JP7185213B2 - Ergothioneine-synthesizing microorganism and method for producing ergothioneine - Google Patents

Description

TECHNICAL FIELD The present invention relates to the creation of an ergothioneine-synthesizing microorganism, and a method for producing ergothioneine using such an ergothioneine-synthesizing microorganism.

Ergothioneine was discovered as a sulfur-containing amino acid isolated from Claviceps purpurea and found to exist in the living bodies of plants and animals. However, plants and animals cannot synthesize ergothioneine, and ergothioneine in vivo is believed to be derived from ergothioneine synthesized by microorganisms such as basidiomycetes. In particular, it is also contained in some basidiomycete mushrooms, such as edible mushrooms such as oyster mushroom, shiitake mushroom, maitake mushroom, and king oyster mushroom, and is known to be contained particularly in Tamogitake mushrooms. Ergothioneine has high antioxidant properties and is reported to have elastase inhibitory action and tyrosinase inhibitory action, and is particularly noted in the beauty and food fields such as whitening and wrinkle prevention. In addition, ergothioneine has been found to be involved in the bio-oxidative defense system, and its application in the medical field has been attempted.

As methods for producing ergothioneine, extraction from basidiomycetes such as Tamogitake, chemical synthesis, and fermentation using microorganisms have been attempted. Extraction from basidiomycetes such as Tamogitake takes a long time to obtain raw materials and is not suitable for mass production. Chemical synthesis is suitable for mass production, but it requires the use of expensive synthetic reagents and has the problem of high purification costs (Patent Document 1). Fermentation methods using microorganisms include fermentation using bacteria and yeast capable of assimilating C1 compounds (Patent Document 2, Non-Patent Document 1), and fermentation using microorganisms overexpressing ergothioneine biosynthetic genes ( Patent document 3) is mentioned. However, it is difficult to obtain a sufficient amount of ergothioneine in fermentation using bacteria or yeast that have not been transfected with genes. The limit was to obtain a production amount of about /L.

Japanese Patent Application Laid-Open No. 2006-160748 WO2016/104437 WO2017/150304

PLOS One 2014 vol. 9(5) e97774

An object of the present invention is to provide a method for producing ergothioneine with a higher yield than the known methods for producing ergothioneine.

The present inventors have conducted intensive research in a method for producing ergothioneine for the purpose of increasing the yield, and have found that ergothioneine is produced using a reaction pathway different from the reaction pathway known in bacteria so far. Thus, the inventors have found that the yield of ergothioneine increases, and completed the present invention.

Accordingly, the present invention relates to the following inventions:
[1] A step of culturing a cysteine-producing microorganism into which a prokaryote-derived ergothioneine-synthesizing gene encoding an enzyme that catalyzes the reaction of cysteine and hercynine to hercynylcysteine sulfoxide is introduced into the microorganism. , a method for the production of ergothioneine.
[2] The enzyme encoded by the ergothioneine synthesis gene catalyzes the reaction of cysteine and hercynine to form hercinylcysteine sulfoxide, while the reaction of γ-glutamylcysteine and hercynine to hercinyl-γ-glutamyl-cysteine sulfoxide. The production method according to item 1, which does not catalyze the reaction.
[3] The production method according to item 1 or 2, wherein a gene encoding a protein that catalyzes the reaction from hercinyl-γ-glutamyl-cysteine sulfoxide to hercinylcysteine sulfoxide is not introduced into the microorganism.
[4] The production method according to any one of items 1 to 3, wherein the ergothioneine-synthesizing gene is an egtB-like gene derived from bacteria belonging to the genus Methylobacterium.
[5] Any one of items 1 to 4, wherein the cysteine-producing microorganism is Escherichia coli having at least one gene selected from the group consisting of mutant cysE, mutant serA, mutant cysB, and ydeD genes. 1. The manufacturing method according to item 1.
[6] The production method according to any one of items 1 to 5, wherein the cysteine-producing strain is further deficient in the metJ gene.
[7] The production method according to any one of items 1 to 6, wherein the medium for culturing the microorganism is a histidine-added medium.
[8] A microorganism that is a cysteine-producing strain and has an ergothioneine-producing ability into which a prokaryote-derived ergothioneine-synthesizing gene encoding an enzyme that catalyzes hercinylcysteine sulfoxide is introduced from cysteine and hercynine.
[9] The enzyme encoded by the ergothioneine synthesis gene catalyzes the reaction of cysteine and hercynine to form hercinylcysteine sulfoxide, while the reaction of γ-glutamylcysteine and hercynine to hercinyl-γ-glutamyl-cysteine sulfoxide. 9. Microorganism according to item 8, which does not catalyze a reaction.
[10] The microorganism according to item 8 or 9, wherein no gene encoding a protein that catalyzes the reaction from hercinyl-γ-glutamyl-cysteine sulfoxide to hercinylcysteine sulfoxide has been introduced into the microorganism.
[11] The microorganism according to any one of items 8 to 10, wherein the ergothioneine-synthesizing gene is an egtB-like gene derived from bacteria belonging to the genus Methylobacterium.
[12] The microorganism according to item 11, wherein the cysteine-producing microorganism is Escherichia coli having at least one gene selected from the group consisting of mutant cysE, mutant serA, mutant cysB, and ydeD genes.
[13] Escherichia coli having an ergothioneine-producing ability into which an ergothioneine synthesis gene encoding an enzyme that catalyzes hercynylcysteine sulfoxide from cysteine and hercynine is introduced.
[14] Escherichia coli according to item 13, wherein the ergothioneine-synthesizing gene is an egtB-like gene derived from bacteria belonging to the genus Methylogacterium.
[15] The Escherichia coli according to item 13 or 14, further into which an egtD or egtE gene has been introduced.
[16] The Escherichia coli according to any one of items 13 to 15, into which a mutant serA gene, a ydeD gene, and a mutant cysE gene have been introduced.
[17] The Escherichia coli according to any one of items 13 to 16, further deficient in the metJ gene.

