deletions, point mutations, etc.
  • PCR polymerase chain reaction
  • Site-directed mutagenesis can rapidly and efficiently improve the properties and characterization of target proteins expressed by DNA, and is a very useful method in genetic research.
  • the method of introducing site-directed mutagenesis by whole plasmid PCR is simple and effective, and is currently used more frequently.
  • the principle is that a pair of primers (forward and reverse) containing the mutation site is annealed to the template plasmid and then "cycle-extended" with a polymerase.
  • the so-called cyclic extension means that the polymerase extends the primer according to the template, and returns to The 5' end of the primer is terminated, and after repeated heating, annealing and extension cycles, this reaction is different from rolling circle amplification, and multiple tandem copies will not be formed.
  • the extension products of the forward and reverse primers are annealed and paired into a nicked open-circular plasmid.
  • the extension product of Dpn I digestion because the original template plasmid was derived from conventional E. coli, was modified by dam methylation, and was sensitive to Dpn I and was chopped, while the plasmid with the mutated sequence synthesized in vitro was not methylated It is not cut, so it can be successfully transformed in the subsequent transformation, and the clone of the mutant plasmid can be obtained.
  • Error-prone PCR It means PCR under error-prone conditions, that is, PCR technology that easily makes mistakes in the copied DNA sequence, also known as mismatch PCR or error-prone PCR. Specifically, by using low-fidelity TaqDNA polymerase and changing PCR reaction conditions, the fidelity of DNA replication is reduced, and base mismatches are increased during the synthesis of new DNA strands, thereby causing more point mutations in the amplified product. A method for inducing DNA sequence variation in vitro.
  • Error-prone PCR is the simplest and most effective gene in vitro random mutagenesis technology.
  • Guanine (G), cytosine (C) and thymine (T) three oxygen-containing bases have keto and enol tautomers.
  • Adenine (A) and thymine, two nitrogenous bases, have two tautomers, amine and imine.
  • G, C and T mainly exist in the ketone structure, the ratio of the enol structure is extremely low, the nitrogen atoms on the two nitrogen-containing bases A and T mainly exist in the amino group (NH 2 ) state, and the imine group (NH 2 ) exists in the state. ) state exists at a very low rate.
  • the difference in the position of the hydrogen atom between different isomers and the difference in the deviation of the electron cloud at the same position can change the pairing form of the bases, so that mismatches may occur in the replicated daughter chain.
  • mismatches may occur in the replicated daughter chain.
  • thymine exists in a keto structure, it pairs with adenine, and when it exists in an enol structure, it pairs with guanine, so that A can be paired with C, and T can be paired with G. unstable bases Yes, resulting in a mismatch.
  • Taq DNA polymerase has the highest mismatch rate.
  • Taq DNA polymerase is one of the most active thermostable DNA polymerases found. It has 5'-3' exonuclease activity and no 3'-5' exonuclease activity. There is no correction function for nucleotide mismatches, so the probability of mismatches is higher than that of DNA polymerases with 3'-5' proofreading activity.
  • the fidelity of DNA polymerase can be reduced by various methods, including using 4 different concentrations of dNTPs, adding Mn 2+ , increasing the Mg 2+ concentration, etc. The mechanisms by which several mutagenesis methods lead to base changes in the amplified DNA strands vary.
  • MnC1 2 is a mutagenic factor of DNA polymerase.
  • the addition of Mn 2+ can reduce the specificity of the polymerase to the template and increase the mismatch rate; the unbalanced concentration of the four dNTPs can increase the probability of base misincorporation and achieve mismatch ;
  • Mg2+ has the effect of activating Taq enzyme, increasing the concentration of Mg 2+ beyond the normal dosage can stabilize non-complementary base pairs; increasing the dosage of Taq DNA polymerase and increasing the extension time of each cycle can increase the extension of mismatch terminals The probability of decreasing the initial template concentration will increase the proportion of variant templates in subsequent PCR cycles.
  • Saturation mutation is a method to obtain mutants in which the amino acids of the target site are replaced by other 19 amino acids in a short period of time by modifying the coding gene of the target protein. This method is not only a powerful tool for protein-directed engineering, but also an important means for the study of protein structure-function relationships. Saturation mutation often leads to more ideal evolutionary body than single point mutation. For these problems that the site-directed mutagenesis method cannot solve, it is precisely the uniqueness that the saturation mutation method is good at.
  • the mutant plasmid was transformed into E. coli cells as above, and overexpressed in E. coli.
  • the crude enzyme was then obtained by sonicating the cells.
  • the best condition for P450 induction expression 25°C, 0.2mM IPTG and 0.5mM Aminolevulinic acid (ALA) induced overnight.
  • a cytochrome P450 enzyme mutant comprising: (a) a protein with one or more amino acid mutations in the sequence of SEQ ID NO: 1, and the protein has The anti-Markov oxidation activity of cytochrome P450 enzymes; or (b) an amino acid sequence derived from a Bacillus megaterium strain and having more than 80% homology to SEQ ID NO: 1, and an anti-Markov enzyme with cytochrome P450 enzymes oxidative activity.
