WO2022181654A1

WO2022181654A1 - Variant acyl-coa hydrolase

Info

Publication number: WO2022181654A1
Application number: PCT/JP2022/007457
Authority: WO
Inventors: 仁美中村; 匡平磯部; 健司河村; 勝成山田; 元佐分; 佳晃寺岡; 倫史亀田; 仁善池部
Original assignee: 東レ株式会社; 国立研究開発法人産業技術総合研究所
Priority date: 2021-02-25
Filing date: 2022-02-24
Publication date: 2022-09-01
Also published as: JPWO2022181654A1

Abstract

The present invention addresses the problem of improving the capacity for producing 3-hydroxyadipic acid and/or α-hydromconic acid by using variant acyl-CoA hydrolase or bacteria with a nucleic acid that codes for the variant acyl-CoA hydrolase introduced thereto.　Provided is a variant acyl-CoA hydrolase having acyl-CoA-hydrolase activity, comprising a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, or a polypeptide having an amino acid sequence having at least 60% sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and having acyl-CoA hydrolase activity, wherein a portion of the amino acids has been substituted. Or, provided is a microorganism with a nucleic acid that codes for said variant acyl-CoA hydrolase introduced thereto.

Description

Mutant Acyl-CoA Hydrolase

The present invention provides a mutant acyl-CoA hydrolase useful for the production of 3-hydroxyadipic acid and/or α-hydromuconic acid by a microorganism, a microorganism introduced with a nucleic acid encoding the mutant enzyme, and a microorganism using the microorganism. The present invention relates to a method for producing 3-hydroxyadipic acid and/or α-hydromuconic acid.

3-hydroxyadipic acid (IUPAC name: 3-hydroxyhexanedioic acid) and α-hydromuconic acid (IUPAC name: (E)-hex-2-enedioic acid) are dicarboxylic acids with 6 carbon atoms. These can be used as polyesters by polymerizing with polyhydric alcohols, and as raw materials for polyamides by polymerizing with polyvalent amines. In addition, by adding ammonia to these terminals to lactamize them, they can be used as raw materials for polyamide even by themselves.

As a method for producing 3-hydroxyadipic acid or the like using a microorganism, Patent Document 1 discloses introducing a nucleic acid encoding a polypeptide involved in the production of 3-hydroxyadipic acid and α-hydromuconic acid, or expressing the polypeptide. A genetically modified microorganism with enhanced , and a method for producing a substance using the microorganism are disclosed.

Examples of documents related to the biosynthesis of 3-hydroxyadipic acid using microorganisms include Patent Document 2, which relates to the production of 1,3-butadiene using unnatural microorganisms. A reaction to produce 3-hydroxyadipate (3-hydroxyadipate) as a metabolic intermediate from 3-hydroxyadipyl-CoA in the biosynthetic pathway of -butadiene has been described.

Examples of documents related to the biosynthesis of α-hydromuconic acid using microorganisms include Patent Document 3 regarding the production of muconic acid using non-natural microorganisms, which describes production of trans,trans-muconic acid from succinyl-CoA. A reaction is described that produces α-hydromuconic acid (2,3-dehydroadipate) as a metabolic intermediate from 2,3-dehydroadipyl-CoA in the resulting pathway.

In the 3-hydroxyadipic acid or α-hydromuconic acid biosynthetic pathways described in Patent Documents 1 to 3, CoA transferase, acyl-CoA hydrolase, or acyl-CoA synthase (acyl-CoA ligase) converts 3-hydroxyadipic acid. Or it is described as an enzyme that produces α-hydromuconic acid, but in Patent Document 1, 3-hydroxyadipyl-CoA produces 3-hydroxyadipate or 2,3-dehydroadipyl-CoA produces α-hydromuconic acid. It is described that CoA transferase is preferable as the enzyme that does this.

Non-Patent Document 1 describes that Escherichia coli acyl-CoA hydrolase TesB hydrolyzes various acyl-CoAs including adipic acid for the purpose of producing adipic acid using microorganisms. , describes that the enzyme has a wide substrate tolerance and catalyzes the hydrolysis of various acyl-CoAs, making it unsuitable for substance production.

WO2019/107516 Japanese translation of PCT publication No. 2013-535203 U.S. Patent Application Publication No. 2011/0124911A1

Regarding the biosynthetic pathway of 3-hydroxyadipic acid or α-hydromuconic acid, it has been suggested that 3-hydroxyadipic acid or α-hydromuconic acid can be produced by using acyl-CoA hydrolase as described above. , the biosynthesis of 3-hydroxyadipic acid or α-hydromuconic acid using acyl-CoA hydrolase is not recommended, and actual verification aimed at producing 3-hydroxyadipic acid or α-hydromuconic acid has not been performed. do not have.

Therefore, we have found a mutant acyl-CoA hydrolase that exhibits excellent activity in the hydrolysis reaction using 3-hydroxyadipyl-CoA and/or 2,3-dehydroadipyl-CoA as a substrate, and developed a new 3-hydroxyadipic acid. Alternatively, an object is to provide a method for producing 3-hydroxyadipic acid and/or α-hydromuconic acid using microorganisms by establishing a biosynthetic pathway for α-hydromuconic acid.

The present inventors have made intensive studies to achieve the above objects, and as a result, a mutant acyl-CoA hydrolase introduced with a specific amino acid substitution converts 3-hydroxyadipyl-CoA to 3-hydroxyadipate or 2,3 -Dehydroadipyl-CoA was found to exhibit excellent activity to catalyze the hydrolysis reaction to α-hydromuconic acid, leading to the completion of the present invention.

Specifically, the present invention comprises the following (1) to (8).
(1) A mutant acyl-CoA hydrolase in which some amino acids in a polypeptide selected from (i) and (ii) below are substituted, wherein 3-hydroxyadipyl-CoA and/or 2,3 - a mutant acyl-CoA-hydrolase with dehydroadipyl-CoA hydrolysis activity:
(i) a polypeptide having the amino acid sequence of SEQ ID NO: 1; or
(ii) a polypeptide having an amino acid sequence having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 and having acyl-CoA hydrolase activity;
(2) The mutant acyl-CoA hydrolase according to (1), wherein the substitution is of any amino acid in the region that interacts with the acyl-CoA substrate.
(3) the region that interacts with the acyl-CoA substrate corresponds to amino acid residues 30-40, 64-70, 82-89, 202-228 and/or 277-286 in the amino acid sequence of SEQ ID NO: 1; The mutant acyl-CoA hydrolase according to (2), which is a region that
(4) the substitution is L31, Q33, F35, F64, P67, G84, N85, S86, F87, D204, L205, N206, F207, L208, P209, and/or F219 residues in the amino acid sequence of SEQ ID NO: 1; A mutant acyl-CoA hydrolase according to (3), which is a substitution of amino acids corresponding to groups.
(5) A microorganism having a gene encoding the mutant acyl-CoA hydrolase according to any one of (1) to (4).
(6) A method for producing 3-hydroxyadipic acid and/or α-hydromuconic acid, which comprises culturing the microorganism according to (5) in a medium containing a carbon source as a fermentation raw material.
(7) 3-hydroxyadipyl-CoA and/or 2,3-dehydrogen is expressed by expressing a gene encoding an amino acid sequence in which some amino acids are substituted in the amino acid sequence of a polypeptide having acyl-CoA hydrolase activity; A method for producing a mutant acyl-CoA hydrolase, comprising the step of obtaining a polypeptide having adipyl-CoA hydrolysis activity.
(8) The method for producing a mutant acyl-CoA hydrolase according to (7), wherein the substitution is any amino acid substitution in the region that interacts with the acyl-CoA substrate in the polypeptide.
This specification includes the disclosure of Japanese Patent Application No. 2021-028444, which is the basis of priority of this application.

The mutant acyl-CoA hydrolase according to the present invention produces 3-hydroxyadipyl-CoA and/or α-hydromucon in a hydrolysis reaction using 3-hydroxyadipyl-CoA and/or 2,3-dehydroadipyl-CoA as a substrate. Excellent acid productivity. Therefore, since the microorganism according to the present invention has a gene encoding a mutant acyl-CoA hydrolase, it is excellent in productivity of 3-hydroxyadipic acid and/or α-hydromuconic acid, and the productivity of these substances is greatly improved. can be improved.