The microorganism used in the present invention can synthesize ergothioneine without passing through hercinyl-γ-glutamyl-cysteine sulfoxide, and can obtain ergothioneine in high yield.

FIG. 1 is a diagram of phylogenetic analysis of the egtB gene. Figure 2A shows the plasmid map of pQE88egtB. FIG. 2B shows the sequence information of egtB-like genes from Methylobacterium pseudosasicola, Methylobacterium brachiatum, and Methylobacterium radiotolerans introduced into pQE88egtB. FIG. 2C shows sequence information of egtB-like genes derived from Methylobacterium phyllostachyos and Methylobacterium mesophilicum introduced into pQE88egtB. FIG. 3A shows the plasmid map of pCtac-MS-egtD-hisMS-egtE. FIG. 3B shows the sequence information of egtD and egtE genes derived from Mycobacterium smegmatis introduced into pCtac-MS-egtD-hisMS-egtE. FIG. 4A shows the plasmid map of pDES. FIG. 4B shows the sequence information of the serA gene and ydeD gene introduced into pDES. FIG. 4C shows the sequence information of the cysE gene introduced into pDES. FIG. 5 shows the amount of ergothioneine produced by ergothioneine-producing strains introduced with egtB-like genes derived from Methylobacterium brachiatum, Methylobacterium mesophilicum, Methylobacterium phyllostachyos, Methylobacterium pseudosasicolar, and Methylobacterium radiotolerans (FIG. 5A). For comparison, the amount of ergothioneine produced by an ergothioneine-producing control strain introduced with the egtABCDE gene of Mycobacterium smegmatis is also shown (Fig. 5B). FIG. 6 is a diagram showing the catalytic activity of ergothioneine-producing strains introduced with egtB-like genes derived from Methylobacterium brachiatum, Methylobacterium mesophilicum, Methylobacterium phyllostachyos, Methylobacterium pseudosasicolar, and Methylobacterium radiotolerans, using cell lysates.

The present invention provides a cysteine-producing microorganism into which a prokaryote-derived ergothioneine-synthesizing gene encoding an enzyme that catalyzes the reaction of cysteine and hercynine to hercynylcysteine sulfoxide is introduced, as well as the microorganism. The present invention relates to a method for producing ergothioneine, including a step of culturing.

The microorganism of the present invention is transfected with a prokaryotic ergothioneine synthesis gene encoding an enzyme that catalyzes the reaction of cysteine and hercynine to hercynylcysteine sulfoxide. In a more preferred embodiment, the microorganism of the present invention does not contain a gene encoding a protein that catalyzes the reaction from hercinyl-γ-glutamyl-cysteine sulfoxide to hercinylcysteine sulfoxide.

Conventionally known ergothioneine-producing microorganisms include egtA, egtB, egtC, egtD and egtE, which are ergothioneine-synthesizing genes found in bacteria such as Mycobacteria and Streptomyces. egtA, egtB, egtC, egtD and egtE for microorganisms carrying all or part of the genes encoding proteins that are transgenic or have the same activity as the protein originally encoded by these genes. obtained by introducing a part of The reaction schemes catalyzed by the proteins encoded by these genes are as follows. Conventional egtB catalyzes the reaction of hercinin and γ-glutamylcysteine as substrates to form hercinyl-γ-glutamyl-cysteine sulfoxide. The present inventors have found problems in the conventional method that the reaction involves multiple steps and that γ-glutamylcysteine, which is a precursor of glutathione, is used for ergothioneine production. Moreover, conventionally, it has been necessary to modify a histidine-producing strain to obtain an ergothioneine-producing strain.

