WO2021095635A1 - Vanillin production method - Google Patents

Abstract

Provided is a vanillin production method. The present invention enables production of vanillin by using, under a condition in which the concentration of ammonia is reduced, a microorganism having the ability to produce vanillin.

Description

How to make vanillin

The present invention relates to a method for producing vanillin using a microorganism.

Vanillin is the main component of the scent of vanilla, and is used as a fragrance in foods and drinks and perfumes. Vanillin is mainly produced by extraction from natural products or chemical synthesis.

Attempts have also been made to produce vanillin by biotechnology. For example, vanillin can be produced from a vanillin precursor such as vanillic acid by utilizing a microorganism such as Corynebacterium glutamicum.

Ammonia is used in culturing microorganisms, for example, as a nitrogen source or for pH adjustment. However, ammonia may cause inhibition of microbial growth at high concentrations (Non-Patent Documents 1 and 2). For example, Corynebacterium glutamicum is not inhibited by 100 mM ammonia (Non-Patent Document 1), but is inhibited by 1 M ammonia (Non-Patent Document 2). In addition, inhibition of the growth of Corynebacterium glutamicum by ammonia is remarkable in glutamate dehydrogenase (GDH) -deficient strains (Non-Patent Documents 1 and 2). Further, it is known that in Corynebacterium glutamicum proline-producing bacteria, the production of proline is improved when the addition of ammonia causes growth inhibition (Non-Patent Document 3).

An object of the present invention is to develop a novel technique for improving the production of vanillin by microorganisms and to provide an efficient method for producing vanillin.

The present inventors have found that the production of vanillin by the microorganism is improved by reducing the ammonia concentration during the production of vanillin using the microorganism, and completed the present invention.

That is, the present invention can be exemplified as follows.
[1]
It ’s a method of making vanillin.
Including the production of vanillin using microorganisms capable of producing vanillin,
A method in which the production is carried out under conditions where the ammonia concentration is reduced.
[2]
The method according to the method, wherein the ammonia concentration during the production is 700 mM or less.
[3]
The method according to the method, wherein the ammonia concentration during the production is 400 mM or less.
[4]
The method according to the method, wherein the ammonia concentration during the production is 100 mM or less.
[5]
The method, wherein the production comprises converting the vanillin precursor to vanillin using the microorganism.
[6]
The method, wherein the conversion comprises culturing the microorganism in a medium containing the precursor and producing and accumulating vanillin in the medium.
[7]
The method, wherein the conversion comprises causing the bacterial cells of the microorganism to act on the precursor in the reaction solution and producing and accumulating vanillin in the reaction solution.
[8]
The method as described above, wherein the cells are used in the form of a culture solution of the microorganism, cells recovered from the culture solution, processed products thereof, or a combination thereof.
[9]
The method, wherein the precursor is vanillic acid.
[10]
The method, wherein the precursor is produced using a microorganism capable of producing the precursor.
[11]
The method, which further comprises producing the precursor using a microorganism capable of producing the precursor prior to the production of the vanillin.
[12]
The method, wherein the precursor is produced under conditions where the ammonia concentration is reduced.
[13]
The precursor is used in the form of a material containing the precursor.
The method as described above, wherein the material is a culture or reaction solution containing the precursor, a supernatant separated from the culture solution or reaction solution, a processed product thereof, or a combination thereof.
[14]
The method according to the method, wherein the content of ammonia in the material is 300% or less as a molar ratio to the content of the precursor in the material.
[15]
The method, wherein the production of vanillin comprises culturing a microorganism capable of producing the vanillin in a medium containing a carbon source, and producing and accumulating vanillin in the medium.
[16]
The method, further comprising recovering vanillin.
[17]
The method, wherein the microorganism capable of producing the vanillin is a bacterium or yeast.
[18]
The method, wherein the microorganism capable of producing the vanillin is a coryneform bacterium or a bacterium of the Enterobacteriaceae family.
[19]
The method as described above, wherein the microorganism capable of producing the vanillin is a bacterium belonging to the genus Corynebacterium or a bacterium belonging to the genus Escherichia.
[20]
The method, wherein the microorganism capable of producing the vanillin is Corynebacterium glutamicum or Escherichia coli.
[21]
The method, wherein the microorganism capable of producing vanillin has been modified to increase the activity of aromatic carboxylic acid reductase and / or phosphopantetinyltransferase as compared to the unmodified strain.
[22]
The method, wherein the microorganism capable of producing the vanillin has been modified such that the activity of vanillate demethylase and / or alcohol dehydrogenase is reduced as compared to the unmodified strain.

The method described herein is a method for producing vanillin, which comprises producing vanillin using a microorganism capable of producing vanillin, and the production is carried out under conditions in which the ammonia concentration is reduced. , May be the method. In addition, the method described herein is a method for improving the production of vanillin, which comprises producing vanillin using a microorganism capable of producing vanillin, the production of which has reduced the ammonia concentration. It may be a method carried out under conditions. Vanillin can be specifically produced from carbon sources and / or vanillin precursors, for example, utilizing microorganisms capable of producing vanillin.

The vanillin precursor can be produced, for example, by utilizing a microorganism capable of producing the vanillin precursor. Vanillin precursors can be specifically produced from carbon sources and / or additional precursors of vanillin precursors, for example, utilizing microorganisms capable of producing vanillin precursors.

Vanillin or vanillin precursor is also referred to as "target substance".

Hereinafter, a method for producing a target substance (that is, vanillin or a vanillin precursor) by utilizing a microorganism having an ability to produce a target substance (that is, vanillin or a vanillin precursor) will be described. Unless otherwise specified, the following description of the method for producing the target substance is a description of the method described in the present specification (for example, a method for producing vanillin) and the method described in the present specification (for example, a method for producing vanillin). Also serves as an explanation of the method for producing the vanillin precursor used in the above.

<1> Microorganism The microorganism used for producing the target substance is a microorganism having an ability to produce the target substance. The ability to produce a target substance is also called "target substance producing ability".

<1-1> Microorganisms capable of producing a target substance “Microorganisms capable of producing a target substance” may mean a microorganism capable of producing a target substance.

The "microorganism capable of producing the target substance" may mean a microorganism capable of producing the target substance by fermentation when the microorganism is used in the fermentation method. That is, the “microorganism capable of producing a target substance” may mean, for example, a microorganism capable of producing a target substance from a carbon source. Specifically, the “microorganism capable of producing a target substance” means that, for example, when cultured in a medium (for example, a medium containing a carbon source), the target substance is produced and accumulated in the medium to the extent that it can be recovered. May mean a microorganism that can.

The "microorganism capable of producing a target substance" may mean a microorganism capable of producing a target substance by biological conversion when the microorganism is used in a biological conversion method. That is, the “microorganism capable of producing a target substance” may mean, for example, a microorganism capable of producing a target substance from a precursor of the target substance. Specifically, the “microorganism capable of producing a target substance” is a medium that can produce and recover the target substance when cultured in, for example, a medium (for example, a medium containing a precursor of the target substance). It may mean a microorganism that can accumulate in it. Further, the “microorganism capable of producing the target substance” is specifically, for example, in the reaction solution to the extent that the target substance can be produced and recovered when it is allowed to act on the precursor of the target substance in the reaction solution. It may mean a microorganism that can accumulate in.

A microorganism capable of producing a target substance may be capable of accumulating a target substance in an amount of 0.01 g / L or more, 0.05 g / L or more, or 0.09 g / L or more in a medium or a reaction solution, for example.

"Target substance" means vanillin or vanillin precursor. The "vanillin precursor" may mean a compound that can be converted to vanillin by biological conversion using a microorganism. Examples of vanillin precursors include protocatechuic acid, vanillic acid, and protocatechuic aldehyde. Vanillin precursors include, in particular, vanillic acid. The microorganism may be capable of producing only one target substance, or may be capable of producing two or more target substances. Further, the microorganism may be able to produce the target substance from one kind of target substance precursor, or may be able to produce the target substance from two or more kinds of target substance precursors.

When the target substance is a compound that can take the form of a salt, the target substance may be obtained as a free form, a salt, or a mixture thereof. That is, unless otherwise specified, the "target substance" may mean a free target substance, a salt thereof, or a mixture thereof. Examples of the salt include an ammonium salt, a sodium salt, and a potassium salt. Examples of the salt of the target substance include salts other than ammonium salts. As the salt of the target substance, one kind of salt may be used, or two kinds or more kinds of salts may be used in combination.

The microorganism used for producing the target substance or the microorganism used as the parent strain for constructing the target substance is not particularly limited. Examples of microorganisms include bacteria and yeast.

Bacteria include bacteria belonging to the family Enterobacteriaceae and coryneform bacteria.

Bacteria belonging to the family Enterobacteriaceae include Escherichia, Enterobacter, Pantoea, Klebsiella, Serratia, Erwinia, and Photolabdas. Examples include bacteria belonging to the genus (Photorhabdus), Providencia, Salmonella, Morganella and the like. Specifically, according to the classification method used in the NCBI (National Center for Biotechnology Information) database (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347). Bacteria classified in the Department of Enterobacteriaceae can be used.

Bacteria of the genus Escherichia are not particularly limited, but examples thereof include bacteria classified into the genus Escherichia according to a classification known to microbiology experts. Examples of Escherichia bacteria include the books of Neidhardt et al. (Backmann, B. J. 1996. Derivations and Genotypes of some mutant derivatives of Escherichia coli K-12, p. 2460-2488. Table 1. In F. D. Neidhardt. (ed.), Escherichia coli and Salmonella Cellular and Molecular Biology / Second Edition, American Society for Microbiology Press, Washington, DC). Examples of the bacterium belonging to the genus Escherichia include Escherichia coli. Specifically, as Escherichia coli, for example, Escherichia coli K-12 strain such as W3110 strain (ATCC 27325) and MG1655 strain (ATCC 47076); Etc. Escherichia coli B strain; and their derivatives.

Bacteria of the genus Enterobacter include, but are not particularly limited, bacteria classified into the genus Enterobacter according to a classification known to microbiology experts. Examples of Enterobacter genus bacteria include Enterobacter agglomerans and Enterobacter aerogenes. Specific examples of Enterobacter agglomerans include the Enterobacter agglomerans ATCC12287 strain. Specific examples of Enterobacter aerogenes include Enterobacter aerogenes ATCC13048 strain, NBRC12010 strain (Biotechonol Bioeng. 2007 Mar 27; 98 (2) 340-348), and AJ110637 strain (FERM BP-10955). .. Examples of Enterobacter bacteria include those described in European Patent Application Publication No. EP0952221. Some Enterobacter agglomerans are classified as Pantoea agglomerans.

Bacteria belonging to the genus Pantoea include, but are not particularly limited, bacteria classified into the genus Pantoea according to a classification known to microbiology experts. Examples of Pantoea bacteria include Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Specifically, as Pantoea ananatis, for example, Pantoea ananatis LMG20103 strain, AJ13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ13601 strain (FERM BP-7207), SC17 strain (FERM BP-6614) -11091), SC17 (0) strain (VKPM B-9246), and SC17sucA strain (FERM BP-8646). Some Enterobacter and Elvinia bacteria have been reclassified into the genus Pantoea (Int. J. Syst. Bacteriol., 39, 337-345 (1989); Int. J. Syst. Bacteriol. , 43, 162-173 (1993)). For example, some Enterobacter agglomerans have recently been reclassified into Pantoea agglomerans, Pantoea ananatis, Pantoea stewarty, etc. based on 16S rRNA sequencing (Int. J. Syst. Bacteriol). ., 39, 337-345 (1989)). Bacteria of the genus Pantoea may also include bacteria thus reclassified into the genus Pantoea.

Examples of Erwinia bacteria include Erwinia amylovora and Erwinia carotovora. Examples of the bacterium belonging to the genus Klebsiella include Klebsiella planticola.

Bacteria belonging to the family Enterobacteriaceae have been reclassified into multiple families by comprehensive comparative genome analysis in recent years (Adelou M. et al., Genome-based phylogeny and taxonomy of the'Enterobacteriales': proposalal. for Enterobacterales ord. Nov. Divided into the families Enterobacteriaceae, Erwiniaceae fam. Nov., Pectobacteriaceae fam. Nov., Yersiniaceae fam. Nov., Hafniaceae fam. Nov., Morganellaceae . J. Syst. Evol. Microbiol., 2016, 66: 5575-5599). However, in the present specification, bacteria conventionally classified in the family Enterobacteriaceae are treated as bacteria belonging to the family Enterobacteriaceae.

Examples of coryneform bacteria include bacteria belonging to genera such as Corynebacterium, Brevibacterium, and Microbacterium.

Specific examples of the coryneform bacterium include the following species.
Corynebacterium acetoacidophilum
Corynebacterium acetoglutamicum
Corynebacterium alkanolyticum
Corynebacterium callunae
Corynebacterium crenatum
Corynebacterium glutamicum
Corynebacterium lilium
Corynebacterium melassecola
Corynebacterium thermoaminogenes (Corynebacterium efficiens)
Corynebacterium herculis
Brevibacterium divaricatum (Corynebacterium glutamicum)
Brevibacterium flavum (Corynebacterium glutamicum)
Brevibacterium immariophilum
Brevibacterium lactofermentum (Corynebacterium glutamicum)
Brevibacterium roseum
Brevibacterium saccharolyticum
Brevibacterium thiogenitalis
Corynebacterium ammoniagenes (Corynebacterium stationis)
Brevibacterium album
Brevibacterium cerinum
Microbacterium ammoniaphilum

Specific examples of the coryneform bacterium include the following strains.
Corynebacterium acetoacidophilum ATCC 13870
Corynebacterium acetoglutamicum ATCC 15806
Corynebacterium alkanolyticum ATCC 21511
Corynebacterium callunae ATCC 15991
Corynebacterium crenatum AS1.542
Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734
Corynebacterium lilium ATCC 15990
Corynebacterium melassecola ATCC 17965
Corynebacterium efficiens (Corynebacterium thermoaminogenes) AJ12340 (FERM BP-1539)
Corynebacterium herculis ATCC 13868
Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC 14020
Brevibacterium flavum (Corynebacterium glutamicum) ATCC 13826, ATCC 14067, AJ12418 (FERM BP-2205)
Brevibacterium immariophilum ATCC 14068
Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC 13869
Brevibacterium roseum ATCC 13825
Brevibacterium saccharolyticum ATCC 14066
Brevibacterium thiogenitalis ATCC 19240
Corynebacterium ammoniagenes (Corynebacterium stationis) ATCC 6871, ATCC 6872
Brevibacterium album ATCC 15111
Brevibacterium cerinum ATCC 15112
Microbacterium ammoniaphilum ATCC 15354

Bacteria belonging to the genus Corynebacterium were previously classified into the genus Brevibacterium, but bacteria currently integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1991)) are also included. included. In addition, Corynebacterium stationis includes bacteria that were previously classified as Corynebacterium ammoniagenes, but have been reclassified into Corynebacterium stationis by 16S rRNA nucleotide sequence analysis, etc. (Int. J. .Syst. Evol. Microbiol., 60, 874-879 (2010)).

The yeast may be budding yeast or fission yeast. The yeast may be a diploid yeast or a diploid or higher polyploid yeast. Yeasts include the genus Saccharomyces cerevisiae and the genus Pichia ciferrii, the genus Pichia sydowiorum, and the genus Pichia pastoris (Pichia pastoris). ), Yeasts belonging to the genus Pichia such as Candida utilis, the genus Pichia such as Hansenula polymorpha, and the genus Pichia such as Schizosaccharomyces pombe. Can be mentioned.

These strains can be sold from, for example, the American Type Culture Collection (address P.O.Box 1549, Manassas, VA20108, United States of America or atcc.org). That is, a registration number corresponding to each strain is assigned, and this registration number can be used for distribution (see atcc.org/). The registration number for each strain can be found in the American Type Culture Collection catalog. In addition, these strains can be obtained, for example, from the depositary institution where each strain was deposited.

The microorganism may be one that originally has the ability to produce the target substance, or may be one that has been modified to have the ability to produce the target substance. A microorganism having a target substance-producing ability can be obtained, for example, by imparting the target substance-producing ability to the above-mentioned microorganism, or by enhancing the target substance-producing ability of the above-mentioned microorganism.

The method of imparting or enhancing the target substance production capacity is not particularly limited. As a method for imparting or enhancing the target substance-producing ability, for example, a known method can be used. Methods for imparting or enhancing the productivity of the target substance are, for example, WO2018 / 099687, WO2018 / 079686, WO2018 / 079685, WO2018 / 099684, WO2018 / 079683, WO2017 / 073701, WO2018 / 079705, US2018-0334693A, and US2019- It is disclosed in 0161776A.

Hereinafter, a method for imparting or enhancing the target substance-producing ability will be specifically illustrated. In addition, any of the modifications for imparting or enhancing the target substance-producing ability as illustrated below may be used alone or in combination as appropriate.

The target substance can be produced by the action of enzymes involved in the biosynthesis of the target substance. Such an enzyme is also referred to as a "target substance biosynthetic enzyme". Therefore, the microorganism may have a target substance biosynthetic enzyme. In other words, the microorganism may have a gene encoding a target substance biosynthetic enzyme. Such a gene is also referred to as a "target substance biosynthesis gene". The microorganism may be one that originally has a target substance biosynthesis gene, or may be one into which a target substance biosynthesis gene has been introduced. Techniques for introducing genes are described herein.

In addition, the ability of microorganisms to produce the target substance can be improved by increasing the activity of the target substance biosynthetic enzyme. That is, as a method for imparting or enhancing the target substance-producing ability, there is a method for increasing the activity of the target substance biosynthetic enzyme. That is, the microorganism may be modified so as to increase the activity of the target substance biosynthetic enzyme. The activity of one target substance biosynthetic enzyme may be increased, or the activity of two or more target substance biosynthetic enzymes may be increased. Techniques for increasing the activity of proteins (enzymes, etc.) are described herein. The activity of a protein (enzyme, etc.) can be increased, for example, by increasing the expression of the gene encoding the protein.