  • mutants provided in this example on the basis of maintaining the cytochrome P450 enzymatic activity, through the mutation of one or more amino acids, or through the homology of the mutated sequence with the sequence of the wild-type Bacillus megaterium strain at 80% % or more to further improve the enzymatic activity and/or the selectivity of anti-Markov oxidation.
  • the mutant has any one or more of 1-14, preferably 2-14, more preferably 3-14, further preferably 10-14, on the sequence of SEQ ID NO: 1 amino acid mutation, and the mutant has anti-Markov oxidation activity of cytochrome P450 enzyme.
  • the mutant is derived from Bacillus megaterium strain, and has more than 85% homology with SEQ ID NO: 1, preferably more than 90%, more preferably more than 95%, further preferably 99% homology and has the anti-Markov oxidation activity of cytochrome P450 enzymes; more preferably any one or more of 1 to 14 mutants (for example, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1), preferably 2 to 14, more preferably 3 to 14, further preferably 10 to 14 amino acid mutations.
  • the P450 enzyme mutant is a mutant having amino acid mutations as shown in Tables 1 to 4 on the basis of SEQ ID NO: 1.
  • the catalytic activity and/or selectivity of these mutants catalyzing the inverse Markov oxidation reaction of olefin compounds were significantly improved compared with the wild type.
  • a DNA molecule encoding any one of the above-mentioned P450 enzyme mutants is also provided.
  • the encoded P450 enzyme mutants have the advantages of high selectivity and significantly improved catalytic activity.
  • a recombinant plasmid is also provided, and the recombinant plasmid is linked with the above-mentioned DNA molecule.
  • the DNA molecule can encode any one of the above-mentioned P450 enzyme mutants with high selectivity and/or significantly improved catalytic activity.
  • the specific sequence is selected from the sequences in Table 1-Table 4 or the nucleotide sequences in which amino acid sequences at other sites are substituted, added or mutated with these sequences on the premise of maintaining the above amino acid site changes.
  • the recombinant plasmid is selected from one of the following: pET-22b(+), pET-21b(+), pET-3a(+), pET-3d(+), pET-11a(+ ), pET-12a(+), pET-14b, pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET- 21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a (+), pET-29a(+),
  • a host cell is also provided, and the host cell contains any of the above-mentioned recombinant plasmids.
  • Specific host cells can be prokaryotic cells or eukaryotic cells, preferably eukaryotic cells are yeast cells. More preferably, the above-mentioned host cells are competent cells, and more preferably, the competent cells are Escherichia coli BL21 cells or Escherichia coli W3110.
  • a method for preparing a carbonyl compound or an alcohol compound comprising: using any one of the above cytochrome P450 enzyme mutants to catalyze olefin compounds Carry out a direct inverse Markov oxidation reaction to form carbonyl compounds or alcohol compounds wherein R represents an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group.
  • R represents an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group with 1-20 carbon atoms, more preferably , R represents an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group with 1-10 carbon atoms; preferably, Substitution means substitution by halogen atom, nitrogen atom, sulfur atom, hydroxyl, nitro, cyano, methoxy, ethoxy, carboxyl, carboxymethyl, carboxyethyl or methylenedioxy; preferably, alkene
  • the compounds are styrene compounds substituted or unsubstituted at any position on the benzene ring, and the reaction is generate The inverse Markov oxidation reaction of , wherein,
  • halogen substitution is chlorine atom substitution.
  • the olefinic compound is any of the following:
  • the above-mentioned parent is SEQ ID NO: 1; the multiples of activity and selectivity increase are represented by +, + represents an increase of 0-1 times, ++ represents an increase of 1-2 times, +++ represents an increase of 2-3 times, ++ ++ means 3-5 times improvement, +++++ means 5-10 times improvement.
  • the selectivity of the present invention/the selectivity of the anti-Markov oxidation is defined as: % of aldehyde products in the product of anti-Markov oxidation/[(% of aldehyde products + % of epoxy products].
  • the above-mentioned parent is SEQ ID NO: 1; the multiples of activity and selectivity increase are represented by +, + represents an increase of 0-1 times, ++ represents an increase of 1-2 times, +++ represents an increase of 2-3 times, ++ ++ means 3-5 times improvement, +++++ means 5-10 times improvement.
  • the above-mentioned parent is SEQ ID NO: 1; the multiples of activity and selectivity increase are represented by +, + represents an increase of 0-1 times, ++ represents an increase of 1-2 times, +++ represents an increase of 2-3 times, ++ ++ means 3-5 times improvement, +++++ means 5-10 times improvement.
  • the above-mentioned parent is SEQ ID NO: 1; the multiples of activity and selectivity increase are represented by +, + represents an increase of 0-1 times, ++ represents an increase of 1-2 times, +++ represents an increase of 2-3 times, ++ ++ means 3-5 times improvement, +++++ means 5-10 times improvement.
  • any other combination of mutation sites disclosed in this application, as well as the repetition of mutation sites on other P450 enzymes with higher homology, may also have better effects.

