WO2006043555A1

WO2006043555A1 - Reductase mutant for forming biopolymer

Info

Publication number: WO2006043555A1
Application number: PCT/JP2005/019125
Authority: WO
Inventors: Souichi Morikawa; Yuji Okubo; Takahisa Nakai; Keiji Matsumoto
Original assignee: Kaneka Corporation
Priority date: 2004-10-19
Filing date: 2005-10-18
Publication date: 2006-04-27
Also published as: JPWO2006043555A1

Abstract

A method of improving the ability to utilize a coenzyme and the catalytic activity of an enzyme, which is a wild type PhbB enzyme originating in Ralstonia eutropha or an enzyme having a sequential homology of at least 30% therewith, by mutating amino acid residues located at positions steric structurally equivalent to Met-12, Gly-13, Ala-32, Cys-34, Gly-35, Pro-36, Asn-37, Ser-38, Pro-39, Arg-40, Arg-41 and Ala-57 of the wild type PhbB enzyme. A method of producing a 3-hydroxyacyl-CoA and an optically active alcohol by using the PhbB enzyme mutant obtained by the above method as a catalyst. A method of producing a polyhydroxyalkanonic acid which involves the step of culturing and proliferating transformed cells containing a vector having a DNA encoding the above enzyme mutant or the step of reacting the enzyme mutant with a 3-ketoacyl-CoA.

Description

Specification

Reductase variants for biopolymer production

Technical field

[0001] The present invention reduces poly (3-ketoalkanoic acid-coenzyme A complexes (hereinafter referred to as 3_ketosacyl-CoA) to produce poly (3-hydroxyalkanoic acid), which is a biopolymer. Hereinafter, various 3-hydroxyalkanoic acid-coenzyme A complexes (hereinafter referred to as 3-hydroxyacyl-CoA), which are monomers constituting the 3-hydroxyalkanoic acid), can be provided. (1) It relates to an enzyme modification method for controlling the catalytic activity of a reductase by modifying the coenzyme utilization ability of CoA reductase. The present invention also provides a 3-ketoasinole-CoA reductase mutant having an enzyme activity superior to that of the wild-type enzyme obtained by the modified method, a DNA encoding the enzyme mutant, and the DNA. The present invention relates to a vector, a transformed cell transformed with this vector, a method for producing the enzyme variant, and a method for producing polyhydroxyalkanoic acid using the enzyme variant or the transformed cell.

Background art

[0002] Polyhydroxyalkanoic acid has been discovered as a polyester-type organic molecular polymer produced by microorganisms. In recent years, it has been industrially produced as a biodegradable environmentally friendly material or biocompatible material, and has a wide variety. Attempts have been made to use it in various industries. The monomer unit constituting the polyhydroxyalkanoic acid is a general name 3-hydroxyalkanoic acid, specifically 3-hydroxybutyric acid, 3-hydroxyvaleric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctane. A polymer molecule is formed by homopolymerization or copolymerization of an acid or a longer alkyl chain, 3-hydroxyalkane acid. It is known that the properties of polyhydroxyalkanoic acid as a polymer, that is, the physicochemical properties such as melting point, glass transition point, crystallization rate, and elongation strength differ depending on the content of monomer species constituting the polymer (non- Patent Document 1).

[0003] Some of the reaction pathways for biosynthesis of polyhydroxyalkanoic acids in microorganisms have already been elucidated. A group of enzymes specifically involved in polyhydroxyalkanoic acid biosynthesis are: It consists of two groups: Pha enzymes that use various hydroxyalkanoic acid monomers as substrates, and Phb enzymes that mainly use hydroxybutyric acid monomers as substrates. Individual enzymes constituting these enzyme groups are enzyme families having similar amino acid sequences for each microorganism species, but the details of amino acid sequences are different (Non-patent Document 1).

[0004] In order to industrially produce and utilize polyhydroxyalkanoic acid, it is necessary to develop technology for increasing production by microorganisms. In this requirement, the amount of polyester produced can be increased by replacing the enzymes that make up the polyester biosynthetic pathway described above with corresponding enzymes from other microorganisms or by mutating wild-type enzymes. Attempts have been made and certain results have been obtained (Patent Document 1, Non-Patent Documents 2 and 3).

However, in order to carry out practical industrial production of polyhydroxyalkanoic acid, it is necessary to improve the production amount and efficiency of microorganisms by further improving these enzyme groups. In particular, by improving 3-ketoacinole-CoA reductase (hereinafter referred to as PhbB enzyme) that produces 3-hydroxyacyl-CoA, which is a monomer raw material that constitutes polyhydroxyalkanoic acid, as a monomer raw material, It is necessary to improve the production volume and thus the polymer production volume.