Although the present invention will be described in more detail below, the present invention is not limited to the following embodiments, and can be implemented with various modifications within the scope of the gist of the present invention.

Hereinafter, in the present specification, 3-hydroxyadipic acid is abbreviated as 3HA, α-hydromuconic acid as HMA, 3-hydroxyadipyl-CoA as 3HA-CoA, and 2,3-dehydroadipyl-CoA as HMA-CoA. There is

1. Mutant Acyl-CoA Hydrolase The mutant acyl-CoA hydrolase described herein is a mutant acyl-CoA hydrolase in which some amino acids in a polypeptide selected from (i) and (ii) below are substituted. be.
(i) a polypeptide having the amino acid sequence of SEQ ID NO: 1;
(ii) a polypeptide having an amino acid sequence having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 and having acyl-CoA hydrolase activity;
As used herein, the term "mutant" refers to a known protein or gene identified as a "wild type" that has been modified.

Acyl-CoA hydrolase belongs to the family of hydrolases, especially those that act on thioester bonds, and is an enzyme that catalyzes a chemical reaction that produces CoA and carboxylate using acyl-CoA and water as substrates. As used herein, the enzymatic activity of acyl-CoA hydrolase can be determined by 3HA-CoA and/or HMA-CoA hydrolysis activity. The 3HA-CoA and/or HMA-CoA hydrolysis activity is the production of 3HA and/or HMA obtained by culturing a microorganism that expresses acyl-CoA hydrolase and has the function of producing 3HA-CoA and/or HMA-CoA. from the amount of CoA, 3HA and/or HMA produced by mixing purified acyl-CoA hydrolase and 3HA-CoA and/or HMA-CoA in solution, or 3HA-CoA and/or HMA- It can be determined from the consumption of CoA.

The mutant acyl-CoA hydrolase described herein is a polypeptide in which some amino acids of the polypeptide selected from (i) and (ii) below are substituted:
(i) a polypeptide having the amino acid sequence of SEQ ID NO: 1, namely acyl-CoA hydrolase from E. coli (TesB);
(ii) a polypeptide having an amino acid sequence exhibiting 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 and having acyl-CoA hydrolase activity;
Preferred is a mutant acyl-CoA hydrolase having improved 3HA-CoA and/or HMA-CoA hydrolysis activity due to the above substitutions.

The mutant acyl-CoA hydrolase described herein is a mutant acyl-CoA hydrolase in which some amino acids are substituted, and is characterized by having 3HA-CoA and/or HMA-CoA hydrolysis activity. . Specifically, it has 3HA-CoA and/or HMA-CoA hydrolysis activity under conditions of 30° C., preferably higher 3HA than before substitution, ie, the corresponding wild-type acyl-CoA hydrolase. - 3HA having CoA and/or HMA-CoA hydrolysis activity, more preferably 1.1 to 100 times the specific activity, still more preferably 1.5 to 50 times, particularly preferably 2.0 to 20 times the specific activity - It is characterized by having CoA and/or HMA-CoA hydrolysis activity.

A polypeptide having an amino acid sequence having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 has 60% or more amino acid sequence identity with that identified as TesB, and acyl-CoA Refers to a polypeptide presumed to have a function or structure similar to that of hydrolase. The acyl-CoA hydrolase used in the invention described herein has a sequence identity of 60% or more, 65% or more, 70% or more, 80% or more, 90% or more, to the amino acid sequence of SEQ ID NO: 1. % or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Hereinafter, in this specification, a polypeptide having an amino acid sequence showing sequence identity with the amino acid sequence of SEQ ID NO: 1 and having acyl-CoA hydrolase activity may be referred to as a "homolog".

Examples of polypeptides having an amino acid sequence having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 include Serratia grimesii-derived acyl-CoA hydrolase (NCBI Protein ID: WP_037425284, SEQ ID NO: 2), Hafnia psycholerans-derived acyl-CoA hydrolase (NCBI Protein ID: WP_188474315, SEQ ID NO: 3), Pseudomonas aeruginosa-derived acyl-CoA hydrolase (NCBI Protein ID: MXH36765, SEQ ID NO: 4), Acinetobacter baumanii-derived acyl-CoA hydrolase (NCBI Pro SST03463, SEQ ID NO: 5), Shimwellia blattae-derived acyl-CoA hydrolase (NCBI Protein ID: WP_002438960, SEQ ID NO: 6), Enterobacter cloacae-derived acyl-CoA hydrolase (NCBI Protein ID: WP_063144757, SEQ ID NO: 7), etc. .

As used herein, the term “sequence identity” refers to an optimal alignment of two amino acid sequences or nucleotide sequences with or without gaps, and the overlapping entire amino acid sequences ( It means the ratio (percentage) of identical amino acids or bases to the base sequence (including the amino acid serving as the translation initiation point) or the base sequence (including the initiation codon), and is calculated according to formula (1). Sequence identity can be easily checked using BLAST (Basic Local Alignment Search Tool), an algorithm widely used in this field. For example, BLAST is available to anyone from websites such as NCBI (National Center for Biotechnology Information) and KEGG (Kyoto Encyclopedia of Genes and Genomes), and can easily check sequence identity using default parameters. .
Sequence identity (%) = number of matches (ignoring gaps)/shorter sequence length (length without gaps) x 100 Equation (1)
According to the formula (1), when the sequence identity between the amino acid sequences described in SEQ ID NOS: 1 to 7 is calculated using the function of Genetyx (% Identity Matrix), the sequence identity value is the highest with the amino acid sequence of SEQ ID NO: 1 The low value for SEQ ID NO: 5 is 61.18%, and the amino acid sequences set forth in SEQ ID NOs: 1-7 have at least 60% or more sequence identity with each other. Table 1 shows the results of sequence identity calculations using Genetyx. In addition, in the following Table 1, the leftmost number indicates the sequence number.

The mutant acyl-CoA hydrolases described herein are characterized by having a polypeptide having the amino acid sequence of SEQ ID NO: 1, or having an amino acid sequence exhibiting 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, A mutant acyl-CoA hydrolase in which some amino acids in a polypeptide having acyl-CoA hydrolase activity are substituted, and the mutant acyl-CoA hydrolase has 3HA-CoA and/or HMA-CoA hydrolysis activity is mentioned. Mutation by amino acid substitution of the mutant acyl-CoA hydrolase is not particularly limited as long as the mutant acyl-CoA hydrolase having the mutation has 3HA-CoA and/or HMA-CoA hydrolysis activity, but acyl-CoA substrate Preferred are amino acid substitutions in regions that interact with The number of regions substituted with amino acids that interact with the acyl-CoA substrate is not particularly limited, and may be one region or multiple regions. The number of amino acid substitutions in the region that interacts with the acyl-CoA substrate is not particularly limited as long as the mutant acyl-CoA hydrolase having the mutation has 3HA-CoA and/or HMA-CoA hydrolysis activity, but 1 to several is preferably 1 to 10, specifically preferably 1 to 10, preferably 1 to 6, more preferably 1 to 5, further preferably 1 to 4, even more preferably 1 to 3, 1 to 2 are more preferred, and 1 is particularly preferred.

The mutant acyl-CoA hydrolase described herein preferably has amino acids substituted from the wild type in the region that interacts with the acyl-CoA substrate. As used herein, the term "interaction" refers to the mutual influence of proteins, low-molecular-weight compounds, and the like, and includes, for example, the effects of electrostatic action, intermolecular force, and hydrogen bonding. The means for determining the region of acyl-CoA hydrolase that interacts with the acyl-CoA substrate is not particularly limited, but the following means can be used, for example. From the co-crystal structure information of wild-type TesB (SEQ ID NO: 1) and the HMA-CoA substrate, the region of TesB that interacts with the substrate can be identified (details will be described later). Then, in the acyl-CoA hydrolase of interest, ie, the homologue of TesB, the region corresponding to that region of SEQ ID NO: 1 is determined as the substrate-interacting region.

A co-crystal of TesB and HMA-CoA, that is, a complex crystal, can be prepared in the step of preparing an aqueous protein solution (A), the crystallization step (B), and the step of binding a substrate to the protein in the crystal (C). A particularly preferred preparation method includes step A for preparing a target protein aqueous solution containing TesB or TesB containing the amino acid sequence of SEQ ID NO: 8 in the sequence listing at a concentration of 1 to 20 mg / mL, and a 1 to 5 M sodium chloride aqueous solution. A 10 to 200 mM sodium acetate aqueous solution is added to the above TesB aqueous solution to grow TesB crystals and salt out, and the TesB crystals precipitated after the B step are immersed in a 1 to 10 mM HMA-CoA solution to obtain crystals. Attaching HMA-CoA to TesB in step C.