The target substance can be produced, for example, from a carbon source and / or a precursor of the target substance. Thus, examples of target substance biosynthetic enzymes include enzymes that catalyze the conversion of carbon sources and / or precursors to target substances. For example, 3-dehydroshikimic acid, an intermediate in the vanillin biosynthetic pathway, can be produced by part of the shikimate pathway. Part of the shikimate pathway is 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthase (3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase; DAHP synthase), 3-dehydroquinate synthase. It may include steps catalyzed by (3-dehydroquinate synthase) and 3-dehydroquinate dehydratase. 3-Dehydroshikimic acid can be converted to protocatechuic acid by the action of 3-dehydroshikimate dehydratase (DHSD). Protocatechuic acid is converted to vanillic acid or protocatechualdehyde by the action of O-methyltransferase (OMT) or aromatic carboxylic acid reductase (ACAR), respectively. Can be converted. Vanillic acid or protocatechuic aldehyde can be converted to vanillin by the action of ACAR or OMT, respectively. That is, specific examples of the target substance biosynthetic enzyme include DAHP synthase, 3-dehydroquinate synthase, 3-dehydroquinate dehydrata, DHSD, OMT, and ACAR.

"3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase (DAHP synthase)" means D-erythrose 4-phosphate and phosphoenols. It may mean a protein having an activity of catalyzing the reaction of converting pyruvate to D-arabino-hepturonic acid-7-phosphate (DAHP) and phosphoric acid (EC 2.5.1.54, etc.). This activity is also called "DAHP synthase activity". The gene encoding DAHP synthase is also referred to as "DAHP synthase gene". Examples of DAHP synthase include AroF, AroG, and AroH proteins encoded by the aroF, aroG, and aroH genes, respectively. Of these, the AroG protein can function as the major DAHP synthase. Examples of DAHP synthases such as AroF, AroG, and AroH proteins include those of various organisms such as bacteria of Enterobacteriaceae and coryneform bacteria. Specific examples of DAHP synthase include AroF, AroG, and AroH proteins of E. coli such as E. coli K-12 MG1655 strain.

"3-dehydroquinate synthase" may mean a protein having an activity of catalyzing a reaction of dephosphorylating DAHP to produce 3-dehydroquinate (EC 4.2.3.4, etc.). .. This activity is also referred to as "3-dehydroquinate synthase activity". The gene encoding 3-dehydroquinate synthase is also referred to as "3-dehydroquinate synthase gene". Examples of 3-dehydroquinate synthase include AroB protein encoded by the aroB gene. Examples of 3-dehydroquinate synthases such as AroB protein include those of various organisms such as bacteria of Enterobacteriaceae and coryneform bacteria. Specific examples of 3-dehydroquinate synthase include AroB protein of E. coli such as E. coli K-12 MG1655 strain.

"3-dehydroquinate dehydratase" may mean a protein having an activity of catalyzing the reaction of dehydrating 3-dehydroquinate to produce 3-dehydroshikimic acid (EC 4.2.1.10). etc). This activity is also referred to as "3-dehydroquinate dehydratase activity". The gene encoding 3-dehydroquinate dehydratase is also referred to as "3-dehydroquinate dehydratase gene". Examples of 3-dehydroquinate dehydratase include the AroD protein encoded by the aroD gene. Examples of 3-dehydroquinate dehydratases such as AroD protein include those of various organisms such as bacteria of Enterobacteriaceae and coryneform bacteria. Specific examples of 3-dehydroquinate dehydratase include AroD protein of E. coli such as E. coli K-12 MG1655 strain.

"3-dehydroshikimate dehydratase (DHSD)" may mean a protein having an activity of catalyzing the reaction of dehydrating 3-dehydroshikimate to produce protocatechuic acid (EC 4.2. 1.118 etc.). This activity is also called "DHSD activity". The gene encoding DHSD is also referred to as the "DHSD gene". DHSD includes the AsbF protein encoded by the asbF gene. Examples of DHSD such as AsbF protein include those of various organisms such as Bacillus thuringiensis, Neurospora crassa, and Podospora pauciseta such as Bacillus thuringiensis BMB171 strain.

Expression of genes encoding shikimate pathway enzymes (DAHP synthase, 3-dehydroquinate synthase, 3-dehydroquinate dehydratase, etc.) is suppressed by the tyrosine repressor TyrR encoded by the tyrR gene. Therefore, the activity of enzymes in the shikimate pathway can also be increased by reducing the activity of the tyrosine repressor TyrR. Examples of the tyrosine repressor TyrR include those of various organisms such as bacteria of Enterobacteriaceae and coryneform bacteria. Specific examples of the tyrosine repressor TyrR include the E. coli TyrR protein of the E. coli K-12 MG1655 strain.

"O-methyltransferase (OMT)" may mean a protein having an activity of catalyzing a reaction of methylating a hydroxyl group of a substrate in the presence of a methyl group donor (EC 2.1.1.68, etc.). ). This activity is also called "OMT activity". The gene encoding OMT is also called the "OMT gene". OMT may have the required substrate specificity, depending on the type of biosynthetic pathway in which the substance of interest is produced. For example, when the method for producing the target substance includes conversion of protocatechuic acid to vanillic acid, at least OMT using protocatechuic acid as a substrate can be used. Further, for example, when the method for producing the target substance includes conversion of protocatechuic aldehyde to vanillin, at least OMT using protocatechuic aldehyde as a substrate can be used. That is, "O-methyltransferase (OMT)" specifically refers to methylating protocatechuic acid and / or protocatechuic aldehyde in the presence of a methyl group donor to produce vanillic acid and / or vanillin. It may mean a protein having an activity of catalyzing a reaction to be produced (that is, methylation of a hydroxyl group at the meta position). OMT may usually use both protocatechuic acid and protocatechuic aldehyde as substrates, but is not limited to this. Methyl group donors include S-adenosylmethionine (SAM).

OMTs include OMTs of various organisms, such as OMTs of Homo sapiens (Hs) (GenBank Accession No. NP_000745, NP_009294), OMTs of Arabidopsis thaliana (GenBank Accession No. NP_200227, NP_009294), and OMTs of Fragaria xananassa (GenBank). No. AAF28353), and various OMTs of mammals, plants, and microorganisms exemplified in WO2013 / 022881. Four transcription variants and two OMT isoforms are known for the Homo sapiens OMT gene. OMT also includes OMT of Bacteroidetes phylum bacteria (ie, bacteria belonging to the Bacteroidetes phylum) (WO2018 / 079683). Examples of Bacteroidetes phylum bacteria include bacteria belonging to the genus Niastella, Terrimonas, Chitinophaga, etc. (International Journal of Systematic and Evolutionary Microbiology (2007), 57, 1828-1833). Examples of Niastella bacterium include Niastella koreensis. The OMT further includes the mutant OMT described in WO2013 / 022881 or WO2018 / 079683.

"Aromatic carboxylic acid reductase (ACAR)" is a reaction that reduces vanillic acid and / or protocatechuic acid in the presence of an electron donor and ATP to produce vanillin and / or protocatechuic acid. It may mean a protein having a catalytic activity (EC 1.2.99.6, etc.). This activity is also called "ACAR activity". The gene encoding ACAR is also called "ACAR gene". ACAR may, but is not limited to, both vanillic acid and protocatechuic acid as substrates. That is, ACAR may have the required substrate specificity depending on the type of biosynthetic pathway in which the target substance is produced. For example, when the method for producing the target substance includes conversion of vanillic acid to vanillin, at least ACAR using vanillic acid as a substrate can be used. Further, for example, when the method for producing the target substance includes conversion of protocatechuic acid to protocatechuic acid, at least ACAR using protocatechuic acid as a substrate can be used. Examples of electron donors include NADH and NADPH. Examples of electron donors include NADPH.

ACAR includes ACAR of various microorganisms such as Nocardia sp. NRRL 5646 strain, Actinomyces sp., Clostridium thermoaceticum, Aspergillus niger, Corynespora melonis, Coriolus sp., Neurospora sp. (J. Biol. Chem. 2007, Vol. 282, No.1, p478-485). The Nocardia sp. NRRL 5646 strain is classified as Nocardia iowensis. ACAR also includes ACAR of other Nocardia bacteria such as Nocardia brasiliensis and Nocardia vulneris. Examples of Nocardia brasiliensis include Nocardia brasiliensis ATCC 700358 strain. Examples of ACAR include ACAR of Gordonia bacterium such as Gordonia effusa, ACAR of Novosphingobium bacterium such as Novosphingobium malaysiense, and ACAR of Coccoyxa algae such as Coccoyxa subellipsoidea (WO2018 / 079705). The nucleotide sequence of the ACAR gene of Gordonia effusa is shown in SEQ ID NO: 1, and the amino acid sequence of ACAR encoded by the gene is shown in SEQ ID NO: 2.

ACAR can become an active enzyme by being phosphopantetinylated (J. Biol. Chem. 2007, Vol. 282, No. 1, p478-485). Therefore, the activity of ACAR can be increased by increasing the activity of an enzyme that catalyzes the phosphopantetinylation of proteins (also referred to as "phosphopantetinylation enzyme"). That is, as a method for imparting or enhancing the target substance-producing ability, a method for increasing the activity of the phosphopantetinylating enzyme can be mentioned. That is, the microorganism may be modified to increase the activity of the phosphopantetinylating enzyme. Examples of the phosphopantetheinyl transferase include phosphopantetheinyl transferase (PPT).

The "phosphopantetheinyl transferase (PPT)" may mean a protein having an activity of catalyzing the reaction of converting ACAR to phosphopantetinyl in the presence of a phosphopantetheinyl group donor. This activity is also called "PPT activity". The gene encoding PPT is also called "PPT gene". Examples of the phosphopantetinyl group donor include coenzyme A (CoA). Examples of PPT include EntD protein encoded by the entD gene. Examples of PPTs such as EntD protein include those of various organisms. Specific examples of PPT include E. coli EntD protein such as E. coli K-12 MG1655 strain. The nucleotide sequence of the entD gene of the E. coli K-12 MG1655 strain is shown in SEQ ID NO: 3, and the amino acid sequence of the EntD protein encoded by the gene is shown in SEQ ID NO: 4. Specifically, the PPTs are Nocardia brasiliensis PPT, Nocardia farcinica IFM10152 PPT (J. Biol. Chem. 2007, Vol. 282, No. 1, pp. 478-485), and C. glutamicum PPT (PPT). App. Env. Microbiol. 2009, Vol.75, No. 9, pp.2765-2774) can also be mentioned. Examples of C. glutamicum include the above-exemplified strains such as C. glutamicum ATCC 13032 strain and ATCC 13869 strain.

In addition, as a method for imparting or enhancing the target substance-producing ability, a substance other than the target substance (for example, a substance produced as an intermediate during the production of the target substance or a substance used as a precursor of the target substance). Examples include a method of increasing the activity of the uptake system of. That is, the microorganism may be modified to increase the activity of such an uptake system. The "substance uptake system" may mean a protein having a function of taking up a substance from the outside of the cell into the cell. This activity is also referred to as "substance uptake activity". A gene encoding such an uptake system is also referred to as an "uptake system gene". Examples of such an uptake system include a vanillic acid uptake system and a protocatechuic acid uptake system. Examples of the vanillic acid uptake system include the VanK protein encoded by the vanK gene (M. T. Chaudhry, et al., Microbiology, 2007. 153: 857-865). Examples of the protocatechuic acid uptake system include the PcaK protein encoded by the pcaK gene (M. T. Chaudhry, et al., Microbiology, 2007. 153: 857-865). Examples of the vanillic acid uptake system such as VanK protein and the vanillic acid uptake system such as PcaK protein include those of various organisms such as coryneform bacteria. Specific examples of the vanillic acid uptake system include the VanK protein of C. glutamicum such as C. glutamicum ATCC 13032 strain and ATCC 13869 strain. Specific examples of the protocatechuic acid uptake system include C. glutamicum PcaK protein (NCgl1031 protein) such as C. glutamicum ATCC 13032 strain and ATCC 13869 strain. The nucleotide sequence of the vanK gene (NCgl2302) of the C. glutamicum ATCC 13869 strain is shown in SEQ ID NO: 5, and the amino acid sequence of the VanK protein encoded by the gene is shown in SEQ ID NO: 6.

Further, as a method for imparting or enhancing the target substance-producing ability, there is a method of reducing the activity of enzymes involved in the by-product of substances other than the target substance. Substances other than such target substances are also referred to as "by-products". Such an enzyme is also referred to as a "by-product-producing enzyme". Examples of by-product producing enzymes include enzymes involved in the assimilation of the target substance and enzymes that catalyze the reaction of branching from the biosynthetic pathway of the target substance to produce a substance other than the target substance. Techniques for reducing the activity of proteins (enzymes, etc.) are described herein. The activity of a protein (enzyme, etc.) can be reduced, for example, by disrupting the gene encoding the protein. For example, in coryneform bacteria, vanillin has been reported to be metabolized and assimilated in the order of vanillin → vanillic acid → protocatechuic acid (Current Microbiology, 2005, Vol.51, p59-65). That is, specific examples of by-product-producing enzymes include enzymes that catalyze the conversion of vanillin to protocatechuic acid and enzymes that catalyze the further metabolism of protocatechuic acid. Such enzymes include vanillate demethylase, protocatechuate 3,4-dioxygenase, and reaction products of protocatechuate 3,4-dioxygenase as succinyl CoA and acetyl CoA. Examples include various enzymes (Appl. Microbiol. Biotechnol., 2012, Vol.95, p77-89) that further decompose up to. In addition, vanillin can be converted to vanillyl alcohol by the action of alcohol dehydrogenase (Kunjapur AM. Et al., J. Am. Chem. Soc., 2014, Vol.136, p11644-11654. Hansen EH. Et al., App. Env. Microbiol., 2009, Vol.75, p2765-2774.). That is, as a by-product producing enzyme, specifically, alcohol dehydrogenase (ADH) can be mentioned. In addition, 3-dehydroshikimic acid, which is an intermediate of the vanillin biosynthetic pathway, can be converted to shikimic acid by the action of shikimate dehydrogenase. That is, as a by-product producing enzyme, specifically, shikimate dehydrogenase can be mentioned.

"Vanillate demethylase" may mean a protein having an activity of catalyzing a reaction of demethylating vanillic acid to produce protocatechuic acid. This activity is also called "vanillate demethylase activity". The gene encoding vanillate demethylase is also referred to as "vanillate demethylase gene". Examples of vanillate demethylase include the VanAB protein encoded by the vanAB gene (Current Microbiology, 2005, Vol.51, p59-65). The vanA and vanB genes encode subunit A and subunit B of vanillate demethylase, respectively. When reducing the vanillate demethylase activity, for example, both vanAB genes may be disrupted, or only one of the vanillate genes may be disrupted. Examples of vanillate demethylase such as VanAB protein include those of various organisms such as bacteria of Enterobacteriaceae and coryneform bacteria. Specific examples of vanillate demethylase include VanAB proteins of C. glutamicum such as C. glutamicum ATCC 13032 strain and ATCC 13869 strain. The nucleotide sequences of the vanAB gene of the C. glutamicum ATCC 13869 strain are shown in SEQ ID NOs: 7 and 9, and the amino acid sequences of the VanAB protein encoded by the gene are shown in SEQ ID NOs: 8 and 10, respectively. The vanAB gene usually constitutes the vanK gene and the vanABK operon. Therefore, the vanABK operons may be collectively destroyed (for example, deleted) in order to reduce the vanillate demethylase activity. In that case, the vanK gene may be introduced into the microorganism again. For example, when vanillic acid existing outside the cells is used and the vanABK operons are collectively destroyed (for example, deleted), it is preferable to introduce the vanK gene again.

"Protocatechuate 3,4-dioxygenase" means a protein having an activity of catalyzing a reaction of oxidizing protocatechuate to produce β-carboxycis and cis-muconic acid. It's okay. This activity is also referred to as "protocatechuate 3,4-dioxygenase activity". The gene encoding protocatechuate 3,4-dioxygenase is also referred to as "protocatechuate 3,4-dioxygenase gene". Examples of protocatechuate 3,4-dioxygenase include the PcaGH protein encoded by the pcaGH gene (Appl. Microbiol. Biotechnol., 2012, Vol.95, p77-89). The pcaG and pcaH genes encode the α and β subunits of protocatechuate 3,4-dioxygenase, respectively. When reducing the protocatechuate 3,4-dioxygenase activity, for example, both of the pcaGH genes may be disrupted, or only one of them may be disrupted. Examples of protocatechuate 3,4-dioxygenase such as PcaGH protein include those of various organisms such as bacteria of Enterobacteriaceae and coryneform bacteria. Specific examples of protocatechuate 3,4-dioxygenase include PcaGH proteins of C. glutamicum such as C. glutamicum ATCC 13032 strain and ATCC 13869 strain.

"Alcohol dehydrogenase (ADH)" may mean a protein having an activity of catalyzing a reaction of reducing an aldehyde to produce an alcohol in the presence of an electron donor (EC 1.1.1.1, EC 1.1). 1.2, EC 1.1.1.71, etc.). This activity is also called "ADH activity". The gene encoding ADH is also referred to as the "ADH gene". Examples of electron donors include NADH and NADPH.

Examples of ADH include those having an activity of catalyzing a reaction of reducing vanillin to produce vanillyl alcohol in the presence of an electron donor. This activity is also referred to as "vanillyl alcohol dehydrogenase activity". In addition, ADH having vanillyl alcohol dehydrogenase activity is also particularly referred to as "vanillyl alcohol dehydrogenase".

