WO2022210228A1 - MODIFIED α-ISOPROPYLMALATE SYNTHASE - Google Patents

Abstract

The present disclosure provides a novel IPMS mutant which can impart, to various microorganisms, the ability to produce a derivative including L-leucine or a precursor thereof. The IPMS mutant contains at least two amino acid substitutions selected from the following (a') to (c') on the basis of the amino acid sequence set forth in SEQ ID NO: 2: (a') substitution of the amino acid residue corresponding to the glycine residue at position 530 with an amino acid residue other than a glycine residue; (b') substitution of the amino acid residue corresponding to the glycine residue at position 532 with an amino acid residue other than a glycine residue; and (c') substitution of the amino acid residue corresponding to the alanine residue at position 535 with an amino acid residue other than an alanine residue.

Description

Modified α-Isopropyl Malate Synthase

The present invention provides a modified α-isopropylmalate synthase with reduced feedback inhibition by leucine, a polynucleotide encoding the modified α-isopropylmalate synthase, a genetically modified microorganism into which the polynucleotide has been introduced, and the gene The present invention relates to a method for producing a desired substance using modified microorganisms.
The present application is based on Japanese Patent Application No. 2021 filed with the Japan Patent Office on March 29, 2021. The priority is claimed based on Japanese Patent Application No. 2021-054521, and the content of the above Japanese patent application is incorporated herein for all purposes. Additionally, the disclosure of each prior art document referred to in this application is also incorporated herein for all purposes.

L-leucine is a type of essential amino acid, an expensive amino acid that is widely used in pharmaceuticals, foods, feed additives, industrial chemicals, etc., and is mainly produced using microorganisms. Specifically, fermentative production of branched-chain amino acids including L-leucine is mainly carried out using Escherichia microorganisms or Corynebacterium microorganisms. It is known that 2-ketoisocaproic acid is biosynthesized as a precursor starting from pyruvic acid through various steps. In such a pathway of L-leucine biosynthesis, α-isopropylmalate synthase (2-isopropylmalate synthase, α-isopropylmalate synthase, EC 2.3.3.13; hereinafter referred to as "IPMS" ) plays a major role, and genes encoding the enzyme have been isolated from various species, and the nucleotide sequences of the genes and the amino acid sequences of the enzyme proteins in many species have also been identified. has been decided.

By the way, although IPMS plays an important role in the L-leucine biosynthetic pathway, it is known that feedback inhibition often occurs, in which the enzymatic activity is inhibited by the final product L-leucine produced. Conventionally, such feedback inhibition has been a problem in the industrial production of L-leucine using microorganisms, and various techniques have been developed to solve this problem.

For example, Patent Document 1 discloses a microorganism transformed with a DNA encoding α-isopropylmalate synthase that has L-leucine-producing ability and has been released from inhibition by L-leucine, and L - A method for the production of leucine is disclosed. In the technique described in Patent Document 1, "α-isopropylmalate synthase whose inhibition by L-leucine has been released" is cloned from a mutant strain of E. coli having L-leucine-producing ability. A mutant IPMS encoded by a mutant leuA gene, characterized by having predetermined amino acid substitutions relative to the corresponding wild-type IPMS. In Patent Document 1, L-leucine-producing strains were actually obtained by transforming a given Escherichia coli strain with several mutant IPMS-encoding nucleic acids, and for each L-leucine-producing strain obtained, The results of evaluating the specific activity of IPMS, the degree of feedback inhibition by L-leucine, and the ability to produce L-leucine are described.

Furthermore, Patent Document 2 discloses a mutant IPMS in which the amino acid sequence of the wild-type IPMS encoded by the LeuA gene derived from Corynebacterium glutamicum is mutated to aspartic acid at the 553rd tyrosine residue. and a fermentation method for producing L-leucine or ketoisocaproate (KIC) using a microorganism introduced with the nucleotide, and the mutant IPMS is L-leucine, etc. It is mentioned that feedback inhibition of enzyme activity by is reduced. More specifically, in Patent Document 2, a genetically modified strain was prepared by introducing a gene encoding the mutant IPMS (mutant leuA gene) into Corynebacterium glutamicum ATCC13032 strain, and the genetically modified strain , Using the wild-type ATCC13032 strain as a control, KIC and L-leucine production tests were conducted, and it was confirmed that the genetically modified strain was endowed with the ability to produce these substances compared to the wild-type ATCC13032 strain. ing.

Furthermore, Patent Document 3 also discloses a mutant IPMS having a predetermined amino acid substitution for an IPMS encoded by a LeuA gene derived from Corynebacterium glutamicum, and a recombinant Corynebacterium glutamicum introduced with a nucleic acid encoding the same. A strain is disclosed, as well as a method for producing L-leucine using the recombinant strain. In Patent Document 3, the amino acid substitutions possessed by the above-mentioned mutant IPMS are the substitution of histidine for arginine at position 494 and the substitution of aspartic acid for glycine at position 497 in the amino acid sequence of IPMS used as a reference in the same document. and that leucine at position 499 is substituted with valine. However, in the amino acid sequence of the wild-type IPMS (LeuA gene protein) possessed by Corynebacterium glutamicum, the 494th, 497th and 499th amino acids are It is different from each amino acid, and although Patent Document 3 is written in Chinese, there are many unclear points about its disclosure.