Specifically, TesB or TesB containing the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing is dissolved in an aqueous buffer solution. The aqueous buffer solution includes, for example, Tris buffer and phosphate buffer, and Tris buffer is preferred. The protein concentration is preferably 0.1-20 mg/mL, more preferably 1-10 mg/mL.

The vapor diffusion method is preferable for the crystallization process, and for example, the hanging drop method and the sitting drop method can be used. Specifically, less than about 10 μL of the protein solution may be mixed with the reservoir solution, the protein/reservoir solution and the reservoir may be sealed with a cover glass or the like, and allowed to stand until crystals grow.

The reservoir solution includes, for example, a precipitant and an aqueous buffer solution known in the art, and a combination of the precipitant and an aqueous buffer solution may be used. As the precipitant, sodium chloride and polyethylene glycol are preferred, and sodium chloride is more preferred. The concentration of sodium chloride is preferably 1-10M, more preferably 1-5M.

The buffer solution is preferably a 1-1000 mM commercially available buffer solution, more preferably a 10-200 mM sodium acetate solution. The temperature for growing crystals is preferably 3 to 25°C, more preferably 10 to 20°C. The crystal growth time is preferably 1 day to 1 year, more preferably 3 days to 3 weeks.

After that, the TesB crystals are added to the HMA-CoA solution and allowed to stand, forming a complex of TesB and HMA-CoA. The substrate concentration is preferably 1-50 mM, more preferably 1-10 mM. The standing time is preferably 1 to 60 minutes, more preferably 1 to 30 minutes. The temperature for standing is preferably 3 to 25°C, more preferably 10 to 20°C.

The crystal has a resolution of at least 10 Å or less, preferably 4.0 Å or less, more preferably 3.4 Å or less, and particularly preferably 2.2 Å or less when subjected to X-ray crystal structure analysis. It is preferable to have the quality to give.

The amino acid residues targeted for amino acid substitution introduction in the mutant acyl-CoA hydrolase described herein were obtained by molecular dynamics (MD) method using the co-crystal structure of TesB and HMA-CoA substrate. Among them, the Adaptive Lambda Square Dynamics (ALSD) method, which is an improved MD simulation method that promotes structural changes in the selected substrate binding region, is preferably used. Details of the ALSD method are described in Journal of Computational Chemistry, 35, 39-50 (2014).

When selecting the target amino acid residue for amino acid substitution introduction in the mutant acyl-CoA hydrolase described herein by the ALSD method, the structural change of the substrate HMA-CoA is promoted to form a complex structure with TesB. comprehensively explore. Specifically, among the three-dimensional structures obtained from the simulation, a complex structure in which the active site of TesB and the reactive site of HMA-CoA are in contact is selected, and each amino acid residue in TesB is combined with HMA-CoA. was calculated. Similar calculations were performed for 3HA-CoA, which is a different substrate, and the contact rate with 3HA-CoA was calculated. In order to propose a mutant TesB with increased selectivity for each substrate, the contact rate varies greatly depending on the substrate. Amino acid residues are selected as substitution candidates. That is, amino acid residues having a high contact rate with 3HA-CoA complexes and a low contact rate with HMA-CoA are selected, and substitutions of these amino acid residues are selected. In this MD calculation method, all amino acid residues in the TesB crystal structure are assigned an N score, which indicates the relative contact rate with each substrate, for each simulation. In the co-crystal structure of TesB and HMA-CoA substrate obtained in the invention described in this specification, TesB is a tetramer (total number of residues: 1144). Amino acid residues with significantly different contact rates depending on the substrate show a high N-score. A high N score is 0 or more, preferably 0.1 or more, more preferably 0.2 or more, more preferably 0.3 or more, and 0.4 or more is particularly preferred. Amino acid residues serving as substitution candidates are selected from two rounds of simulation. An amino acid residue whose N score is within the top 10 in one calculation result and within the top 30 in the two calculation results is selected as a substitution candidate.

In the multiple alignment of acyl-CoA hydrolases having the amino acid sequences of SEQ ID NOs: 1 to 7, the regions interacting with the acyl-CoA substrate and the preferred amino acid substitution sites selected by the MD calculation method are shown in Table 2. That is, the region that interacts with the acyl-CoA substrate corresponds to amino acid residues 30-40, 64-70, 82-89, 202-228 and/or 277-286 in the amino acid sequence of SEQ ID NO:1. are preferably regions, among these regions L31, Q33, F35, F64, P67, G84, N85, S86, F87, D204, L205, N206, F207, L208, P209 in the amino acid sequence of SEQ ID NO: 1, and It is more preferably an amino acid corresponding to the F219 residue, more preferably an amino acid corresponding to the N206 and/or Q33 residue in the amino acid sequence of SEQ ID NO:1. The amino acid after substitution is not particularly limited as long as the mutant acyl-CoA hydrolase after substitution has 3HA-CoA and/or HMA-CoA hydrolysis activity, but the amino acid substitution maintains the polarity of the amino acid residue. It is preferred that the substitution is That is, it is preferable to replace a hydrophobic amino acid residue with another hydrophobic residue, and a hydrophilic amino acid residue with another hydrophilic residue. At this time, glycine can be classified as either hydrophobic or hydrophilic. In addition, when the amino acid residue to be substituted has an aromatic side chain, it is preferable to substitute a residue that maintains polarity or substitute another aromatic side chain. For example, amino acid substitutions of N206 and Q33 residues to the hydrophilic amino acids lysine, arginine, histidine, aspartic acid, glutamic acid, glycine, serine, threonine, cysteine, tyrosine, glutamine or asparagine are preferred, among which the base Substitution with lysine, arginine, and histidine, which are natural amino acids, is more preferred. Table 3 shows preferred substitution positions and preferred substitution amino acid candidates in the amino acid sequence of SEQ ID NO:1.

The gene encoding the acyl-CoA hydrolase is not particularly limited as long as it is a nucleotide sequence that can be translated into the amino acid sequences set forth in SEQ ID NOs: 1 to 7 or homologous amino acid sequences thereof, and codons corresponding to each amino acid (standard genetic code) can be determined by reference. At that time, the nucleotide sequence may be redesigned with codons commonly used for the host microorganism used herein. Also, for enzyme purification, etc., a nucleotide sequence that can be translated into the amino acid sequence that serves as the tag sequence may be added.

A specific example of the nucleotide sequence of the gene encoding the polypeptide having the amino acid sequence of SEQ ID NO: 1 is the nucleotide sequence of SEQ ID NO: 9.

2. Microorganisms Expressing Mutant Acyl-CoA Hydrolase The microorganisms described herein are microorganisms having a gene encoding any of the mutant acyl-CoA hydrolases described in the section "1. Mutant acyl-CoA hydrolase". is. The microorganisms described herein are characterized by containing a gene capable of expressing a mutant acyl-CoA hydrolase, and are capable of hydrolyzing 3HA-CoA and/or HMA-CoA. Preferably, it has increased 3HA-CoA and/or HMA-CoA hydrolysis compared to a corresponding microorganism having a gene encoding an acyl-CoA hydrolase without amino acid substitutions. Specifically, for example, in a microbial culture at 30° C., the specific production activity of 3HA and/or HMA is 1.05 to 10 times, particularly 1.1 to 5 times, more than that of the wild type. preferably has 1.2 to 3 times higher hydrolytic capacity.

The method for expressing the mutant acyl-CoA hydrolase in the microorganism described herein is not particularly limited. Introduction of a gene encoding a mutant acyl-CoA hydrolase using a replicable expression vector, introduction of a gene encoding a mutant acyl-CoA hydrolase using techniques such as homologous recombination onto the genome of a microorganism, or Specific examples include methods such as replacing a gene encoding an endogenous acyl-CoA hydrolase with a gene encoding a mutant acyl-CoA hydrolase. Introduction of a gene encoding a mutant acyl-CoA hydrolase using a technique such as homologous recombination, or introduction of a gene encoding a mutant acyl-CoA hydrolase using an expression vector capable of autonomous replication in microorganisms is preferred.