Patent Document 1: JP 2002-199890

Non-Patent Document 1: Journal of Japan Oil Chemists' Society, 1999, 48 卷, 1353-1364

Non-Patent Document 2: Fermentation Handbook (Kyoritsu Shuppan), 2001, 374-378

Non-Patent Document 3: J. Bacteriol., 1998, 180 卷, 6459-6467

Disclosure of the invention

Problems to be solved by the invention

[0005] The present invention is a rational enzyme modification for improving the ability to use a coenzyme and catalytic activity of a PhbB enzyme and improving the production of 3-hydroxyacyl-CoA, which is a raw material for polyhydroxyalkanoic acid. The purpose is to provide a method. The present invention also provides a PhbB enzyme mutant that has improved the catalytic activity of a wild-type enzyme by the modification method, a DNA encoding the PhbB enzyme mutant, and a transformed cell into which the enzyme has been introduced. Alternatively, a method for producing 3-hydroxyacyl-CoA and polyhydroxyalkanoic acid using transformed cells is proposed. The purpose is to provide.

Means for solving the problem

[0006] As a result of intensive studies to solve the above-mentioned problems, the present inventors have newly developed an enzyme modification method that can improve the catalytic activity of the PhbB enzyme. The production of 3-hydroxylacyl_CoA, an in-vivo raw material, was efficiently carried out, and a PhbB enzyme mutant was successfully produced, thereby completing the present invention.

[0007] That is, the present invention is an enzyme modification method for converting the catalytic activity of an enzyme, wherein one or a plurality of arbitrary amino acid residues at a preselected site of a PhbB enzyme are substituted, inserted or deleted. Alternatively, the present invention relates to an enzyme modification method characterized by controlling the magnitude of the binding energy of nicotinamide coenzyme molecules and controlling the reduction reaction activity catalyzed by the enzyme by combining them.

Here, nicotinamide coenzyme molecules are β-nicotinamide adenine dinucleotide (ΝΑ DH), β nicotinamide adenine dinucleotide phosphate (NADPH), and their oxidized forms (hereinafter, unless otherwise specified) Called coenzyme).

[0008] The present invention also relates to a 3 keto-asinole CoA reductase comprising the amino acid sequence represented by SEQ ID NO: 1 (PhbB enzyme), an enzyme modification method for an enzyme having 30% or more sequence homology with the enzyme, The present invention relates to an enzyme variant modified by a method and a method of using the enzyme variant.

[0009] Further, the present invention is a PhbB enzyme having a sequence homology of 30% or more, preferably 50% or more, more preferably 70% or more with respect to the PhbB enzyme consisting of the amino acid sequence represented by SEQ ID NO: 1. And the coenzyme molecule binding site is Met-12, Gly_13, Ala_32, Cys-34, Gly-35, among the amino acid residue sites of PhbB enzyme consisting of amino acid sequence IJ represented by SEQ ID NO: 1. Enzyme modification that performs mutation treatment on amino acid residues that occupy three-dimensional structures equivalent to Pro-36, Asn-37, Ser-38, Pro-39, Arg-40, Arg-41, and Ala-57 Regarding the method.

[0010] The present invention also relates to the above-described enzyme modification method, which is a wild-type PhbB enzyme derived from the above-mentioned PhbB enzyme strength S, Ralstonia eutropha.

Furthermore, the present invention provides an enzyme that is obtained from a wild-type PhbB enzyme by substitution, insertion or deletion of amino acid residues, or a combination thereof, and is superior to the enzyme activity exhibited by the wild-type enzyme. More preferably, the present invention relates to a PhbB enzyme variant, wherein the wild-type PhbB enzyme is a PhbB enzyme derived from Ralstonia eutropha.

[0011] Furthermore, the present invention relates to a PhbB enzyme mutant obtained by the enzyme modification method, a DNA encoding the enzyme mutant, a vector having the DNA, and a transformed cell transformed with the vector.

[0012] Furthermore, the present invention relates to a method for producing a PhbB enzyme mutant comprising a step of culturing and proliferating the transformed cell.

Furthermore, the present invention relates to a method for producing an optically active alcohol comprising a step of reacting the enzyme mutant with various ketones.

Furthermore, the present invention relates to a method for producing 3-hydroxyasynole CoA, which comprises a step of reacting the enzyme mutant or the transformed cell with 3-ketoacinole CoA.

Furthermore, the present invention relates to a method for producing polyhydroxyalkanoic acid, comprising the step of culturing and growing the transformed cell or the step of reacting the enzyme mutant with 3-ketosil-CoA.

Hereinafter, the present invention will be described in detail.

First of all, in this specification, “3-ketoacyl-CoA reductase” means 3-ketoacyl-CoA as a reaction substrate and nicotinamide coenzyme as a reducing agent. An enzyme that catalyzes the reaction of asymmetric reduction of CoA to the corresponding 3-hydroxylacyl-CoA, commonly referred to as the PhbB enzyme.