Furthermore, Patent Document 4 discloses a novel mutant polypeptide having IPMS activity, a polynucleotide encoding the same, a microorganism containing the polypeptide, and a method for producing L-leucine by culturing the microorganism. there is In Patent Document 4, the "novel mutant polypeptide having IPMS activity" more specifically refers to the 558th arginine in the wild-type IPMS amino acid sequence encoded by the Corynebacterium glutamicum-derived LeuA gene. A residue is substituted with an amino acid residue other than arginine, or glycine at position 561 (residue is substituted with an amino acid residue other than glycine), and is defined as a variant polypeptide having IPMS activity In Patent Document 4, IPMS mutants that were actually produced and strains into which the IPMS mutants were introduced are IPMS mutants that possess amino acid substitutions of R558H, R558A, R558Q, G561D, G561R, and G561Y, respectively, and IPMS mutants having amino acid substitutions by a combination of R55H and G561D, and Corynebacterium glutamicum recombinant strains into which these mutants were respectively introduced, and these recombinant strains have L-leucine relative to the wild type. It has been confirmed that the productivity of

JP-A-2001-37494 Japanese Patent Publication No. 2015-514431 Chinese Patent Application Publication No. 104480058 Japanese Patent Publication No. 2020-503045

An object of the present invention is to provide novel IPMS mutants that can impart to various microorganisms the ability to produce derivatives containing L-leucine and its precursors.

As a result of intensive research to solve the above problems, by appropriately combining multiple independent mutations of IPMS that are resistant to feedback inhibition to L-leucine, a remarkable L-leucine that could not be obtained by a single mutation An increase in productivity was observed, leading to the completion of the present invention.

That is, according to aspects of the present invention, the following are provided.
[1] A modification in which L-leucine feedback inhibition is reduced compared to a protein comprising an amino acid sequence defined in any of the following (A) to (C) and consisting of the amino acid sequence shown in SEQ ID NO:2 Type α-isopropylmalate synthase:
(A) an amino acid sequence having at least two substitutions selected from the following (a) to (c) in the amino acid sequence shown in SEQ ID NO: 2;
(B) an amino acid sequence having a sequence identity of 60% or more to the amino acid sequence shown in SEQ ID NO: 2 and containing at least two substitutions selected from (a) to (c) below;
(C) the amino acid sequence shown in SEQ ID NO: 2, containing at least two substitutions selected from the following (a) to (c), and one or more at sites excluding the sites where the at least two substitutions are located amino acid sequences in which amino acids have been deleted, substituted, inserted or added;
(a) replacement of the 530th glycine residue with an amino acid residue other than a glycine residue;
(b) replacement of the 532nd glycine residue with an amino acid residue other than a glycine residue; and (c) replacement of the 535th alanine residue with an amino acid residue other than an alanine residue.

[2] The modified α-isopropyl malate according to [1], wherein the amino acid sequence defined in (B) above has a sequence identity of 84% or more to the amino acid sequence shown in SEQ ID NO:2. Synthase.
[3] The modified α according to [1] or [2], wherein the amino acid sequence defined in (B) above has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2. - isopropyl malate synthase.
[4] The modified α-isopropyl malate synthase according to any one of [1] to [3], containing all the substitutions defined in (a) to (c) above.
[5] The modified α-isopropylmalate synthase of [4], comprising the amino acid sequence defined in (A) or (C) above.

[6] having at least one amino acid mutation that reduces L-leucine feedback inhibition compared to the corresponding wild-type 2-isopropyl malate synthase;
A modified α-isopropyl malate synthase in which the at least one amino acid mutation comprises at least two substitutions selected from the following (a′) to (c′) based on the amino acid sequence shown in SEQ ID NO: 2:
(a') substitution of an amino acid residue corresponding to the 530th glycine residue with an amino acid residue other than the glycine residue;
(b′) substitution of the amino acid residue corresponding to the 532nd glycine residue with an amino acid residue other than the glycine residue; and (c′) alanine of the amino acid residue corresponding to the 535th alanine residue Substitutions to amino acid residues other than residues.

[7] The modified α-isopropyl malate synthase of [6], wherein the at least one amino acid mutation includes all of the substitutions defined in (a') to (c') above.
[8] Any one of [1] to [7], including an amino acid sequence having at least two substitutions in the amino acid sequence of a wild-type 2-isopropylmalate synthase derived from a bacterium belonging to the genus Corynebacterium The modified α-isopropyl malate synthase described in .

[9] The modified α-isopropyl according to any one of [1] to [8], wherein the at least two substitutions are at least two substitutions selected from (d) to (f) below. Malate synthase:
(d) aspartic acid residue, glutamic acid residue, lysine residue, alanine residue, valine residue, glutamine residue, cysteine residue of the 530th glycine residue or an amino acid residue corresponding to the glycine residue , proline, arginine, leucine, isoleucine, threonine or histidine residues;
(e) aspartic acid residue, alanine residue, valine residue, proline residue, leucine residue, isoleucine residue, threonine residue of the 532nd glycine residue or an amino acid residue corresponding to the glycine residue or substitution with a histidine residue; and (f) a threonine residue, serine residue, valine residue, lysine residue, aspartic acid residue of the 535th alanine residue or an amino acid residue corresponding to the alanine residue groups, phenylalanine residues, glycine residues, leucine residues, isoleucine residues or histidine residues.

[10] The modified α-isopropylmalate synthase of [9], containing all the substitutions defined in (d) to (f) above.
[11] The modified α-isopropylmalate synthase of [10], comprising the amino acid sequence defined in (A) or (C) above.
[12] The modified α-isopropyl according to any one of [1] to [11], wherein the at least two substitutions are at least two substitutions selected from (g) to (j) below. Malate synthase:
(g) replacement of the 530th glycine residue or an amino acid residue corresponding to the glycine residue with an aspartic acid residue;
(h) substitution of the 532nd glycine residue or an amino acid residue corresponding to the glycine residue with an aspartic acid residue; and (j) the 535th alanine residue or an amino acid corresponding to the alanine residue Substitution of residues for threonine, valine, glycine, leucine or isoleucine residues.

[13] The modified α-isopropylmalate synthase of [12], containing all the substitutions defined in (g) to (j) above.
[14] The modified α-isopropylmalate synthase of [13], comprising the amino acid sequence defined in (A) or (C) above.
[15] A polynucleotide encoding the modified α-isopropylmalate synthase of any one of [1] to [14].