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Abstract

提供了一种细胞色素P450酶突变体及其应用。通过定向进化的手段对来源于Bacillus megaterium野生型菌株的P450酶活和反马尔科夫氧化的选择性进行蛋白质改造，提高了酶的活性和选择性，开发出了一系列可用于工业化生产的P450酶突变体。

Description

细胞色素P450酶突变体及其应用 技术领域
本发明涉及酶技术领域，具体而言，涉及一种细胞色素P450酶突变体及其应用。
背景技术
催化烯烃类原料的反马尔科夫氧化反应可以简化很多重要化工原料的合成路线。对烯烃类化合物进行直接的反马尔科夫氧化反应生成相应的羰基化合物是有机合成中十分重要的挑战，这个过程中往往需要高效的催化剂参与。目前的合成方法中，使用贵金属作为催化剂存在转化效率和对映选择性低，需要多步催化，三废多等问题[G.Dong,P.Teo,Z.K.Wickens,R.H.Grubbs,Primary alcohols from terminal olefins:Formal anti-Markovnikov hydration via triple relay catalysis.Science 333,1609–1612(2011)]。
细胞色素P450单加氧酶(P450s)是一类亚铁血红素依赖的酶家族，以氧气作为氧化剂，能够在温和的条件下选择性地活化C-H键，催化多种传统化学方法难以实现的合成反应，包括烯烃的氧化反应，在精细化学和药物及其代谢物合成方面具有极大的应用潜力。来源于Bacillus megaterium的P450(BM3)属于自给自足型的单加氧酶，即参与电子传递的氧化还原蛋白伴侶与P450氧化酶部分融合在一条肽链上。这种融合重组的结构大大提高了电子传递效率和氧化反应的电子耦合效率，BM3也是目前催化效率较高的P450酶之一。
但目前尚无高效催化烯烃类化合物进行反马尔科夫氧化反应的BM3酶的报道。
发明内容
本发明的主要目的在于提供一种细胞色素P450酶突变体及其应用，以解决现有技术中尚无高效催化烯烃类化合物进行反马尔科夫氧化反应的P450酶。
为了实现上述目的，根据本发明的一个方面，提供了一种细胞色素P450酶突变体，该突变体包括：(a)在SEQ ID NO：1的序列上发生一个或多个氨基酸突变的蛋白，且蛋白具有细胞色素P450酶的反马尔科夫氧化活性；或者(b)来源于Bacillus megaterium菌株，且与SEQ ID NO：1具有80％以上同源性的氨基酸序列，且具有细胞色素P450酶的反马尔科夫氧化活性。
进一步地，突变体为在SEQ ID NO：1的序列上发生1～14个中任意一个或多个，优选2～14个，更优选3～14个，进一步优选10～14个氨基酸突变的蛋白，且蛋白具有细胞色素P450酶的反马尔科夫氧化活性。
进一步地，突变体为来源于Bacillus megaterium菌株，且与SEQ ID NO：1具有85％以上，优选为90％以上，更优选为95％以上，进一步优选为99％以上同源性，且具有细胞色素P450 酶的反马尔科夫氧化活性；进一步优选突变体在SEQ ID NO：1基础上发生1～14个中任意一个或多个，优选2～14个，更优选3～14个，进一步优选10～14个氨基酸突变。
进一步地，突变体是在SEQ ID NO：1基础上发生下氨基酸突变：
V79A+F332A
V79A+F332A+A75V
V79A+F332A+A75F
V79A+F332A+L76A
V79A+F332A+A83F
V79A+F332A+A83V
V79A+F332A+F88A
V79A+F332A+F88V
V79A+F332A+T89A
V79A+F332A+T89V
V79A+F332A+A265V
V79A+F332A+A265F
V79A+F332A+T269V
V79A+F332A+T269A
V79A+F332A+T269F
V79A+F332A+A329V
V79A+F332A+A329F
V79A+F332A+A331F
V79A+F332A+A331V
V79A+F332A+L438A
V79A+F332A+L438F
V79A+F332A+C63Y
V79A+F332A+C63Y+S107C
V79A+F332A+C63Y+S107C+Q129L
V79A+F332A+C63Y+S107C+Q129+E229K
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L T428I+N320I
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L T428I+S165G
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L T428I+F78L
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A331P
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A331G
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A331D
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329L
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A75R
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329Y+A331P
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329L+A331P
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+A331F
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+A331R
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+A331D
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+R48H
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+Y52G
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+Q74L
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+T269P
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+S333H
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+S333M
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+M355F
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+M355K