[0014] In addition, 3-ketoasyl-CoA is 3_ketobutanol_CoA, 3-ketopentanoylol-CoA, 3-ketohexanoyl_CoA, or 3-ketofatty acid_CoA having a longer alkyl chain, each alone Or a mixture of these compounds.

3-Hydroxylacyl CoA means 3-hydroxybutanol _CoA, 3-hydroxypentanoylol CoA, 3-hydroxyhexanol _CoA, or a longer alkyl chain length 3-hydroxy fatty acid CoA. A compound or a mixture of these compounds. [0015] In the present specification, amino acids, peptides, and proteins are represented by the following abbreviations adopted by the IUPAC- IUB Biochemical Nomenclature Committee (CBN). Unless otherwise specified, the amino acid residue sequences of peptides and proteins are expressed from the N-terminus to the C-terminus from the left end to the right end, and in the N-terminal order. The connection of amino acid residues is represented by “one”. For example, Ala_Gly_Leu. When the same amino acid residue continues, it is expressed, for example, as (Ala) 3, which is synonymous with Ala-Ala-Ala.

A == Ala = 'maranine, C = Cys = cystine,

D = = Asp = = aspartic acid, E = Glu = glutamic acid,

F = ^: Phe =: Phenenolealanine, G = Gly = glycine,

H = = His = seven stidines, I = Ile = isoleucine,

K = ^: Lys =: Lysine, L = Leu = Leucine,

M = = Met = = methionine, N = Asn = asparagine,

P = Pro = proline, Q = Gin = glutamine,

R = Arg =: Anoleginine, 3 = 36 = Serine,

T = Thr =: Threonine, V = Val = Valine,

W == Trp == Tributophane, 丫 = Ding = Chiguchi Shin,

B = ^: Asx = = Asp or Asn, 2 = 01 = 0111 or 0111,

X =: Xaa =: Any amino acid.

[0017] In describing the PhbB enzyme mutant in this specification, the nomenclature (original amino acid; position; substituted amino acid) is applied for easy reference. Thus, for example, the substitution of tyrosine for aspartic acid at position 64 is indicated as Tyr64Asp or Y64D. Multiple mutations are expressed by separating them with a slash mark ("Z"). For example, S41AZY64D indicates substitution of serine at position 41 with alanine and tyrosine at position 64 with aspartic acid.

[0018] In the present specification, an enzyme "mutant" has an amino acid sequence in which at least one amino acid contained in the amino acid sequence of the enzyme is substituted, inserted or deleted, and has at least the activity of the enzyme. A modified enzyme that retains a portion. [0019] Amino acid mutations used in the design of mutants include amino acid substitutions and amino acid insertions or deletions. Amino acid substitution refers to substituting the original peptide with one or more amino acids. An amino acid insertion refers to the insertion of one or more amino acids into the original peptide chain. Deletion of amino acids refers to the deletion of one or more amino acids from the original peptide.

[0020] Hereinafter, protein amino acid mutations for obtaining a desired mutant will be described.

The method for performing amino acid substitution or the like is not limited to the force including changing the codon of the DNA sequence encoding the amino acid in a technique using chemical synthesis or genetic engineering.

[0021] In order to improve the catalytic activity of the PhbB enzyme, the present inventors designed an amino acid mutation that can enhance the binding ability between the enzyme and the coenzyme. This design consists of identifying the binding site for the coenzyme molecule of the PhbB enzyme, determining the amino acid residue that interacts with the coenzyme molecule in the vicinity of the binding site, and the coenzyme molecule for the determined residue. It includes a step of designing a mutation to control the magnitude of the binding energy.

[0022] The step of specifying the coenzyme molecule binding site of the PhbB enzyme is a result of multiple alignment of the amino acid sequences of related enzymes having high homology with the enzyme and known steric structures. And the results of modeling the three-dimensional structure of the enzyme were analyzed.

The step of determining amino acid residues that interact with the coenzyme molecule of the PhbB enzyme was performed by modeling the three-dimensional structure of the enzyme and analyzing its three-dimensional structure.

The step of designing the mutation for controlling the magnitude of the binding energy of the coenzyme molecule with respect to the determined residue is a method of calculating an optimized solution of the multiple mutant protein amino acid sequence (Japanese Patent Laid-Open No. 2001-184381), etc. Was carried out by computational chemical analysis.

[0023] As a result of these design work, amino acid sequences of various PhbB enzyme mutants that improve the coenzyme binding ability of the wild-type PhbB enzyme and can improve the enzyme catalytic activity were determined. Various PhbB enzyme mutants having the amino acid sequence were obtained by genetic engineering techniques, and the enzyme activity of these mutants was verified. As a result, 3-hydroxylacyl_CoA production was confirmed. We succeeded in obtaining an efficient mutant of PhbB enzyme.

[0024] Embodiments of the present invention are specifically shown below.