これにより、上述のｅｇｔＡ、ｅｇｔＢ、ｅｇｔＣ、ｅｇｔＤ及びｅｇｔＥの遺伝子がそれぞれコードするタンパク質が触媒する上述のスキーム１の反応のうち、ｅｇｔＡ、ｅｇｔＢ及びｅｇｔＣ遺伝子がコードするタンパク質が触媒する反応に関与する遺伝子のうち、１つ、場合により２つ又は全ての遺伝子を含まない場合でも、エルゴチオネインを合成することができる。また、本発明で導入されるｅｇｔＢ様遺伝子にコードされる酵素は、システインを基質として用いることができることから、本発明ではシステイン生産株を元にして遺伝子導入を行うことが可能になった。 The microorganism of the present invention contains a prokaryote-derived ergothioneine-synthesizing gene that encodes an enzyme (egtB-like enzyme) that catalyzes the reaction of cysteine and hercynine to hercynylcysteine sulfoxide using cysteine and hercynine as substrates, as described in the scheme below. genetically introduced.

As a result, among the reactions in Scheme 1 catalyzed by the proteins encoded by the egtA, egtB, egtC, egtD and egtE genes, the reaction catalyzed by the proteins encoded by the egtA, egtB and egtC genes is involved. Ergothioneine can be synthesized even if one, possibly two or all of the genes are absent. In addition, since the enzyme encoded by the egtB-like gene introduced in the present invention can use cysteine as a substrate, it has become possible to perform gene introduction based on cysteine-producing strains in the present invention.

In another aspect of the present invention, the microorganism of the present invention encodes a protein having the activity of the protein encoded by the egtC gene, that is, a protein that catalyzes the reaction from hercinyl-γ-glutamyl-cysteine sulfoxide to hercinylcysteine sulfoxide. It is not necessary to introduce the gene to

In a further aspect of the present invention, the microorganism of the present invention is introduced with a gene encoding a protein having the activity of the protein encoded by the egtA gene, that is, a protein that catalyzes the reaction from glutamic acid and cysteine to γ-glutamylcysteine. It doesn't have to be.

In yet another aspect of the present invention, the microorganism of the present invention catalyzes the reaction of proteins having the activity of the protein encoded by the egtB gene, ie, γ-glutamylcysteine and hercynin, to hercynyl-γ-glutamyl-cysteine-sulfoxide. A gene encoding a protein that does not need to be introduced. As used herein, the expression "no introduced gene" does not mean that the endogenously contained same gene is not included.

An ergothioneine synthetic gene encoding an enzyme that catalyzes the reaction of cysteine and hercynine to hercynylcysteine sulfoxide may be a gene derived from any microorganism. By way of example, such synthetic genes can also be obtained from bacteria of the genus Methylobacterium, cyanobacteria of the genus Microcystis, Basidiomycetes, Neurospora and yeast. Among them, from the viewpoint of gene introduction into Escherichia coli, prokaryotic genes are preferable, and prokaryotic genes other than cyanobacteria are more preferable. Examples of such prokaryotes include bacteria belonging to the genus Methylobacterium. can be used. The ergothioneine synthesis gene, which encodes an enzyme that catalyzes the reaction of cysteine and hercynine to hercynylcysteine sulfoxide, is used to catalyze the reaction of γ-glutamylcysteine and hercynine to hercynyl-γ-glutamylcysteine sulfoxide. In order to distinguish it from egtB, which is an ergothioneine synthesis gene, it can be referred to as a hercinyl-γ-glutamyl-cysteine-sulfoxide non-producing egtB-like gene. We will simply refer to the egtB-like gene as the egtB-like gene. Examples of the protein encoded by the egtB-like gene derived from Methylobacterium pseudosasicola include a protein having the amino acid sequence of SEQ ID NO: 2, and the protein encoded by the egtB-like gene derived from Methylobacterium brachiatum includes the protein having the amino acid sequence of SEQ ID NO: 4, Methylobacterium radiotolerans. As the protein encoded by the egtB-like gene derived from Methylobacterium phyllostachyos, the protein consisting of the amino acid sequence of SEQ ID NO: 6, the protein encoded by the egtB-like gene derived from Methylobacterium phyllostachyos, the protein consisting of the amino acid sequence of SEQ ID NO: 8, and the egtB derived from Methylobacterium mesophilicum Examples of proteins encoded by the like genes include a protein consisting of the amino acid sequence of SEQ ID NO:10.

In another aspect of the invention, the protein encoded by the egtB-like gene contains amino acid Mutant proteins with mutations may be included. A mutein may comprise additions, deletions and/or substitutions of one or several amino acids to at least one amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8 and 10. Further, in another aspect, the mutein has at least 30%, preferably at least 40%, more preferably at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8 and 10. at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 97% %, even more preferably at least 98% identity.