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+L182I
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+L182M
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+A331F+S333Q
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+A331F+S333V
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+A331F+W91M
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+A331F+S90C
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+A331F+L18V
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+A331F+S90V
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+A331F+S90G
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+S333H+L182A
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+S333H+L182G
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+S333H+L182M
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+S333H+L182F
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+S333H+A331P
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+S333H+S331S
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+S333M+M355W
V79A+F332A+C63Y+S107C+Q129+E229K+Q388L+T428I+F78L+I175S+A329K+S333M+M355G
。
为了实现上述目的，根据本发明的一个方面，提供了一种DNA分子，编码上述任一种突变体。
为了实现上述目的，根据本发明的一个方面，提供了一种重组质粒，该重组质粒连接有上述的DNA分子。
进一步地，重组质粒选自如下任意一种：pET-21b(+)、pET-22b(+)、pET-3a(+)、pET-3d(+)、pET-11a(+)、pET-12a(+)、pET-14b、pET-15b(+)、pET-16b(+)、pET-17b(+)、pET-19b(+)、pET-20b(+)、pET-21a(+)、pET-23a(+)、pET-23b(+)、pET-24a(+)、pET-25b(+)、pET-26b(+)、pET-27b(+)、pET-28a(+)、pET-29a(+)、pET-30a(+)、pET-31b(+)、pET-32a(+)、pET-35b(+)、pET-38b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42a(+)、pET-43a(+)、pET-43b(+)、pET-44a(+)、pET-49b(+)、pQE2、pQE9、pQE30、pQE31、pQE32、pQE40、pQE70、pQE80、pRSET-A、pRSET-B、pRSET-C、pGEX-5X-1、pGEX-6p-1、pGEX-6p-2、pBV220、pBV221、pBV222、pTrc99A、pTwin1、pEZZ18、pKK232-8、pUC-18以及pUC-19。
为了实现上述目的，根据本发明的一个方面，提供了一种宿主细胞，该宿主细胞含有上述任一种重组质粒。
进一步地，宿主细胞为原核细胞或真核细胞，优选真核细胞为酵母细胞。
进一步地，宿主细胞为感受态细胞，优选感受态细胞为大肠杆菌BL21细胞或大肠杆菌W3110。
为了实现上述目的，根据本发明的一个方面，提供了一种羰基化合物或醇类化合物的制备方法，该制备方法包括：采用上述任一种细胞色素P450酶突变体催化烯烃类化合物
Figure PCTCN2021089667-appb-000001
进行直接的反马尔科夫氧化反应生成羰基化合物
Figure PCTCN2021089667-appb-000002
和醇类化合物
Figure PCTCN2021089667-appb-000003
其中，R表示任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基。
进一步地，R表示碳原子数为1-20的任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基；优选的，R表示碳原子数为1-10的任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基；优选的，取代是指被卤素原子、氮原子、硫原子、羟基、硝基、氰基、甲氧基、乙氧基、羧基、羧甲基、羧乙基或亚甲二氧基取代；优选的，烯烃类化合物为苯环上任意位置被取代或未被取代的苯乙烯类化合物，反应为
Figure PCTCN2021089667-appb-000004
生成
Figure PCTCN2021089667-appb-000005
的反马尔科 夫氧化反应，其中，R 1表示苯环上任意位置取代为卤素、硝基、甲基或甲氧基；优选的，卤素取代为氯原子取代；更优选地，烯烃类化合物为如下任一种：
Figure PCTCN2021089667-appb-000006
及
Figure PCTCN2021089667-appb-000007
应用本发明的技术方案，通过定向进化的手段对野生酶进行蛋白质改造，提高了酶的活性和选择性，开发出可用于工业化生产的P450酶。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。
由于目前尚无高效催化烯烃类化合物进行反马尔科夫氧化反应的BM3酶的报道，为改善这一状况，本申请的发明人通过酶筛选发现BM3具有催化烯烃类化合物进行反马尔科夫氧化反应的活力，但是其活性较低，反马尔科夫氧化的选择性较差。为了进一步提高其催化反应活性和/或其选择性，发明人通过定向进化的手段对野生酶进行蛋白质改造，提高了酶的活性和选择性，开发出可用于工业化生产的P450酶。
本发明的发明人通过定向进化的方法提高来源于Bacillus megaterium野生型菌株的P450酶活和反马尔科夫氧化的选择性，降低酶的使用量。首先通过全质粒PCR的方式在野生型P450酶SEQ ID NO：1上引入突变位点，对突变体进行活性和选择性检测，挑选活性和选择性(即醛在总产品中占的比例)提高的突变体。
P450酶催化的反马尔科夫氧化反应式如下：
Figure PCTCN2021089667-appb-000008