[16] A microorganism into which the polynucleotide of [15] has been introduced.
[17] A microorganism comprising the modified α-isopropylmalate synthase of any one of [1] to [14].
[18] The microorganism of [16] or [17], which is a coryneform bacterium.
[19] The microorganism of [18], which is Corynebacterium glutamicum.

[20] The microorganism according to any one of [16] to [19], wherein wild-type α-isopropylmalate synthase activity is reduced or inactivated.

[21] (I) Producing a target substance by culturing or reacting the microorganism according to any one of [16] to [20] in a predetermined medium or reaction medium; and (II) the object recovering material;
A method of producing a desired substance, comprising

[22] The target substance is (2S)-2-isopropyl malate, 2-isopropyl malate, (2R,3S)-3-isopropyl malate, (2S)-2-isopropyl-3-oxosuccinate, The method of [21], which is 4-methyl-2-oxopentanoate or L-leucine or a metabolite via these compounds on the biosynthetic pathway.

[23] The method of [21] or [22], wherein the target substance is L-leucine or a metabolite derived therefrom.

According to the present invention, IPMS mutants that are highly resistant to feedback inhibition can be utilized and used to produce L-leucine and its derivatives, including its precursors, with high yield and productivity. can.

FIG. 2 is a diagram for use in explaining metabolic pathways that microorganisms may have in embodiments of the present invention.

An aspect of the present invention is a novel mutant polypeptide with IPMS activity. That is, according to this aspect, a modified α-isopropylmalate synthase with reduced L-leucine feedback inhibition is provided, as specified by the components shown in [1] or [6] above. More specifically, the novel mutant polypeptide comprises at least two of glycine, glycine, and alanine at positions 530, 532, and 535 from the N-terminus of a polypeptide consisting of the amino acid sequence of UniprotKB:P42455, or at least two of the amino acid residues corresponding to are replaced with different amino acid residues. Said mutant polypeptide is at least more resistant to feedback inhibition than the polypeptide having IPMS activity of UniprotKB:P42455.

Here, the amino acid sequence of UniprotKB:P42455 is shown in Table 1 below.

UniprotKB: The amino acid sequence of P42455 is the amino acid sequence of wild-type IPMS derived from Corynebacterium glutamicum ATCC13032/DSM20300/BCRC11384/JCM1318/LMG3730/NCIMB10025 strains. In the sequence listing, the nucleotide sequence containing the region (CDS) encoding the wild-type IPMS and the amino acid sequence of the wild-type IPMS in the genome sequence of Corynebacterium glutamicum ATCC13032 strain are listed in order of sequence numbers. 1,2.

"Feedback inhibition" in the present invention means that the final product of the metabolic system becomes an inhibitor and inhibits the reaction at the initial stage of the metabolic system. In the present invention, it means that L-leucine or a derivative thereof inhibits the activity of IPMS that mediates the first step of its biosynthetic pathway. is widely known. Therefore, when simply referring to "feedback inhibition", inhibitors against IPMS are not limited to L-leucine. Releasing feedback inhibition of IPMS can improve L-leucine productivity compared to others. However, when referring to "L-leucine feedback inhibition", it means that IPMS is subject to feedback inhibition by L-leucine, and when saying "L-leucine feedback inhibition is reduced" or the like, it means that IPMS of interest is , means that at least the feedback inhibition by L-leucine is reduced or eliminated. The modified α-isopropylmalate synthase described above has a property of reduced L-leucine feedback inhibition, and, although not necessarily essential, reduced feedback inhibition by L-leucine derivatives or other metabolites. It may have properties.

In the present invention, "highly resistant to feedback inhibition" means maintaining enzyme activity without being inhibited even in the presence of high concentrations of inhibitors. Resistance to feedback inhibition can be assessed by comparing IPMS activity in the presence of inhibitor to activity in the absence of inhibitor.

IPMS in the present invention is a reaction of 2-ketoisovalerate (3-methyl-2-oxobutanoate) with acetyl CoA to form isopropylmalate, which is one of the precursors of L-leucine. It is an enzyme that converts More specifically, IPMS, as shown in FIG. -isopropylmalate], that is, the reaction represented by the reaction formula of 3-methyl-2-oxobutanoate + acetyl CoA + H ₂ O = (2S)-2-isopropylmalate + CoA + H ⁺ and its enzymatic activity is defined as EC number 2.3.3.13.

The IPMS in the present invention may be derived from any organism as long as it is a modified enzyme having the conversion activity and reduced feedback inhibition, but is preferably an enzyme derived from a coryneform bacterium. More preferably, it may be an IPMS derived from Corynebacterium glutamicum. It is not limited. Further, said IPMS has at least 80%, 84%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with the amino acid sequence of UniprotKB:P42455 (SEQ ID NO: 2) may include a polypeptide having For example, an amino acid sequence having such sequence homology or sequence identity and showing an effect corresponding to IPMS may have an amino acid sequence in which a part of the sequence is deleted, modified, substituted or added. Needless to say, it is included in the present invention. Moreover, when using an amino acid sequence other than UniprotKB:P42455, the homologous amino acid residue to be mutated can be estimated by aligning with UniprotKB:P42455. Common programs can be used for alignment, such as Clustal and BLAST. Alignments may be conformational superpositions, for which reason they can be evaluated, for example, by utilizing molecular graphics tools such as PyMOL.

In the present invention, the term "coryneform bacterium" refers to a group of microorganisms defined in Burgeys Manual of Determinative Bacteriology (Vol. 8, p.599, 1974).
More specifically, coryneform bacteria include Corynebacterium spp., Brevibacterium spp., Arthrobacter spp., Mycobacterium spp., Micrococcus ) genus, Microbacterium genus, and the like.
For more details, reference can be made to the definitions described in WO2019156152A1.

"Mutation", "mutant" or "mutant" in the present invention refers to genes, polypeptides, enzymes, or microorganisms possessing them, in which the base sequence of a gene is altered and the amino acid sequence is accordingly altered. . IPMS mutant or mutant IPMS means a mutant in which the amino acid sequence of IPMS is changed by one or more amino acids compared to the wild type, and the activity and the degree of feedback inhibition to L-leucine and its derivatives are changed. do.