Examples of ADH include YqhD protein, NCgl0324 protein, NCgl0313 protein, NCgl2709 protein, NCgl0219 protein, and NCgl2382 protein encoded by the yqhD gene, NCgl0324 gene, NCgl0313 gene, NCgl2709 gene, NCgl0219 gene, and NCgl2382 gene, respectively. Both the yqhD gene and the NCgl0324 gene encode vanillyl alcohol dehydrogenase. Examples of such ADH include those of various organisms such as bacteria of Enterobacteriaceae and coryneform bacteria. The yqhD gene can be found, for example, in bacteria of the Enterobacteriaceae family, such as E. coli. The NCgl0324 gene, NCgl0313 gene, NCgl2709 gene, NCgl0219 gene, and NCgl2382 gene can be found in coryneform bacteria such as C. glutamicum, for example. That is, as ADH, specifically, YqhD protein of E. coli such as E. coli K-12 MG1655 strain can be mentioned. Specific examples of ADH include NCgl0324 protein, NCgl0313 protein, NCgl2709 protein, NCgl0219 protein, and NCgl2382 protein of C. glutamicum such as C. glutamicum ATCC 13032 strain and ATCC 13869 strain. The nucleotide sequences of the NCgl0324 gene, NCgl0313 gene, and NCgl2709 gene of the C. glutamicum ATCC 13869 strain are shown in SEQ ID NOs: 11, 13, and 15, and the amino acid sequences of the proteins encoded by the genes are shown in SEQ ID NOs: 12, 14, and 16. Each is shown. The activity of one ADH may be reduced, or the activity of two or more ADHs may be reduced. For example, the activity of one or more of the NCgl0324, NCgl2709, and NCgl0313 proteins may be reduced. In particular, at least the activity of the NCgl0324 protein may be reduced.

“Shikimate dehydrogenase” may mean a protein having an activity of catalyzing a reaction of reducing 3-dehydrogenic acid to produce shikimic acid in the presence of an electron donor (EC 1.1. 1.25 mag). This activity is also referred to as "shikimate dehydrogenase activity". The gene encoding shikimate dehydrogenase is also referred to as "shikimate dehydrogenase gene". Examples of electron donors include NADH and NADPH. Examples of shikimate dehydrogenase include the AroE protein encoded by the aroE gene. Examples of shikimate dehydrogenase such as AroE protein include those of various organisms such as bacteria of Enterobacteriaceae and coryneform bacteria. Specific examples of shikimate dehydrogenase include AroE proteins of E. coli such as E. coli K-12 MG1655 strain.

Further, as a method for imparting or enhancing the target substance-producing ability, there is a method for increasing the activity of L-cysteine biosynthetic enzyme (WO2018 / 099687).

"L-Cysteine biosynthesis enzyme" may mean a protein involved in L-cysteine biosynthesis. The gene encoding the L-cysteine biosynthetic enzyme is also referred to as "L-cysteine biosynthetic gene". Examples of the L-cysteine biosynthetic enzyme include proteins involved in the utilization of sulfur. Proteins involved in the utilization of sulfur include CysIXHDNYZ protein and Fpr2 protein encoded by the cysIXHDNYZ gene and fpr2 gene, respectively. The CysIXHDNYZ protein is particularly involved in the reduction of inorganic sulfur compounds such as sulfates and sulfites. The Fpr2 protein may be particularly involved in electron transfer for the reduction of sulfites. Examples of the L-cysteine biosynthetic enzyme include O-acetylserine (thiol) -lyase. Examples of O-acetylserine (thiol) -lyase include CysK protein encoded by the cysK gene. Examples of the L-cysteine biosynthetic enzyme include those of various organisms such as bacteria of Enterobacteriaceae and coryneform bacteria. Specific examples of the L-cysteine biosynthetic enzyme include CysIXHDNYZ protein, Fpr2 protein, and CysK protein of C. glutamicum such as C. glutamicum ATCC 13032 strain and ATCC 13869 strain. The activity of one L-cysteine biosynthetic enzyme may be increased, or the activity of two or more L-cysteine biosynthetic enzymes may be increased. For example, the activity of one or more of the CysIXHDNYZ protein, Fpr2 protein, and CysK protein may be increased, and the activity of one or more of the CysIXHDNYZ protein and Fpr2 protein may be increased.

The activity of L-cysteine biosynthetic enzyme is, for example, by increasing the expression of genes encoding L-cysteine biosynthetic enzyme (ie, L-cysteine biosynthetic genes such as cysIXHDNYZ gene, fpr2 gene, cysK gene). Can be increased.

The expression of the L-cysteine biosynthesis gene can be increased, for example, by modifying (eg, increasing or decreasing) the activity of the expression regulator of the gene. That is, the expression of the L-cysteine biosynthesis gene can be increased, for example, by increasing the activity of a positive expression regulator (eg, activator) of the gene. In addition, the expression of the L-cysteine biosynthesis gene can be increased, for example, by reducing the activity of a negative expression regulator (eg, repressor) of the gene. Such regulators are also referred to as "regulatory proteins." A gene encoding such a regulatory factor is also referred to as a "regulatory gene".

Examples of such activators include CysR protein and SsuR protein encoded by the cysR gene and the ssuR gene, respectively. Increased activity of the CysR protein can increase the expression of one or more of the cysIXHDNYZ gene, fpr2 gene, and ssuR gene. In addition, increased activity of the SsuR protein can increase the expression of genes involved in the utilization of organic sulfur compounds. Examples of such activators include those of various organisms such as Enterobacteriaceae bacteria and Coryneform bacteria. Specific examples of such activators include CysR protein and SsuR protein of C. glutamicum such as C. glutamicum ATCC 13032 strain and ATCC 13869 strain. The activity of one or both of the CysR protein and the SsuR protein may be increased. For example, at least the activity of the CysR protein may be reduced. The activity of such an activator can be increased, for example, by increasing the expression of the gene encoding the activator.

Examples of such a repressor include the McbR protein encoded by the mcbR gene. Decreased activity of the McbR protein can increase the expression of one or more of the cysR and ssuR genes, which in turn increases the expression of one or more of the cysIXHDNYZ and fpr2 genes. Can be. Examples of such repressors include those of various organisms such as Enterobacteriaceae bacteria and Coryneform bacteria. Specific examples of such repressors include McbR proteins of C. glutamicum such as C. glutamicum ATCC 13032 strain and ATCC 13869 strain. The activity of such a repressor can be reduced, for example, by reducing the expression of the gene encoding the repressor, or by disrupting the gene encoding the repressor.

That is, specifically, the activity of the L-cysteine biosynthetic enzyme is increased, for example, by increasing the expression of one or more of the cysIXHDNYZ gene, fpr2 gene, cysR gene, and ssuR gene. Can be done. That is, "increased activity of L-cysteine biosynthetic enzyme" means, for example, increased expression of one or more of the cysIXHDNYZ gene, fpr2 gene, cysR gene, and ssuR gene. Good. For example, at least the expression of the cysR gene may be increased. Also, for example, the expression of all of these genes may be increased. Expression of one or more of the cysIXHDNYZ gene, fpr2 gene, and ssuR gene may be increased by increasing the expression of the cysR gene.

Further, as a method for imparting or enhancing the target substance-producing ability, there is a method for reducing the activity of NCgl2048 protein (WO2018 / 079686).

"NCgl2048 protein" may mean a protein encoded by the NCgl2048 gene. Examples of NCgl2048 protein include those of various organisms such as bacteria of Enterobacteriaceae and coryneform bacteria. Specific examples of the NCgl2048 protein include the NCgl2048 protein of C. glutamicum such as the C. glutamicum ATCC 13869 strain. Regarding the conservative variant, the "original function of NCgl2048 protein" may mean the function of a protein having the amino acid sequence of NCgl2048 protein of C. glutamicum ATCC 13869 strain, and by reducing the activity in microorganisms. It may mean the property of increasing the production of the target substance.

Further, as a method for imparting or enhancing the target substance-producing ability, there is a method for reducing the activity of enolase (WO2018 / 079685).

"Enolase" is a protein that has the activity of catalyzing the reaction of dehydrating 2-phospho-D-glyceric acid to produce phosphoenolpyruvic acid. May mean (EC 4.2.1.11, etc.). This activity is also referred to as "enolase activity". enolase is also referred to as "phosphopyruvate hydratase". The gene encoding enolase is also referred to as "enolase gene". Examples of the enolase include an Eno protein encoded by the eno gene. Examples of enolases such as Eno proteins include those of various organisms such as Enterobacteriaceae bacteria and coryneform bacteria. Specific examples of the enolase include the Eno protein of C. glutamicum such as the C. glutamicum ATCC 13869 strain.

In addition, as a method for imparting or enhancing the target substance-producing ability, there is a method for increasing the activity of S-adenosyl-L-homocysteine hydrolase (WO2018 / 079684). ..

"S-adenosyl-L-homocysteine hydrolase" is a hydrolysis of S-adenosyl-L-homocysteine (SAH) and L-. It may mean a protein having an activity of catalyzing the reaction of producing homocysteine and adenosine (EC 3.3.1.1, etc.). This activity is also referred to as "S-adenosyl-L-homocysteine hydrolase activity". S-adenosyl-L-homocysteine hydrolase is also called "adenosylhomocysteinase". The gene encoding S-adenosyl-L-homocysteine hydrolase is also referred to as "S-adenosyl-L-homocysteine hydrolase gene". Examples of the S-adenosyl-L-homocysteine hydrolase include the SahH protein encoded by the sahH gene. Examples of S-adenosyl-L-homocysteine hydrolase such as SahH protein include those of various organisms such as yeast, Streptomyces genus bacteria, and coryneform bacteria. Specific examples of the S-adenosyl-L-homocysteine hydrolase include SahH proteins of C. glutamicum such as C. glutamicum ATCC 13032 strain and ATCC 13869 strain.

Further, as a method for imparting or enhancing the target substance-producing ability, there is a method for reducing the activity of L-serine deaminase (US2018-0334693A).

"L-serine deaminase" may mean a protein having an activity of catalyzing the reaction of converting L-serine to pyruvic acid and ammonia (EC 4.3.1.17, etc.). This activity is also referred to as "L-serine deaminase activity". L-serine deaminase is also referred to as "L-serine ammonia-lyase". The gene encoding L-serine deaminase is also referred to as "L-serine deaminase gene". Examples of the L-serine deaminase include the SdaA protein encoded by the sdaA gene. Examples of L-serine deaminase such as SdaA protein include those of various organisms such as bacteria of Enterobacteriaceae and coryneform bacteria. Specific examples of the L-serine deaminase include the SdaA protein of C. glutamicum such as the C. glutamicum ATCC 13869 strain.

In addition, as a method for imparting or enhancing the target substance-producing ability, the activity of AICAR formyltransferase / IMP cyclohydrolase is reduced, or AICAR has a mutation described in US 2018-0334693A. A method of modifying the gene encoding formyltransferase / IMP cyclohydrolase can be mentioned (US2018-0334693A).

"AICAR formyltransferase / IMP cyclohydrolase" may mean AICAR formyltransferase and / or IMP cyclohydrolase, that is, one or both of AICAR formyltransferase and IMP cyclohydrolase. "AICAR formyltransferase" is 5-amino-1- (5-phospho-D-ribosyl) imidazole (5-amino-1- (5-phospho-D-ribosyl) imidazole). -4-carboxamide; AICAR and 10-formyltetrahydrofolate, 5-formamido-1- (5-phospho-D-ribosyl) imidazole-4-carboxamide (5-formamido-1- (5)) -phospho-D-ribosyl) imidazole-4-carboxamide; may mean a protein having an activity of catalyzing the reaction of converting FAICAR) to tetrahydrofolate (EC 2.1.2.3, etc.). This activity is also referred to as "AICAR formytransferase activity". “IMP cyclohydrolase” may mean a protein having an activity of catalyzing a reaction of dehydrating FAICAR to produce IMP (EC 3.5.4.10, etc.). This activity is also called "IMP cyclohydrolase activity". The gene encoding AICAR formyltransferase / IMP cyclohydrolase is also referred to as "AICAR formyltransferase / IMP cyclohydrolase gene". AICAR formyltransferase and IMP cyclohydrolase may be encoded as bifunctional enzymes. Therefore, "AICAR formyltransferase / IMP cyclohydrolase" specifically refers to a bifunctional AICAR formyltransferase / IMP cyclohydrolase, that is, a protein having both AICAR formyltransferase activity and IMP cyclohydrolase activity. It may mean. Examples of AICAR formyltransferase / IMP cyclohydrolase include PurH protein, which is a bifunctional AICAR formyltransferase / IMP cyclohydrolase encoded by the purH gene. Examples of AICAR formytransferase / IMP cyclohydrolase such as PurH protein include those of various organisms such as bacteria of Enterobacteriaceae and coryneform bacteria. Specific examples of AICAR formyltransferase / IMP cyclohydrolase include PurH protein of C. glutamicum such as C. glutamicum ATCC 13869 strain.

In addition, as a method for imparting or enhancing the target substance-producing ability, the activity of D-3-phosphoglycerate dehydrogenase (3-PGDH) is increased, or feedback inhibition by L-serine is performed. There is a method of modifying a microorganism to have a gene encoding 3-PGDH resistant to 3 (US2018-0334693A).

"D-3-phosphoglycerate dehydrogenase (3-PGDH)" is a 3-phosphoglyceric acid that oxidizes 3-phosphoglyceric acid in the presence of an electron acceptor. It may mean a protein having an activity of catalyzing a reaction for producing hydroxypyruvic acid (3-phosphohydroxylpyruvic acid) (EC 1.1.1.95, etc.). This activity is also referred to as "3-PGDH activity". Examples of electron acceptors include NAD ⁺ and NADP ⁺ . The gene encoding 3-PGDH is also referred to as "3-PGDH gene". Examples of 3-PGDH include the SerA protein encoded by the serA gene. Examples of 3-PGDH such as SerA protein include those of various organisms such as bacteria of Enterobacteriaceae and coryneform bacteria. Specific examples of 3-PGDH include SerA protein of B. flavum such as B. flavum ATCC 14067 strain. Specific examples of 3-PGDH include SerA protein of E. coli such as E. coli K-12 MG1655 strain. Examples of 3-PGDH resistant to feedback inhibition by L-serine include those disclosed in US 2018-0334693A. Specific examples of 3-PGDH resistant to feedback inhibition by L-serine include the SerA * protein of the B. flavum AJ13327 strain encoded by the serA * gene (US2018-0334693A).

The genes and proteins used for breeding microorganisms capable of producing the target substance include, for example, the type of the target substance, the type of biosynthetic pathway in which the target substance is produced, and the type and activity of the protein inherently possessed by the microorganism. It can be appropriately selected according to the various conditions of. For example, when vanillin is produced by bioconversion from protocatechuic acid, the activity of one or more of the OMT, ACAR, PPT, and protocatechuic acid uptake systems may be increased, in particular. Also, for example, when vanillin is produced by bioconversion from vanillic acid, the activity of one or more of the ACAR, PPT, and vanillic acid uptake systems may be increased, in particular. When vanillin is produced by bioconversion from vanillic acid, the activity of at least ACAR and / or PPT may be increased, more particularly. When vanillin is produced by bioconversion from vanillic acid, the activity of at least ACAR and PPT may be increased, more particularly. Also, especially when vanillin is produced by bioconversion from protocatechuic aldehyde, the activity of OMT may be increased.

The genes and proteins used for breeding microorganisms capable of producing a target substance may have, for example, known base sequences and amino acid sequences (including those exemplified above), respectively. Known nucleotide sequences and amino acid sequences include those exemplified above, WO2018 / 099687, WO2018 / 079686, WO2018 / 079685, WO2018 / 099684, WO2018 / 079683, WO2017 / 073701, WO2018 / 079705, US2018-0334693A, and US2019. -0161776 A can be mentioned. Unless otherwise specified, the expression "a gene or protein has a base sequence or an amino acid sequence" may mean that the gene or protein contains the base sequence or the amino acid sequence, and the gene or protein has the base sequence or the amino acid sequence. The case consisting of an amino acid sequence may also be included.

The gene and protein used for breeding a microorganism capable of producing a target substance may be a conservative variant of the gene and protein having a known base sequence and amino acid sequence, respectively. "Conservative variant" means a variant that retains its original function. Conservative variants include, for example, homologues and artificial variants of genes and proteins having known nucleotide and amino acid sequences.

"Maintaining original function" means that a variant of a gene or protein has a function (eg, activity or property) that corresponds to the function (eg, activity or property) of the original gene or protein. .. By "maintaining the original function" of a gene is meant that a variant of the gene encodes a protein that retains its original function. For example, "maintaining the original function" of an ACAR gene means that a variant of the gene encodes a protein with ACAR activity. In addition, "maintaining the original function" of ACAR means that the protein variant has ACAR activity. The activity of each protein is measured, for example, by the method described in WO2018 / 099687, WO2018 / 079686, WO2018 / 079685, WO2018 / 099684, WO2018 / 079683, WO2017 / 073701, WO2018 / 079705, US2018-0334693A, or US2019-0161776A. can do.

The following is an example of a conservative variant.

Homologs of genes or proteins used for breeding microorganisms capable of producing the target substance can be easily obtained from public databases by, for example, BLAST search or FASTA search using a known base sequence or amino acid sequence as a query sequence. it can. In addition, for the homologue of a gene used for breeding a microorganism capable of producing a target substance, for example, an oligonucleotide prepared based on a known base sequence using a chromosome of an organism such as a coryneform bacterium as a template is used as a primer. It can be obtained by PCR.

Genes used for breeding microorganisms capable of producing a target substance are replaced with one or several amino acids at one or several positions in a known amino acid sequence, respectively, as long as the original function is maintained. , Which may encode a protein having an amino acid sequence deleted, inserted or added. For example, the encoded protein may have its N-terminus and / or C-terminus extended or shortened. The above "1 or several" differs depending on the position and type of the amino acid residue in the protein structure, but specifically, for example, 1 to 50, 1 to 40, 1 to 30, etc. It preferably means 1 to 20, more preferably 1 to 10, still more preferably 1 to 5, and particularly preferably 1 to 3.