That is, among the amino acid residue sites of the PhbB enzyme consisting of the amino acid sequence shown in SEQ ID NO: 1, Met-12, Gly-13, Ala-32, Cys-34, Gly-35, Pro-36, Asn-3 7 Ser_38, Pro_39, Arg_40, Arg_41, Ala_57, by substituting, inserting or deleting one or more amino acids, inserting or deleting them, or combining them, the ability of the enzyme to bind to the coenzyme This enzyme modification method is characterized by obtaining a PhbB enzyme mutant with improved catalytic activity.

Moreover, it is a PhbB enzyme mutant obtained by the modification method.

[0025] The PhbB enzyme consisting of the amino acid sequence represented by SEQ ID NO: 1 is a wild-type PhbB enzyme derived from Ralstonia eutropha.

In addition, the catalytic activity of the enzyme can be obtained, for example, by performing an enzyme-catalyzed reaction using acetacetyl CoA as a substrate and NADPH or NADH as a coenzyme, and measuring the change in coenzyme concentration during the reaction process by absorbance analysis. By applying the measured measurement data to the Michaelis-Menten equation.

[0026] Preferably, the amino acid residue part of the PhbB enzyme comprising the amino acid sequence represented by SEQ ID NO: 1 is selected, Met-12 is Sed, Gly-13 is Arg, and Ala-32 is Vali. Cys-34 to Asp, Gly-35 to lie or Arg, Pro-36 to Ser, Asn-37 to Lys, Ser-38 to Glu, Pro-39 to Asp, Arg-40 By performing at least one amino acid substitution of Ala or Glu, Arg-41 to Ala, and Ala-57 to Tyr, the ability to bind the enzyme to the coenzyme is improved, and the catalytic activity is increased. An improved PhbB enzyme variant.

[0027] More preferred is a PhbB enzyme or a PhbB enzyme variant consisting of the amino acid sequences represented by SEQ ID NOs: 2, 3, 4, 5 and 6, respectively.

Table 1 shows a comparison between the amino acid sequences of the mutants designed for these mutants and the amino acid sequence of the wild-type PhbB enzyme.

[0028] [Table 1] Amino acid group

Enzyme jsn ¾ ■ = ■ position E

BC Tari Φ

1 2 1 3 32 34 35 36 37 38 39 40 41 57 Wild type 1 Met Gly Ala Cys Gly Pro Asn Ser Pro Arg Arg Ala mutant M-3 2 Met Gly Ala Cys Arg Ser ys Glu Asp Glu Arg Ala mutant M-4 3 Met Gly Ala Cys Gly Pro Asn Ser Asp Ala Ala Ala variant M-8 4 Arg Ala Cys Gly Pro Asn Ser Asp Ala Ala Ala variant M-9 5 Met Gly Val Asp lie Pro Asn Ser Pro Ala Ala Tyr mutant M-1 1 6 Met Gly Ala Cys Gly Pro Asn Ser Asp Ala Ala Tyr

[0029] Further, in the present invention, a 3-ketocil having a sequence homology of 30% or more, preferably 50% or more, more preferably 70% or more with respect to the PhbB enzyme consisting of the amino acid sequence represented by SEQ ID NO: 1. — CoA reductase, Met-12, Gly-13, Ala-32, Cys-34, Gly-35, Pro-36, Asn-37, Ser-38, Pro-39, Arg-40, Arg-41, The catalytic activity is improved by substituting, inserting or deleting one or more amino acids for amino acid residues that occupy the same position as that of the amino acid residue of Ala 57. An enzyme modification method characterized by obtaining a reductase mutant is also included.

Moreover, the PhbB enzyme variant obtained by the said enzyme modification method is also mentioned.

[0030] Sequence homology can be determined using the programs FASTA (Perason WR et al., Genomics, 46, 24 —36 (1997)) and BLAST (Altschul, Stephen F. et al., Nucleic Acids Res. 25, 3389— 3402. (1997)) can be determined by amino acid sequence homology analysis. The amino acid mutation for improving catalytic activity according to the present invention is also effective for an enzyme having sufficient sequence homology to the PhbB enzyme consisting of the amino acid sequence represented by SEQ ID NO: 1.

[0031] Furthermore, embodiments of the present invention include DNA encoding the PhbB enzyme variant obtained by the enzyme modification method, a vector having the DNA, and a transformed cell transformed with the vector.

[0032] In order to obtain DNA encoding the PhbB enzyme variant of the present invention, site-specific mutation introduction into the DNA encoding the wild-type PhbB enzyme is carried out as follows. PCR can be used.

In other words, the introduction of mutations by recombinant DNA technology, for example, the presence of appropriate restriction enzyme recognition sequences on both sides of the target site where mutations are desired in the wild-type PhbB enzyme gene. In this case, the restriction enzyme is used to cleave it, remove the region containing the site where mutation is desired, and then insert the DNA fragment mutated into the target site by chemical synthesis or the like. Can do.