As used herein, when a gene encoding a mutant acyl-CoA hydrolase to be expressed is incorporated into an expression vector, the expression vector is composed of a promoter, a ribosome binding sequence, a gene encoding a protein to be expressed, and a transcription termination sequence. is preferred.

When integrating a gene encoding a mutant acyl-CoA hydrolase into the genome of a microorganism, the nucleic acid for integration into the genome consists of a promoter, a ribosome binding sequence, a gene encoding a protein to be expressed, and a transcription termination sequence. It is preferable to incorporate a gene encoding a mutant acyl-CoA hydrolase in a form that replaces the wild-type acyl-CoA hydrolase or its homologue. It may also contain a gene that controls promoter activity.

In the present specification, the promoter used is not particularly limited as long as it allows the enzyme to be expressed in the microorganism, and examples thereof include gap promoter, trp promoter, lac promoter, tac promoter, T7 promoter and the like.

In the present specification, when an expression vector is used to introduce a gene or express a protein, it is not particularly limited as long as it can autonomously replicate in the microorganism. Examples include pBBR1MCS vector, pBR322 vector, pMW vector, pET vector. , pRSF vectors, pCDF vectors, pACYC vectors, and derivatives of the vectors described above.

In this specification, site-specific homologous recombination is used to introduce a gene or express a protein using a nucleic acid for genome integration. Although the method of site-specific homologous recombination is not particularly limited, for example, a method using λ Red recombinase and sacB gene (see, for example, Biosci.Biotechnol.Biochem.2007;71(12):2905-2911), λ Red recombinase and methods using FLP recombinase (see, eg, Proc. Natl. Acad. Sci. USA 2000;97(12):6640-6645).

The method of introducing the expression vector or the nucleic acid for genome integration is not particularly limited as long as it is a method of introducing the nucleic acid into the microorganism. , the calcium ion method (see, eg, J. Mol. Biol. 1970;53(1):159-162).

The microorganism used herein is not particularly limited as long as it is a genetically modified microorganism having a gene encoding a mutant acyl-CoA hydrolase, but is preferably a microorganism capable of producing 3HA and/or HMA. Specifically, the genus Serratia, the genus Escherichia, the genus Hafnia, the genus Corynebacterium, the genus Bacillus, the genus Streptomyces, the genus Cupriavidus, the genus Acinetobacter, the genus Alcaligenes, the genus Nocardioides, the genus Brevibacterium, the genus Delftiabacteria, and the genus Delftiabacteria, It is preferably a microorganism that is more selected, more preferably a microorganism belonging to the genus Serratia or Escherichia, and particularly preferably a microorganism belonging to the genus Escherichia.

When the microorganism described herein has the ability to produce 3HA and/or HMA, it is characterized by having a productivity of 3HA and/or HMA that is superior to conventional microorganisms that do not possess mutant acyl-CoA hydrolase. Here, "excellent 3HA and/or HMA productivity" means higher accumulation than a microbial strain that expresses only a wild-type acyl-CoA hydrolase that has not been mutated under the same host microorganism and fermentation conditions. It means producing 3HA and/or HMA in concentration and/or yield.

In the method for producing 3HA and/or HMA by microorganisms described herein, the 3HA yield is calculated according to formula (2). HMA yield is calculated by replacing 3HA in formula (2) with HMA.
Yield (%) = 3HA (mol)/carbon source consumption (mol) x 100 Formula (2)
3HA and HMA are produced by the reaction pathway shown in Scheme 1 below, and during the fermentative production of these organic acids, a microbial strain that expresses an enzyme that catalyzes reaction A, reaction B, reaction C, reaction D, and reaction E. to use.

Scheme 1 above shows examples of the reaction pathways required to produce 3HA and/or HMA. Here, reaction A represents a reaction that produces 3-oxoadipyl-CoA and coenzyme A from acetyl-CoA and succinyl-CoA. Reaction B shows the reaction to produce 3-hydroxyadipyl-CoA from 3-oxoadipyl-CoA. Reaction C shows a reaction to produce 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA. Reaction D shows the reaction to form 3HA from 3-hydroxyadipyl-CoA. Reaction E shows the reaction to produce HMA from 2,3-dehydroadipyl-CoA.

Specific examples of enzymes that catalyze these reactions include an acyltransferase as the enzyme that catalyzes reaction A, 3-oxoadipyl-CoA reductase as the enzyme that catalyzes reaction B, and enoyl-CoA hydratase as the enzyme that catalyzes reaction C. Enzymes that catalyze D and reaction E include acyl-CoA hydrolases.

A specific example of a gene encoding an enzyme that catalyzes reaction A is acyltransferase pcaF (NCBI Gene ID: 1041755, SEQ ID NO: 10) derived from Pseudomonas putida strain KT2440.

A specific example of a gene encoding an enzyme that catalyzes reaction B is 3-oxoadipyl-CoA reductase (NCBI Gene ID: JMPQ01000047.1, SEQ ID NO: 11) derived from Serratia marcescens ATCC 13880 strain.

A specific example of a gene encoding an enzyme that catalyzes reaction C is enoyl-CoA hydratase paaF (NCBI Gene ID: 1046932, SEQ ID NO: 12) derived from Pseudomonas putida strain KT2440.

Specific examples of genes encoding enzymes that catalyze reactions D and E include TesB (NCBI Gene ID: HXW89_RS08820, SEQ ID NO: 9) derived from Escherichia coli MG1655 strain.

The genes encoding the enzymes that catalyze reactions A to C may be genes that are conventionally possessed by microorganisms, or may be introduced artificially. The method for introducing the gene is not particularly limited, and a method of introducing the gene into an autonomously replicable expression vector into the microorganism, a method of incorporating the gene into the genome of the microorganism, or the like can be used.

3. Method for producing 3-hydroxyadipic acid and/or α-hydromuconic acid The method for producing 3HA and/or HMA described herein is the same as described in “2. The method is characterized by including a step of culturing the microorganism in a medium containing a carbon source as a fermentation raw material. The mutant acyl-CoA hydrolase expressed in the microbial cell can be isolated from the microbial cell, purified, and used to produce 3HA and/or HMA. The method for producing 3HA and/or HMA described herein is, for example, 1.05- to 10-fold, particularly 1.1- to 5-fold higher than when using a microorganism having a wild-type acyl-CoA hydrolase. It is preferred to have a 3HA-CoA and/or HMA-CoA hydrolysis that is 1.2- to 3-fold higher.

In the method for producing 3HA and/or HMA described herein, the genetically modified microorganism is cultured in a medium, preferably a liquid medium, containing a carbon source usable by ordinary microorganisms as a fermentation raw material, and organic acid to manufacture. In addition to a carbon source that can be used by the genetically modified microorganism, a medium containing a nitrogen source, inorganic salts, and, if necessary, organic micronutrients such as amino acids and vitamins in appropriate amounts is used. Either a natural medium or a synthetic medium can be used as long as it contains the above nutrients.

A fermentation raw material is a raw material that can be metabolized by the genetically modified microorganism. The term “metabolism” refers to the conversion of a chemical substance taken by a microorganism from outside the cell or produced by another chemical substance inside the cell into another chemical substance by an enzymatic reaction. Sugars can be preferably used as the carbon source. In addition to sugar, any substance that can be used as a single carbon source for the growth of the genetically modified microorganism can be preferably used. Specific examples of preferred carbon sources include monosaccharides such as glucose, fructose, galactose, mannose, xylose, and arabinose, disaccharides such as sucrose to which these monosaccharides are bound, polysaccharides, and starch saccharification solutions containing these sugars. , molasses, cellulose-containing biomass saccharification liquid, and the like.

Also, when producing 3HA and/or HMA, 3HA and/or HMA can be produced by adding a carbon source such as succinic acid in addition to the sugars listed above.

The carbon sources listed above may be used alone or in combination. In the addition of the carbon source, the concentration of the carbon source in the medium is not particularly limited, and can be appropriately set according to the type of carbon source. .1 to 100 g/L.

Nitrogen sources include, for example, ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other auxiliary organic nitrogen sources such as oil cakes, soybean hydrolysates, casein hydrolysates, other amino acids, Vitamins, corn steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, various fermented cells and hydrolysates thereof can be used. The nitrogen source concentration in the medium is not particularly limited, but is preferably 0.1 to 50 g/L.