The above-mentioned substitution, deletion, insertion, or addition of one or several amino acids is a conservative mutation in which the function of the protein is maintained normally. A typical conservative mutation is a conservative substitution. Conservative substitutions are polar amino acids between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid. In some cases, between Gln and Asn, between Lys, Arg and His if it is a basic amino acid, and between Asp and Glu if it is an acidic amino acid, if it is an amino acid with a hydroxyl group. Is a mutation that replaces each other between Ser and Thr. Substitutions that are considered conservative substitutions include, specifically, Ala to Ser or Thr substitutions, Arg to Gln, His or Lys substitutions, Asn to Glu, Gln, Lys, His or Asp substitutions. Asp to Asn, Glu or Gln replacement, Cys to Ser or Ala replacement, Gln to Asn, Glu, Lys, His, Asp or Arg replacement, Glu to Gly, Asn, Gln, Lys or Asp Substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Lys to Asn, Glu, Gln, His or Arg, Met to Ile, Leu, Val or Phe, Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala Substitutions include Thr to Ser or Ala, Trp to Phe or Tyr, Tyr to His, Phe or Trp, and Val to Met, Ile or Leu. In addition, the above-mentioned amino acid substitutions, deletions, insertions, additions, or inversions are naturally occurring mutations (mutants or variants) such as those based on individual differences or species differences of the organism from which the gene is derived. Also includes those caused by.

In addition, the genes used for breeding microorganisms capable of producing the target substance, for example, 50% or more, 65% or more, and 80, respectively, with respect to the entire known amino acid sequence as long as the original functions are maintained. It may be a gene encoding a protein having an amino acid sequence having an identity of% or more, preferably 90% or more, more preferably 95% or more, further preferably 97% or more, and particularly preferably 99% or more.

In addition, genes used for breeding microorganisms capable of producing a target substance can be prepared from known base sequences, for example, all or part of known base sequences, as long as the original functions are maintained. It may be a DNA that hybridizes with a complementary sequence to the DNA under stringent conditions. The "stringent condition" refers to a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. To give an example, DNAs having high identity, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, particularly preferably 99. 60 ° C, 1 × SSC, 0.1% SDS, which is a condition in which DNAs having an identity of% or more hybridize with each other and DNAs having a lower identity do not hybridize with each other, or a normal Southern hybridization washing condition. Conditions of washing once, preferably 2-3 times, preferably at a salt concentration and temperature corresponding to 60 ° C., 0.1 × SSC, 0.1% SDS, more preferably 68 ° C., 0.1 × SSC, 0.1% SDS. be able to.

As described above, the probe used for the above hybridization may be a part of the complementary sequence of the gene. Such a probe can be prepared by PCR using an oligonucleotide prepared based on a known base sequence as a primer and a DNA fragment containing the above-mentioned gene as a template. For example, as a probe, a DNA fragment having a length of about 300 bp can be used. When a DNA fragment having a length of about 300 bp is used as the probe, the conditions for washing the hybridization include 50 ° C., 2 × SSC, and 0.1% SDS.

Further, since the codon degeneracy differs depending on the host, the gene used for breeding a microorganism capable of producing a target substance may be a gene in which an arbitrary codon is replaced with a codon equivalent thereto.

The "identity" between amino acid sequences is calculated by blastp using the default settings of Scoring Parameters (Matrix: BLOSUM62; GapCosts: Presence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment). It means the identity between amino acid sequences. In addition, "identity" between base sequences means the identity between base sequences calculated by blastn using the default setting Scoring Parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear). To do.

<1-2> Method for increasing protein activity A method for increasing protein activity will be described below.

"Increased protein activity" may mean that the activity of the protein is increased as compared with the unmodified strain. "Increased protein activity" may specifically mean that the per-cell activity of the protein is increased relative to the unmodified strain. The "activity of a protein per cell" may mean the average value of the activity of the protein per cell. The unmodified strain is also referred to as a "non-modified microorganism" or a "non-modified microorganism strain". The term "unmodified strain" as used herein may mean a control strain that has not been modified to increase the activity of the target protein. Examples of the unmodified strain include a wild strain and a parent strain. Specific examples of the unmodified strain include a reference strain (type strain) of each microbial species. Further, as the unmodified strain, specifically, the strain exemplified in the description of the microorganism can be mentioned. That is, in one embodiment, the activity of the protein may be increased compared to the reference strain (ie, the reference strain of the species to which the microorganism capable of producing the target substance belongs). Moreover, in another embodiment, the activity of the protein may be increased as compared with the C. glutamicum ATCC 13869 strain. Moreover, in another embodiment, the activity of the protein may be increased as compared with the C. glutamicum ATCC 13032 strain. Moreover, in another embodiment, the activity of the protein may be increased as compared with the E. coli K-12 MG1655 strain. It should be noted that "increasing protein activity" is also referred to as "enhancing protein activity". "Increased protein activity" means, more specifically, that the number of molecules of the protein per cell is increased as compared to the unmodified strain, and / or per molecule of the protein. It may mean that the function is increasing. That is, the "activity" in the case of "increasing the activity of a protein" means not only the catalytic activity of the protein but also the transcription amount (mRNA amount) or the translation amount (protein amount) of the gene encoding the protein. You may. The “number of molecules of a protein per cell” may mean the average value of the number of molecules of the protein per cell. In addition, "increasing the activity of a protein" means not only increasing the activity of the protein in a strain that originally has the activity of the target protein, but also that the strain of the protein that originally does not have the activity of the target protein has the same protein. It also includes imparting activity. Further, as long as the activity of the protein is increased as a result, the activity of the target protein originally possessed by the host may be reduced or eliminated, and then the activity of the suitable target protein may be imparted.

The degree of increase in protein activity is not particularly limited as long as the protein activity is increased as compared with the unmodified strain. The activity of the protein may be increased, for example, 1.2 times or more, 1.5 times or more, 2 times or more, or 3 times or more that of the unmodified strain. When the unmodified strain does not have the activity of the target protein, the protein may be produced by introducing a gene encoding the protein. For example, the activity of the protein is measured. It may be produced to the extent possible.

Modifications that increase the activity of the protein can be achieved, for example, by increasing the expression of the gene encoding the protein. "The expression of a gene is increased" may mean that the expression of the gene is increased as compared with an unmodified strain such as a wild strain or a parent strain. Specifically, "the expression of a gene is increased" may mean that the expression level of the gene per cell is increased as compared with the unmodified strain. The “expression level of a gene per cell” may mean the average value of the expression level of the gene per cell. "Increased gene expression" means, more specifically, an increase in the amount of transcription (mRNA) of a gene and / or an increase in the amount of translation (amount of protein) of a gene. It's okay. It should be noted that "increased gene expression" is also referred to as "enhanced gene expression". The expression of the gene may be increased, for example, 1.2 times or more, 1.5 times or more, 2 times or more, or 3 times or more that of the unmodified strain. In addition, "increasing gene expression" means not only increasing the expression level of the gene in the strain in which the target gene is originally expressed, but also in the strain in which the target gene is not originally expressed. Expression of the same gene is also included. That is, "the expression of a gene is increased" may mean, for example, introducing the gene into a strain that does not carry the target gene and expressing the gene.

Increased gene expression can be achieved, for example, by increasing the number of copies of the gene.

The increase in the number of copies of a gene can be achieved by introducing the gene into the host chromosome. For example, homologous recombination can be used to introduce a gene into a chromosome (Miller, J. H. Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). As a gene transfer method using homologous recombination, for example, the Red-driven integration method (Datsenko, K.A, and Wanner, B. L. Proc. Natl. Acad. Sci. U S A. 97 : 6640-6645 (2000)), etc., a method using a linear DNA, a method using a plasmid containing a temperature-sensitive origin of replication, a method using a conjugation-transmissible plasmid, a suicide vector having no origin of replication that functions in the host. Examples include a method using a plasmid and a transduction method using a phage. Only one copy of the gene may be introduced, or two or more copies may be introduced. For example, a large number of copies of a gene can be introduced into a chromosome by performing homologous recombination targeting a sequence having a large number of copies on the chromosome. Sequences in which a large number of copies are present on a chromosome include repetitive DNA sequences and inverted repeats present at both ends of a transposon. In addition, homologous recombination may be performed by targeting an appropriate sequence on the chromosome such as a gene unnecessary for the production of the target substance. The gene can also be randomly introduced onto the chromosome using a transposon or Mini-Mu (Japanese Patent Laid-Open No. 2-109985, US5,882,888, EP805867B1).

To confirm that the target gene was introduced on the chromosome, Southern hybridization using a probe having a sequence complementary to all or part of the gene was used, or a primer prepared based on the sequence of the gene was used. It can be confirmed by PCR or the like.

In addition, an increase in the number of copies of a gene can also be achieved by introducing a vector containing the same gene into the host. For example, a DNA fragment containing a target gene can be linked to a vector that functions in the host to construct an expression vector for the gene, and the host can be transformed with the expression vector to increase the number of copies of the gene. it can. The DNA fragment containing the target gene can be obtained, for example, by PCR using the genomic DNA of the microorganism having the target gene as a template. As the vector, a vector capable of autonomous replication in the host cell can be used. The vector may be a multicopy vector. Also, in order to select transformants, the vector may have markers such as antibiotic resistance genes. The vector may also include a promoter or terminator for expressing the inserted gene. The vector may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a cosmid, a phagemid or the like. Specific examples of vectors capable of autonomous replication in Enterobacteriaceae bacteria such as Escherichia coli include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, and pSTV29 (all available from Takara Bio). pACYC184, pMW219 (Nippon Gene), pTrc99A (Pharmacia), pPROK vector (Clontech), pKK233-2 (Clontech), pET vector (Novagen), pQE vector (Kiagen), pColdTFDNA ( TaKaRa), pACYC-based vector, broad host range vector RSF1010. Specific examples of vectors capable of autonomous replication in coryneform bacteria include pHM1519 (Agric. Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, 2901-). 2903 (1984)); A plasmid having an improved drug resistance gene; pCRY30 (Japanese Patent Laid-Open No. 3-210184); pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX (Japanese Patent Laid-Open No. 2-72876, US Pat. No. 5,185,262) PCRY2 and pCRY3 (Japanese Patent Laid-Open No. 1-191686); pAJ655, pAJ611, and pAJ1844 (Japanese Patent Laid-Open No. 58-192900); pCG1 (Japanese Patent Laid-Open No. 57-134500); pCG2 (Japanese Patent Laid-Open No. 58-35197); pCG4 and pCG11 (Japanese Patent Laid-Open No. 57-183799); pPK4 (US Patent No. 6,090,597); pVK4 (Japanese Patent Laid-Open No. 9-322774); pVK7 (Japanese Patent Laid-Open No. 10-215883); pVK9 (WO2007 / 046389); pVS7 (WO2013 / 069634) ; PVC7 (Japanese Patent Laid-Open No. 9-070291) can be mentioned. Further, as a vector capable of autonomous replication in a coryneform bacterium, specifically, a variant of pVC7 such as pVC7H2 can be mentioned (WO2018 / 179834).

When introducing a gene, the gene may be expressed by the host. Specifically, the gene may be retained so as to be expressed under the control of a promoter that functions in the host. "Promoter functioning in a host" may mean a promoter having promoter activity in the host. The promoter may be a host-derived promoter or a heterologous promoter. The promoter may be a promoter unique to the gene to be introduced, or may be a promoter of another gene. As the promoter, for example, a stronger promoter as described herein may be used.

A terminator for transcription termination can be placed downstream of the gene. The terminator is not particularly limited as long as it functions in the host. The terminator may be a host-derived terminator or a heterogeneous terminator. The terminator may be a unique terminator of the gene to be introduced, or may be a terminator of another gene. Specific examples of the terminator include a T7 terminator, a T4 terminator, an fd phage terminator, a tet terminator, and a trpA terminator.

Vectors, promoters, and terminators that can be used in various microorganisms are described in detail in, for example, "Basic Microbiology Course 8 Genetic Engineering, Kyoritsu Shuppan, 1987", and they can be used.

In addition, when introducing two or more genes, it is sufficient that each gene is retained in the host so that it can be expressed. For example, each gene may be all retained on a single expression vector or all on a chromosome. In addition, each gene may be separately retained on a plurality of expression vectors, or may be separately retained on a single or a plurality of expression vectors and on a chromosome. In addition, an operon may be composed of two or more genes and introduced. When "introducing two or more genes", for example, when introducing genes encoding two or more proteins (eg, enzymes), a single protein complex (eg, enzyme complex) When introducing a gene encoding each of the two or more subunits constituting it, or a combination thereof may be introduced.

The gene to be introduced is not particularly limited as long as it encodes a protein that functions in the host. The gene to be introduced may be a host-derived gene or a heterologous gene. The gene to be introduced can be obtained by PCR using, for example, a primer designed based on the base sequence of the gene, genomic DNA of an organism having the gene, a plasmid carrying the gene, or the like as a template. Further, the gene to be introduced may be totally synthesized, for example, based on the base sequence of the gene (Gene, 60 (1), 115-127 (1987)). The acquired gene can be used as it is or after being appropriately modified. That is, the variant can be obtained by modifying the gene. The gene can be modified by a known method. For example, a site-specific mutation method can be used to introduce a desired mutation into a target site of DNA. That is, for example, site-specific mutagenesis can modify the coding region of a gene such that the encoded protein comprises substitutions, deletions, insertions, and / or additions of amino acid residues at specific sites. .. As a site-specific mutation method, a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, H. A. Eds., Stockton press (1989); Carter, P., Meth. In Enzymol., 154, 382 (1987)) and methods using phage (Kramer, W. and Frits, H. J., Meth. In Enzymol., 154, 350 (1987); Kunkel, T. A. et al., Meth . In Enzymol., 154, 367 (1987)). Alternatively, the gene variant may be totally synthesized.

When a protein functions as a complex composed of a plurality of subunits, all of these subunits may be modified or only a part thereof may be modified as long as the activity of the protein is increased as a result. That is, for example, when increasing the activity of a protein by increasing the expression of a gene, the expression of all the genes encoding each of these subunits may be enhanced, or only a part of the expression may be enhanced. Good. Usually, it is preferable to enhance the expression of all genes encoding those subunits. In addition, each subunit constituting the complex may be derived from one organism or two or more different organisms as long as the complex has the function of the target protein. That is, for example, a gene derived from the same organism that encodes a plurality of subunits may be introduced into the host, or a gene derived from a different organism may be introduced into the host.

In addition, an increase in gene expression can be achieved by improving the transcription efficiency of the gene. In addition, an increase in gene expression can be achieved by improving the translation efficiency of the gene. Improvements in gene transcription efficiency and translation efficiency can be achieved, for example, by modifying the expression regulatory sequence. The "expression regulatory sequence" may be a general term for sites that affect gene expression. Expression regulatory sequences include, for example, a promoter, a Shine-Dalgarno (SD) sequence (also referred to as a ribosome binding site (RBS)), and a spacer region between the RBS and the start codon. The expression regulatory sequence can be determined using a promoter search vector or gene analysis software such as GENETYX. These expression regulatory sequences can be modified by, for example, a method using a temperature-sensitive vector or a Red-driven integration method (WO2005 / 010175).

Improvement of gene transcription efficiency can be achieved, for example, by replacing the promoter of the gene on the chromosome with a stronger promoter. A "stronger promoter" may mean a promoter in which transcription of a gene is improved over a wild-type promoter that originally exists. More potent promoters include, for example, the well-known highly expressed promoters T7 promoter, trp promoter, lac promoter, thr promoter, tac promoter, trc promoter, tet promoter, araBAD promoter, rpoH promoter, msrA promoter, and Pm1 derived from Bifidobacterium. Promoters include promoters, PR promoters, and PL promoters. In addition, as stronger promoters that can be used in coryneform bacteria, for example, the artificially redesigned P54-6 promoter (Appl. Microbiol. Biotechnol., 53, 674-679 (2000)), in coryneform bacteria The pta, aceA, aceB, adh, amyE promoters that can be induced with acetic acid, ethanol, pyruvate, etc., and the cspB, SOD, tuf (EF-Tu) promoters (Journal of), which are strong promoters with high expression levels in coryneform bacteria. Biotechnology 104 (2003) 311-323, Appl Environment Microbiol. 2005 Dec; 71 (12): 8587-96.), P2 promoter (US2018-0334693A), P3 promoter (US2018-0334693A), F1 promoter (WO2018 / 179834) , Lac promoter, tac promoter, trc promoter. Further, as a stronger promoter, a highly active form of a conventional promoter may be obtained by using various reporter genes. For example, the activity of the promoter can be enhanced by bringing the -35 and -10 regions within the promoter region closer to the consensus sequence (International Publication No. 00/18935). Examples of the highly active promoter include various tac-like promoters (Katashkina JI et al. Russian Federation Patent application 2006134574). Methods for evaluating the strength of promoters and examples of strong promoters are described in the paper by Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)).

Improvement of gene translation efficiency can be achieved, for example, by replacing the Shine-Dalgarno (SD) sequence (also referred to as ribosome binding site (RBS)) of the gene on the chromosome with a stronger SD sequence. A "stronger SD sequence" may mean an SD sequence in which the translation of mRNA is improved over the originally existing wild-type SD sequence. As a stronger SD sequence, for example, RBS of gene 10 derived from phage T7 can be mentioned (Olins P. O. et al, Gene, 1988, 73, 227-235). In addition, the substitution, insertion, or deletion of several nucleotides in the spacer region between the RBS and the start codon, especially in the sequence immediately upstream of the start codon (5'-UTR), contributes to mRNA stability and translation efficiency. It is known to have a great effect, and the translation efficiency of genes can be improved by modifying these.

Improvement of gene translation efficiency can also be achieved by, for example, modifying codons. For example, the translation efficiency of a gene can be improved by replacing the rare codon present in the gene with a synonymous codon that is used more frequently. That is, the gene to be introduced may be modified to have an optimum codon depending on, for example, the codon usage frequency of the host used. Codon substitution can be performed, for example, by a site-specific mutation method that introduces the desired mutation into the target site of DNA. In addition, the gene fragment in which the codon has been replaced may be totally synthesized. The frequency of codon usage in various organisms is described in the "Codon Usage Database" (http://www.kazusa.or.jp/codon; Nakamura, Y. et al, Nucl. Acids Res., 28, 292 (2000)). It is disclosed in.