Specifically, the mutant polypeptides having IPMS activity of the present invention are amino acid residues 530, 532, and 535 from the N-terminus of a polypeptide consisting of the amino acid sequence of UniprotKB: P42455, glycine, glycine, and alanine, or at least two of the amino acid residues corresponding to those amino acids may be substituted with different amino acid residues. In this case, the another amino acid residue refers to one amino acid residue selected from 19 types of 20 standard amino acids excluding the amino acid residue before mutation in wild-type IPMS.

In addition, the mutant polypeptide includes the amino acid sequence shown in SEQ ID NO: 2 (Table 1) and at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, Polypeptides with 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or identity may be included. . For example, if it is an amino acid sequence that has such sequence homology or sequence identity and shows an effect corresponding to IPMS, in addition to mutations at positions 530, 532, and 535, a part of the sequence is deleted or modified. Also included in the present invention are enzyme variants having substituted or added amino acid sequences. In addition, it is clear to those skilled in the art that the present invention includes those calculated in consideration of the number of amino acids when some amino acids are added or deleted from the N-terminus of UniprotKB:P42455.

In the present invention, "one or more" ("one or The range of "plurality") is not particularly limited, but in certain embodiments, for example, 1 to 400, 1 to 300, 1 to 200, 1 to 100, 1 to 50, 1 30 from . In another embodiment, the range of "one or more" ("one or more") is at least 2 or more, preferably 2 to 20, more preferably 2 to 10, even more preferably 2 to 5 particularly preferably 2 to 4, 2 to 3, for example 2.

In addition, in the modified α-isopropylmalate synthase according to [6] above, the “corresponding wild-type 2-isopropylmalate synthase (α-isopropylmalate synthase)” may be interpreted literally, and the present invention In the modified α-isopropyl malate synthase according to, wild-type 2-isopropyl malate synthase (α-isopropyl malate synthase) that serves as a reference for reducing L-leucine feedback inhibition. That is, the modified α-isopropylmalate synthase may reduce the degree of L-leucine feedback inhibition compared to the reference wild-type 2-isopropylmalate synthase (α-isopropylmalate synthase) at least 1 have one amino acid mutation, and the at least one amino acid mutation contains at least two of the amino acid substitutions defined in (a'), (b') and (c') above Enough.

Furthermore, in the modified α-isopropyl malate synthase according to [6] above, the meanings of the “corresponding amino acid residues” described in (a′), (b′) and (c′) are SEQ ID NO: 2, the substitution site is specified in the IPMS amino acid sequence to be mutated. That is, the "corresponding amino acid residues" in the above (a'), (b') and (c') are specifically ClustalW and ClustalX (Bioinformatics, Vol. 23, Issue 21, 2008 11 Month, pp. 2947-2948; Bioinformatics, Volume 23, Issue 21, 1, November 2007, pp 2947-2948), the amino acid sequence of IPMS to be mutagenized for the amino acid sequence shown in SEQ ID NO: 2. Aligned one-to-one with each amino acid residue shown in (a'), (b') and (c') above when one-to-one alignment (pairwise alignment) is performed based on identity say amino acids.

In certain embodiments, the modified α-isopropyl malate synthase of the present invention is 、Ｇ５３０Ｆ／Ｇ５３２Ｄ／Ａ５３５Ｔ、Ｇ５３０Ｎ／Ｇ５３２Ｄ／Ａ５３５Ｔ、Ｇ５３０Ｃ／Ｇ５３２Ｄ／Ａ５３５Ｔ、Ｇ５３０Ｐ／Ｇ５３２Ｄ／Ａ５３５Ｔ、Ｇ５３０Ｒ／Ｇ５３２Ｄ／Ａ５３５Ｔ、Ｇ５３０Ｌ／Ｇ５３２Ｄ／Ａ５３５Ｔ、Ｇ５３０Ｉ／Ｇ５３２Ｄ／Ａ５３５Ｔ、Ｇ５３０Ｔ／Ｇ５３２Ｄ／Ａ５３５Ｔ、Ｇ５３０Ｈ ／Ｇ５３２Ｄ／Ａ５３５Ｔ、Ｇ５３０Ｄ／Ｇ５３２Ｅ／Ａ５３５Ｔ、Ｇ５３０Ｄ／Ｇ５３２Ｋ／Ａ５３５Ｔ、Ｇ５３０Ｄ／Ｇ５３２Ａ／Ａ５３５Ｔ、Ｇ５３０Ｄ／Ｇ５３２Ｖ／Ａ５３５Ｔ、Ｇ５３０Ｄ／Ｇ５３２Ｆ／Ａ５３５Ｔ、Ｇ５３０Ｄ／Ｇ５３２Ｎ／Ａ５３５Ｔ、Ｇ５３０Ｄ／Ｇ５３２Ｃ／Ａ５３５Ｔ、Ｇ５３０Ｄ／Ｇ５３２Ｐ ／Ａ５３５Ｔ、Ｇ５３０Ｄ／Ｇ５３２Ｌ／Ａ５３５Ｔ、Ｇ５３０Ｄ／Ｇ５３２Ｉ／Ａ５３５Ｔ、Ｇ５３０Ｄ／Ｇ５３２Ｔ／Ａ５３５Ｔ、Ｇ５３０Ｄ／Ｇ５３２Ｈ／Ａ５３５Ｔ、Ｇ５３０Ｄ／Ｇ５３２Ｄ／Ａ５３５Ｓ、Ｇ５３０Ｄ／Ｇ５３２Ｄ／Ａ５３５Ｖ、Ｇ５３０Ｄ／Ｇ５３２Ｄ／Ａ５３５Ｋ、Ｇ５３０Ｄ／Ｇ５３２Ｄ／Ａ５３５Ｄ , G530D/G532D/A535N, G530D/G532D/A535F, G530D/G532D/A535C, G530D/G532D/A535G, G530D/G532D/A535L, G530D/G532D/A535I, and G530D/G532D/A535H Contains triple mutations.