As inorganic salts used for culturing the genetically modified microorganism, for example, phosphate, magnesium salt, calcium salt, iron salt, manganese salt, etc. can be added and used as appropriate.

The culture conditions for genetically modified microorganisms for producing organic acids are the medium having the above-mentioned composition, culture temperature, stirring speed, pH, aeration amount, planting amount, etc., depending on the type of genetically modified microorganism and external conditions. Then, adjust or select and set as appropriate. If foaming occurs in the liquid culture, antifoaming agents such as mineral oil, silicone oil and surfactants can be appropriately added to the medium.

After the organic acid is produced in a recoverable amount in the culture of the genetically modified microorganism, the produced product can be recovered. Recovery of the produced product, for example, isolation, can be carried out according to a general method of stopping the culture when the amount of accumulation has increased appropriately and collecting the fermentation product from the culture. . Specifically, after separating the cells by centrifugation, filtration, etc., the product is isolated from the culture by column chromatography, ion exchange chromatography, activated carbon treatment, crystallization, membrane separation, distillation, etc. be able to. More specifically, a method of adding an acidic component to the salt of the product to recover the precipitate, a method of concentrating the culture using a reverse osmosis membrane, an evaporator, etc. to remove water and increase the concentration of the product is recovered by a distillation operation, or crystals of the product and/or a salt of the product are precipitated by cooling crystallization or adiabatic crystallization, and the product and/or the product are obtained by centrifugation, filtration, etc. A method of obtaining a crystal of a salt of a substance, a method of adding alcohol to a culture to convert a sugar product into an ester, recovering the ester of the product by distillation, and obtaining the product by hydrolysis, etc. Examples include, but are not limited to. Moreover, these recovery methods can be appropriately selected and optimized according to the physical properties of the product.

4. Method for Producing Mutant Acyl-CoA Hydrolase The method for producing a mutant acyl-CoA hydrolase described herein (hereinafter also referred to as the “enzyme production method”) comprises It is characterized by including a step of expressing a gene encoding an amino acid sequence in which some of the amino acids are substituted. Preferably, the enzyme production method described herein yields an acyl-CoA hydrolase with higher 3-hydroxyadipyl-CoA and/or 2,3-dehydroadipyl-CoA hydrolysis activity than said polypeptide. .

In the enzyme production method described herein, the amino acid substitution in the polypeptide is preferably substitution of any amino acid in the region that interacts with the acyl-CoA substrate.

The method for producing an enzyme described herein preferably comprises any of the microorganisms described in the section “2. including the step of culturing under. Unless otherwise contradicted, the detailed culture conditions conform to those described in the section "Method for producing 3.3-hydroxyadipic acid and/or α-hydromuconic acid".

The enzyme production method described herein preferably includes a step of isolating and purifying mutant acyl-CoA from a culture medium in which microorganisms are cultured. Specifically, the mutant acyl-CoA hydrolase is obtained by, for example, collecting the cells from a culture solution obtained by culturing the microorganism in a liquid medium by centrifugation, disrupting the cells, and then subjecting them to ammonium sulfate precipitation and gel chromatography. It can be produced by performing known purification such as. The method for producing the enzyme described herein is not limited to specific means as long as the mutant acyl-CoA hydrolase can be obtained, and any known means can be used.

The present invention will be explained more specifically by the following examples. However, the scope of the invention shall not be limited by this example.

(Reference Example 1) Preparation of plasmid for expressing acyl-CoA hydrolase A vector pCDF-1b (manufactured by Novagen) capable of autonomous replication in E. coli was cleaved with KpnI to obtain pCDF-1b/KpnI. To amplify the gene encoding TesB (SEQ ID NO: 1), an acyl-CoA hydrolase from Escherichia coli str. K-12 substr. Using the genomic DNA of the MG1655 strain as a template, primers for PCR amplification of the full length of the acyl-CoA hydrolase gene tesB (NCBI GeneID: HXW89_RS08820, SEQ ID NO: 9) were designed (SEQ ID NOS: 13 and 14), and a PCR reaction was performed according to a conventional method. gone. The resulting fragment and pCDF-1b/KpnI were ligated using the In-Fusion HD Cloning Kit and introduced into E. coli strain DH5α. The plasmid was extracted from the obtained recombinant strain, and the plasmid whose nucleotide sequence was confirmed by a conventional method was designated as pCDF-1b::tesB.

(Reference Example 2) Preparation of Plasmid for Expressing Mutant Acyl-CoA Hydrolase A QuickChange site-directed mutagenesis kit (manufactured by New England Biolabs) was used to introduce an amino acid substitution into an acyl-CoA hydrolase. Using pCDF-1b::tesB as a template, primers containing sequences of amino acid residues to be substituted were designed (SEQ ID NOS: 15 to 21), and the full length of the plasmid was amplified by PCR according to a conventional method. The resulting fragment was treated with KLD Enzyme Mix (manufactured by New England Biolabs) and introduced into E. coli strain DH5α. The plasmid was extracted from the obtained recombinant strain, and the plasmid whose base sequence was confirmed by a conventional method was pCDF-1b::tesB-N206K, pCDF-1b::tesB-N206H, pCDF-1b::tesB-N206G, pCDF-1b::tesB-N206R and pCDF-1b::tesB-Q33R. A plasmid for expressing TesB-N206R-Q33R with two amino acid residues substituted was prepared using a fragment amplified by a PCR reaction using pCDF-1b::tesB-N206R as a template, and A plasmid whose nucleotide sequence was confirmed was designated as pCDF-1b::tesB-N206R-Q33R.

(Reference Example 3) Preparation of plasmid for expression of 3HA-CoA synthase A vector pBBR1MCS-2 (ME Kovach, (1995), Gene 166: 175-176) capable of autonomous replication in E. coli was cleaved with XhoI to obtain pBBR1MCS- 2/XhoI was obtained. To incorporate a constitutive expression promoter into the vector, Escherichia coli (E. coli) str. K-12 substr. Using the genomic DNA of MG1655 as a template, primers for PCR amplification of the upstream region 200b (SEQ ID NO: 22) of gapA (NCBI GeneID: NC_000913.3) were designed (SEQ ID NOS: 23 and 24), and a PCR reaction was performed according to a conventional method. rice field. The obtained fragment and pBBR1MCS-2/XhoI were ligated using In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.). It was introduced into E. coli DH5α. The plasmid was extracted from the obtained recombinant strain, and the plasmid whose nucleotide sequence was confirmed by a conventional method was named pBBR1MCS-2::Pgap. pBBR1MCS-2::Pgap was subsequently cut with ScaI to give pBBR1MCS-2::Pgap/ScaI. In order to amplify the gene encoding the enzyme that catalyzes reaction A, primers were designed for PCR amplification of the full-length acyltransferase gene pcaF (SEQ ID NO: 10) using the genomic DNA of Pseudomonas putida KT2440 strain as a template (SEQ ID NO: 25, 26), PCR reaction was carried out according to a standard method. The resulting fragment and pBBR1MCS-2::Pgap/ScaI were ligated using the In-Fusion HD Cloning Kit and introduced into E. coli strain DH5α. The plasmid was extracted from the obtained recombinant strain, and the plasmid whose nucleotide sequence was confirmed by a conventional method was named pBBR1MCS-2::AT. pBBR1MCS-2::AT was then cut with ScaI to give pBBR1MCS-2::AT/ScaI. In order to amplify the nucleic acid encoding the 3-oxoadipyl-CoA reductase that catalyzes reaction B, to PCR amplify the full length of the 3-oxoadipyl-CoA reductase gene (SEQ ID NO: 11) using the genomic DNA of Serratia marcescens ATCC 13880 strain as a template were designed (SEQ ID NOS: 27 and 28), and a PCR reaction was performed according to a conventional method. The obtained fragment and pBBR1MCS-2::AT/ScaI were ligated using In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.). It was introduced into E. coli DH5α. The plasmid was extracted from the obtained recombinant strain, and the plasmid whose base sequence was confirmed by a conventional method was named pBBR1MCS-2::AT3OR-kanR. Subsequently pBBR1MCS-2::AT3OR-kanR was cut with SapI to give pBBR1MCS-2::AT3OR-kanR/SapI. Primers for PCR amplification of the full-length chloramphenicol resistance gene (SEQ ID NO: 36) were designed (SEQ ID NOS: 37, 38) and PCR reactions were performed according to the information. The resulting fragment and pBBR1MCS-2::AT3OR-kanR/SapI were ligated using the In-Fusion HD Cloning Kit and introduced into E. coli strain DH5α. The plasmid was extracted from the obtained recombinant strain, and the plasmid whose nucleotide sequence was confirmed by a conventional method was named pBBR1MCS-2::AT3OR.