In addition, an increase in gene expression can also be achieved by amplifying a regulator that increases gene expression, or by deleting or weakening a regulator that decreases gene expression.

The above-mentioned method for increasing gene expression may be used alone or in any combination.

Further, modifications that increase the activity of the protein can also be achieved by, for example, enhancing the specific activity of the protein. The enhancement of specific activity may also include desensitization to feedback inhibition. That is, when a protein is subject to feedback inhibition by metabolites, the activity of the protein can be increased by mutating the gene or protein in the host so that the feedback inhibition is desensitized. Unless otherwise specified, the "desensitization of feedback inhibition" may include a case where the feedback inhibition is completely canceled and a case where the feedback inhibition is reduced. Further, "the feedback inhibition is desensitized" (that is, the feedback inhibition is reduced or canceled) is also referred to as "tolerance to the feedback inhibition". Proteins with enhanced specific activity can be obtained by searching for, for example, various organisms. In addition, a highly active form may be obtained by introducing a mutation into a conventional protein. The mutation introduced may be, for example, one or several amino acids substituted, deleted, inserted, and / or added at one or several positions of the protein. The mutation can be introduced by, for example, the site-specific mutation method as described above. Further, the introduction of the mutation may be carried out by, for example, a mutation treatment. Mutation treatment includes X-ray irradiation, ultraviolet irradiation, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethylmethanesulfonate (EMS), and methylmethanesulfonate (MMS). ) And other treatments with a mutagen. Alternatively, the DNA may be treated directly with hydroxylamine in vitro to induce random mutations. The enhancement of specific activity may be used alone or in combination with the above-mentioned method for enhancing gene expression.

The transformation method is not particularly limited, and a conventionally known method can be used. For example, a method of treating recipient cells with calcium chloride to increase DNA permeability, as reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol. 1970, 53, 159-162) and methods of preparing competent cells from proliferative cells and introducing DNA, as reported for Bacillus subtilis (Duncan, C. H., Wilson, G. A. and Young, F. E., 1977. Gene 1: 153-167) can be used. Alternatively, the cells of a DNA-recepting bacterium, as known for Bacillus subtilis, actinomycetes, and yeast, are transformed into a protoplast or spheroplast that readily incorporates the recombinant DNA to turn the recombinant DNA into a DNA-receptive bacterium. How to introduce (Chang, S. and Choen, S. N., 1979. Mol. Gen. Genet. 168: 111-115; Bibb, M. J., Ward, J. M. and Hopwood, O. A. 1978. Nature 274: 398-400; Hinnen, A., Hicks, J. B. and Fink, G. R. 1978. Proc. Natl. Acad. Sci. USA 75: 1929-1933) can also be applied. Alternatively, the electric pulse method (Japanese Patent Laid-Open No. 2-207791) as reported for coryneform bacteria can also be used.

The increase in protein activity can be confirmed by measuring the activity of the protein.

The increase in protein activity can also be confirmed by confirming that the expression of the gene encoding the protein has increased. The increase in gene expression can be confirmed by confirming that the transcription amount of the gene has increased and that the amount of protein expressed from the gene has increased.

Confirmation that the transcription amount of the gene has increased can be confirmed by comparing the amount of mRNA transcribed from the gene with an unmodified strain such as a wild strain or a parent strain. Methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR, microarray, RNA-seq, etc. (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory, etc. Press, Cold Spring Harbor (USA), 2001). The amount of mRNA (for example, the number of molecules per cell) may be increased, for example, 1.2 times or more, 1.5 times or more, 2 times or more, or 3 times or more that of the unmodified strain.

Confirmation that the amount of protein has increased can be confirmed by Western blotting using an antibody (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The amount of protein (eg, number of molecules per cell) may be increased, for example, 1.2 times or more, 1.5 times or more, 2 times or more, or 3 times or more that of the unmodified strain.

The above-mentioned method for increasing the activity of a protein can be used for enhancing the activity of an arbitrary protein or enhancing the expression of an arbitrary gene.

<1-3> Method for reducing protein activity A method for reducing protein activity will be described below.

"Reduced protein activity" may mean that the activity of the protein is reduced as compared with the unmodified strain. "Reduced protein activity" may specifically mean that the per-cell activity of the protein is reduced as compared to the unmodified strain. The "activity of a protein per cell" may mean the average value of the activity of the protein per cell. The unmodified strain is also referred to as a "non-modified microorganism" or a "non-modified microorganism strain". The term "unmodified strain" as used herein may mean a control strain that has not been modified to reduce the activity of the target protein. Examples of the unmodified strain include a wild strain and a parent strain. Specific examples of the unmodified strain include a reference strain (type strain) of each microbial species. Further, as the unmodified strain, specifically, the strain exemplified in the description of the microorganism can be mentioned. That is, in one embodiment, the activity of the protein may be reduced as compared with the reference strain (that is, the reference strain of the species to which the microorganism capable of producing the target substance belongs). Moreover, in another embodiment, the activity of the protein may be reduced as compared with the C. glutamicum ATCC 13869 strain. In another embodiment, the activity of the protein may be reduced as compared to the C. glutamicum ATCC 13032 strain. Moreover, in another embodiment, the activity of the protein may be reduced as compared with the E. coli K-12 MG1655 strain. The "decrease in protein activity" may include the case where the activity of the protein is completely eliminated. More specifically, "reduced protein activity" means that the number of molecules of the protein per cell is reduced as compared with the unmodified strain, and / or per molecule of the protein. It may mean that the function is deteriorated. That is, the "activity" in the case of "reducing the activity of a protein" means not only the catalytic activity of the protein but also the transcription amount (mRNA amount) or the translation amount (protein amount) of the gene encoding the protein. You may. The “number of molecules of a protein per cell” may mean the average value of the number of molecules of the protein per cell. The fact that "the number of molecules of a protein per cell is reduced" may include the case where the protein does not exist at all. In addition, "the function per molecule of the protein is reduced" may include the case where the function per molecule of the protein is completely lost. The degree of decrease in protein activity is not particularly limited as long as the protein activity is decreased as compared with the unmodified strain. The activity of the protein may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain.

Modifications that reduce the activity of the protein can be achieved, for example, by reducing the expression of the gene encoding the protein. "The expression of a gene is reduced" may mean that the expression of the gene is reduced as compared with an unmodified strain such as a wild strain or a parent strain. Specifically, "reduced gene expression" may mean that the per-cell expression level of the gene is reduced as compared with the unmodified strain. The “expression level of a gene per cell” may mean the average value of the expression level of the gene per cell. "Reduced gene expression" means, more specifically, a decrease in the transcription amount (mRNA amount) of a gene and / or a decrease in the translation amount (protein amount) of a gene. It's okay. "Reduced expression of a gene" may include the case where the gene is not expressed at all. It should be noted that "decreased gene expression" is also referred to as "weakened gene expression". Gene expression may be reduced, for example, to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain.

The decrease in gene expression may be due to, for example, a decrease in transcription efficiency, a decrease in translation efficiency, or a combination thereof. Decreased gene expression is the modification of expression regulatory sequences such as the promoter of a gene, the Shine-Dalgarno (SD) sequence (also called the ribosome binding site (RBS)), and the spacer region between the RBS and the start codon. Can be achieved by. When the expression regulatory sequence is modified, the expression regulatory sequence is modified by 1 or more bases, 2 or more bases, or 3 or more bases. A decrease in gene transcription efficiency can be achieved, for example, by replacing the promoter of the gene on the chromosome with a weaker promoter. A "weaker promoter" may mean a promoter in which transcription of a gene is weaker than the native wild-type promoter. Weaker promoters include, for example, inducible promoters. Weaker promoters include, for example, the P4 promoter (US2018-0334693A) and the P8 promoter (US2018-0334693A). That is, an inducible promoter can function as a weaker promoter under non-inducible conditions (eg, in the absence of an inducer). In addition, a part or all of the expression regulatory sequence may be deleted. In addition, a decrease in gene expression can also be achieved by, for example, manipulating factors involved in expression control. Examples of factors involved in expression control include small molecules (inducing substances, inhibitors, etc.), proteins (transcription factors, etc.), nucleic acids (siRNA, etc.) involved in transcription and translation control. The reduction in gene expression can also be achieved, for example, by introducing a mutation into the coding region of the gene that reduces the expression of the gene. For example, gene expression can be reduced by replacing codons in the coding region of a gene with synonymous codons that are used less frequently in the host. Also, for example, gene disruption as described herein can reduce gene expression itself.

Further, modifications that reduce the activity of the protein can be achieved, for example, by destroying the gene encoding the protein. "Gene disruption" may mean that the gene is modified so that it does not produce a normally functioning protein. "Do not produce a normally functioning protein" means that no protein is produced from the gene, or that the gene produces a protein whose per-molecule function (eg, activity or property) is reduced or eliminated. May also be included.

Gene destruction can be achieved, for example, by deleting (destroying) a gene on a chromosome. "Gene deletion" may mean deletion of part or all of the coding region of a gene. Furthermore, the entire gene may be deleted, including the sequences before and after the coding region of the gene on the chromosome. As long as reduced protein activity can be achieved, the regions to be deleted are the N-terminal region (ie, the region encoding the N-terminal side of the protein), the internal region, and the C-terminal region (ie, the region encoding the C-terminal side of the protein). It may be any region such as. In general, the longer the region to be deleted, the more reliable the gene can be inactivated. The region to be deleted is, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more of the total length of the coding region of the gene. Alternatively, it may be a region having a length of 95% or more. Further, it is preferable that the reading frames of the sequences before and after the region to be deleted do not match. A reading frame mismatch can result in a frameshift downstream of the region to be deleted.

Gene disruption can be, for example, by introducing an amino acid substitution (missense mutation) into the coding region of a gene on a chromosome, by introducing a stop codon (nonsense mutation), or by adding or deleting one or two bases (a missense mutation). It can also be achieved by introducing a frameshift mutation, etc. (Journal of Biological Chemistry 272: 8611-8617 (1997), Proceedings of the National Academy of Sciences, USA 95 5511-5515 (1998), Journal of Biological Chemistry 26 116 , 20833-20839 (1991)).

Also, gene disruption can be achieved, for example, by inserting another base sequence into the coding region of the gene on the chromosome. The insertion site may be any region of the gene, but the longer the base sequence to be inserted, the more reliable the gene can be inactivated. Further, it is preferable that the reading frames do not match in the arrangement before and after the insertion site. A frameshift can occur downstream of the insertion site due to a mismatch in the leading frames. The other base sequence is not particularly limited as long as it reduces or eliminates the activity of the encoded protein, and examples thereof include a marker gene such as an antibiotic resistance gene and a gene useful for the production of a target substance.

Gene disruption may be performed so that the amino acid sequence of the encoded protein is deleted (deleted). In other words, modifications that reduce the activity of a protein are specifically made by deleting the amino acid sequence (a part or all of the region of the amino acid sequence) of the protein, specifically, one of the amino acid sequences (one of the amino acid sequences). This can be achieved by modifying the gene to encode a protein lacking part or all regions. In addition, "deletion of amino acid sequence of protein" may mean deletion of a part or all region of amino acid sequence of protein. Further, "deletion of amino acid sequence of protein" may mean that the original amino acid sequence does not exist in the protein, and may include the case where the original amino acid sequence is changed to another amino acid sequence. That is, for example, a region changed to another amino acid sequence by a frame shift may be regarded as a deleted region. Deletion of the amino acid sequence typically shortens the overall length of the protein, but the overall length of the protein may not change or may be extended. For example, by deleting a part or all of the coding region of a gene, the region encoded by the deleted region can be deleted in the amino acid sequence of the encoded protein. Further, for example, by introducing a stop codon into the coding region of a gene, the region encoded by the region downstream from the introduction site can be deleted in the amino acid sequence of the encoded protein. Further, for example, a frameshift in a gene coding region can cause a region encoded by the frameshift site to be deleted. Regarding the position and length of the region to be deleted in the deletion of the amino acid sequence, the description of the position and length of the region to be deleted in the deletion of the gene can be applied mutatis mutandis.

Modifying a gene on a chromosome as described above is, for example, producing a disrupted gene modified so as not to produce a normally functioning protein, and transforming the host with a recombinant DNA containing the disrupted gene. This can be achieved by substituting the wild gene on the chromosome with the disruptive gene by causing homologous recombination between the disruptive gene and the wild gene on the chromosome. At that time, if the recombinant DNA contains a marker gene according to a trait such as auxotrophy of the host, it is easy to operate. Destructive genes include genes lacking part or all of the coding region of a gene, genes with missense mutations, genes with nonsense mutations, genes with frameshift mutations, transposons and marker genes. Examples include the inserted gene. Even if the protein encoded by the disrupted gene is produced, it has a three-dimensional structure different from that of the wild-type protein, and its function is reduced or lost. Gene disruption by gene substitution using such homologous recombination has already been established, and a method called "Red-driven integration" (Datsenko, K.A, and Wanner, B.L.Proc) . Natl. Acad. Sci. U S A. 97: 6640-6645 (2000)), Red driven integration method and λ phage-derived excision system (Cho, E. H., Gumport, R. I., Gardner, J A method using linear DNA such as a method combined with F. J. Bacteriol. 184: 5200-5203 (2002) (see WO2005 / 010175), or a method using a plasmid containing a temperature-sensitive replication origin, There are a method using a plasmid capable of conjugation and transmission, a method using a suicidal vector having no replication origin that functions in the host, and the like (US Patent No. 6303383, JP-A 05-007491).

Further, modifications that reduce the activity of the protein may be performed, for example, by mutation treatment. Mutation treatment includes X-ray irradiation, ultraviolet irradiation, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethylmethanesulfonate (EMS), and methylmethanesulfonate (MMS). ) And other treatments with a mutagen.

When a protein functions as a complex composed of a plurality of subunits, all of these subunits may be modified or only a part thereof may be modified as long as the activity of the protein is reduced as a result. That is, for example, all of the genes encoding these subunits may be destroyed, or only a part of them may be destroyed. In addition, when a plurality of isozymes are present in a protein, all the activities of those isozymes may be reduced or only a part of the activities may be reduced as long as the activity of the protein is reduced as a result. That is, for example, all of the genes encoding these isozymes may be destroyed, or only a part of them may be destroyed.

The above-mentioned method for reducing protein activity may be used alone or in any combination.

The decrease in protein activity can be confirmed by measuring the activity of the protein.

The decrease in protein activity can also be confirmed by confirming that the expression of the gene encoding the protein has decreased. The decrease in gene expression can be confirmed by confirming that the transcription amount of the gene has decreased and that the amount of protein expressed from the gene has decreased.

Confirmation that the transcription amount of the gene has decreased can be confirmed by comparing the amount of mRNA transcribed from the gene with that of the unmodified strain. Examples of methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR, microarray, RNA-seq, etc. (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The amount of mRNA (eg, number of molecules per cell) may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain.

Confirmation that the amount of protein has decreased can be confirmed by Western blotting using an antibody (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The amount of protein (eg, number of molecules per cell) may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain.

It can be confirmed that the gene has been disrupted by determining the base sequence of a part or all of the gene, the restriction enzyme map, the total length, etc., depending on the means used for the disruption.

The above-mentioned method for reducing the activity of a protein can be used to reduce the activity of an arbitrary protein or the expression of an arbitrary gene.

<2> Method for Producing Target Substance The target substance can be produced by utilizing a microorganism capable of producing the target substance. That is, the method for producing the target substance may be a method including a step of producing the target substance using a microorganism capable of producing the target substance. The process of producing a target substance by using a microorganism capable of producing the target substance is also referred to as a "target substance manufacturing process".

<2-1> Fermentation method The target substance can be produced, for example, by fermentation using a microorganism capable of producing the target substance. That is, one aspect of the method for producing the target substance may be a method for producing the target substance by fermentation using microorganisms. This aspect is also referred to as a "fermentation method". In addition, the process of producing a target substance by fermentation using microorganisms is also referred to as "fermentation process". That is, the target substance manufacturing step may include, for example, a fermentation step. Further, the target substance manufacturing step may be carried out by, for example, a fermentation step.

The fermentation process can be carried out by culturing microorganisms. Specifically, in the fermentation step, the target substance can be produced from a carbon source. That is, the fermentation step may be, for example, a step of culturing the microorganism in a medium (for example, a medium containing a carbon source) and producing and accumulating the target substance in the medium. That is, the fermentation method may be a method for producing a target substance, which comprises culturing a microorganism in a medium (for example, a medium containing a carbon source) and producing and accumulating the target substance in the medium. In other words, the fermentation step may be, for example, a step of producing a target substance from a carbon source using a microorganism.

The medium used is not particularly limited as long as microorganisms can grow and the target substance is produced. As the medium, for example, a normal medium used for culturing microorganisms such as bacteria and yeast can be used. The medium may contain a medium component such as a carbon source, a nitrogen source, a phosphoric acid source, a sulfur source, and various other organic components and inorganic components, if necessary. The type and concentration of the medium component may be appropriately set according to various conditions such as the type of microorganism used.