In addition, the introduction of site-specific mutation by PCR involves the use of a mutation primer in which a mutation of interest is introduced into a desired site to be introduced into the wild-type PhbB enzyme gene, and the sequence of one end of the gene. A mutation primer that amplifies one side of the gene with an amplification primer and a primer complementary to the mutation primer and the other of the gene. Amplification primer containing no mutation at the terminal site of the other, amplify the other side, anneal the two obtained amplification fragments, and then PCR with the two amplification primers It can be carried out.

[0033] The vector of the present invention can be obtained by ligating (inserting) the DNA encoding the aforementioned PhbB enzyme mutant into an appropriate vector.

The transformed cell of the present invention can be obtained by introducing the recombinant vector of the present invention into a host so that the gene of the present invention can be expressed.

The vector for inserting the gene is not particularly limited as long as it can autonomously replicate in the host, and plasmid DNA or phage DNA can be used as the vector. For example, when using Escherichia coli as a host, plasmid DNA such as pBR322, pUC18, and pBluescript II, phage DNA such as EMBL3, Μ13, and λ gtl l; and YEpl3 when using yeast as the host the YCp50 like; in the case of using a plant cell as the host cell, pB 1121, the ρΒΙΙΟΙ like; the case of using animal cells as the host, can be used pcDNAI, the pcDNAl / a m _p such as a vector.

[0034] The host is not particularly limited, and examples thereof include bacteria such as Escherichia coli, yeast, plant cells, and animal cells.

[0035] The transformed cell of the present invention can be obtained by introducing the vector into a host cell. Examples of the method for introducing a recombinant vector into bacteria include a method using calcium ions and an electopore position method. Examples of the method for introducing the recombinant vector into yeast include the electopore position method, the free mouth plast method, and the lithium acetate method. As a method for introducing a recombinant vector into a plant cell, for example, Examples include the Globataterum infection method, the particle gun method, and the polyethylene glycol method. Examples of the method for introducing a recombinant vector into animal cells include the electoporation method and the calcium phosphate method.

More specifically, transformed cells that produce polyhydroxyalkanoic acid are obtained by a method described in a method for transforming E. coli (JP 2002-199890) or a method for transforming yeast (WO03Z033707). Obtainable.

[0036] Next, the method for producing a PhbB enzyme variant of the present invention includes the step of culturing and growing the transformed cell, that is, using the transformed cell.

Specifically, the transformed cells are cultured in a medium, and the enzyme variant of the present invention is produced and accumulated in the culture (cultured cells or culture supernatant), and the enzyme variant is collected from the culture. By doing so, the PhbB enzyme mutant of the present invention can be produced.

[0037] The method of culturing the transformed cell of the present invention in a medium is performed according to a usual method used for culturing a host. Examples of the medium for culturing transformed cells obtained by using bacteria such as E. coli as a host include complete medium or synthetic medium such as LB medium, M9 medium and the like. In addition, by culturing aerobically for 6 to 24 hours at a culture temperature of 20 to 40 ° C., the enzyme variant of the present invention can be accumulated in the microbial cells.

Subsequently, the enzyme mutant can be recovered by, for example, centrifuging the culture obtained by the above-described culture method (cells are crushed with a sonicator or the like).

[0038] For purification of the enzyme mutant of the present invention, the recovered enzyme mutant is used alone or appropriately combined with affinity mouth chromatography, cation or anion exchange chromatography, gel filtration and the like. Can be done by.

Confirmation that the obtained purified substance is the target enzyme can be carried out by an ordinary method such as SDS polyacrylamide gel electrophoresis, Western blotting or the like.

[0039] Next, the method for producing an optically active alcohol of the present invention uses the above enzyme mutant. Specifically, the enzyme mutant and various asymmetric ketones are reacted under appropriate conditions. It is a method including the process to make.

Appropriate reaction conditions include, for example, reaction conditions described in enzyme modification methods and oxidoreductase mutants (Japanese Patent Laid-Open No. 2003-804653). [0040] Further, the method for producing 3-hydroxyasinole CoA uses the above-mentioned enzyme mutant or transformed cell. Specifically, the enzyme mutant or transformed cell and 3-keto-asinole CoA are combined with each other. It is a method including a step of reacting under appropriate conditions. In this method, 3-hydroxyasinole-CoA can be produced as an enzyme reaction product. Appropriate reaction conditions include, for example, the reaction conditions described in the literature on acetacetyl CoA reductase (FEMS Microbiology Letters vol. 52 (1988) pp. 259-264).

[0041] Further, the method for producing a polyhydroxyalkanoic acid of the present invention includes the step of using the transformed cell, that is, the step of culturing and growing the transformed cell.

Specifically, in the method for producing the polyhydroxyalkanoic acid, the transformed cells obtained by the above-described method are mixed with an appropriate medium, that is, a sufficient amount of sugar such as gnolecose, saturated fatty acid glyceride and the like. Polyhydroxyalkanoic acid, which is a polyester, can be produced by culturing in a medium containing the same.