(Reference Example 4) Preparation of Dehydratase Expression Plasmid pMW119 (manufactured by Nippon Gene) was cleaved with SacI to obtain pMW119/SacI. To incorporate a constitutive expression promoter into the vector, E. coli str. K-12 substr. Using the genomic DNA of MG1655 as a template, primers for PCR amplification of the upstream region 200b (SEQ ID NO: 22) of gapA (NCBI Gene ID: NC_000913.3) were designed (SEQ ID NOS: 29 and 30), and a PCR reaction was performed according to a conventional method. gone. The resulting fragment and pMW119/SacI were ligated using In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.). It was introduced into E. coli DH5α. The plasmid was extracted from the obtained recombinant strain, and the plasmid whose base sequence was confirmed by a conventional method was designated as pMW119::Pgap. pMW119::Pgap was subsequently cut with SphI to give pMW119::Pgap/SphI. In order to amplify the gene encoding the enzyme that catalyzes reaction C, primers were designed for PCR amplification of the full-length enoyl-CoA hydratase gene paaF (SEQ ID NO: 12) using the genomic DNA of Pseudomonas putida strain KT2440 as a template ( SEQ ID NOS: 31, 32), PCR reaction was performed according to a conventional method. The obtained fragment and pMW119::Pgap/SphI were ligated using In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.). It was introduced into E. coli DH5α. The plasmid was extracted from the obtained recombinant strain, and the base sequence was confirmed by a conventional method. The resulting plasmid was named pMW119::EH.

(Reference Example 5) Construction of Plasmids for Expression of 3HA-CoA Synthase and Acyl-CoA Hydrolase pBBR1MCS-2::AT3OR was cleaved with MfeI and KpnI to obtain pBBR1MCS-2::AT3OR/MfeI_KpnI. To amplify the gene encoding TesB (SEQ ID NO: 1), pCDF-1b::tesB was used as a template for PCR amplification of the full length of the acyl-CoA hydrolase gene tesB (NCBI GeneID: HXW89_RS08820, SEQ ID NO: 9) gene. Primers were designed (SEQ ID NOs: 39 and 40), and PCR reaction was carried out according to a standard method. The resulting fragment and pBBR1MCS-2::AT3OR/MfeI_KpnI were ligated using In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.). It was introduced into E. coli DH5α. The plasmid was extracted from the obtained recombinant strain, and the plasmid whose nucleotide sequence was confirmed by a conventional method was named pBBR1MCS-2::AT3OR-tesB.

(Reference Example 6) Preparation of plasmids for expression of 3HA-CoA synthase and mutant acyl-CoA hydrolase To amplify the gene encoding TesB-N206R (SEQ ID NO: 41), pCDF-1b::tesB-N206R was used. As a template, primers for PCR amplification of the full-length acyl-CoA hydrolase gene tesB-N206R mutant acyl-CoA hydrolase gene were designed (SEQ ID NOs: 39 and 40), and PCR reaction was carried out according to a conventional method. The resulting fragment and pBBR1MCS-2::AT3OR/MfeI_KpnI were ligated using In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.). It was introduced into E. coli DH5α. The plasmid was extracted from the resulting recombinant strain, and the plasmid whose base sequence was confirmed by a conventional method was named pBBR1MCS-2::AT3OR-tesB-N206R.

(Reference Example 7) Expression and Purification of Acyl-CoA Hydrolase Acyl-CoA hydrolase and mutant acyl-CoA hydrolase were produced by E. coli BL21 (DE3) strain into which pCDF-1b::tesB or pCDF-1b::tesB-N206R was introduced. expressed in Specifically, 50 mL of LB medium (Bacto tryptone (manufactured by Difco Laboratories) 10 g / L, Bacto yeast extract (manufactured by Difco Laboratories) 5 g / L, sodium chloride 5 g / L) is inoculated with one platinum loop of the E. coli, Shaking culture was performed at 30° C. and 120 rpm. 10 mL of the culture solution was added to 1 L of LB medium in a 2 L Sakaguchi flask, and cultured with shaking at 37° C. and 120 rpm. When the OD600 reached 0.25 to 0.35, the temperature setting of the shaking culture machine was lowered to 18°C, and shaking culture was carried out. When the OD600 reached about 0.6, isopropyl-β-D-1 thiogalactopyranoside (IPTG) (manufactured by Sigma-Aldrich) was added to a final concentration of 500 μM, and the mixture was heated at 18° C. for another 20 hours. Cultured with shaking.

The acyl-CoA hydrolase expressed in E. coli was purified using His Bind Kits (manufactured by Novagen). The enzyme solution obtained by His-bind resin purification was concentrated using Amicon Ultra (manufactured by Merck Millipore) UF membrane (MW 10000) and replaced with storage buffer (20 mM Tris-HCl, 100 mM NaCl, pH 8.0). . Purified enzyme solutions were analyzed for purity by polyacrylamide gel electrophoresis (SDS-PAGE), and enzyme concentrations were determined by the Bio-Rad protein assay (Bradford method).

(Reference Example 8) Synthesis of HMA-CoA Substrate 3-Hydroxyadipate-3,6-lactone was produced by the method described in WO2016/068108. Reagents used in the synthesis of HMA-CoA were manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. unless otherwise specified. Dissolve 80 mg (0.56 mmol) of 3-hydroxyadipic acid-3,6-lactone in 2 mL of dichloromethane, add 5 μL (0.05 mmol) of N,N-dimethylformamide and 90 μL (1.0 mmol) of oxalyl chloride, and stir at room temperature. Stirred for 30 minutes. After completion of the reaction, the mixture was concentrated with a rotary evaporator, and 2 mL of tetrahydrofuran was added to the obtained liquid to obtain an acyl-chloride solution (0.28M).

9.6 mg (0.012 mmol, manufactured by Sigma-Aldrich) of Coenzyme A was dissolved in 0.5 mL of 0.5 M sodium hydrogen carbonate solution, 0.5 mL of acyl-chloride solution was added, and the mixture was stirred at room temperature. After 15 minutes, 1N hydrochloric acid was added to adjust the pH of the reaction mixture to 2-3. After completion of the reaction, it was concentrated with a rotary evaporator, and 3-hydroxyadipic acid-3,6-lactone-CoA was crudely purified with an ODS column (Purif-pack ODS 20, 30 μm, Shoko Scientific) (methanol: 0.1% formic acid in water, gradient = 0-25% A, 10 min). The resulting eluate was concentrated on a rotary evaporator and 0.5M sodium bicarbonate was added. After 16 hours, a 50% formic acid aqueous solution was added until the pH reached 2-3, filtered through a 0.22 μm filter, and then purified by HPLC.

[Purification of HMA-CoA by HPLC]
HPLC: 1260 Infinity (manufactured by Agilent Technologies)
Column: Synergi hydro-RP (manufactured by Phenomenex), length 250 mm, inner diameter 4.6 mm, particle size 4 μm
Mobile phase A: 50 mM potassium phosphate (pH 5.4)
Mobile phase B: acetonitrile gradient (5-20% B, 10 min)
Flow rate: 1.0 mL/min Column temperature: 40°C
HMA-CoA purified by HPLC under the above conditions was concentrated by a rotary evaporator, potassium phosphate was removed by a Strata C18-E column (manufactured by Phenomenex), concentrated by a rotary evaporator, and dried under vacuum to obtain HMA-CoA 2 .7 mg was obtained. The NMR spectrum of the obtained HMA-CoA was as follows.
¹ H-NMR (400 MHz, DO): δ 0.79 (s, 3H), 0.92 (s, 3H), δ 2.39-2.42 (m, 2H), δ 2.51-2.56 (m , 2H), δ2.80-2.83 (m, 2H), δ2.98-3.01 (m, 2H), δ3.30-3.33 (m, 3H), δ3.42-3.45 (m, 2H), δ 3.55-3.59 (m, 1H), δ 3.83-3.86 (m, 1H), 4.02 (s, 1H), δ 4.24 (s, 2H), 4.58 (d, 1H), 4.75-4.94 (m, 3H), δ 5.85 (d, 1H), δ 6.21 (d, 1H), δ 6.91-6.97 (m, 1H) δ 8.42 (s, 1H), δ 8.67 (s, 1H).