The amino acid sequence that the modified α-isopropylmalate synthase of the present invention can have is obtained by BLAST known to those skilled in the art for the amino acid sequence shown in SEQ ID NO: 2 (wild-type IPMS of Corynebacterium glutamicum ATCC13032 strain). Analysis can be performed and design can be made based on the amino acid sequences of various IPMS homologues retrieved thereby. BLAST analysis of the amino acid sequence shown in SEQ ID NO: 2 revealed, for example, Corynebacterium deserti-derived IPMS (Accession No. WP_053543926.1, sequence identity 94.64%), Corynebacterium efficiens-derived IPMS (Accession No. WP_006768473.1, sequence identity 94.64%). sequence identity 90.26%), IPMS derived from Corynebacterium halotolerans (Accession No. WP_015399748.1, sequence identity 82.62%), IPM derived from Corynebacterium ammoniagenes (Accession No. WP_040354747.1, sequence identity 79%) , Corynebacterium maris-derived IPMS (Accession No. WP_041631981.1, sequence identity 79.87%), Corynebacterium hadale-derived IPMS (Accession No. WP_095538136.1, sequence identity 76.02%), etc. can. Based on the amino acid sequences of these IPMS homologues, amino acid sequences that the modified α-isopropylmalate synthase according to the present invention may have can be designed. Please note that these Accession No. The amino acid sequences identified by are incorporated herein by reference. In addition, for these amino acid sequences, the above (a'), (b') and (c'), or the above (d), (e) and (f), or the above (g), (h) and ( A mutant polypeptide having an amino acid sequence obtained by making at least two of each of the amino acid substitutions shown in j) is specified herein as an embodiment of the modified α-isopropylmalate synthase according to the present invention. It is what is done.

In some embodiments, the modified α-isopropylmalate synthase of the present invention is eukaryotic, prokaryotic, fungal, bacterial, archaeal, coryneform, corynebacterium (e.g., Corynebacterium glutamicum, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium halotolerans, Corynebacterium ammoniagenes, Corynebacterium maris, Corynebacterium hadale) in the amino acid sequences of the wild-type α-(c′), Alternatively, it has an amino acid sequence obtained by performing at least two of the amino acid substitutions shown in (d), (e) and (f) above, or (g), (h) and (j) above.

Another aspect of the invention is a polynucleotide encoding said mutant polypeptide.
The polynucleotide may have any sequence as long as it contains a nucleotide sequence that encodes the mutant polypeptide or modified α-isopropylmalate synthase of the present invention.

The polynucleotide of the wild-type leuA gene before mutagenesis may be a sequence amplified by PCR from genomic DNA prepared from an organism having the leuA gene, or a nucleotide sequence designed based on known genetic information is artificially synthesized. good too. Any combination of codons may be used as long as they encode the same polypeptide, and the codon usage frequency, secondary structure, etc. may be taken into account in the design.

The method of introducing mutations into the wild-type polynucleotide may be in vitro or in vivo. Methods for introducing mutations in vitro include, but are not limited to, PCR-based methods using mutated primers, and random mutation-introducing methods by performing PCR under error-prone conditions. Methods for introducing mutations in vivo include, but are not limited to, a method of spontaneously mutating by repeating passages in culture, and a method of treatment with a mutagen such as ultraviolet light or an alkylating agent.

According to yet another aspect of the present invention, there is provided a microorganism containing the mutant polypeptide (modified α-isopropylmalate synthase) or polynucleotide described above. In certain embodiments, the microorganisms of the present invention are capable of producing derivatives containing L-leucine and its precursors. Additionally, in some embodiments, the microorganisms of the present invention are microorganisms, including eukaryotic microorganisms such as, for example, fungi, prokaryotes, bacteria, archaea, corynebacterium, coryneform bacteria. The precursor of L-leucine mentioned above corresponds to all intermediates in the leucine biosynthetic pathway after 2-ketoisovaleric acid such as isopropylmalic acid and 2-ketoisocaproic acid. Specifically, FIG. Contains various metabolites that make up the L-leucine biosynthetic pathway shown in . Furthermore, derivatives of L-leucine include all substances that can be biosynthesized from L-leucine and its precursors. These include, but are not limited to, 3-hydroxyisovaleric acid derived from 2-ketoisocaproic acid, 3-methylbutanol, isoamylacetic acid, and the like.

The microorganisms in the present invention include those artificially produced by transformation and naturally occurring ones.

For example, the microorganism may be a microorganism transformed with a vector containing a polynucleotide encoding the mutant polypeptide (modified α-isopropylmalate synthase). In some embodiments, the microorganisms include microorganisms that express the mutant polypeptide (modified α-isopropylmalate synthase) by various known methods.

In a specific embodiment, the polynucleotide encoding the mutant polypeptide (modified α-isopropylmalate synthase) may be introduced into the microorganism in a form capable of being expressed in host cells. In another embodiment, in the microorganism, a polynucleotide encoding the mutant polypeptide (modified α-isopropylmalate synthase) is converted into a mutant polypeptide (modified α-isopropylmalate synthase) in host cells. Synthase) may be introduced in a form that does not express. In addition, when the transformed gene (polynucleotide encoding the mutant polypeptide) is introduced in a form that expresses the mutant polypeptide (modified α-isopropyl malate synthase) in host cells , may be integrated within a chromosome or located extrachromosomally. Further, when the gene is introduced in a form in which it is expressed in host cells, the polynucleotide encoding the mutant polypeptide (modified α-isopropylmalate synthase) may be in any form. may have the form of DNA or RNA.