More specifically, for example, by the method described in the method using transformed E. coli (JP-A-11 243956), the method using transformed yeast (JP-A 2003-833707), etc. Hydroxyalkanoic acids can be produced.

[0042] Incidentally, the polyhydroxyalkanoic acid S, the following general formula (1):

[0043] [Chemical 1]

(In the formula, R represents an alkyl group having 1 to 20 carbon atoms, and n represents the number of monomer units of the polymer. The carbon number of the alkyl group may be the same or different in the polymer. However, it is preferably a compound represented by

[0045] Examples of the alkyl group having 1 to 20 carbon atoms of R include a methyl group, an ethyl group, an n-propyl group, an iso group. A propyl group, a butyl group, a pentyl group, a hexyl group, an octyl group, a decyl group, a tetradecyl group, an octadecyl group, etc. are mentioned, and a methyl group or an n-propyl group is preferable.

[0046] The intracellular content of the polyester accumulated in the cells and the composition of the polyester were determined by the method of Kato et al. (Appl. Microbiol. Biotechnol., 45 卷, 363 (1996); Bull. Chem. Soc., 69 After extraction from cultured cells using an organic solvent such as chlorohonorem, the extract can be measured and analyzed by subjecting it to gas chromatography, NMR, or the like.

The invention's effect

[0047] By using the enzyme variant of the present invention as a catalyst, 3-hydroxycyl-CoA and optically active alcohol can be produced with high efficiency. Further, by using the enzyme mutant or transformed cell of the present invention, it becomes possible to produce polyhydroxyalkanoic acid with high efficiency.

BEST MODE FOR CARRYING OUT THE INVENTION

[0048] Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.

[Example l] PhbB enzyme three-dimensional structure modeling and coenzyme binding site analysis

The query BLAS T (Altschul, Stephen F. et al., Nucleic Acids Res. 25, using the amino acid sequence of the wild-type PhbB enzyme derived from Ralstonia eu tropha consisting of the amino acid sequence shown in SEQ ID NO: 1 as a query, 3389-3402 (1 997)) was used to search and extract highly homologous amino acid sequences from protein primary structure data listed in the Protein Data Bank (PDB). Among the obtained amino acid sequences, gnorecose dehydrogenase (PDB code: 1G CO) was selected as the sequence having the highest homology with the wild-type PhbB enzyme, and the atomic coordinate data of the enzyme (1GCO) listed in the PDB As a template, the amino acids using Swiss-PDUV iewer (Swiss Institute of Biomormatics (SI B), ExPASy Molecular Biology Server (http://www.expasy.ch)) Residue substitution, insertion, and deletion operations were performed to obtain a three-dimensional structural model of wild-type PhbB enzyme (synonymous with atomic coordinate data). Next, with coenzyme An operation of loading certain NADH or NADPH into the above three-dimensional structure model was performed to obtain a three-dimensional structure model of a complex of a wild-type PhbB enzyme and a coenzyme (NADH or NADPH). Using the three-dimensional structure model (atomic coordinate data) of the obtained PhbB enzyme coenzyme complex, the amino acid residues present in the vicinity of ribose 2 '_H of NADH or ribose 2'-phosphate of NADPH are Residue numbers 11-: 17, residue numbers 32-44, and residue number 57 were identified. By using the method for calculating the optimized solution of the multiple mutant protein amino acid sequence (Japanese Patent Laid-Open No. 2001-184381), the amino acid residue positions that can be expected to improve NADH binding ability from the identified amino acid residue positions Residue species and amino acid residue positions and residue species that are expected to improve NA DPH binding ability were selected and used as the amino acid sequence of the Ph bB enzyme mutant. The PhbB enzyme mutated amino acid sequence is as shown in SEQ ID NOs: 2 to 6, and the comparison between the mutated amino acid sequence group and the wild-type amino acid sequence is as shown in Table 1 above.

[Example 2] Gene encoding wild-type PhbB enzyme (SEQ ID NO: 1)

The wild type phbB gene consisting of the base sequence shown in SEQ ID NO: 7 was fully synthesized and amplified with primers consisting of the base sequences shown in SEQ ID NOs: 8 and 9. The wild-type phbB gene was cloned into the Ndel / Pstl site of plasmid pUCNT-2 in which the Hindlll site in the multi-cooling site of plasmid pUCNT (described in W094 / 03613) was destroyed by the blunt end method.

[0051] (Example 3) Gene encoding mutant M-3 (SEQ ID NO: 2)

Overlapping PCR reaction was performed by mixing 150 pmoles of the synthetic gene consisting of the nucleotide sequences set forth in SEQ ID NOs: 10 and 11. As the polymerase, pyrobest manufactured by Takara was used, and the attached buffer and dNTP were used. The reaction volume was 0.03 ml. The reaction conditions were 96 ° C for 5 minutes once, 96 ⁰ 2 minutes '60 ⁰ times 30 less' 72 ⁰ times 30 times less cyclone 12 times. About 15 Obp of DNA was excised by agarose electrophoresis and digested with Ndel and Hindlll. This gene fragment was cloned into pUCNT-2 containing the wild-type gene cleaved with the same enzyme to create a mutant M-3 gene.