The carbon source is not particularly limited as long as the microorganism can assimilate and the target substance is produced. Specific examples of the carbon source include sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, waste sugar honey, starch hydrolyzate, and biomass hydrolyzate, acetic acid, citric acid, and succinic acid. , Organic acids such as gluconic acid, alcohols such as ethanol, glycerol and crude glycerol, and fatty acids. As the carbon source, a plant-derived raw material can be used in particular. Plants include, for example, corn, rice, wheat, soybeans, sugar cane, beets and cotton. Examples of plant-derived raw materials include organs such as roots, stems, trunks, branches, leaves, flowers, and seeds, plants containing them, and decomposition products of those plant organs. The form of use of the plant-derived raw material is not particularly limited, and for example, any form such as an unprocessed product, a squeezed juice, a crushed product, and a refined product can be used. Further, pentose such as xylose, hexose such as glucose, or a mixture thereof can be obtained from, for example, plant biomass and used. Specifically, these saccharides can be obtained by subjecting plant biomass to treatments such as steam treatment, concentrated acid hydrolysis, dilute acid hydrolysis, hydrolysis with enzymes such as cellulase, and alkali treatment. Since hemicellulose is generally more easily hydrolyzed than cellulose, hemicellulose in plant biomass may be hydrolyzed in advance to release pentose, and then cellulose may be hydrolyzed to produce pentose. Good. Further, xylose may be supplied by, for example, having a microorganism possess a conversion route from hexose such as glucose to xylose and converting it from hexose. As the carbon source, one type of carbon source may be used, or two or more types of carbon sources may be used in combination.

The concentration of the carbon source in the medium is not particularly limited as long as the microorganism can grow and the target substance is produced. The concentration of the carbon source in the medium may be, for example, as high as possible without inhibiting the production of the target substance. The initial concentration of carbon source in the medium may be, for example, 5-30 w / v% or 10-20 w / v%. In addition, a carbon source may be added to the medium as appropriate. For example, a carbon source may be added to the medium in response to the decrease or depletion of the carbon source as the fermentation progresses. The carbon source may be temporarily depleted as long as the target substance is finally produced, but the culture should be carried out so that the carbon source is not depleted or the carbon source is not depleted continuously. May be preferable.

Specific examples of the nitrogen source include ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen sources such as peptone, yeast extract, meat extract and soybean protein decomposition products, ammonia and urea. Ammonia gas or aqueous ammonia used for pH adjustment may be used as a nitrogen source. As the nitrogen source, one type of nitrogen source may be used, or two or more types of nitrogen sources may be used in combination.

Specific examples of the phosphoric acid source include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphoric acid polymers such as pyrophosphoric acid. As the phosphoric acid source, one kind of phosphoric acid source may be used, or two or more kinds of phosphoric acid sources may be used in combination.

Specific examples of the sulfur source include inorganic sulfur compounds such as sulfates, thiosulfates and sulfites, and sulfur-containing amino acids such as cysteine, cystine and glutathione. As the sulfur source, one type of sulfur source may be used, or two or more types of sulfur sources may be used in combination.

As various other organic components and inorganic components, specifically, for example, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium and calcium; vitamin B1, vitamin B2, vitamin B6 and nicotine. Vitamins such as acids, nicotinic acid amides and vitamin B12; amino acids; nucleic acids; organic components such as peptone, casamino acid, yeast extract and soybean proteolytic products containing these can be mentioned. As various other organic components and inorganic components, one type of component may be used, or two or more types of components may be used in combination.

Further, when an auxotrophic mutant strain that requires nutrients such as amino acids for growth is used, the medium may preferably contain such nutrients. The medium may also contain components used in the production of the substance of interest. Specific examples of such components include methyl group donors (eg, SAM) and precursors thereof (eg, methionine).

The culture conditions are not particularly limited as long as the microorganism can grow and the target substance is produced. Culturing can be carried out under normal conditions used for culturing microorganisms such as bacteria and yeast, for example. The culture conditions may be appropriately set according to various conditions such as the type of microorganism used.

Culturing can be performed using a liquid medium. At the time of culturing, for example, those in which microorganisms are cultured in a solid medium such as agar medium may be directly inoculated into a liquid medium, or those in which microorganisms are seed-cultured in a liquid medium are inoculated into a liquid medium for main culture. You may. That is, the culture may be divided into a seed culture and a main culture. In that case, the culture conditions of the seed culture and the main culture may or may not be the same. The target substance may be produced at least during the main culture period. The amount of microorganisms contained in the medium at the start of culturing is not particularly limited. For example, a seed culture solution having OD660 = 4 to 100 may be inoculated at the start of culture in an amount of 0.1% by mass to 100% by mass or 1% by mass to 50% by mass with respect to the medium for main culture.

Culturing can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof. The medium at the start of culturing is also referred to as "initial medium". Further, a medium added to a culture system (for example, a fermenter) in fed-batch culture or continuous culture is also referred to as "fed-batch medium". In addition, adding a fed-batch medium to a culture system in fed-batch culture or continuous culture is also referred to as "fed-batch". When the culture is divided into a seed culture and a main culture, the culture forms of the seed culture and the main culture may or may not be the same. For example, both seed culture and main culture may be carried out in batch culture, seed culture may be carried out in batch culture, and main culture may be carried out in fed-batch culture or continuous culture.

Various components such as a carbon source may be contained in the initial medium, the fed-batch medium, or both. That is, in the process of culturing, various components such as a carbon source may be added to the medium alone or in any combination. All of these components may be added once or multiple times, or may be added continuously. The type of component contained in the initial medium may or may not be the same as the type of component contained in the fed-batch medium. Further, the concentration of each component contained in the initial medium may or may not be the same as the concentration of each component contained in the fed-batch medium. In addition, two or more fed-batch media having different types and / or concentrations of the components contained may be used. For example, when multiple feedings are performed intermittently, the types and / or concentrations of the components contained in each fed-batch medium may or may not be the same.

Culturing may be carried out under aerobic conditions, for example. The “aerobic condition” may mean a condition in which the dissolved oxygen concentration in the medium is 0.33 ppm or more, or 1.5 ppm or more. Specifically, the oxygen concentration may be controlled to, for example, about 1 to 50% or about 5% with respect to the saturated oxygen concentration. The culture can be carried out, for example, by aeration culture or shaking culture. The pH of the medium may be, for example, pH 3-10 or pH 4.0-9.5. During culturing, the pH of the medium can be adjusted as needed. The pH of the medium is various alkaline or acidic substances such as ammonia gas, aqueous ammonia, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, etc. Can be adjusted using. The pH of the medium may be adjusted, in particular, with a substance other than ammonia, such as ammonia gas or aqueous ammonia. The culture temperature may be, for example, 20 to 45 ° C, or 25 ° C to 37 ° C. The culture period may be, for example, 10 hours to 120 hours. Culturing may continue, for example, until the carbon source in the medium is consumed or until the microbial activity is depleted.

By culturing the microorganism under such conditions, the target substance accumulates in the medium.

The production of the target substance can be confirmed by a known method used for detecting or identifying the compound. Such techniques include, for example, HPLC, UPLC, LC / MS, GC / MS, NMR. These methods can be used alone or in combination as appropriate. These techniques can also be used to determine the concentration of various components present in the medium.

The generated target substance can be recovered as appropriate. That is, the fermentation method may further include a step of recovering the target substance. This process is also referred to as a "recovery process". The recovery step may be a step of recovering the target substance from the culture solution, specifically, the medium. The target substance can be recovered by a known method used for separating and purifying the compound. Examples of such a method include an ion exchange resin method, a membrane treatment method, a precipitation method, an extraction method, a distillation method, and a crystallization method. Specifically, the target substance can be recovered by extraction with an organic solvent such as ethyl acetate, or by steam distillation. These methods can be used alone or in combination as appropriate.

If the target substance precipitates in the medium, it can be recovered by, for example, centrifugation or filtration. Further, the target substance precipitated in the medium may be isolated at the same time after the target substance dissolved in the medium is crystallized.

In addition to the target substance, the target substance to be recovered may contain other components such as microbial cells, medium components, water, and metabolic by-products of microorganisms. The purity of the recovered target substance is, for example, 30% (w / w) or more, 50% (w / w) or more, 70% (w / w) or more, 80% (w / w) or more, 90% ( It may be w / w) or more, or 95% (w / w) or more.

<2-2> Biological conversion method The target substance can also be produced, for example, by biological conversion using a microorganism capable of producing the target substance. That is, another aspect of the method for producing the target substance may be a method for producing the target substance by biological conversion using a microorganism. This aspect is also referred to as a "biological conversion method". In addition, the process of producing a target substance by biological conversion using microorganisms is also referred to as "biological conversion step". That is, the target substance manufacturing step may include, for example, a biological conversion step. Further, the target substance manufacturing step may be carried out by, for example, a biological conversion step.

Specifically, in the biological conversion step, the target substance can be produced from a precursor of the target substance. More specifically, in the biological conversion step, the target substance can be produced by converting a precursor of the target substance into the target substance using a microorganism. That is, the biological conversion step may be a step of converting a precursor of a target substance into the target substance using a microorganism.

The precursor of the target substance is also simply referred to as "precursor". Precursors include intermediates in the biosynthetic pathway of the target substance (eg, those mentioned in connection with the description of the target substance biosynthetic enzyme). The vanillin precursor is as described above. That is, examples of vanillin precursors include protocatechuic acid, vanillic acid, and protocatechuic aldehyde. Vanillin precursors include, in particular, vanillic acid. Vanillic acid precursors include protocatechuic acid. Protocatechuic acid precursors include protocatechuic acid. As the precursor, one kind of precursor may be used, or two or more kinds of precursors may be used in combination. When the precursor is a compound that can take the form of a salt, the precursor may be used as a free form, as a salt, or as a mixture thereof. That is, unless otherwise specified, "precursor" may mean a precursor of a free form, a salt thereof, or a mixture thereof. Examples of the salt include an ammonium salt, a sodium salt, and a potassium salt. Examples of the precursor salt include salts other than ammonium salts. As the precursor salt, one type of salt may be used, or two or more types of salts may be used in combination.

As the precursor, a commercially available product may be used, or one obtained by appropriately producing the precursor may be used. The method for producing the precursor is not particularly limited, and for example, a known method can be used. The precursor can be produced, for example, by a chemical synthesis method, an enzymatic method, a biological conversion method, a fermentation method, an extraction method, or a combination thereof. That is, for example, a precursor of a target substance utilizes an enzyme (also referred to as "precursor-producing enzyme") that catalyzes the conversion reaction of the target substance from the further precursor to the precursor of the target substance. Can be manufactured from the body. Further, for example, a precursor of a target substance can be produced from a carbon source or from such a further precursor by utilizing a microorganism capable of producing a precursor. The “microorganism capable of producing a precursor” may mean a microorganism capable of producing a precursor. "Precursor-producing microorganism" may specifically mean a microorganism capable of producing a precursor of a substance of interest from a carbon source and / or from such an additional precursor. .. For microorganisms capable of producing precursors, the description of microorganisms capable of producing target substances can be applied mutatis mutandis. Further, with respect to the production of a precursor using a microorganism capable of producing a precursor, the description regarding the production of a target substance using a microorganism capable of producing a target substance can be applied mutatis mutandis. For example, as a method for producing protocatechuic acid by an enzymatic method or a biological conversion method, a method of converting paracresol to protocatechuic acid using Pseudomonas putida KS-0180 (Japanese Patent Laid-Open No. 7-75589), NADH A method for converting parahydroxybenzoic acid to protocatechuic acid using a dependent parahydroxybenzoic acid hydroxylase (Japanese Patent Laid-Open No. 5-244941), a trait into which a gene involved in the reaction for producing protocatechuic acid from terephthalic acid has been introduced. A method for producing protocatechuic acid by culturing the transformant in a medium to which terephthalic acid has been added (Japanese Patent Laid-Open No. 2007-104942), having protocatechuic acid producing ability and decreased or deficient protocatechuic acid 5-position oxidase activity. Examples thereof include a method for producing protocatechuic acid from its precursor using the above-mentioned microorganism (Japanese Patent Laid-Open No. 2010-207094). Further, as a method for producing protocatechuic acid by the fermentation method, a method for producing protocatechuic acid using acetic acid as a carbon source using a bacterium belonging to the genus Brevibacterium (Japanese Patent Laid-Open No. 50-89592) and 3 -A method for producing protocatechuic acid from glucose as a carbon source using a bacterium belonging to the genus Escherichia or Klebsiella into which a gene encoding dihydroshikimic acid dehydrogenase has been introduced (US Pat. No. 5,272,073). Can be mentioned. Further, protocatechuic aldehyde can be produced by an enzymatic method using ACAR or a biological conversion method using a microorganism having ACAR, using protocatechuic acid as a precursor. In addition, vanillic acid can be produced by an enzymatic method using OMT or a biological conversion method using a microorganism having OMT using protocatechuic acid as a precursor (J. Am. CHm. Soc., 1998, Vol. 120). In addition, vanillic acid _{can be produced by a biological conversion method using Pseudomonas sp. AV 10} strain using ferulic acid as a precursor (J. App. Microbiol., 2013, Vol.116, p903-910). The precursor may be one produced as described above. For example, a vanillin precursor such as vanillic acid may be produced by utilizing a microorganism capable of producing a vanillin precursor. The biotransformation method may further include the step of producing a precursor. Specifically, the biological conversion method may include a step of producing a precursor as described above. For example, when the target substance is vanillin, the biological conversion method may particularly include a step of producing a vanillin precursor such as vanillic acid by utilizing a microorganism capable of producing a vanillin precursor. The produced precursor can be used as it is or after being appropriately subjected to treatments such as concentration, dilution, drying, dissolution, fractionation, sterilization, extraction and purification, and then used in a biological conversion method. That is, as the precursor, for example, a refined product purified to a desired degree may be used, or a material containing the precursor may be used. For example, vanillin precursors such as vanillic acid may be used, in particular, in the form of materials containing vanillin precursors. The material containing the precursor is not particularly limited as long as the microorganism can utilize the precursor. Specific examples of the material containing the precursor include a culture solution or reaction solution containing the precursor, a supernatant separated from the culture solution or reaction solution, a concentrate thereof (for example, a concentrate), and a diluted product (for example, a concentrate). For example, a treated product such as a diluted solution) and a dried product can be mentioned.

In one embodiment, the biological conversion step can be carried out by culturing a microorganism capable of producing a target substance. This aspect is also referred to as "the first aspect of the biological conversion method". That is, the biological conversion step may be, for example, a step of culturing the microorganism in a medium containing a precursor of the target substance and converting the precursor into the target substance. Specifically, the biological conversion step may be a step of culturing the microorganism in a medium containing a precursor of the target substance and producing and accumulating the target substance in the medium.

The medium used is not particularly limited as long as it contains a precursor of the target substance, microorganisms can grow, and the target substance is produced. The culture conditions are not particularly limited as long as the microorganism can grow and the target substance is produced. Regarding the culture in the first aspect of the biological conversion method, the description of the culture in the fermentation method (for example, the description of the medium and the culture conditions) is described in the same embodiment except that the medium contains a precursor of the target substance. Can be applied mutatis mutandis.

The precursor may be contained in the medium for the entire period of the culture, or may be contained in the medium only for a part of the period of the culture. That is, "culturing a microorganism in a medium containing a precursor" does not require that the precursor be contained in the medium during the entire period of culturing. For example, the precursor may or may not be contained in the medium from the beginning of the culture. If the precursor is not contained in the medium at the start of the culture, the precursor is added to the medium after the start of the culture. The timing of addition can be appropriately set according to various conditions such as culture time. For example, the precursor may be added to the medium after the microorganism has fully grown. In either case, the precursor may be added to the medium as appropriate. For example, the precursor may be added to the medium according to the decrease or depletion of the precursor with the production of the target substance. The means for adding the precursor to the medium is not particularly limited. For example, the precursor can be added to the medium by feeding the fed-batch medium containing the precursor into the medium. Further, for example, by co-culturing a microorganism having a target substance-producing ability and a microorganism having a precursor-producing ability, a microorganism having a precursor-producing ability is made to produce a precursor in a medium, and thus the precursor is used as a medium. It can also be added. The "component" in the case of "adding a certain component to the medium" may also include those produced or regenerated in the medium. These adding means may be used alone or in combination as appropriate. The concentration of the precursor in the medium is not particularly limited as long as the microorganism can use the precursor as a raw material for the target substance. The precursor concentration in the medium is, for example, 0.1 g / L or more, 1 g / L or more, 2 g / L or more, 5 g / L or more, 10 g / L or more, or It may be 15 g / L or more, 200 g / L or less, 100 g / L or less, 50 g / L or less, or 20 g / L or less, or a combination thereof. The precursor may or may not be contained in the medium at the concentrations illustrated above for the entire duration of the culture. The precursor may be contained in the medium at the above-exemplified concentration at the start of culturing, or may be added to the medium at the above-exemplified concentration after the start of culturing. When the culture is divided into a seed culture and a main culture, the target substance may be produced at least during the main culture period. Therefore, the precursor may be contained in the medium at least during the main culture, that is, during the entire main culture or a part of the main culture. That is, the precursor may or may not be contained in the medium during the seed culture period. In such a case, the description about the culture (for example, "culture period (culture period)" or "culture start") can be read as that for the main culture.

In another embodiment, the biological conversion step can be carried out by utilizing the bacterial cells of a microorganism capable of producing the target substance. This aspect is also referred to as "the second aspect of the biological conversion method". That is, the biological conversion step may be, for example, a step of converting a precursor of the target substance in the reaction solution into the target substance by utilizing the bacterial cells of the microorganism. Specifically, the biological conversion step may be a step of causing the bacterial cells of the microorganism to act on the precursor of the target substance in the reaction solution to generate and accumulate the target substance in the reaction solution. The biological conversion step carried out using such cells is also referred to as "conversion reaction".

Microbial cells are obtained by culturing microorganisms. The culture method for obtaining the bacterial cells is not particularly limited as long as the microorganism can grow. At the time of culturing to obtain the cells, the precursor may or may not be contained in the medium. Further, at the time of culturing for obtaining bacterial cells, the target substance may or may not be produced in a medium. Regarding the culture for obtaining the cells in the second aspect of the biological conversion method, the description of the culture in the fermentation method (for example, the description of the medium and the culture conditions) can be applied mutatis mutandis.