In yet another aspect of the invention, the egtB-like gene is mutated so long as it maintains similar activity to the egtB-like protein, ie, catalytic activity for the reaction of cysteine and hercynine to hercynylcysteine-sulfoxide. may contain a mutated gene. Mutant genes may include additions, deletions and/or substitutions of one or several nucleotides to at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7 and 9. Furthermore, in another aspect, the mutant gene has at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 97% %, even more preferably at least 98% identity.

Microorganisms according to the present invention contain a gene encoding a protein that catalyzes the reaction from γ-glutamylcysteine and hercynin to hercynyl-γ-glutamyl-cysteine-sulfoxide, that is, if the egtB gene has not been introduced, hercynyl-γ -Does not produce glutamyl-cysteine-sulfoxide or produces only small amounts. Therefore, the microorganism of the present invention can also be called a non-hercinyl-γ-glutamyl-cysteine-sulfoxide-producing microorganism. Hercinyl-γ-glutamyl-cysteine-sulfoxide non-producing microorganisms can be determined by measuring the catalytic activity of the microorganism. Specifically, the catalytic activity can be determined by adding cysteine and/or γ-glutamylcysteine, which are reaction substrates, to the cell lysate supernatant of the microorganism and detecting the reaction product. A non-hercinyl-γ-glutamyl-cysteine-sulfoxide-producing microorganism can be determined if hercinyl-γ-glutamyl-cysteine-sulfoxide is absent after addition of the reaction substrate. In the ergothioneine-producing strains of the present invention, hercinylcysteine sulfoxide is detected after addition of the reaction substrate while hercinyl-γ-glutamyl-cysteine-sulfoxide is absent.

In yet another aspect, non-hercinyl-γ-glutamyl-cysteine-sulfoxide-producing microorganisms can be determined by measuring the content of hercinyl-γ-glutamyl-cysteine-sulfoxide in a culture. The content of hercinyl-γ-glutamyl-cysteine-sulfoxide in the culture can be determined by a contract analysis of the sulfur index of Anatech. Preferably, the non-hercinyl-γ-glutamyl-cysteine-sulfoxide-producing microorganism contains hercinyl-γ-glutamyl-cysteine-sulfoxide below the detection limit, optionally 50 μg/ml or less, for example 5 μg/ml or less. If it is

Since both cysteine and histidine are raw materials for ergothioneine synthesis, the microorganism according to the present invention is preferably a production strain with improved production of either histidine or cysteine, or both. Additionally, in another aspect, either histidine or cysteine, or both, can be added to the medium. From the viewpoint of introducing an egtB-like gene that uses cysteine and hercynin as substrates, it is preferable to use a cysteine-producing strain. A cysteine-producing strain means a bacterium capable of producing and accumulating a higher amount of the amino acid of interest than the wild strain or parental strain. A cysteine-producing strain may be one modified to have the ability to produce the desired amino acid using mutation methods or recombinant DNA technology.

The cysteine-producing strain may be any strain as long as it has increased cysteine production compared to the wild strain, and can be produced by any method known in the technical field. For example, acquisition of auxotrophic mutants, analog-resistant strains or metabolic control mutants, creation of recombinant strains with enhanced expression of L-cysteine biosynthetic enzymes, etc. Conventionally, coryneform bacteria or Escherichia bacteria (Amino Acid Fermentation, Gakkai Shuppan Center Co., Ltd., first edition published on May 30, 1986, pp. 77-100). Here, in the breeding of cysteine-producing bacteria, properties such as auxotrophy, analog resistance, and metabolic control mutation may be imparted singly or in combination of two or three or more. Also, the expression of cysteine biosynthetic enzymes may be enhanced either singly or in combination of two or more. Furthermore, imparting properties such as auxotrophy, analog resistance, and metabolic control mutation may be combined with enhancement of biosynthetic enzymes.

In order to obtain auxotrophic mutants with cysteine-producing ability, cysteine analog-resistant strains, or metabolic regulatory mutants, parent strains or wild-type strains are subjected to normal mutation treatment, that is, irradiation with X-rays or ultraviolet rays, or N-methyl -N'-Nitro-N-nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS) treatment with a mutagen, etc., and among the resulting mutant strains, auxotrophy, analog resistance, or metabolism It can be obtained by selecting those exhibiting control mutations and having the ability to produce L-amino acids.