本发明所提供的细胞色素P450酶突变体可催化烯烃类底物生成醛，醛可以进一步被辅酶还原成醇。
来源于Bacillus megaterium的野生型P450酶序列SEQ ID NO：1如下：
Figure PCTCN2021089667-appb-000009
Figure PCTCN2021089667-appb-000010
以野生型P450酶的序列SEQ ID NO：1为模板，设计了27对定点突变引物(A75V,A75F,L76A,L76I,L76F,V79A,V79L,V79F,A83F,A83V,F88A,F88V,T89A,T89V,A265V,A265F,T269V,T269A,T269F,A329V,A329F,A331F,A331V,F332A,F332V,L438A,L438F)，利用定点突变手段，以pET-22b(+)为表达载体，获得带有目的基因的突变质粒。
其中，定点突变：是指通过聚合酶链式反应(PCR)等方法向目的DNA片段(可以是基因组，也可以是质粒)中引入所需变化(通常是表征有利方向的变化)，包括碱基的添加、删除、点突变等。定点突变能迅速、高效的提高DNA所表达的目的蛋白的性状及表征，是基因研究工作中一种非常有用的手段。
利用全质粒PCR引入定点突变的方法简单有效，是目前使用比较多的手段。其原理是，一对包含突变位点的引物(正、反向)，和模板质粒退火后用聚合酶“循环延伸”，所谓的循环延伸是指聚合酶按照模版延伸引物，一圈后回到引物5’端终止，再经过反复加热退火延伸的循环，这个反应区别于滚环扩增，不会形成多个串联拷贝。正反向引物的延伸产物退火后配对成为带缺刻的开环质粒。Dpn I酶切延伸产物，由于原来的模版质粒来源于常规大肠杆菌，是经dam甲基化修饰的，对Dpn I敏感而被切碎，而体外合成的带突变序列的质粒由于没有甲基化而不被切开，因此在随后的转化中得以成功转化，即可得到突变质粒的克隆。
在单点突变获得性状提高的突变体基础上，可对有益的氨基酸位点进行组合，以获得性状更优的突变体。
获得活性和反马尔科夫氧化的选择性大幅提高的P450突变体后，使用易错PCR的方法对其进行随机突变，构建高质量的突变体库，开发合适的高通量筛选方法，对文库进行筛选，获得性状进一步提高的突变体。
易错PCR：意为易错条件下的PCR,即容易使复制出的DNA序列出现错误的PCR技术，又称错配PCR或倾向错误PCR。具体是指通过利用低保真度TaqDNA聚合酶和改变PCR反应条件，降低DNA复制的保真度，在新DNA链合成过程中增加碱基错配，从而使扩增产物出现较多点突变的一种体外诱导DNA序列变异的方法。
易错PCR是目前最简单、有效的基因体外随机诱变技术，其原理是：碱基的异构为错配提供了可能，组成DNA的4种碱基都有互变异构体存在，其中鸟嘌呤(G)、胞嘧啶(C)和胸腺嘧啶(T)3种含氧碱基有酮式和烯醇式两种互变异构体。腺嘌呤(A)和胸腺嘧啶两种含氮碱基，有胺式、亚胺式两种互变异构体。G、C和T主要以酮式结构存在，烯醇式结构的比率极低，A和T两种含氮碱基上的氮原子主要以氨基(NH 2)状态存在，以亚胺基(NH)状态存在的比率极低。不同同分异构体之间氢原子位置的不同及同一位置电子云偏离方向的不同，可使得碱基的配对形式发生改变，这样在复制后的子链上就可能出现错配。例如当胸腺嘧啶以酮式结构存在时，与腺嘌呤配对，而以烯醇式结构存在时，与鸟嘌呤配对，这样就出现了A能配上C，T能配上G的不稳定碱基对，从而造成错配。
在已知的几种耐热DNA聚合酶中，Taq DNA聚合酶的错配率最高。Taq DNA聚合酶是发现的耐热DNA聚合酶中活性最高的一种，具有5'-3'外切酶活性，不具3'-5'外切酶活性，因此在合成中对某些单核苷酸错配没有校正功能，所以比有3'-5'校对活性的DNA聚合酶发生错配的概率较高。DNA聚合酶的保真性可以通过多种方法来降低，包括使用4种浓度不同dNTP、添加Mn 2+、提高Mg 2+浓度等。几种诱变方法导致扩增DNA链碱基变异的机理各不相同。MnC1 2是DNA聚合酶的诱变因子，加入Mn 2+可以降低聚合酶对模板的特异性，提高错配率；4种dNTPs浓度的不平衡可以提高碱基错误掺入的概率，实现错配；Mg2+具有激活Taq酶的作用，增加Mg 2+浓度，使之超过正常用量，能稳定非互补的碱基对；提高Taq DNA聚合酶用量、增加每个循环延伸时间，可以增加错配终端延伸的概率；降低起始模板浓度，会使后面PCR循环的变异模板比例增加。
通过对易错PCR构建的突变体库进行筛选，获得活性和反马尔科夫氧化的选择性进一步提高的P450突变体。又设计了31条饱和突变引物(R48，R52，N71，L72，S73，Q74，A75，L76，F82，A83，D85，G86，F88，T89，S90，W91，R148，S165，H172，P173，F174，L182，I264，A265，T269，P327、A329，A331，S333，M355，L440)，进一步进行突变体进化，以获得活性和反马尔科夫氧化的选择性最优的突变。
饱和突变是通过对目的蛋白的编码基因进行改造，短时间内获取靶位点氨基酸分别被其它19种氨基酸替代的突变体的一种方法。此方法不仅是蛋白质定向改造的强有力工具，而且是蛋白质结构－功能关系研究的重要手段。饱和突变往往能获得比单点突变更为理想的进化体。而对于定点突变方法不能解决的这些问题，恰恰是饱和突变方法所擅长的独特之处。
上述将突变质粒转化至大肠杆菌细胞内，在大肠杆菌中过量表达。然后通过超声破碎细胞的方法获得粗酶。P450诱导表达最佳条件：25℃，0.2mM IPTG和0.5mM Aminolevulinic acid(ALA)诱导过夜。
在上述研究结果的基础上，申请人提出了本申请的方案。在一种典型的实施方式中，提供了一种细胞色素P450酶突变体，该突变体包括：(a)在SEQ ID NO：1的序列上发生一个或多个氨基酸突变的蛋白，且蛋白具有细胞色素P450酶的反马尔科夫氧化活性；或者(b)来源 于Bacillus megaterium菌株，且与SEQ ID NO：1具有80％以上同源性的氨基酸序列，且具有细胞色素P450酶的反马尔科夫氧化活性。
该实施例中所提供的突变体，在保持细胞色素P450酶活性的基础上，通过发生一个或多个氨基酸的突变，或者通过突变后的序列与野生型Bacillus megaterium菌株序列的同源性在80％以上，来进一步提升酶反应活性和/或提升反马尔科夫氧化的选择性。
采用本申请前述的各种诱变方法获得的突变体，只要满足上述条件，均在本申请的保护范围内。
在一种优选的实施例中，突变体在SEQ ID NO：1的序列上发生1～14个中任意一个或多个，优选2～14个，更优选3～14个，进一步优选10～14个氨基酸突变，且该突变体具有细胞色素P450酶的反马尔科夫氧化活性。
在一种优选的实施例中，突变体为来源于Bacillus megaterium菌株，且与SEQ ID NO：1具有85％以上，优选为90％以上，更优选为95％以上，进一步优选为99％同源性，且具有细胞色素P450酶的反马尔科夫氧化活性；进一步优选突变体1～14个中任意一个或多个(比如可以是14、13、12、11、10、9、8、7、6、5、4、3、2或1个)，优选2～14个，更优选3～14个，进一步优选10～14个氨基酸突变。
在一种更优选的实施例中，P450酶突变体是在SEQ ID NO：1基础上发生如表1至表4所示的氨基酸突变的突变体。这些突变体催化烯烃类化合物进行反马尔科夫氧化反应的催化活性和/或选择性均较野生型显著提高。