The bacterial cells may be used in the conversion reaction as they are contained in the culture medium (specifically, the medium), or may be recovered from the culture solution (specifically, the medium) and used in the conversion reaction. In addition, the bacterial cells may be appropriately treated and then used in the conversion reaction. That is, examples of the bacterial cells include a culture solution of microorganisms, bacterial cells recovered from the culture solution, and processed products thereof. That is, the bacterial cells may be used, for example, in the form of a culture solution of microorganisms, bacterial cells recovered from the culture solution, processed products thereof, or a combination thereof. In other words, examples of the bacterial cells include bacterial cells contained in a culture solution of a microorganism, bacterial cells recovered from the culture solution, and bacterial cells contained in a processed product thereof. That is, the bacterial cells may be used, for example, in the form of bacterial cells contained in a culture solution of a microorganism, bacterial cells recovered from the culture solution, bacterial cells contained in a processed product thereof, or a combination thereof. .. Examples of the treated product include those obtained by subjecting the bacterial cells (for example, the bacterial cells contained in the culture or the bacterial cells recovered from the culture) to the treatment. The cells of these embodiments may be used alone or in combination as appropriate.

The method for recovering the bacterial cells from the culture solution is not particularly limited, and for example, a known method can be used. Such techniques include, for example, natural sedimentation, centrifugation and filtration. Alternatively, a flocculant may be used. These methods may be used alone or in combination as appropriate. The recovered cells can be appropriately washed using a suitable medium. In addition, the recovered cells can be appropriately resuspended using an appropriate medium. Examples of the medium that can be used for washing or suspension include an aqueous medium (aqueous solvent) such as water or an aqueous buffer solution.

Examples of the treatment of the bacterial cells include dilution, concentration, immobilization treatment on a carrier such as acrylamide and carrageenan, freeze-thaw treatment, and treatment for increasing the permeability of the membrane. The permeability of the membrane can be enhanced by utilizing, for example, a surfactant or an organic solvent. These processes may be used alone or in combination as appropriate.

The bacterial cells used in the conversion reaction are not particularly limited as long as they have the ability to produce the target substance. It is preferable that the bacterial cells maintain their metabolic activity. "Maintaining metabolic activity" may mean that the cells have the ability to assimilate the carbon source to produce or regenerate the substance necessary for the production of the target substance. Examples of such substances include electron donors such as ATP, NADH and NADP, and methyl group donors such as SAM. The cells may or may not have the ability to grow.

The conversion reaction can be carried out in an appropriate reaction solution. Specifically, the conversion reaction can be carried out by allowing the cells and the precursor to coexist in an appropriate reaction solution. The conversion reaction may be carried out in a batch manner or in a columnar manner. In the case of the batch type, for example, the conversion reaction can be carried out by mixing the bacterial cells and the precursor of the microorganism in the reaction solution in the reaction vessel. The conversion reaction may be carried out by allowing it to stand, or by stirring or shaking. In the case of the column type, the conversion reaction can be carried out, for example, by passing a reaction solution containing a precursor through a column packed with immobilized cells. Examples of the reaction solution include an aqueous medium (aqueous solvent) such as water and an aqueous buffer solution.

The reaction solution may contain components other than the precursor, if necessary, in addition to the precursor. Ingredients other than precursors include electron donors such as ATP, NADH and NADPH, methyl group donors such as SAM, metal ions, buffers, surfactants, organic solvents, carbon sources, phosphoric acid sources, and various other media. Ingredients are mentioned. That is, for example, a medium containing a precursor may be used as the reaction solution. That is, for the reaction solution in the second aspect of the biological conversion method, the description of the medium in the first aspect of the biological conversion method can be applied mutatis mutandis. The type and concentration of the components contained in the reaction solution may be appropriately set according to various conditions such as the type of precursor to be used and the mode of the bacterial cells to be used.

The conditions of the conversion reaction (dissolved oxygen concentration, pH of the reaction solution, reaction temperature, reaction time, concentration of various components, etc.) are not particularly limited as long as the target substance is produced. The conversion reaction can be carried out under the usual conditions used for substance conversion using microbial cells such as quiescent cells. The conditions of the conversion reaction may be appropriately set according to various conditions such as the type of microorganism used. The conversion reaction may be carried out under aerobic conditions, for example. The “aerobic condition” may mean a condition in which the dissolved oxygen concentration in the reaction solution is 0.33 ppm or more, or 1.5 ppm or more. Specifically, the oxygen concentration may be controlled to, for example, about 1 to 50% or about 5% with respect to the saturated oxygen concentration. The pH of the reaction solution may be, for example, usually 6.0 to 10.0, or 6.5 to 9.0. The reaction temperature may be, for example, usually 15-50 ° C, 15-45 ° C, or 20-40 ° C. The reaction time may be, for example, 5 minutes to 200 hours. In the case of the column method, the flow rate of the reaction solution may be, for example, a rate such that the reaction time is within the range of the reaction time exemplified above. Further, the conversion reaction can also be carried out under culture conditions such as normal conditions used for culturing microorganisms such as bacteria and yeast. In the conversion reaction, the cells may or may not grow. That is, with respect to the conversion reaction in the second aspect of the biological conversion method, the description of the culture in the first aspect of the biological conversion method is applied mutatis mutandis, except that the cells may or may not grow in the same aspect. it can. In such a case, the culture conditions for obtaining the bacterial cells and the conditions for the conversion reaction may or may not be the same. The concentration of the precursor in the reaction solution is, for example, 0.1 g / L or more, 1 g / L or more, 2 g / L or more, 5 g / L or more, 10 g / L or more in terms of the weight of the free form. , Or 15 g / L or more, 200 g / L or less, 100 g / L or less, 50 g / L or less, or 20 g / L or less, or a combination thereof. Good. The concentration of the cells in the reaction solution may be, for example, 1 or more, 300 or less, or a combination thereof in terms of optical density (OD) at 600 nm.

In the process of conversion reaction, bacterial cells, precursors, and other components may be added to the reaction solution alone or in any combination. For example, the precursor may be added to the reaction solution according to the decrease or depletion of the precursor due to the production of the target substance. These components may be added once or multiple times, or may be added continuously.

The means for adding various components such as precursors to the reaction solution is not particularly limited. All of these components can be added to the reaction solution by adding them directly to the reaction solution. Further, for example, by co-culturing a microorganism having a target substance-producing ability and a microorganism having a precursor-producing ability, a microorganism having a precursor-producing ability can generate a precursor in a reaction solution, thereby reacting the precursor. It can also be added to the liquid. Further, for example, components such as ATP, electron donor, and methyl group donor may all be produced or regenerated in the reaction solution, or may be produced or regenerated in the microbial cell, and are heterologous cells. It may be generated or regenerated by interconjugation. For example, when the metabolic activity is maintained in the microbial cell, a carbon source can be used to generate or regenerate components such as ATP, an electron donor, and a methyl group donor in the microbial cell. For example, specifically, the microorganism may have an enhanced ability to produce or regenerate SAM, and the SAM produced or regenerated by the microorganism may be used in the conversion reaction. The generation or regeneration of SAM can be further enhanced in combination with other techniques for generating or reproducing SAM. As a method for producing or regenerating ATP, for example, a method of supplying ATP from a carbon source using a bacterium belonging to the genus Corynebacterium (Hori, Het al., Appl. Microbiol. Biotechnol. 48 (6): 693-698 (1997)), ATP regeneration method using yeast cells and glucose (Yamamoto, S et al., Biosci. Biotechnol. Biochem. 69 (4): 784-789 (2005)), phospho Method of regenerating ATP using enolpyrbic acid and pyruvate kinase (C. Aug'e and Ch. Gautheron, Tetrahedron Lett. 29: 789-790 (1988)), using polyphosphate and polyphosphate kinase Examples include a method of regenerating ATP (Murata, K et al., Agric. Biol. Chem. 52 (6): 1471-1477 (1988)). The "component" in the case of "adding a certain component to the reaction solution" may also include those produced or regenerated in the reaction solution.

Further, the reaction conditions may be uniform from the start to the end of the conversion reaction, or may change in the process of the conversion reaction. The fact that "the reaction conditions change in the process of the conversion reaction" is not limited to the temporal changes of the reaction conditions, but may also include the spatial changes of the reaction conditions. "The reaction conditions change spatially" means that, for example, when the conversion reaction is carried out by a column type, the reaction conditions such as the reaction temperature and the cell density are different depending on the position on the flow path. You can do it.

By carrying out the biological conversion step in this way, a culture solution (specifically, a medium) or a reaction solution containing the target substance can be obtained. Confirmation that the target substance has been produced and recovery of the target substance can both be carried out in the same manner as in the fermentation method described above. That is, the biological conversion method may further include a recovery step (for example, a step of recovering the target substance from the culture solution (specifically, the medium) or the reaction solution). In addition to the target substance, the target substance to be recovered may contain other components such as microbial cells, medium components, reaction solution components, water, and metabolic by-products of microorganisms. The purity of the recovered target substance is, for example, 30% (w / w) or more, 50% (w / w) or more, 70% (w / w) or more, 80% (w / w) or more, 90% ( It may be w / w) or more, or 95% (w / w) or more.

<2-3> Reduction of Ammonia Concentration In the method described in the present specification (for example, a method for producing vanillin), the production of vanillin is carried out under the condition that the ammonia concentration is reduced. That is, in the method described in the present specification, the vanillin production step is carried out under the condition that the ammonia concentration is reduced.

Vanillin production is improved by producing vanillin under conditions where the ammonia concentration is reduced. Specifically, by producing vanillin under the condition that the ammonia concentration is reduced, the vanillin production is improved as compared with the case where the vanillin is produced under the condition that the ammonia concentration is not reduced. The "condition for reducing the ammonia concentration" is also referred to as the "ammonia reduction condition". "Conditions that do not reduce the ammonia concentration" are also referred to as "control conditions". Vanillin production may be improved, for example, by improving the growth of microorganisms used in the vanillin production process, by improving the amount of vanillin produced per cell of the microorganisms used in the vanillin production process, or by a combination thereof.

"Ammonia concentration" means the concentration of ammonia in the medium or reaction solution (that is, in the case of vanillin production, the medium or reaction solution in which vanillin production is carried out). "Ammonia" is a general term for _{ammonia molecules (NH 3} ) and ammonium ions (NH ₄ ^+). That is, the "ammonia concentration" specifically refers to the concentration of _{ammonia molecules (NH 3} ) in the medium or reaction solution (that is, in the case of the production of vanillin, the medium or reaction solution in which the production of vanillin is carried out). It means the total concentration of ammonium ions _(NH ^{4 +).}

The degree of reduction of ammonia concentration is not particularly limited as long as vanillin production is improved. The ammonia concentration during vanillin production (that is, the ammonia concentration under the ammonia reduction conditions) is appropriately determined according to various conditions such as the type of microorganism used in the vanillin production process, the length of the vanillin production process, and the desired vanillin production amount. Can be set.

"Vanillin production or vanillin production process is carried out under conditions of reduced ammonia concentration" or "vanillin production or vanillin production process is carried out in a medium or reaction solution with reduced ammonia concentration" means that vanillin is produced. It may mean that the ammonia concentration at the time of is in a predetermined range. During the production of vanillin, ammonia may or may not be contained in the medium or reaction solution (ie, in the case of vanillin production, the medium or reaction solution in which the production of vanillin is carried out).

The ammonia concentration during the production of vanillin (that is, the ammonia concentration under the ammonia reduction condition) is, for example, 0 mM or more, 0.1 mM or more, 0.3 mM or more, 0.5 mM or more, 0.7 mM or more, 1 mM or more, 1. 5 mM or more, 2 mM or more, 2.5 mM or more, 3 mM or more, 3.5 mM or more, 4 mM or more, 4.5 mM or more, 5 mM or more, 5.5 mM or more, 6 mM or more, 6.5 mM or more, 7 mM or more, 7.5 mM or more , 8 mM or more, 8.5 mM or more, 9 mM or more, 9.5 mM or more, or 10 mM or more, 700 mM or less, 600 mM or less, 500 mM or less, 400 mM or less, 300 mM or less, 200 mM or less, 100 mM or less, 50 mM or less, It may be 20 mM or less, or 10 mM or less, and may be a consistent combination thereof. Specifically, the ammonia concentration during the production of vanillin (that is, the ammonia concentration under the ammonia reduction condition) may be, for example, 1 to 700 mM, 1 to 400 mM, or 1 to 100 mM.

Ammonia concentration may be reduced during the entire period of the vanillin production process, or may be reduced only during a part of the vanillin production process. That is, for example, when the production of vanillin is carried out by fermentation using microorganisms, the ammonia concentration may be reduced during the entire fermentation process, or may be reduced only during a part of the fermentation process. Good. Further, for example, when the production of vanillin is carried out by biological conversion using microorganisms, the ammonia concentration may be reduced during the entire period of the biological conversion step, and is reduced only during a part of the biological conversion step. May be. That is, "the production of vanillin or the vanillin production process is carried out under the condition that the ammonia concentration is reduced" or "the production of vanillin or the vanillin production process is carried out in the medium or the reaction solution in which the ammonia concentration is reduced" means that the ammonia is produced. It suffices if the concentration is reduced during some of the vanillin production steps (eg, fermentation or bioconversion) and the ammonia concentration is reduced during the entire vanillin production process (eg, fermentation or bioconversion). It doesn't need to be.

The ammonia concentration may be reduced to the above-exemplified concentration during the entire period of the vanillin production process, or may be reduced to the above-exemplified concentration only during a part of the vanillin production process. That is, for example, when the production of vanillin is carried out by fermentation using microorganisms, the ammonia concentration may be reduced to the above-exemplified concentration during the entire fermentation process, and only during a part of the fermentation process. It may be reduced to the above-exemplified concentration. Further, for example, when the production of vanillin is carried out by biological conversion using microorganisms, the ammonia concentration may be reduced to the above-exemplified concentration during the entire period of the biological conversion step, and is a part of the biological conversion step. The concentration may be reduced to the above-exemplified concentration only during the period. That is, "the production of vanillin or the process of producing vanillin is carried out under the condition that the ammonia concentration is reduced to a certain concentration" or "the production of vanillin or the step of producing vanillin is carried out in a medium or a reaction solution having a certain ammonia concentration". It is sufficient that the ammonia concentration is within the range of the concentration during a part of the vanillin production step (for example, fermentation step or biological conversion step), and the ammonia concentration is in the vanillin production step (for example, fermentation step or biological conversion step). ) Does not need to be within the range of the concentration for the entire period.

The "partial period" is not particularly limited as long as vanillin production improves. The "partial period" can be appropriately set according to various conditions such as the type of microorganism used in the vanillin production process, the length of the vanillin production process, and the desired vanillin production amount. "Partial period" is, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more of the entire period of the vanillin production process (for example, fermentation process or biological conversion process). , 97% or more, or 99% or more. Further, the "partial period" is, for example, 10 hours or more, 15 hours or more, 20 hours or more, 30 hours or more, 40 hours or more, 50 in the vanillin production process (for example, fermentation process or biological conversion process). The period may be hours or longer, 70 hours or longer, 100 hours or longer, or 150 hours or longer.

Further, the ammonia concentration during the production of vanillin (that is, the ammonia concentration under the ammonia reduction conditions) may be reduced to the above-exemplified concentration as an average value over the entire period of the vanillin production process, for example. That is, "the production of vanillin or the process of producing vanillin is carried out under the condition that the ammonia concentration is reduced to a certain concentration" or "the production of vanillin or the process of producing vanillin is carried out in a medium or a reaction solution having a certain concentration of ammonia". May mean that the average value of the ammonia concentration throughout the entire vanillin production process is within that concentration range. The "average value of ammonia concentration over the entire period of the vanillin production process" is not particularly limited as long as it reflects the fluctuation of the ammonia concentration over the entire period of the vanillin production process. For example, the entire period of the vanillin production process is not limited. It may mean the average value of ammonia concentration measured every 60 minutes, every 30 minutes, every 20 minutes, or every 10 minutes throughout.

The means for reducing the ammonia concentration is not particularly limited. Ammonia concentration is, for example, reducing the initial concentration of ammonia (ie, the concentration of ammonia in the medium or reaction solution at the beginning of the vanillin production process), the flow of ammonia (ie, the medium or reaction after the start of the vanillin production process). The amount of ammonia supplied to the liquid) may be reduced, or a combination thereof may be used.

The initial concentration of ammonia in the production of vanillin (that is, the initial concentration of ammonia under ammonia reduction conditions) is, for example, 0 mM or more, 0.1 mM or more, 0.3 mM or more, 0.5 mM or more, 0.7 mM or more, 1 mM. 1.5 mM or more, 2 mM or more, 2.5 mM or more, 3 mM or more, 3.5 mM or more, 4 mM or more, 4.5 mM or more, 5 mM or more, 5.5 mM or more, 6 mM or more, 6.5 mM or more, 7 mM or more, It may be 7.5 mM or more, 8 mM or more, 8.5 mM or more, 9 mM or more, 9.5 mM or more, or 10 mM or more, 700 mM or less, 600 mM or less, 500 mM or less, 400 mM or less, 300 mM or less, 200 mM or less, 100 mM or less. , 50 mM or less, 20 mM or less, or 10 mM or less, and may be a consistent combination thereof. Specifically, the initial concentration of ammonia in the production of vanillin (that is, the initial concentration of ammonia under the ammonia reduction condition) may be, for example, 1 to 700 mM, 1 to 400 mM, or 1 to 100 mM.

The amount of ammonia used in the production of vanillin (that is, the amount of ammonia used under the ammonia reduction conditions) is, for example, 300% or less, 250% or less, 200% or less, 150 as a molar ratio to the amount of vanillin precursor used. % Or less, 100% or less, 70% or less, 50% or less, 30% or less, 20% or less, 15% or less, 10% or less, 5% or less, 2% or less, or 1% or less.

The amount of ammonia used in the production of vanillin (that is, the amount of ammonia used under ammonia reduction conditions) is, for example, 300% or less, 250% or less, 200% or less, 150% as the molar ratio to the amount of carbon source used. Below, it may be 100% or less, 70% or less, 50% or less, 30% or less, 20% or less, 15% or less, 10% or less, 5% or less, 2% or less, or 1% or less.