A specific example of a modification that enhances cysteine-producing ability is a transformant transformed with multiple cysE alleles encoding feedback inhibition-resistant serine acetyltransferase (SAT) (U.S. Patent No. 6,218,168), E. coli W3110 with an overexpressed gene encoding a protein suitable for excreting toxic substances into cells (U.S. Pat. No. 5,972,663), cysteine desulfhydrase E. coli strain with decreased activity (Japanese Patent Application Laid-Open No. 11-155571), transformant with increased activity of the positive transcription factor of the cysteine regulon encoded by the cysB gene (WO01/27307), feedback inhibition by serine strains belonging to the genus Escherichia, such as transformants of a mutant serA gene encoding a phosphoglycerate dehydrogenase that does not undergo cytotoxicity. Also included are transformants transformed with the ydeD gene, which is a transporter. Furthermore, in order to enhance cysteine-producing ability, at least one protein selected from the group consisting of glpE protein, yeeD protein, pspE protein, sseA protein, yceA protein, and sirA protein (hereinafter referred to as "thiosulfate pathway activation (sometimes referred to as "protein") may be modified to overexpress.

The bacteria belonging to the family Enterobacteriaceae used in the production method of the present invention may be modified so that the activity of O-acetylserine sulfhydrylase B is reduced. Such modified strains have significantly different cysteine-producing abilities depending on whether the carbon source of the medium is glucose or glycerol. Therefore, cysteine-producing ability can be easily adjusted by changing the carbon source of the medium.

O-acetylserine sulfhydrylase B is an enzyme that works in the thiosulfate pathway, which is one of the cysteine biosynthetic pathways, in bacteria belonging to the family Enterobacteriaceae, using O-acetylserine and thiosulfate as substrates. It is an enzyme having an activity to synthesize S-sulfocysteine (cysteine precursor). In this respect, O-acetylserine sulfhydrylase B is not particularly limited. Specifically, in the case of Escherichia coli, for example, proteins expressed from the Escherichia coli cys M gene can be mentioned.

Histidine-producing strains include microorganisms with reduced or eliminated histidine ammonia lyase activity or reduced or eliminated histidine ammonia lyase gene expression.

A microorganism transfected with an ergothioneine synthesis gene encoding an enzyme that catalyzes the reaction of cysteine and hercynine to hercynylcysteine sulfoxide, wherein the reaction of hercynyl-γ-glutamyl-cysteine sulfoxide to hercynylcysteine sulfoxide As a method for obtaining a microorganism that does not contain a gene encoding a protein that catalyzes the ergothioneine synthesis gene, it may be obtained by introducing an egtB-like gene into a bacterium that does not have an ergothioneine-synthesizing gene. In yet another aspect, a method for obtaining such a microorganism includes transfecting an egtB-like gene and knocking out the egtC gene in a microorganism originally having an ergothioneine synthesis gene.

When the egtB-like gene is introduced into a microorganism that does not have the ergothioneine-synthesizing gene, it is preferable to introduce the egtD and egtE genes simultaneously or sequentially. The egtD and egtE genes may be derived from the same species as the origin of the egtB-like gene, or may be derived from different species. As an example, the egtD and egtE genes from Mycobacterium smegmatis can be used.

The enzyme encoded by the egtB-like gene catalyzes the reaction of hercynin and cysteine to hercynylcysteine sulfoxide, but does not catalyze the reaction of γ-glutamylcysteine and hercynin to hercynyl-γ-glutamyl-cysteine sulfoxide. In E. coli, γ-glutamylcysteine is the substrate for glutathione production. Glutathione is an antioxidant and contributes to reducing oxidative stress in cells. Generally, in fermentation culture, gene-introduced bacteria are exposed to strong stress during culture, which makes continuous culture difficult. Therefore, by using an egtB-like gene instead of egtB, which is an enzyme that uses γ-glutamylcysteine as a substrate, there is no competition for γ-glutamylcysteine in E. coli, which is preferable in terms of resistance to oxidative stress. . As a result, long-term continuous culture can be achieved compared to microorganisms introduced with the egtB gene, which is an enzyme that uses γ-glutamylcysteine as a substrate.

Microorganisms used in the present invention may be of any type, and examples include enterobacteria, actinomycetes, ascomycetes, and basidiomycetes. Enteric bacteria may include Escherichia, Enterobacter, Pantoea, Klebsiella, Serratia, Erwinia, Salmonella, Morganella, and the like. Microorganisms such as Streptomyces, Actinomyces, Mycobacterium, and Corynebacterium may be used as actinomycetes. From the viewpoint of increasing the amount of fermentation production, it is preferable to use Escherichia coli (E. coli) as the enterobacterium. Yeast may include the genera Saccharomyces, Schizosaccharomyces, Candida, Aspergillus, Penicillium, and Neurospora.

In the present invention, gene transfer is performed using any method commonly used in the technical field, depending on the type of microorganism to be introduced. Gene introduction is enabled by introducing into microorganisms an expression vector in which an ergothioneine synthesizing gene is integrated in an expressible manner downstream of a promoter sequence.