在本发明一种典型的实施方式中，还提供了一种DNA分子，该DNA分子编码上述任一种P450酶突变体。编码的上述P450酶突变体具有选择性高和催化活性显著提高的优势。
在本发明一种典型的实施方式中，还提供了一种重组质粒，该重组质粒连接有上述DNA分子。该DNA分子能够编码上述任一种选择性高，且/或催化活性显著提高的P450酶突变体。具体序列选自表1-表4中的序列或者与这些序列在保持上述氨基酸位点变化的前提下，其他位点的氨基酸序列发生取代、添加或缺失突变的核苷酸序列。
上述重组质粒中，任意能够用于表达上述羟化酶的DNA分子的重组质粒均适用于本发明。在本发明的优选实施例中，重组质粒选自如下之一：pET-22b(+)、pET-21b(+)、pET-3a(+)、pET-3d(+)、pET-11a(+)、pET-12a(+)、pET-14b、pET-15b(+)、pET-16b(+)、pET-17b(+)、pET-19b(+)、pET-20b(+)、pET-21a(+)、pET-23a(+)、pET-23b(+)、pET-24a(+)、pET-25b(+)、pET-26b(+)、pET-27b(+)、pET-28a(+)、pET-29a(+)、pET-30a(+)、pET-31b(+)、pET-32a(+)、pET-35b(+)、pET-38b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42a(+)、pET-43a(+)、pET-43b(+)、pET-44a(+)、pET-49b(+)、pQE2、pQE9、pQE30、pQE31、pQE32、pQE40、pQE70、pQE80、pRSET-A、pRSET-B、pRSET-C、pGEX-5X-1、pGEX-6p-1、pGEX-6p-2、pBV220、pBV221、pBV222、pTrc99A、pTwin1、pEZZ18、pKK232-8、pUC-18以及pUC-19。
在本发明一种典型的实施方式中，还提供了一种宿主细胞，该宿主细胞含有上述任一种重组质粒。具体的宿主细胞可以为原核细胞或真核细胞，优选真核细胞为酵母细胞。更优选地，上述宿主细胞为感受态细胞，进一步优选感受态细胞为大肠杆菌BL21细胞或大肠杆菌W3110。
在本发明一种典型的实施方式中，还提供了一种羰基化合物或醇类化合物的制备方法，该制备方法包括：采用上述任一种细胞色素P450酶突变体催化烯烃类化合物
Figure PCTCN2021089667-appb-000011
进行直接的反马尔科夫氧化反应生成羰基化合物
Figure PCTCN2021089667-appb-000012
或醇类化合物
Figure PCTCN2021089667-appb-000013
其中，R表示任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基。
优选地，R表示碳原子数为1-20的任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基，更优选的，R表示碳原子数为1-10的任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基；优选的，取代是指被卤素原子、氮原子、硫原子、羟基、硝基、氰基、甲氧基、乙氧基、羧基、羧甲基、羧乙基或亚甲二氧基取代；优选的，烯烃类化合物为苯环上任意位置被取代或未被取代的苯乙烯类化合物，反应为
Figure PCTCN2021089667-appb-000014
生成
Figure PCTCN2021089667-appb-000015
的反马尔科夫氧化反应，其中，R 1表示苯环上任意位置取代为卤素、硝基、甲基或甲氧基。
优选的，卤素取代为氯原子取代。
更优选地，烯烃类化合物为如下任一种：
Figure PCTCN2021089667-appb-000016
及
Figure PCTCN2021089667-appb-000017
下面结合具体的实施例来进一步说明本申请的有益效果。需要说明的是，下述实施例中用到的原料包括：
原料1：
Figure PCTCN2021089667-appb-000018
苯乙烯Styrene CasNo:100-42-5。
原料2：
Figure PCTCN2021089667-appb-000019
4-氯苯乙烯4-Chlorostyrene CasNo:1073-67-2。
原料3：
Figure PCTCN2021089667-appb-000020
4-甲基苯乙烯4-MethylstyreneCAS号:622-97-9
原料4：
Figure PCTCN2021089667-appb-000021
4-(三氟甲基)苯乙烯4-(Trifluoromethyl)styrene CasNo:402-50-6。
实施例1
10mL的玻璃瓶中分别加入1.5mM的原料1、原料2、原料3和原料4，加入1eq NADP +，20eq葡萄糖，3wt葡萄糖脱氢酶，0.1g P450酶，用Tris-HCl buffer(pH 8.0,100mM)补至4mL，混匀，于30℃，200rpm摇床反应3h。反应结束后，加入2mL乙酸乙酯，充分混合后进行离心，12000rpm离心5min，取上层，HPLC检测，波长210nm。部分突变体反应特性如表1所示。
表1：
Figure PCTCN2021089667-appb-000022
上述母本为SEQ ID NO:1；活性和选择性提高的倍数用+表示，+表示提高0-1倍，++表示提高1-2倍，+++表示提高2-3倍，++++表示提高3-5倍，+++++表示提高5-10倍。本发明中的选择性/反马尔科夫氧化的选择性定义为：反马尔科夫氧化产物中的醛产品％/[(醛产品％+环氧产品％]。
实施例2
10mL的玻璃瓶中分别加入1.5mM的原料1、原料2、原料3和原料4，加入1eq NADP +，20eq葡萄糖，3wt葡萄糖脱氢酶，0.1g P450酶，用Tris-HCl buffer(pH 8.0,100mM)补至4mL，混匀，于30℃，200rpm摇床反应3h。反应结束后，加入2mL乙酸乙酯，充分混合后进行离心，12000rpm离心5min，取上层，HPLC检测，波长210nm。部分突变体反应特性如表2所示。
表2：
Figure PCTCN2021089667-appb-000023
上述母本为SEQ ID NO:1；活性和选择性提高的倍数用+表示，+表示提高0-1倍，++表示提高1-2倍，+++表示提高2-3倍，++++表示提高3-5倍，+++++表示提高5-10倍。
由表2结果可以看出，运用随机突变(易错PCR)的定向进化方法使得突变体活性和反马尔科夫氧化的选择性大幅提高。下一步可继续饱和突变的进化方法，进一步提高突变体活性和选择性。
实施例3
10mL的玻璃瓶中分别加入3mM的原料1、原料2、原料3和原料4，加入0.5eq NADP +，10eq葡萄糖，1wt葡萄糖脱氢酶，0.1g P450酶，用Tris-HCl buffer(pH 8.0,100mM)补至2mL，混匀，于40℃，200rpm摇床反应8h。反应结束后，加入2mL乙酸乙酯，充分混合后进行离心，12000rpm离心5min，取上层，HPLC检测，波长210nm。部分突变体反应特性如表3所示。
表3：
Figure PCTCN2021089667-appb-000024