(Reference Example 9) MD simulation of wild-type TesB and substrate A composite crystal structure of TesB and HMA-CoA containing the amino acid sequence shown in SEQ ID NO: 8, or TesB and 3HA in which HMA-CoA in the structure is replaced with 3HA-CoA An MD simulation using the ALDS method was performed using the -CoA composite crystal structure. The contact rate between the TesB amino acid residue and the substrate was calculated for each simulation, and the variation in the contact rate due to different substrates for each amino acid residue was expressed as an N score. Each simulation was performed twice, and Table 4 shows amino acid residues with an N score of 0 or more calculated in any of the simulations. The residue number of each amino acid residue is the number corresponding to the amino acid sequence of SEQ ID NO:1.

(Reference Example 10) MD simulation of mutant TesB-N206R and substrate N A score was calculated. The structure of TesB-N206R was prepared by substituting arginine in silico for the asparagine corresponding to the N206 residue of TesB (SEQ ID NO: 8) in the composite crystal structure of TesB and HMA-CoA in Reference Example 8. Each simulation was performed twice, and Table 5 shows amino acid residues with an N score of 0 or more calculated in any of the simulations. The residue number of each amino acid residue is the number corresponding to the amino acid sequence of SEQ ID NO:1.

(Comparative Example 1) Cultivation of E. coli strain carrying gene encoding wild-type TesB 33) and the genes encoding pyruvate kinases PykF and PykA (SEQ ID NOS:34, 35) are deleted and pBBR1MCS-2::AT3OR, pMW119::EH and pCDF-1b::tesB are retained. Escherichia coli strain HMS174(DE3) was used.

LB medium containing chloramphenicol 15 µg/mL adjusted to pH 7, ampicillin 100 µg/mL and streptomycin 50 µg/mL (Bacto tryptone (manufactured by Difco Laboratories) 10 g/L, Bacto yeast extract (manufactured by Difco Laboratories) 5 g/ml One platinum loop of the E. coli strain was inoculated into 5 mL of L, sodium chloride 5 g/L, and cultured with shaking at 30° C. and 120 rpm for 18 hours. 0.05 mL of the culture solution was placed in a test tube and medium I containing 15 μg/mL chloramphenicol, 100 μg/mL ampicillin and 50 μg/mL streptomycin adjusted to pH 6.5 (glucose 10 g/L, ammonium sulfate 1 g/L , potassium phosphate 50 mM, magnesium sulfate 0.025 g/L, iron sulfate 0.0625 mg/L, manganese sulfate 2.7 mg/L, calcium chloride 0.33 mg/L, sodium chloride 1.25 g/L, Bacto tryptone2. 5 g/L, Bacto yeast extract 1.25 g/L) and cultured with shaking at 30° C. and 120 rpm for 24 hours.

The supernatant obtained by centrifuging the cells from the culture medium was subjected to membrane treatment using Millex-GV (0.22 μm, PVDF, manufactured by Merck), and the permeate was analyzed by LC-MS/MS. Table 6 shows the results of quantitative analysis of 3HA and HMA accumulated in the culture supernatant.

[Conditions for quantitative analysis of 3HA and HMA by LC-MS/MS]
HPLC: 1290 Infinity (manufactured by Agilent Technologies)
Column: Synergi hydro-RP (manufactured by Phenomenex), length 100 mm, inner diameter 3 mm, particle size 2.5 μm
Mobile phase: 0.1% formic acid aqueous solution/methanol = 70/30
Flow rate: 0.3 mL/min Column temperature: 40°C
LC detector: DAD (210 nm)
MS/MS: Triple-Quad LC/MS (manufactured by Agilent Technologies)
Ionization method: ESI negative mode

(Comparative Example 2) Cultivation of a Serratia bacterial strain carrying a gene encoding a wild-type TesB As a control strain carrying the E. coli-derived wild-type acyl-CoA hydrolase TesB set forth in SEQ ID NO: 1, the glucose transporter PtsG ( Serratia grimesii NBRC13537 harboring pBBR1MCS-2::AT3OR-tesB with deletion of the genes encoding pyruvate kinase PykF and PykA (SEQ ID NOs:44, 45) and the genes encoding pyruvate kinase PykF and PykA (SEQ ID NOs:44, 45). used the stock.

LB medium containing 25 µg/mL of kanamycin adjusted to pH 7 (Bacto tryptone (manufactured by Difco Laboratories) 10 g/L, Bacto yeast extract (manufactured by Difco Laboratories) 5 g/L, sodium chloride 5 g/L), the E. coli One platinum loop of the strain was inoculated and cultured with shaking at 30° C. and 120 rpm for 18 hours. 0.05 mL of the culture solution was placed in a test tube and medium I containing 25 μg/mL of kanamycin adjusted to pH 6.5 (glucose 10 g/L, ammonium sulfate 1 g/L, potassium phosphate 50 mM, magnesium sulfate 0.025 g/L , iron sulfate 0.0625 mg/L, manganese sulfate 2.7 mg/L, calcium chloride 0.33 mg/L, sodium chloride 1.25 g/L, Bacto tryptone 2.5 g/L, Bacto yeast extract 1.25 g/L) It was added to 5 mL and cultured with shaking at 30° C. and 120 rpm for 24 hours.

The supernatant obtained by centrifuging the cells from the culture medium was subjected to membrane treatment using Millex-GV (0.22 μm, PVDF, manufactured by Merck), and the permeate was analyzed by LC-MS/MS. The analysis conditions for LC-MS/MS were the same as in Comparative Example 1. Table 7 shows the results of quantitative analysis of 3HA and HMA accumulated in the culture supernatant.

(Comparative Example 3) Evaluation of enzymatic activity of wild-type TesB Enzymatic activity of wild-type TesB purified by the method described in Reference Example 7 was measured. Specifically, 100 μM substrate (3HA-CoA or HMA-CoA) and 0.175 mg/mL were added to a solution containing 50 mM Tris-HCl, 50 mM sodium chloride, 10 mM magnesium chloride and 1 mM tris(2-carboxyethyl)phosphine. of wild-type TesB was added and the reaction was carried out in a total volume of 100-200 μL. After 2, 5, 15, 30, and 60 minutes from the start of the reaction, 20 μL of the reaction solution was stopped with 5 μL of formic acid aqueous solution (50% v/v), left on ice for 10 minutes, and then centrifuged to remove the precipitate (13, 000 rpm, 10 min, 4° C.), and the supernatant was analyzed by LC-MS/MS. The analysis conditions for LC-MS/MS were the same as in Comparative Example 1. Quantitative analysis of 3HA and HMA accumulated in the reaction supernatant was performed, and wild-type TesB hydrolyzed the difference from the amount of 3HA and HMA accumulated in the negative control reaction supernatant to which wild-type TesB was not added. The amount of substrate was used. The kcat/Km values were calculated from the time course of the wild-type TesB response (see Proc. Natl. Acad. Sci. USA 2016;113(14):E2001-10). Table 8 shows the results.

(Example 1) Cultivation of Escherichia coli Strain Having Gene Encoding Mutant TesB-N206K As a strain for evaluating the activity of mutant TesB-N206K, a gene encoding the glucose transporter PtsG (SEQ ID NO: 33) and pyruvate The Escherichia coli HMS174 (DE3) strain carrying pBBR1MCS-2::AT3OR, pMW119::EH and pCDF-1b::tesB-N206K with deletion of the genes encoding the kinases PykF and PykA (SEQ ID NOS:34, 35) was used.

The supernatant obtained by centrifuging the cells from the culture medium was subjected to membrane treatment using Millex-GV (0.22 μm, PVDF, manufactured by Merck), and the permeate was analyzed by LC-MS/MS. The analysis conditions for LC-MS/MS were the same as in Comparative Example 1. Table 6 shows the results of quantitative analysis of 3HA and HMA accumulated in the culture supernatant.