A medium containing a carbon source, a nitrogen source, a sulfur source, inorganic ions and, if necessary, other organic components can be used. Any of those generally used in this technical field can be selected according to the type of microorganism used. When E. coli is used, media such as LB medium, M9 medium, and SM1 medium can be used as examples. In order to express the introduced gene, an inducer such as IPTG is added depending on the type of the introduced plasmid. Furthermore, in a preferred embodiment of the present invention, it is preferred to use a histidine-added medium. The histidine-supplemented medium contains 5 mM or more, preferably 120 mM or more, more preferably 240 mM or more of histidine, and contains 10 M or less, preferably 1 M or less, more preferably 0.5 M or less from the viewpoint of toxicity. Furthermore, in preferred embodiments of the present invention, methionine may be added. The methionine-supplemented medium contains 10 mM or more, preferably 350 mM or more, more preferably 700 mM or more of methionine, and contains 10 M or less, preferably 1 M or less, more preferably 0.5 M or less from the viewpoint of toxicity. When E. coli with low cysteine production is used, cystine may be further added, and the cystine-added medium contains 2.5 mM or more, preferably 60 mM or more, more preferably 120 mM or more, and 500 mM or less, preferably 500 mM or less from the viewpoint of toxicity. contains 250 mM or less.

The culture conditions and culture time can be arbitrarily selected by those skilled in the art according to the culture apparatus to be used and the desired ergothioneine production amount. As an example, culture may be performed under aerobic conditions for hours to weeks. The culture temperature can be appropriately selected according to the strain, and can be selected, for example, between 25°C and 40°C. From the viewpoint of culturing E. coli, 37°C is preferable. It is preferable to control the pH at 5-8 during the culture.

In the present invention, metJ refers to the E. coli methionine repressor gene. In combination with S-adenosylmethionine, metJ suppresses expression of the methionine regulon and enzymes involved in S-adenosylmethionine synthesis. Therefore, deletion of the metJ gene (ΔmetJ) enhances the expression of the methionine regulon, which is suppressed by the methionine repressor. Ergothioneine production can be enhanced by deleting the methionine repressor gene. Deletion of the metJ gene may be performed by any method, for example, the method of Biochem. J. (2006) 396, 227-234 can be used.

In addition to the step of culturing the microorganism, the method for producing ergothioneine of the present invention may further include a step of purifying ergothioneine from the obtained culture supernatant and/or the cells according to a conventional method.

All documents mentioned herein are hereby incorporated by reference in their entirety.

The embodiments of the invention described below are for illustrative purposes only and are not intended to limit the scope of the invention. The technical scope of the present invention is limited only by the description of the claims. Modifications of the present invention, such as additions, deletions and replacements of constituent elements of the present invention, can be made without departing from the gist of the present invention.

Phylogenetic tree analysis
Using the egtB amino acid sequence of M. pseudosasicola as a query, a BlastP search (representative species) was performed on the genomes of representative species in the KEGG database to obtain a group of homologous gene sequences. These sequence groups were subjected to multiple alignment using ClustalX to create a phylogenetic tree (Dendroscope) (Fig. 1). Each gene name is a KEGG gene ID (the first three letters represent the species (see KEGG organisms)). The egtB-like genes of the genus Methylobacterium according to the present invention are the egtB gene of Mycobacterium smegmatis, which is known to have an ergothioneine biosynthetic pathway, and the egtB gene of Microcystis aeruginosa, which has been found to have a novel ergothioneine biosynthetic pathway. , were found to belong to completely different groups in phylogenetic tree analysis. Therefore, the ergothioneine synthesis gene encoding the enzyme that catalyzes the reaction from cysteine and hercynine to hercynylcysteine sulfoxide of the present invention is a strain different from the strain to which the cyanobacterium Microcystis aeruginosa belongs on the phylogenetic tree. , and Mycobacterium smegmatis, which has a known ergothioneine biosynthetic pathway, belongs to a different strain.

Cloning of egtB-like gene Methylobacterium pseudosasicola (NBRC number: 105203), Methylobacterium brachiatum (NBRC number: 103629), Methylobacterium radiotolerans (NBRC number: 15690), Methylobacterium phyllostachyos (NBRC number: 105206) distributed from NBRC (NITE Biological Resource Center) ), and Methylobacterium mesophilicum (NBRC number: 15688), respectively, and cloned into pQE-1a used in J. Agric. Food Chem, 2018, 66, 1191-1196 to create an egtB-like protein expression plasmid (pQE88egtB) was prepared. A plasmid map of the prepared pQE88egtB is shown in FIG. 2A. Sequence information of egtB-like genes derived from Methylobacterium pseudosasicola, Methylobacterium brachiatum, and Methylobacterium radiotolerans, and sequence information of egtB-like genes derived from Methylobacterium phyllostachyos and Methylobacterium mesophilicum introduced into the pQE88egtB plasmid are shown in FIGS. 2B and 2C, respectively.