Figure PCTCN2021089667-appb-000025
上述母本为SEQ ID NO:1；活性和选择性提高的倍数用+表示，+表示提高0-1倍，++表示提高1-2倍，+++表示提高2-3倍，++++表示提高3-5倍，+++++表示提高5-10倍。
实施例4
10mL的玻璃瓶中分别加入3mM的原料1、原料2、原料3和原料4，加入0.5eq NADP+，10eq葡萄糖，1wt葡萄糖脱氢酶，0.1g P450酶，用Tris-HCl buffer(pH 8.0,100mM)补至2mL，混匀，于40℃，200rpm摇床反应8h。反应结束后，加入2mL乙酸乙酯，充分混合后进行离 心，12000rpm离心5min，取上层，HPLC检测，波长210nm。部分突变体反应特性如表4所示。
表4：
Figure PCTCN2021089667-appb-000026
上述母本为SEQ ID NO:1；活性和选择性提高的倍数用+表示，+表示提高0-1倍，++表示提高1-2倍，+++表示提高2-3倍，++++表示提高3-5倍，+++++表示提高5-10倍。
实施例5
在原料1、原料2、原料3和原料4被P450突变体氧化生成醛类产品的反应体系中，加入10U ADH(西格玛公司)，1mM NADP +，总体积1％的异丙醇，混匀，于40℃，200rpm摇床反应1h。反应结束后，加入2mL乙酸乙酯，充分混合后进行离心，12000rpm离心5min，取上层，HPLC检测，波长210nm。反应式如下，部分突变体反应特性如表5所示。
Figure PCTCN2021089667-appb-000027
表5：
Figure PCTCN2021089667-appb-000028
上述母本为SEQ ID NO:1；*代表醛产品被完全转化为醇产品。
从以上的描述中，可以看出，本发明上述的实施例实现了如下技术效果：通过改造后的突变体的活性和/或选择性均有不同程度的提高。
此外，本申请中公开的突变位点的其它任意组合，以及突变位点在其它同源性较高P450酶上进行重复，也可能有较好的效果。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (15)

  1. 一种细胞色素P450酶突变体，其特征在于，所述突变体在SEQ ID NO：1基础上发生如下氨基酸突变：
    V79A+F332A V79A+F332A+A75V V79A+F332A+A75F V79A+F332A+A76A V79A+F332A+A83F V79A+F332A+A83V V79A+F332A+F88A V79A+F332A+F88V V79A+F332A+T89A V79A+F332A+T89V V79A+F332A+A265V V79A+F332A+A265F V79A+F332A+T269V V79A+F332A+T269A V79A+F332A+T269F V79A+F332A+A329V V79A+F332A+A329F V79A+F332A+A331F V79A+F332A+A331V V79A+F332A+L438A
    V79A+F332A+L438F V79A+F332A+C63Y V79A+F332A+C63Y+S107C V79A+F332A+C63Y+S107C+Q129L V79A+F332A+C63Y+S107C+Q129L+E229K V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L T428I+N320I V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L T428I+S165G V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L T428I+F78L V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A331P V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A331G V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A331D V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329L V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A75R V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329Y+A331P V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329L+A331P V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+A331F V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+A331R V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+A331D V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+R48H V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+Y52G V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+Y52G V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+T269P V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+S333H V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+S333M V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+M355F V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+M355K V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+L182I V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+L182M V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+A331F+S333Q V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+A331F+S333V V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+A331F+W91M V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+A331F+S90C