In some embodiments, the microorganism according to the present invention expresses the mutant polypeptide (modified α-isopropylmalate synthase) and, in addition, 3-methyl-2-oxobutanoate , (2S)-2-isopropyl maleate, 2-isopropyl maleate, (2R,3S)-3-isopropyl maleate, (2S)-2-isopropyl-3-oxosuccinate, 4-methyl-2-oxo It has a biosynthetic pathway that biosynthesizes pentanoate and L-leucine in this order. In another embodiment, the microorganism according to the present invention expresses the mutant polypeptide (modified α-isopropylmalate synthase), and at least one of these compounds on the biosynthetic pathway It has one or more biosynthetic pathways that biosynthesize metabolites derived from one.

Furthermore, in some embodiments, the microorganism according to the present invention may have reduced or inactivated wild-type α-isopropylmalate synthase activity. Thus, when the wild-type α-isopropylmalate synthase activity is reduced or inactivated, the microorganism has an adverse effect on the production of the target substance due to the L-leucine feedback inhibition exhibited by the wild-type enzyme. Expression of the modified α-isopropylmalate synthase in which the feedback inhibition is reduced while being eliminated allows the metabolic reaction to the target substance to proceed efficiently. As a result, according to such microorganisms, it is possible to produce the target substance with particularly high yield and productivity. Specifically, the reduction or inactivation of wild-type α-isopropylmalate synthase activity in microorganisms is performed by leuA present on the genome or its homolog gene (wild-type α-isopropylmalate synthase-encoding gene) or regulation of gene expression thereof. It can be realized by a technique such as disruption of the region (e.g. promoter region).

According to still another aspect of the present invention, as described above, (I) producing the target substance by culturing or reacting the microorganism according to the present invention in a predetermined medium or reaction medium; ) recovering the target substance. In a specific embodiment, the method for producing a target substance comprises (I) culturing or reacting coryneform bacteria capable of producing L-leucine among the microorganisms according to the present invention in a medium to produce L-leucine or metabolize L-leucine. (b) recovering the L-leucine or metabolite produced in step (II) from the microorganism or culture medium; .

"Culturing" generally means growing the microorganism under suitably controlled environmental conditions, and "cultivating or reacting" in the present invention has such a general meaning. but not limited to the culture of, for example, culture or reaction modes that can realize the production of the target substance by allowing at least a part of the metabolism of the microorganism to function without substantial growth of the microorganism. do. The culturing or reaction process in the present invention can be carried out by employing suitable media/reaction media and culturing conditions known in the art. Such a culture/reaction process can be easily adjusted and used according to the strain selected by those skilled in the art. Specifically, the culture or reaction, ie step (I), may be, but is not limited to, batch, continuous and fed-batch culture.

Furthermore, in step (I), the culture or reaction conditions are not limited as long as the microorganism according to the present invention can produce the target substance. When producing , it is generally preferred that step (I) is carried out under microaerobic or aerobic conditions. In some embodiments, step (I) is performed at a concentration of dissolved oxygen (DO) of said predetermined culture medium or reaction medium from about 0 to about 60 ppm, preferably from about 0 ppm to about 40 ppm, such as from about 0 ppm to about 8.0 ppm. Carry out step (I) so that the range is 0 ppm. In another embodiment, step (I) is carried out such that the dissolved oxygen concentration (DO) ranges from about 0 ppm to about 2 ppm, preferably from about 0 to about 1 ppm, more preferably from 0 to 0.5 ppm. may Further, in certain embodiments, the ORP (oxidation-reduction potential) is about -500 mV to about 700 mV, about -500 mV to about 550 mV, about -400 mV to about 500 mV, about -400 mV to about 400 mV, about Perform step (I) to a range of 350 mV, from about -300 mV to about 300 mV, from about -250 mV to about 200 mV, or from about -250 mV to about 100 mV. In general, when using microorganisms that can be cultured at high density, such as coryneform bacteria, if step (I) is performed under microaerobic or aerobic conditions with air aeration and agitation, , the DO and ORP of the medium or reaction medium decrease with the growth of the microorganism, and after sufficient growth, the DO value becomes almost zero and the ORP value decreases to the range of about -300 mV to about -200 mV. . However, the DO may not be zero depending on the type and properties of the microorganisms used and various conditions such as stirring throughout the period, and the present invention is not limited by the above numerical range.

In certain embodiments, the target substance is (2S)-2-isopropylmalate, 2-isopropylmalate, (2R,3S)-3-isopropylmalate, (2S)-2-isopropyl-3-oxo Succinate, 4-methyl-2-oxopentanoate or L-leucine, or metabolites via these compounds on the biosynthetic pathway. As shown in FIG. 1, the compounds from (2S)-2-isopropylmalate to L-leucine are metabolites in the L-leucine biosynthetic pathway that microorganisms may have. As illustrated in FIG. 1, L-leucine, the final product in the L-leucine biosynthetic pathway, leads to 3-methyl-2-oxobutanoate + acetyl-CoA + H ₂ O = (2S)-2-isopropyl Although feedback inhibits the activity of wild-type IPMS that can catalyze the reaction represented by the reaction formula of malate + CoA + H ⁺ , the present invention provides L-leucine feedback in addition to/instead of wild-type IPMS. Since the modified α-isopropylmalate synthase having inhibition resistance is employed as a component, the target substance can be produced with high yield and productivity.

In the method for producing a target substance according to the present invention, the method for recovering the target substance produced in step (I) (for example, L-leucine or a downstream metabolite derived therefrom) is not particularly limited. , recovering the target substance (e.g. L-leucine or its precursor, derivative or derivative) from the microorganism or medium or reaction medium using a suitable method known in the art, depending on the culture/reaction method be able to. For example, centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, etc., can be used to recover the target substance from the microorganism or culture medium or reaction medium using suitable methods known in the art. In addition, the recovery step according to (II) includes a step of recovering a fraction containing the target substance or a crude product. may further include a step of purifying the

The present application will be described in more detail below with examples. However, these examples are for the purpose of illustratively describing the present application, and the present application is not limited to these examples.