The amino acid and nucleic acid sequences of the egtB-like gene were as follows:

Cloning of egtA-egtE genes
J.Agric. Food Chem, 2018, 66, 1191-1196 used in pCF1s-MsD, introduced the egtE gene of Mycobacterium smegmatis (JCM6386) used in the same document, pCtac-MS-egtD-hisMS-egtE plasmid was prepared. A plasmid map of the prepared pCtac-MS-egtD-hisMS-egtE is shown in FIG. 3A. The egtD and egtE sequences introduced into the pCtac-MS-egtD-hisMS-egtE plasmid are shown in FIG. 3B.

As a control, the egtABCDE gene was amplified from Mycobacterium smegmatis and integrated into the pCF1s plasmid to prepare the pCtac-MS-egtABCDE plasmid.

JpDES Plasmid Preparation The pDES plasmid is a pACYC184 plasmid (stock Nippon Gene), serA gene mutated so that the 410th amino acid (threonine) is converted to a stop codon, ydeD gene, and 167th amino acid (threonine) to alanine. The cysE gene mutated to be converted has a structure inserted under the control of the OmpA promoter. The mutant serA gene and mutant cysE alleviate feedback inhibition, and the ydeD gene promotes cysteine excretion to the outside of the cell. Therefore, a high cysteine-producing strain can be obtained by introducing the pDES plasmid into Escherichia coli. A plasmid map of the prepared pDES is shown in FIG. 4A. Sequence information of the serA gene and ydeD gene, and sequence information of the cysE gene introduced into pDES are shown in FIGS. 4B and 4C.

Production of ergothioneine-producing strains As pQEegtB plasmids, plasmids introduced with egtB-like genes derived from Methylobacterium pseudosasicola, Methylobacterium brachiatum, Methylobacterium radiotolerans, Methylobacterium phyllostachyos, and Methylobacterium mesophilicum were used, and pCtac-MS-egtD-hisMS-egtE was used. The plasmid and pDES plasmid were each introduced into ΔmetJ Escherichia coli to produce an ergothioneine-producing strain. ΔmetJ E. coli was created by deleting the metJ gene in the wild strain BW25113 using the first crossover method. Further, as controls, ergothioneine-producing control strains into which the MS-egtABCDE-pDES plasmid and pDES plasmid were introduced were prepared.

Preparation of ergothioneine An ergothioneine-producing strain was cultured for 24 hours in SM1 medium supplemented with 3% glucose (with CaCO ₃ : see the table below) to obtain a preculture solution. The preculture solution was added to the same SM1 medium so that OD562=0.1, and main culture was started at 30° C. and 200 rpm. After culturing for 6 hours, 0.1 mM IPTG was added as an inducer, and 10 mM sodium thiosulfate, 5 mM histidine and 6 mM methionine were added as substrates, and culture was further performed. At 96 hours, 120 hours, 144 hours, 168 hours and 192 hours after culturing, cultures were obtained, and ergothioneine contained in the cultures was measured as follows.

Determination of the amount of ergothioneine The amount of ergothioneine in the culture was measured by a contract analysis (Anatech). A diagram comparing the production amount of an ergothioneine-producing strain introduced with egtB derived from Methylobacterium pseudosasicola, Methylobacterium brachiatum, Methylobacterium radiotolerans, Methylobacterium phyllostachyos, and Methylobacterium mesophilicum and an ergothioneine-producing control strain introduced with egtABCDE derived from Mycobacterium smegmatis. It is shown in FIG. In the control strain, no growth was observed after 144 hours of culture. Therefore, the amount of ergothioneine produced in the control strain at 144 hours was compared with that of the ergothioneine-producing strain at 192 hours.

Measurement of catalytic activity of cultures
Cultures of ergothioneine-producing strains introduced with egtB derived from Methylobacterium pseudosasicola, Methylobacterium brachiatum, Methylobacterium radiotolerans, Methylobacterium phyllostachyos, and Methylobacterium mesophilicum, and E. coli strains into which the overexpressed gene was not introduced as a negative control were centrifuged to separate the cells. collected. The collected cells were subjected to a sonication treatment to obtain a cell lysate supernatant. A catalytic reaction measurement solution shown in the table below was mixed with cell lysate supernatant (1 mg (protein)/ml) and incubated at 30° C. for 2 hours. The product species of the reaction solution containing cysteine or γ-glutamylcysteine as the reaction substrate species was analyzed by LC-ESI-MS. The results are shown in FIG.