The amount of ammonia used in the production of vanillin (that is, the amount of ammonia used under the ammonia reduction conditions) is, for example, 300% or less, 250% or less, 200 as the molar ratio to the total amount of vanillin precursor and carbon source used. % Or less, 150% or less, 100% or less, 70% or less, 50% or less, 30% or less, 20% or less, 15% or less, 10% or less, 5% or less, 2% or less, or 1% or less. Good.

“Amount of a certain component used” means the initial content of the component (that is, the content of the component in the medium or reaction solution at the start of the vanillin production process) and the fed-batch amount of the component (that is, vanillin production). It means the total amount of the component) supplied to the medium or reaction solution after the start of the process. The "fed-batch amount of a certain component" may mean, in particular, the fed-batch amount of the component in the period from the start of the vanillin production process to the sufficient production of vanillin. "Sufficient vanillin is produced" means, for example, that the vanillin concentration in the medium or reaction solution is 90% or more, 95% or more, 97% or more of the vanillin concentration in the medium or reaction solution at the end of the vanillin production process. Or it may mean reaching a concentration of 99% or higher. Further, "sufficiently produced vanillin" means that, for example, the vanillin production amount is 90% or more, 95% or more, 97% or more, or 99% or more of the vanillin production amount from the start to the end of the vanillin production process. May mean reaching the quantity of.

The ammonia concentration may be reduced, in particular, by reducing the amount of ammonia used with the use of the vanillin precursor. For example, when the vanillin precursor is used in the form of a composition that may contain ammonia, the use of the vanillin precursor (specifically, the composition) by reducing the amount of ammonia contained in the composition. The amount of ammonia used can be reduced. That is, by reducing the amount of ammonia contained in such a composition, the introduction of ammonia into the medium or reaction solution due to the use of the vanillin precursor (specifically, the use of the composition) is reduced. Therefore, the amount of ammonia used can be reduced. Such compositions include vanillin precursors produced using microorganisms. Specific examples of such a composition include a material containing a vanillin precursor produced by utilizing a microorganism. The materials containing the vanillin precursor are as described above. Specific examples of the material containing the vanillin precursor include a culture solution or reaction solution containing the vanillin precursor, a supernatant separated from the culture solution or reaction solution, a concentrate thereof (for example, a concentrate), and a dilution thereof. Examples thereof include processed products such as products (for example, diluents) and dried products. The content of the vanillin precursor in such a composition is, for example, 1% (w / w) or more, 3% (w / w) or more, 5% (w / w) or more, 7% (w / w). It may be 10% (w / w) or more, 15% (w / w) or more, or 20% (w / w) or more, 95% (w / w) or less, 90% (w / w) or more. ) Or less, 80% (w / w) or less, 70% (w / w) or less, 60% (w / w) or less, 50% (w / w) or less, 40% (w / w) or less, 30% It may be (w / w) or less, 20% (w / w) or less, or 10% (w / w) or less, and may be a consistent combination thereof. The content of the vanillin precursor in such a composition is, for example, 1% (w / w) or more and 3% (w / w) or more as the amount of the vanillin precursor with respect to the total amount of the composition excluding water. Even if it is 5% (w / w) or more, 7% (w / w) or more, 10% (w / w) or more, 15% (w / w) or more, or 20% (w / w) or more. Well, 95% (w / w) or less, 90% (w / w) or less, 80% (w / w) or less, 70% (w / w) or less, 60% (w / w) or less, 50% ( It may be w / w) or less, 40% (w / w) or less, 30% (w / w) or less, 20% (w / w) or less, or 10% (w / w) or less. It may be a consistent combination.

The vanillin precursor may be produced in a form in which the ammonia content is reduced. The vanillin precursor may be specifically prepared in the form of a composition with a reduced ammonia content. As for the method for producing the vanillin precursor in a form in which the content of ammonia is reduced, the description for the method described in the present specification (for example, the method for producing vanillin) can be applied mutatis mutandis. That is, the production of the vanillin precursor may be carried out under the condition that the ammonia concentration is reduced. "Ammonia concentration" means the concentration of ammonia in the medium or reaction solution (that is, in the case of production of vanillin precursor, the medium or reaction solution in which the production of vanillin precursor is carried out). By producing the vanillin precursor under the condition that the ammonia concentration is reduced, the vanillin precursor can be produced in a form in which the ammonia content is reduced, and thus the amount of ammonia used accompanying the use of the vanillin precursor can be reduced. Can be reduced. In other words, the ammonia concentration in the production of vanillin may be reduced at least by producing the vanillin precursor under the condition that the ammonia concentration is reduced. For example, the amount of ammonia that can be used as a nitrogen source in the production of vanillin precursors may be reduced. Also, for example, the amount of ammonia that can be used for pH adjustment in the production of vanillin precursors may be reduced. pH adjustments include neutralization of protocatechuic acid and / or vanillic acid. That is, specifically, for example, when producing vanillic acid from protocatechuic acid as a raw material, the amount of ammonia that can be used for neutralizing protocatechuic acid and / or vanillic acid may be reduced. In particular, when producing vanillic acid from protocatechuic acid as a raw material, the amount of ammonia that can be used to neutralize protocatechuic acid may be reduced. Specifically, for example, when protocatechuic acid is produced as a raw material, the amount of ammonia that can be used for neutralizing protocatechuic acid may be reduced. The amount of ammonia that can be used for pH adjustment, such as neutralization of protocatechuic acid and / or vanillic acid, is, for example, a pH adjustment substance other than ammonia, for example, sodium hydroxide or potassium hydroxide. It can be reduced by using a substance for adjusting the pH of the above-exemplified medium other than ammonia, such as. In other words, the amount of ammonia that can be used for pH adjustment, such as neutralization of protocatechuic acid and / or vanillic acid, is, for example, a portion or all of the ammonia that can be used for pH adjustment with other pH adjusting substances (eg, for example. It can be reduced by substituting with a substance for adjusting the pH of the above-exemplified medium other than ammonia, such as sodium hydroxide and potassium hydroxide. Examples of ammonia that can be used for pH adjustment include ammonia gas and ammonia water.

The content of ammonia in the composition as described above (for example, a material containing a vanillin precursor) is not particularly limited as long as a reduction in the ammonia concentration during the production of vanillin can be achieved. The content of ammonia in the composition as described above (for example, a material containing a vanillin precursor) is, for example, 300% or less, 250% or less, 200 as a molar ratio to the content of the vanillin precursor in the composition. % Or less, 150% or less, 100% or less, 70% or less, 50% or less, 30% or less, 20% or less, 15% or less, 10% or less, 5% or less, 2% or less, or 1% or less. Good.

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples.

Example: Vanillin production from vanillic acid under ammonia-restricted conditions (1) Analytical conditions Vanillin was quantified by HPLC analysis. The analysis conditions are as shown below.
Mobile phase A: 0.1% Phosphate mobile phase B: Acetonitrile Flow rate: 1.0 ml / min
Column temperature: 40 ° C
Detection: UV 210 nm
Column: Cadenza CD-C18, 4.6 × 150 mm, 3 μm (Imtakt)
Gradient: 0-2 min (B: 5%), 2-15 min (B: 5-25%), 15-16 min (B: 25-60%), 16-20 min (B: 60%) 20.1 -25 min (B: 5%)

Ammonia was quantified by BF-7 (Oji measuring instrument). The analysis conditions are as shown below.
Mobile phase: BF buffer pH 7.0 (Oji measuring instrument) with α-ketoglutaric acid added to a final concentration of 1 mM and NADH added to a final concentration of 1 mM Flow rate: 1 mL / min
Time: 90 sec
Temperature: 37 ℃
Electrodes: Glutamic acid electrode, ammonia electrode (Oji measuring instrument)
Remarks: Since glutamic acid is also measured with the ammonia electrode, the value obtained by subtracting the glutamic acid concentration after measuring the glutamic acid concentration with the glutamic acid electrode was calculated as the ammonia concentration.

OD was measured by UV-1800 (Shimadzu Corporation). The analysis conditions are as shown below.
Wavelength: 600 nm
Cell length: 1 cm

(2) Construction of vanillin-producing strain The plasmid pVK9 :: Ptuf * -Ge_ACAR-entD (Example <3> of WO2018 / 079705) was applied to Corynebacterium glutamicum FKFC14 (C. glutamicum 2256ΔvanABKΔNCgl0324ΔNCgl0313ΔNCgl2709; Example <2> of WO2018 / 079705). Introduced by the electroporation method. FKFC14 is a deficient strain of vanillate demethylase and vanillate uptake gene (vanABK) and alcohol dehydrogenase gene (NCgl0324, NCgl0313, NCgl2709) constructed with C. glutamicum 2256 (ATCC 13869) as the parent strain. The plasmid pVK9 :: Ptuf * -Ge_ACAR-entD is a co-expression plasmid of aromatic carboxylic acid reductase (ACAR) from Gordonia effusa and phosphorpantetheinyl transferase (PPT) from E. coli. The cells CM-Dex SGFC plates (glucose 2.5 g / L, fructose 2.5 g / L, polypeptone 10 g / L, Yeast extract 10 g / L, KH 2 PO 4 1 g / L, MgSO 4 · 7H 2 O 0.4 _{g / L, FeSO 4 · 7H} 2 O 0.01 g / L, MnSO 4 · 7H 2 O 0.01 g / L, succinate disodium _{· 6H 2 O 2 g / L} , sodium gluconate 4 g / L, urea 3 g / L, biotin 10 μg / L, soybean hydrochloric acid hydrolyzate (as total nitrogen) 1.2 g / L, adjusted to pH 7.5 with NaOH, cultivated at 31.5 ° C with canamycin 25 mg / L, Agar 15 g / L) did. The acquired colonies were purified on a CM-Dex SGFC plate and named FKFC14 / pVK9 :: Ptuf * -Ge_ACAR-entD. The obtained strain was cultured in 4 mL of CM-Dex SGFC medium at 31.5 ° C. for about 16 hours, 0.9 mL of the culture solution was mixed with 0.6 mL of 50% glycerol, and stored as a glycerol stock at -80 ° C.

(3) Vanillin production from vanillic acid FKFC14 / pVK9 :: Ptuf * -Ge_ACAR-entD CM-Dex-Km (glucose 5 g / L, polypeptone 10 g / L, yeast extract 10 g / L, KH ₂ PO _{4 1 g / L, MgSO 4 ·} 7H 2 O 0.4 g / L, urea _{3 g / L, FeSO 4 ·} 7H 2 O 0.01 g / L, MnSO 4 · 7H 2 O 0.01 g / L, biotin 10 [mu] g / L, Soybean hydrochloric acid hydrolyzate (as total nitrogen) 1.2 g / L, adjusted to pH 7.5 with KOH, cultivated on a canamycin 25 mg / L, Agar 20 g / L) plate at 20 ° C. for 3 days. CM-Dex-Glc10 medium containing 25 mg / L of canamycin (glucose 10 g / L, polypeptone 10 g / L, yeast extract 10 g / L, KH ₂ PO ₄ 1 g / L, DDL ₄・7H ₂ O 0.4 g / L, urea _{3 g / L, FeSO 4 ·} 7H 2 O 0.01 g / L, MnSO 4 · 7H 2 O 0.01 g / L, biotin 10 [mu] g / L, hydrochloric acid hydrolyzate of soybean (total As nitrogen) 1.2 g / L, adjusted to pH 7.5 with KOH) Inoculated into 50 mL and cultivated with shaking at 31.5 ° C for 18 hours using a Sakaguchi flask. 25 mL of the obtained culture solution was centrifuged, the supernatant was removed, and the mixture was suspended in 0.85% NaCl solution so that OD 600 nm was 15, to obtain a bacterial cell suspension. 1 mL of the cell suspension 9 mL of vanillin production medium (final concentration: vanillic acid _{30 g / L, NH 4 Cl} 0-1000 mM, (NH 4) 2 SO 4 0-500 mM, glucose 60 g / L, polypeptone 10 g / L, Yeast extract 10 g / L, KH ₂ PO ₄ 1 g / L, DDL ₄・ 7H ₂ O 0.4 g / L, FeSO ₄・ 7H ₂ O 0.01 g / L, MnSO ₄・ 7H ₂ O 0.01 g / L, biotin 10 μg / L, adjusted to pH 7.4 with KOH, inoculated into canamycin 25 mg / L), and cultured at 30 ° C. for 48 hours. 8 μL of the culture solution at the start of the culture was mixed with 1 mL of ultrapure water, and the ammonia concentration was measured. In addition, 10 μL of the culture solution at 48 hours of culture and 1 mL of the culture stop solution (1% phosphoric acid, 50% ethanol) were mixed, and the filter filtrate was subjected to HPLC analysis. In addition, 20 μL of the culture solution at 48 hours of culture was mixed with 1 mL of ultrapure water, and the OD was measured. Table 1 shows the amounts of NH ₄ Cl and (NH ₄ ) ₂ SO ₄ added, the ammonia concentration at the start of culture, and the amount of OD and vanillin produced at 48 hours of culture.

As the amount of ammonium salt added decreased, the growth and vanillin production improved. In particular, when the NH ₄ ⁺ addition concentration was 50-600 mM, an increase in vanillin production per OD was observed as compared with the case where the _{NH 4} ^{+ addition concentration was 800-1000 mM.} Since there was no difference in the tendency of the results depending on the type of ammonium salt, it is considered that the improvement in growth and the improvement in vanillin production were due to the reduction in ammonia concentration.

According to the present invention, the production of vanillin by microorganisms can be improved, and vanillin can be efficiently produced.

<Explanation of sequence listing>
SEQ ID NO: 1: Nucleotide sequence of ACAR gene of Gordonia effusa SEQ ID NO: 2: Nucleotide sequence of ACAR protein of Gordonia effusa SEQ ID NO: 3: Nucleotide sequence of entD gene of Escherichia coli MG1655 SEQ ID NO: 4: Nucleotide sequence of EntD protein of Escherichia coli MG1655 SEQ ID NO: 5: Nucleotide sequence of vanK gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 6: Nucleotide sequence of VanK protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 7: Nucleotide sequence of vanA gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 8: Nucleotide sequence of VanA protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 9: Nucleotide sequence of vanB gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 10: VanB protein of Corynebacterium glutamicum 2256 (ATCC 13869) Amino acid sequence SEQ ID NO: 11: Nucleotide sequence of NCgl0324 gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 12: Nucleotide sequence of NCgl0324 protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 13: NCgl0313 gene of Corynebacterium glutamicum 2256 (ATCC 13869) Nucleotide sequence of Corynebacterium glutamicum 2256 (ATCC 13869) NCgl0313 Protein amino acid sequence SEQ ID NO: 15: Nucleotide sequence of NCgl2709 gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 16: NCgl2709 of Corynebacterium glutamicum 2256 (ATCC 13869) Protein amino acid sequence

Claims (22)

1. It ’s a method of making vanillin.
   Including the production of vanillin using microorganisms capable of producing vanillin,
   A method in which the production is carried out under conditions where the ammonia concentration is reduced.
2. The method according to claim 1, wherein the ammonia concentration at the time of the production is 700 mM or less.
3. The method according to claim 1 or 2, wherein the ammonia concentration at the time of the production is 400 mM or less.
4. The method according to any one of claims 1 to 3, wherein the ammonia concentration at the time of the production is 100 mM or less.
5. The method according to any one of claims 1 to 4, wherein the production comprises converting a vanillin precursor into vanillin using the microorganism.
6. The method of claim 5, wherein the conversion comprises culturing the microorganism in a medium containing the precursor and producing and accumulating vanillin in the medium.
7. The method according to claim 5, wherein the conversion comprises causing the bacterial cells of the microorganism to act on the precursor in the reaction solution and producing and accumulating vanillin in the reaction solution.
8. The method according to claim 7, wherein the cells are used in the form of a culture solution of the microorganism, cells recovered from the culture solution, processed products thereof, or a combination thereof.
9. The method according to any one of claims 5 to 8, wherein the precursor is vanillic acid.
10. The method according to any one of claims 5 to 9, wherein the precursor is produced by utilizing a microorganism having an ability to produce the precursor.
11. The method according to any one of claims 5 to 10, further comprising producing the precursor using a microorganism capable of producing the precursor before producing the vanillin.
12. The method according to claim 10 or 11, wherein the production of the precursor is carried out under conditions where the ammonia concentration is reduced.
13. The precursor is used in the form of a material containing the precursor.
    Any one of claims 10 to 12, wherein the material is a culture solution or reaction solution containing the precursor, a supernatant separated from the culture solution or reaction solution, a processed product thereof, or a combination thereof. The method described in.
14. The method according to claim 13, wherein the content of ammonia in the material is 300% or less as a molar ratio to the content of the precursor in the material.
15. The production of vanillin comprises culturing a microorganism capable of producing the vanillin in a medium containing a carbon source, and producing and accumulating vanillin in the medium, according to any one of claims 1 to 4. The method described.
16. The method according to any one of claims 1 to 15, further comprising recovering vanillin.
17. The method according to any one of claims 1 to 16, wherein the microorganism capable of producing vanillin is a bacterium or yeast.
18. The method according to any one of claims 1 to 17, wherein the microorganism capable of producing vanillin is a coryneform bacterium or a bacterium of Enterobacteriaceae.
19. The method according to any one of claims 1 to 18, wherein the microorganism capable of producing vanillin is a bacterium belonging to the genus Corynebacterium or a bacterium belonging to the genus Escherichia.
20. The method according to any one of claims 1 to 19, wherein the microorganism capable of producing the vanillin is Corynebacterium glutamicum or Escherichia coli.
21. Any of claims 1-20, wherein the microorganism capable of producing vanillin has been modified to increase the activity of aromatic carboxylic acid reductase and / or phosphopantetinyltransferase as compared to the unmodified strain. The method according to item 1.
22. The invention according to any one of claims 1 to 21, wherein the microorganism capable of producing vanillin is modified so that the activity of vanillate demethylase and / or alcohol dehydrogenase is reduced as compared with the unmodified strain. the method of.