(1) Preparation of Expression Plasmid Vector The IPMS gene amino acid sequence of coryneform bacterium (UniprotKB: P42455) was obtained, and restriction enzymes NdeI and BamHI recognition sequences were added to the 5′ and 3′ ends by artificial gene synthesis. synthesized DNA. A DNA fragment obtained by cleaving this DNA with NdeI and BamHI was mixed with a fragment obtained by cleaving the expression vector pGE409 (WO2020090016A1) with the same restriction enzymes and ligated with T4 DNA ligase (NEB). The resulting plasmid was named pGE1835.

(2) Mutagenesis Mutagenesis was performed using various predetermined primer pairs and pGE1835 as a template in the same manner as described in WO2019156152A1 to obtain mutagenesis plasmids shown in Table 2 below.

(3) Mutagenesis 2 (permissive evaluation of each residue based on triple mutants)
Mutagenesis was performed using various predetermined primer pairs and pGE1881 as a template by the same method as described in WO2019156152A1.

(4) Preparation of ΔleuA strain Chromosome gene recombination of Corynebacterium glutamicum was performed by the method described in WO2019156152A1.

Plasmids for chromosome gene recombination were obtained by performing PCR using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, using various predetermined primer pairs, and cloning the DNA fragment of pGE209 (WO2019156152A1) cut with KpnI and BamHI. Plasmid vector pGE1781 was obtained by circular ligation using Kit.

pGE1781 was introduced into Corynebacterium glutamicum ATCC13032 strain by electroporation, and ΔleuA strain was produced by the markerless genetic recombination method described in WO2019156152A1.

(5) IPMS Mutant Overexpressing Strain The ΔleuA strain was electroporated with each expression plasmid shown in Tables 3 and 4 and selected with kanamycin to obtain a mutant IPMS overexpressing strain.

(6) Cultivation The resulting transformant was streaked on A agar medium (WO2020090016A1) and cultured at 33° C. for 24 hours. 2, and cultured with shaking at 33° C. and 200 rpm for 24 hours. After the culture was completed, the culture medium was centrifuged to separate the supernatant from the pellet, and the concentration of L-leucine in the supernatant was analyzed by high performance liquid chromatography (WO2020090016A1) according to the method described in (WO2020090016A1). The pellet was suspended in a disruption buffer (50 mM Tris (7.5), 100 mM KCl, 1 mM EDTA), and cells were disrupted with a bead shocker according to the method described in (WO2020090016A1). The protein concentration of the cell lysate was quantified according to the Bradford method. The enzymatic activity of isopropylmalate synthase in cell lysate was 100 mM Tris-HCl (pH 7.5), 2 mM 4-PDS (4,4'-Dithiodipyridine), 2 mM Acetyl-CoA, 2 mM 3 -Methyl-2-oxobutanoic acid was mixed with the cell lysate, and the change in absorbance at 324 nm due to the reaction of liberated CoA with 4-PDS to produce 4-thiopyridine was measured. For measurements of feedback inhibition by L-leucine, 10 mM L-leucine was added to the reaction.

<Production medium>
Glucose 40g, ₍ NH4) _2SO4 20g, urea 5g, _KH2PO4 1g, _{K2HPO4 1g} , _{MgSO4.7H2O 0.25g} , _{MOPS 42g} , _{CaCl2 10mg} , _{FeSO4.7H2O 10} mg, _{MnSO4.H2O 10} mg, _{ZnSO4.7H2O 1} mg, _CuSO4 0.2 mg, _NiCl2.6H2O 0.02 mg, biotin 0.2 mg, thiamine hydrochloride 0.2 mg, PCA 30 mg/L , pH 7.4

(7) Mutant performance test 1
Using strains in which wild-type and mutant IPMS were overexpressed by plasmid introduction into the ΔleuA strain, L-leucine production, IPMS activity, and IPMS residual activity in the presence of L-leucine were compared.

As shown in Table 3, by introducing mutations to residues around the leucine-binding site in IPMS possessed by coryneform bacteria, L-leucine-producing ability is maintained and feedback inhibition resistance to L-leucine is acquired. type IPMS could be obtained. However, when a double mutant was prepared by combining these mutations, the resistance to feedback inhibition against L-leucine was high, but the L-leucine production amount and IPMS activity were greatly reduced. Furthermore, when we created a triple mutant in which all three mutations were introduced, we unexpectedly maintained high IPMS activity and feedback inhibition resistance to L-leucine, and L-leucine which is remarkable compared to the single mutant. An increase in production was observed.

(8) Mutant performance test 2
For each residue of the triple mutant, each residue was replaced with a different amino acid residue to verify whether the same effect could be obtained by mutating to other amino acid residues, and the plasmid was introduced into the ΔleuA strain. Using overexpression strains, L-leucine production, IPMS activity, and IPMS residual activity in the presence of L-leucine were compared. Table 4 shows the results.

All mutants maintained high resistance to L-leucine feedback inhibition, but the L-leucine production amount and IPMS activity differed greatly depending on the mutant type, and high L-leucine productivity was obtained if even an enzyme resistant to feedback inhibition was obtained. It turns out that you don't get it.

G530D, G530E, G530K, G530A, G530V, G530N, G530C, G530P, G530R, G530L, G530I, G530T, G530H, G532D, G532A, G532V, G532P, G532L, G532T, G532T, G532T, G532T, G530D, G530E, G530K, G530A, G530V, G530N, G530C, G530P, G530R, G530L, G530I, G530T, G530H, G532D, G532A, G532V, G532P, G532L, G532T, G532T, G5322 , A535S, A535V, A535K, A535D, A535F, A535G, A535L, A535I, and A535H are expected to improve the performance of IPMS, which is effective in increasing L-leucine production.

The present invention has high industrial applicability in the field of industrial production of various chemical substances.