Claims (17)

In a cysteine-producing prokaryote, an egtB-like gene derived from a bacterium of the genus Methylobacterium that encodes an enzyme that catalyzes the reaction of cysteine and hercynine to hercynylcysteine sulfoxide, and at least an amino acid sequence encoded by the egtB-like gene Culturing a prokaryote into which a mutant gene encoding a mutant protein having 90% identity or a mutant gene consisting of a nucleotide sequence having at least 90% identity to the nucleotide sequence of the egtB-like gene is introduced. A method for producing ergothioneine, comprising steps, wherein the mutant protein encoded by the mutant gene catalyzes the reaction of cysteine and hercynine to hercynylcysteine sulfoxide. The enzyme encoded by the ergothioneine synthesis gene catalyzes the reaction of cysteine and hercynine to form hercinylcysteine sulfoxide, while catalyzing the reaction of γ-glutamylcysteine and hercynine to form hercinyl-γ-glutamyl-cysteine sulfoxide. The manufacturing method according to claim 1, wherein no. 3. The production method according to claim 1, wherein no gene encoding a protein that catalyzes the reaction from hercinyl-γ-glutamyl-cysteine sulfoxide to hercinylcysteine sulfoxide is introduced into the prokaryote. The amino acid sequence encoded by the egtB-like gene is selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, and 10, or the nucleotide sequence of the egtB-like gene is SEQ ID NOS: 1, 3, 5, and 7 , and 9, the production method according to any one of claims 1 to 3. 4. The cysteine-producing strain prokaryote according to any one of claims 1 to 3 , wherein the prokaryote is Escherichia coli having at least one gene selected from the group consisting of mutant cysE, mutant serA, and mutant cysB. Production method. The production method according to any one of claims 1 to 4 , wherein the cysteine-producing strain is further deficient in the metJ gene. The production method according to any one of claims 1 to 5 , wherein the medium for culturing the prokaryote is a histidine-added medium. An egtB -like gene derived from bacteria belonging to the genus Methylobacterium that encodes an enzyme that catalyzes the reaction of cysteine and hercynine to hercynylcysteine sulfoxide in prokaryotes that are cysteine-producing strains, and at least an amino acid sequence encoded by the egtB-like gene A mutant gene encoding a mutant protein having 90% identity, or a mutant gene comprising a nucleotide sequence having at least 90% identity to the nucleotide sequence of the egtB-like gene, and having an ergothioneine-producing ability . , wherein the mutant protein encoded by said mutant gene catalyzes the reaction of cysteine and hercynine to hercynylcysteine sulfoxide . The enzyme encoded by the ergothioneine synthesis gene catalyzes the reaction of cysteine and hercynine to form hercinylcysteine sulfoxide, while catalyzing the reaction of γ-glutamylcysteine and hercynine to form hercinyl-γ-glutamyl-cysteine sulfoxide. 9. The prokaryote of claim 8, which does not. 10. The prokaryote according to claim 8 or 9, wherein no gene encoding a protein that catalyzes the reaction from hercinyl-γ-glutamyl-cysteine sulfoxide to hercinylcysteine sulfoxide has been introduced into said prokaryote. The amino acid sequence encoded by the egtB-like gene is selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, and 10, or the nucleotide sequence of the egtB-like gene is SEQ ID NOS: 1, 3, 5, and 7 , and 9. The prokaryote according to any one of claims 8 to 10. 12. Any one of claims 8 to 11, wherein the cysteine-producing prokaryote is Escherichia coli having at least one gene selected from the group consisting of mutant cysE, mutant serA, mutant cysB, and ydeD genes. A prokaryotic organism according to the paragraph . An egtB-like gene from a bacterium belonging to the genus Methylobacterium that encodes an enzyme that catalyzes the reaction of cysteine and hercynine to hercynylcysteine sulfoxide, and a mutein having at least 90% identity to the amino acid sequence encoded by said egtB-like gene or a mutated gene consisting of a nucleotide sequence having at least 90% identity to the nucleotide sequence of the egtB-like gene, wherein the ergothioneine-producing ability is introduced, wherein the mutated gene encodes mutated protein catalyzes the reaction of cysteine and hercynine to hercynylcysteine sulfoxide . The amino acid sequence encoded by the egtB-like gene is selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, and 10, or the nucleotide sequence of the egtB-like gene is SEQ ID NOS: 1, 3, 5, and 7 , and 9. E. coli according to claim 13. 15. The Escherichia coli according to claim 13 or 14, further into which an egtD or egtE gene has been introduced. The Escherichia coli according to any one of claims 13 to 15, into which a mutant serA gene, a ydeD gene, and a mutant cysE gene have been introduced. The Escherichia coli according to any one of claims 13 to 16, further deficient in the metJ gene.

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