V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+A331F+L18V V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+A331F+S90V V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+A331F+S90G V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+S333H+L182A V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+S333H+L182G V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+S333H+L182M V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+S333H+L182F V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+S333H+A331P V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+S333H+A331S V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+S333M+M355W V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+S333M+M355G
    V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+S333M+A331D V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+S333H+L182F+S73G V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+S333H+L182F+W91F V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+S333H+L182F+P173Q V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+S333H+L182F+P173R V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+S333H+L182F+L72F V79A+F332A+C63Y+S107C+Q129L+E229K+Q388L+T428I+F78L+I175S+A329K+S333H+L182F+S73A
    。
  2. 一种DNA分子，其特征在于，编码权利要求1所述的突变体。
  3. 一种重组质粒，其特征在于，所述重组质粒连接有权利要求2所述的DNA分子。
  4. 根据权利要求3所述的重组质粒，其特征在于，所述重组质粒选自如下任意一种：pET-21b(+)、pET-22b(+)、pET-3a(+)、pET-3d(+)、pET-11a(+)、pET-12a(+)、pET-14b、pET-15b(+)、pET-16b(+)、pET-17b(+)、pET-19b(+)、pET-20b(+)、pET-21a(+)、pET-23a(+)、pET-23b(+)、pET-24a(+)、pET-25b(+)、pET-26b(+)、pET-27b(+)、pET-28a(+)、pET-29a(+)、pET-30a(+)、pET-31b(+)、pET-32a(+)、pET-35b(+)、pET-38b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42a(+)、pET-43a(+)、pET-43b(+)、pET-44a(+)、pET-49b(+)、pQE2、pQE9、pQE30、pQE31、pQE32、pQE40、pQE70、pQE80、pRSET-A、pRSET-B、pRSET-C、pGEX-5X-1、pGEX-6p-1、pGEX-6p-2、pBV220、pBV221、pBV222、pTrc99A、pTwin1、pEZZ18、pKK232-8、pUC-18以及pUC-19。
  5. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求3或4所述的重组质粒。
  6. 根据权利要求5所述的宿主细胞，其特征在于，所述宿主细胞为原核细胞或真核细胞，所述真核细胞为酵母细胞。
  7. 根据权利要求6所述的宿主细胞，其特征在于，所述宿主细胞为感受态细胞。
  8. 根据权利要求7所述的宿主细胞，其特征在于，所述感受态细胞为大肠杆菌BL21细胞或大肠杆菌W3110。
  9. 一种羰基化合物或醇类化合物的制备方法，其特征在于，所述制备方法包括：
    采用权利要求1所述的细胞色素P450酶突变体催化烯烃类化合物
    Figure PCTCN2021089667-appb-100001
    进行直接的反马尔科夫氧化反应生成羰基化合物
    Figure PCTCN2021089667-appb-100002
    和醇类化合物
    Figure PCTCN2021089667-appb-100003
    其中，R表示任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基。
  10. 根据权利要求9所述的制备方法，其特征在于，R表示碳原子数为1-20的任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基。
  11. 根据权利要求10所述的制备方法，其特征在于，R表示碳原子数为1-10的任选取代或未 被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基。
  12. 根据权利要求11所述的制备方法，其特征在于，所述取代是指被卤素原子、氮原子、硫原子、羟基、硝基、氰基、甲氧基、乙氧基、羧基、羧甲基、羧乙基或亚甲二氧基取代。
  13. 根据权利要求9所述的制备方法，其特征在于，所述烯烃类化合物为苯环上任意位置被取代或未被取代的苯乙烯类化合物，反应为
    Figure PCTCN2021089667-appb-100004
    生成
    Figure PCTCN2021089667-appb-100005
    Figure PCTCN2021089667-appb-100006
    的反马尔科夫氧化反应，其中，R 1表示苯环上任意位置取代为卤素、硝基、甲基或甲氧基。
  14. 根据权利要求13所述的制备方法，其特征在于，所述卤素取代为氯原子取代。
  15. 根据权利要求9所述的制备方法，其特征在于，所述烯烃类化合物为如下任一种：
    Figure PCTCN2021089667-appb-100007
    及
    Figure PCTCN2021089667-appb-100008
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