(Example 2) Culture of Escherichia coli Strain Having Gene Encoding Mutant TesB-N206H As a strain for evaluating the activity of mutant TesB-N206H, a gene encoding the glucose transporter PtsG (SEQ ID NO: 33) and pyruvate The Escherichia coli HMS174 (DE3) strain carrying pBBR1MCS-2::AT3OR, pMW119::EH and pCDF-1b::tesB-N206H with deletion of the genes encoding the kinases PykF and PykA (SEQ ID NOS:34, 35) was used.

(Example 3) Culture of Escherichia coli Strain Having Gene Encoding Mutant TesB-N206G As a strain for evaluating the activity of mutant TesB-N206K, a gene encoding the glucose transporter PtsG (SEQ ID NO: 33) and pyruvate The Escherichia coli HMS174 (DE3) strain carrying pBBR1MCS-2::AT3OR, pMW119::EH and pCDF-1b::tesB-N206G with deletion of the genes encoding the kinases PykF and PykA (SEQ ID NOS: 34, 35) was used.

(Example 4) Culture of Escherichia coli Strain Having Gene Encoding Mutant TesB-N206R As a strain for evaluating the activity of mutant TesB-N206R, a gene encoding the glucose transporter PtsG (SEQ ID NO: 33) and pyruvate The Escherichia coli HMS174 (DE3) strain carrying pBBR1MCS-2::AT3OR, pMW119::EH and pCDF-1b::tesB-N206R with deletion of the genes encoding the kinases PykF and PykA (SEQ ID NOS:34, 35) was used.

(Example 5) Cultivation of Escherichia coli Strain Having Gene Encoding Mutant TesB-Q33R As a strain for evaluating the activity of mutant TesB-Q33R, a gene encoding the glucose transporter PtsG (SEQ ID NO: 33) and pyruvate The Escherichia coli HMS174(DE3) strain carrying pBBR1MCS-2::AT3OR, pMW119::EH and pCDF-1b::tesB-Q33R with deletion of the genes encoding the kinases PykF and PykA (SEQ ID NOS:34, 35) was used.

(Example 6) Culture of Escherichia coli Strain Having Gene Encoding Mutant TesB-N206R-Q33R Gene Encoding Glucose Transporter PtsG (SEQ ID NO: 33) as a Mutant TesB-N206R-Q33R Activity Evaluation Strain , and the genes encoding pyruvate kinase PykF and PykA (SEQ ID NOS: 34, 35) have been deleted and carry pBBR1MCS-2::AT3OR, pMW119::EH and pCDF-1b::tesB-N206R-Q33R. The HMS174(DE3) strain was used.

(Example 7) Cultivation of Serratia bacteria strain carrying gene encoding mutant TesB-N206R Serratia glimesi harboring pBBR1MCS-2::AT3OR-tesB-N206R with deletion of the gene encoding the porter PtsG (SEQ ID NO:43) and the genes encoding the pyruvate kinases PykF and PykA (SEQ ID NOS:44, 45) (Serratia grimesii) strain NBRC13537 was used.

(Example 8) Evaluation of enzymatic activity of mutant TesB-N206R The enzymatic activity of mutant TesB-N206R purified by the method described in Reference Example 7 was measured. Specifically, 100 μM substrate (3HA-CoA or HMA-CoA) and 0.175 mg/mL were added to a solution containing 50 mM Tris-HCl, 50 mM sodium chloride, 10 mM magnesium chloride and 1 mM tris(2-carboxyethyl)phosphine. Mutant TesB-N206R was added, and the reaction was carried out in a total volume of 100 to 200 μL. After 2, 5, 15, 30, and 60 minutes from the start of the reaction, 20 μL of the reaction solution was stopped with 5 μL of formic acid aqueous solution (50% v/v), left on ice for 10 minutes, and then centrifuged to remove the precipitate (13, 000 rpm, 10 min, 4° C.), and the supernatant was analyzed by LC-MS/MS. The analysis conditions for LC-MS/MS were the same as in Comparative Example 1. Quantitative analysis of 3HA and HMA accumulated in the reaction supernatant was performed, and the difference between the amount of 3HA and HMA accumulated in the reaction supernatant of a negative control to which wild-type TesB was not added was calculated by adding mutant TesB-N206R. The amount of the decomposed substrate was defined as the amount. The kcat/Km values were calculated from the time course of the mutant TesB-N206R response (see Proc. Natl. Acad. Sci. USA 2016;113(14):E2001-10). Table 8 shows the results.

From the results of Comparative Example 1 and Example 1, the E. coli strain carrying the gene encoding the mutant TesB in which the 206th asparagine residue in the amino acid sequence shown in SEQ ID NO: 1 is substituted with lysine produces wild-type TesB. High production of HMA was shown compared to E. coli strains harboring the encoding gene.

From the results of Comparative Example 1, Example 2, and Example 5, in the amino acid sequence shown in SEQ ID NO: 1, the 206th asparagine residue was substituted with histidine, or the 33rd glutamine was substituted with arginine, encoding mutant TesB. E. coli strains harboring the gene encoding wild-type TesB were shown to produce higher amounts of 3HA than E. coli strains harboring the gene encoding wild-type TesB.

From the results of Comparative Example 1, Example 3, and Example 4, E. coli strains harboring the gene encoding mutant TesB in which the asparagine residue at position 206 in the amino acid sequence shown in SEQ ID NO: 1 is replaced with glycine or arginine showed higher production of 3HA and HMA compared to E. coli strains harboring the gene encoding wild-type TesB.

From the results of Comparative Example 1 and Example 6, the E. coli strain carrying the gene encoding the mutant TesB in which the asparagine residue at position 206 and the glutamine at position 33 in the amino acid sequence shown in SEQ ID NO: 1 are substituted with arginine is , showed higher production of 3HA and HMA compared to E. coli strains harboring the gene encoding wild-type TesB.

From the results of Comparative Example 2 and Example 7, the Serratia glimesi strain carrying the gene encoding the mutant TesB in which the asparagine residue at position 206 in the amino acid sequence shown in SEQ ID NO: 1 is replaced with arginine is the wild type Higher production of 3HA and HMA was shown compared to the Serratia glimesi strain harboring the gene encoding TesB.

From the results of Comparative Example 3 and Example 8, the mutant TesB in which the asparagine residue at position 206 in the amino acid sequence shown in SEQ ID NO: 1 was replaced with arginine had higher 3HA and HMA producing activity than wild-type TesB. was shown to have
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Claims

A mutant acyl-CoA hydrolase in which some amino acids in a polypeptide selected from (i) and (ii) below are substituted, wherein 3-hydroxyadipyl-CoA and/or 2,3-dehydroadipyl - a mutant acyl-CoA hydrolase with CoA hydrolysis activity:
(i) a polypeptide having the amino acid sequence of SEQ ID NO:1; or (ii) a polypeptide having an amino acid sequence having 60% or more sequence identity with the amino acid sequence of SEQ ID NO:1 and having acyl-CoA hydrolase activity.
The mutant acyl-CoA hydrolase according to claim 1, wherein the substitution is of any amino acid in the region that interacts with the acyl-CoA substrate.
The region that interacts with the acyl-CoA substrate corresponds to amino acid residues 30-40, 64-70, 82-89, 202-228 and/or 277-286 in the amino acid sequence of SEQ ID NO: 1 The mutant acyl-CoA hydrolase of claim 2, wherein the mutant acyl-CoA hydrolase is
said substitutions correspond to residues L31, Q33, F35, F64, P67, G84, N85, S86, F87, D204, L205, N206, F207, L208, P209 and/or F219 in the amino acid sequence of SEQ ID NO: 1 4. The mutant acyl-CoA hydrolase according to claim 3, which is a substitution of amino acids to
A microorganism having a gene encoding the mutant acyl-CoA hydrolase according to any one of claims 1 to 4.
A method for producing 3-hydroxyadipic acid and/or α-hydromuconic acid, which comprises culturing the microorganism according to claim 5 in a medium containing a carbon source as a fermentation raw material.
3-hydroxyadipyl-CoA and/or 2,3-dehydroadipyl- by expressing a gene encoding an amino acid sequence in which some amino acids are substituted in the amino acid sequence of a polypeptide having acyl-CoA hydrolase activity A method for producing a mutant acyl-CoA hydrolase, comprising the step of obtaining a polypeptide having CoA hydrolysis activity.
The method for producing a mutant acyl-CoA hydrolase according to claim 7, wherein said substitution is an amino acid substitution in any region of said polypeptide that interacts with an acyl-CoA substrate.