[0052] (Example 4) Gene encoding mutant M-4 (SEQ ID NO: 3)

Using a synthetic gene consisting of the nucleotide sequences set forth in SEQ ID NOs: 10 and 12, mutant M— Mutant M-4 gene was prepared in the same way as when 3 genes were created.

[Example 5] Gene encoding mutant M-8 (SEQ ID NO: 4)

Using a synthetic gene consisting of the nucleotide sequences set forth in SEQ ID NOs: 13 and 12, a mutant M_8 gene was prepared in the same manner as when the mutant M-3 gene was prepared.

[Example 6] Gene encoding mutant M-9 (SEQ ID NO: 5)

Using the synthetic gene consisting of the nucleotide sequences set forth in SEQ ID NOs: 10 and 14, a mutant M_2 gene was prepared in the same manner as when the mutant M-3 gene was prepared. For the pUCNT-2 containing this mutant M-2 gene, using a primer consisting of the base sequence described in SEQ ID NOs: 15 and 16, the amino acid of the mutant M_2 of 57 A mutant M-9 gene in which alanine was converted to tyrosine was created. The quick change method followed the Stratagene protocol.

[Example 7] Gene encoding mutant M-11 (SEQ ID NO: 6)

For pUCNT-2 containing the mutant M-4 gene, the alanine of the amino acid sequence 57 of the mutant M-4 was converted by the quick change method using a primer consisting of the nucleotide sequence of SEQ ID NOs: 15 and 16. A mutant M-11 gene converted to tyrosine was created.

[Example 8] Confirmation of gene sequence

All of the above mutant genes were sequence-checked using DNA Sequencer 310 Genetic Analyzer manufactured by PERIKIN ELMER APPLIED BIO SYSTEMS.

[Example 9] Gene expression and enzyme purification

Each of the plasmids prepared above was used to transform E. coli JM-109 cells (Takara). The resulting JM-109 cells containing either the wild-type phbB gene or the phbB mutant gene were cultured overnight in LB medium containing ampicillin. 1 ml of the main culture was inoculated into 2YT medium (10 ml, containing ampicillin) containing isopropinole 1_thio 1β_D_galactoside (IPTG) (0.2 mM) and cultured at 30 ° C. for 8 hours. After completion of the culture, the cells were collected by centrifugation, and diluted to 0.5 ml of potassium phosphate buffer (10 mM, pH 7.0) containing ethylenediamine tetraacetic acid (EDTA) (0.5 mM) and dithiothreitol (DTT) (ImM). Resuspended. Cells are disrupted by ultrasonic disruption and centrifuged to remove the supernatant fraction. Fractionated into soluble fractions. When each fraction was analyzed by SDS electrophoresis, it was found that all of the supernatant fractions of the cells into which the mutants M-3, 4, 8, 9, 11 were introduced were almost identical to the wild-type PhbB enzyme. Protein expression, which was thought to be induced by IPTG, was observed at a molecular weight of 25,000. The expression level was almost the same as the wild type. Accumulation in the insoluble fraction was not observed in all mutants.

Example 10 Measurement of Mutant Activity

Regarding the activity of these mutants, the activity and coenzyme characteristics in the reduction reaction of Acetoacetyl CoA (manufactured by Sigma) were measured. The composition of the reaction solution was a Tris-HCl buffer solution containing NADH or NADPH (from Le oriental Oriental Yeast Co.) at a concentration of 0.02 to 0.5 mM as a coenzyme and acetocetyl CoA (0.05 mM) as a substrate ( 50 mM, pH 8.0) 0.5 ml, and 0.01 ml of appropriately diluted bacterial cell disruption solution was added to start the reaction. The dilution amount of the cell disruption solution was determined as a sufficient amount to obtain the initial reaction rate by preliminary experiments. The dilution was about 20 to 250 times. The reaction was monitored for 3 minutes using a spectrophotometer and the decrease in absorbance at a wavelength of 340 nm was followed for 3 minutes to measure the initial rate of the reaction. A spectrophotometer cell with an optical path length of about 0.4 cm was used. The initial reaction rate when the coenzyme concentration was changed by 4 to 5 points was plotted and fitted to the Michaelis-Menten equation by the least square method to obtain various parameters. The results are shown in Table 2.