Claims (23)

1. A modified α-type having reduced L-leucine feedback inhibition as compared with a protein comprising the amino acid sequence defined in any of the following (A) to (C) and consisting of the amino acid sequence shown in SEQ ID NO: 2. Isopropyl malate synthase:
   (A) an amino acid sequence having at least two substitutions selected from the following (a) to (c) in the amino acid sequence shown in SEQ ID NO: 2;
   (B) an amino acid sequence having a sequence identity of 60% or more to the amino acid sequence shown in SEQ ID NO: 2 and containing at least two substitutions selected from (a) to (c) below;
   (C) the amino acid sequence shown in SEQ ID NO: 2, containing at least two substitutions selected from the following (a) to (c), and one or more at sites excluding the sites where the at least two substitutions are located amino acid sequences in which amino acids have been deleted, substituted, inserted or added;
   (a) replacement of the 530th glycine residue with an amino acid residue other than a glycine residue;
   (b) replacement of the 532nd glycine residue with an amino acid residue other than a glycine residue; and (c) replacement of the 535th alanine residue with an amino acid residue other than an alanine residue.
2. 2. The modified α-isopropylmalate synthase according to claim 1, wherein the amino acid sequence defined in (B) above has 84% or more sequence identity with the amino acid sequence shown in SEQ ID NO:2.
3. 3. The modified α-isopropylmalate synthase according to claim 1 or 2, wherein the amino acid sequence defined in (B) above is an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2. .
4. The modified α-isopropylmalate synthase according to any one of claims 1 to 3, comprising all the substitutions defined in (a) to (c) above.
5. 5. The modified α-isopropylmalate synthase according to claim 4, comprising the amino acid sequence defined in (A) or (C) above.
6. having at least one amino acid mutation that reduces L-leucine feedback inhibition compared to the corresponding wild-type 2-isopropyl malate synthase;
   A modified α-isopropyl malate synthase in which the at least one amino acid mutation comprises at least two substitutions selected from the following (a′) to (c′) based on the amino acid sequence shown in SEQ ID NO: 2:
   (a') substitution of an amino acid residue corresponding to the 530th glycine residue with an amino acid residue other than the glycine residue;
   (b′) substitution of the amino acid residue corresponding to the 532nd glycine residue with an amino acid residue other than the glycine residue; and (c′) alanine of the amino acid residue corresponding to the 535th alanine residue Substitutions to amino acid residues other than residues.
7. 7. The modified α-isopropylmalate synthase according to claim 6, wherein said at least one amino acid mutation comprises all the substitutions defined in (a') to (c') above.
8. The modified form according to any one of claims 1 to 7, comprising an amino acid sequence having at least two substitutions in the amino acid sequence of a wild-type 2-isopropylmalate synthase derived from a bacterium belonging to the genus Corynebacterium. α-Isopropylmalate synthase.
9. The modified α-isopropylmalate synthase according to any one of claims 1 to 8, wherein the at least two substitutions are at least two substitutions selected from (d) to (f) below:
   (d) aspartic acid residue, glutamic acid residue, lysine residue, alanine residue, valine residue, glutamine residue, cysteine residue of the 530th glycine residue or an amino acid residue corresponding to the glycine residue , proline, arginine, leucine, isoleucine, threonine or histidine residues;
   (e) aspartic acid residue, alanine residue, valine residue, proline residue, leucine residue, isoleucine residue, threonine residue of the 532nd glycine residue or an amino acid residue corresponding to the glycine residue or substitution with a histidine residue; and (f) a threonine residue, serine residue, valine residue, lysine residue, aspartic acid residue of the 535th alanine residue or an amino acid residue corresponding to the alanine residue groups, phenylalanine residues, glycine residues, leucine residues, isoleucine residues or histidine residues.
10. 10. The modified α-isopropylmalate synthase according to claim 9, which contains all the substitutions specified in (d) to (f) above.
11. 11. The modified α-isopropylmalate synthase according to claim 10, comprising the amino acid sequence defined in (A) or (C) above.
12. The modified α-isopropylmalate synthase according to any one of claims 1 to 11, wherein the at least two substitutions are at least two substitutions selected from (g) to (j) below:
    (g) replacement of the 530th glycine residue or an amino acid residue corresponding to the glycine residue with an aspartic acid residue;
    (h) substitution of the 532nd glycine residue or an amino acid residue corresponding to the glycine residue with an aspartic acid residue; and (j) the 535th alanine residue or an amino acid corresponding to the alanine residue Substitution of residues for threonine, valine, glycine, leucine or isoleucine residues.
13. 13. The modified α-isopropylmalate synthase according to claim 12, comprising all the substitutions specified in (g) to (j) above.
14. 14. The modified α-isopropylmalate synthase according to claim 13, comprising the amino acid sequence defined in (A) or (C) above.
15. A polynucleotide encoding the modified α-isopropylmalate synthase according to any one of claims 1 to 14.
16. A microorganism into which the polynucleotide of claim 15 has been introduced.
17. A microorganism comprising the modified α-isopropylmalate synthase according to any one of claims 1-14.
18. 18. The microorganism according to claim 16 or 17, which is a coryneform bacterium.
19. 19. The microorganism of claim 18, which is Corynebacterium glutamicum.
20. The microorganism according to any one of claims 16 to 19, wherein wild-type α-isopropylmalate synthase activity is reduced or inactivated.
21. (I) producing a target substance by culturing or reacting the microorganism according to any one of claims 16 to 20 in a predetermined medium or reaction medium; and (II) recovering the target substance. ,
    A method of producing a desired substance, comprising
22. The target substance is (2S)-2-isopropyl malate, 2-isopropyl malate, (2R,3S)-3-isopropyl malate, (2S)-2-isopropyl-3-oxosuccinate, 4-methyl -2-oxopentanoate or L-leucine or a metabolite via these compounds on the biosynthetic pathway.
23. The method according to claim 21 or 22, wherein the target substance is L-leucine or a metabolite derived therefrom.
    
    

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