[0059] [Table 2]

In Table 2, Vmax and Km are enzyme catalytic constants according to Michaelis Menten's formula. When NADPH or NADH is used as a coenzyme and acetoacetyl CoA is used as a substrate, wild-type PhbB enzyme (SEQ ID NO: 1) and PhbB enzyme are used. The maximum reaction rate and coenzyme binding constant of the mutant (SEQ ID NO: 2 to 6) are shown respectively. The activity value (VmaxZKm) is Based on the change in absorbance (◯ D) 340 nm per minute per ml of enzyme solution under the reaction conditions, it was calculated by the above method using the Michaelis-Menten equation.

N.S. indicates that saturation was not reached in the measured concentration range.

[0061] Activity comparison between wild-type enzyme and enzyme mutant:

As shown in the results in Table 2, the mutants M_4, M_8 and M_11 had improved activity when NADPH was used as a coenzyme compared to the wild type. Mutants M_3, M_4, M-9 and M_11 showed improved activity when NADH was used as a coenzyme compared to the wild type. Industrial applicability

[0062] By using the enzyme variant of the present invention as a catalyst, 3-hydroxycyl-CoA and optically active alcohol can be produced with high efficiency. Further, by using the enzyme mutant or transformed cell of the present invention, it becomes possible to produce polyhydroxyalkanoic acid with high efficiency.

Claims

The scope of the claims

[1] Among the amino acid residue sites of the 3_ketosyl_CoA reductase consisting of the amino acid sequence shown in SEQ ID NO: 1, Met-12, Gly-13, Ala-32, Cys-34, Gly-35, Pro-36 , Asn-37, Ser_38, Pro_39, Arg_40, Arg_41, Ala-57ί, improved catalytic activity by substituting, inserting or deleting one or more amino acids, or combining them. A method for modifying an enzyme, which comprises obtaining a 3-ketoacinole CoA reductase mutant.

[2] The amino acid residue site according to claim 1, wherein the 3-ketoacinole CoA reductase having a sequence homology of 30% or more with respect to the 3-ketocil CoA reductase consisting of the amino acid sequence represented by SEQ ID NO: 1 A 3-ketoacinole CoA reductase variant with improved catalytic activity is obtained by substituting, inserting or deleting one or more amino acids at amino acid residues that occupy the same position as the three-dimensional structure. A method for modifying an enzyme.

[3] A 3-ketoasinoleo CoA reductase mutant obtained by the modification method according to claim 1.

[4] A 3-ketoacinole-CoA reductase mutant obtained by the modification method according to claim 2.

[5] The amino acid residue of the 3-ketosyl-CoA reductase consisting of the amino acid sequence shown in SEQ ID NO: 1 is selected, Met-12 is Sed, Gly-13 is Arg (this, Ala32 is Val, Cys —34 as Asp, Gly—35 lie, and f Arg (This, Pro_ 36, Ser (This, Asn_ 37, Lys (This, Ser_ 38, Glu, Pro—39, Asp, Arg_40 A 3-ketoacinole CoA reductase mutant obtained by substituting at least one selected amino acid with Ala or Glu, Arg_41 with A1a, and Ala-57 with Tyr.

[6] 3_ketocil-CoA reductase consisting of the amino acid sequence represented by SEQ ID NO: 2.

[7] A 3-ketosyl-CoA reductase consisting of the amino acid sequence represented by SEQ ID NO: 3.

[8] A 3-ketosyl-CoA reductase consisting of the amino acid sequence represented by SEQ ID NO: 4.

[9] A 3-ketosyl-CoA reductase consisting of the amino acid sequence represented by SEQ ID NO: 5.

[10] A 3-ketosyl-CoA reductase consisting of the amino acid sequence represented by SEQ ID NO: 6. DNA encoding the enzyme variant according to any one of claims 3 to 5 or the enzyme according to any one of claims 6 to 10:

A vector comprising the DNA according to claim 11.

A transformed cell obtained by transformation with the vector according to claim 12.

A method for producing the 3_ketocil-CoA reductase mutant according to any one of claims 3 to 5 or the enzyme according to any one of claims 6 to 10 using the transformed cell according to claim 13. .

A method for producing an optically active alcohol using the enzyme variant according to any one of claims 3 to 5 or the enzyme according to any one of claims 6 to 10:

A method for producing 3-hydroxylacyl-CoA using the enzyme variant according to any one of claims 3 to 5 or the enzyme according to any one of claims 6 to 10:

A method for producing 3-hydroxyasinole-CoA using the transformed cell according to claim 13. A method for producing polyhydroxyalkanoic acid using the transformed cell according to claim 13. The polyhydroxyalkanoic acid power is represented by the following general formula (1):

(In the formula, R represents an alkyl group having 1 to 20 carbon atoms, and n represents the number of monomer units of the polymer. The carbon number of the alkyl group may be the same or different in the polymer. 19. The method for producing a polyhydroxyalkanoic acid according to claim 18, which is a compound represented by

20. The method for producing a polyhydroxyalkanoic acid according to claim 19, which is a compound in which R in the formula (1) is a methyl group or an n-propyl group.