JP2023001394A - Method for producing vanillin - Google Patents

Abstract

To provide methods for producing vanillin.SOLUTION: Vanillin is produced by using a microorganism capable of producing vanillin under conditions that the concentration of ammonia is reduced.SELECTED DRAWING: None

Description

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

Vanillin is a main component of vanilla scent, and is used as a flavoring agent by blending it in foods, beverages, perfumes, and the like. Vanillin is mainly produced by extraction from natural products or by chemical synthesis.

Attempts have also been made to produce vanillin by biotechnological techniques. For example, vanillin can be produced from vanillin precursors such as vanillic acid using microorganisms such as Corynebacterium glutamicum.

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

Martin TeschBernhard J. et al. Ammonia assimilation in Corynebacterium glutamicum and a glutamate dehydrogenase-deficient mutant. Biotechnology Letters. October 1998, Volume 20, Issue 10, pp 953-957 Mueller T. et al. Ammonium toxicity in bacteria. Curr Microbiol. 2006 May;52(5):400-6. Kazumi ARAKI et al. Fermentative Production of L-Proline with Auxotrophs of Corynebacterium glutamicum. Agricultural and Biological Chemistry. 1975; 39(6) p.1193-1200

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

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

That is, the present invention can be exemplified as follows.
[1]
A method for producing vanillin, comprising:
including producing vanillin using a microorganism capable of producing vanillin,
The method, wherein the production is carried out under conditions with reduced ammonia concentration.
[2]
The above method, wherein the concentration of ammonia during the production is 700 mM or less.
[3]
The above method, wherein the ammonia concentration during the production is 400 mM or less.
[4]
The above method, wherein the concentration of ammonia during the production is 100 mM or less.
[5]
The above method, wherein the producing comprises converting a vanillin precursor into the vanillin using the microorganism.
[6]
The above method, wherein the conversion comprises culturing the microorganism in a medium containing the precursor to produce and accumulate vanillin in the medium.
[7]
The above-described method, wherein the conversion includes allowing cells of the microorganism to act on the precursor in the reaction solution to produce and accumulate vanillin in the reaction solution.
[8]
The above method, wherein the cells are used in the form of a culture solution of the microorganism, cells collected from the culture solution, processed products thereof, or a combination thereof.
[9]
The above method, wherein the precursor is vanillic acid.
[10]
The above method, wherein the precursor is produced using a microorganism capable of producing the precursor.
[11]
The above method, further comprising, prior to producing the vanillin, producing the precursor using a microorganism capable of producing the precursor.
[12]
The above method, wherein the preparation of the precursor is carried out under conditions with a reduced concentration of ammonia.
[13]
The precursor is used in the form of a material containing the precursor,
The above method, 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.
[14]
The above 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 above method, wherein the production of vanillin comprises culturing a microorganism capable of producing vanillin in a medium containing a carbon source to produce and accumulate vanillin in the medium.
[16]
The above method, further comprising recovering vanillin.
[17]
The above method, wherein the microorganism capable of producing vanillin is bacteria or yeast.
[18]
The above method, wherein the microorganism capable of producing vanillin is a coryneform bacterium or a bacterium of the Enterobacteriaceae family.
[19]
The above method, 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 microorganism capable of producing vanillin is Corynebacterium glutamicum (
Corynebacterium glutamicum) or Escherichia coli.
[21]
The above method, wherein the microorganism capable of producing vanillin has been modified such that the activity of aromatic carboxylic acid reductase and/or phosphopantetheinyltransferase is increased compared to an unmodified strain.
[22]
The above method, wherein the microorganism capable of producing vanillin has been modified such that vanillate demethylase and/or alcohol dehydrogenase activity is reduced compared to an unmodified strain.

ADVANTAGE OF THE INVENTION According to this invention, production of vanillin by microorganisms can be improved and vanillin can be manufactured efficiently.

The method described herein is a method for producing vanillin, comprising producing vanillin using a microorganism having the ability to produce vanillin, wherein the production is carried out under conditions in which the concentration of ammonia is reduced. , is the method. Vanillin can be specifically produced from a carbon source and/or a vanillin precursor, for example, using a microorganism capable of producing vanillin.

A vanillin precursor can be produced, for example, using a microorganism capable of producing a vanillin precursor. Vanillin precursors can be produced from a carbon source and/or further precursors of vanillin precursors, in particular utilizing, for example, microorganisms having the ability to produce vanillin precursors.

Vanillin or vanillin precursors are also referred to as "target substances".

A method for producing a target substance (ie, vanillin or vanillin precursor) using a microorganism capable of producing the target substance (ie, vanillin or vanillin precursor) will be described below. Unless otherwise specified, the following description of the method for producing the target substance is the description of the method described herein (i.e., the method for producing vanillin) and the method described herein (i.e., the method for producing vanillin). It also serves as an explanation of the method for producing the vanillin precursor used in the above.

<1> Microorganisms Microorganisms used to produce the target substance are those that have the ability to produce the target substance. The ability to produce the target substance is also called "target substance production capacity".

<1-1> Microorganisms Capable of Producing the Target Substance “Microorganisms capable of producing the target substance” may mean microorganisms capable of producing the target substance.

The term "microorganism capable of producing a target substance" may mean a microorganism capable of producing a target substance by fermentation when the same microorganism is used in a fermentation method. That is, "a microorganism capable of producing a desired substance" may mean, for example, a microorganism capable of producing a desired substance from a carbon source. Specifically, for example, when cultured in a medium (e.g., a medium containing a carbon source), the "microorganism capable of producing the target substance" produces the target substance and accumulates it in the medium to the extent that it can be recovered. may mean a microorganism capable of

The term "microorganism capable of producing a target substance" may mean a microorganism capable of producing a target substance by biotransformation when the same microorganism is used in a biotransformation method. That is, "a 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, for example, when cultured in a medium (for example, a medium containing a precursor of the target substance), the "microorganism having the ability to produce the target substance" can produce and recover the target substance. may refer to microorganisms that are capable of accumulating in Further, the term "microorganism capable of producing the target substance" specifically means, for example, when reacting with a precursor of the target substance in the reaction solution, the target substance is produced and collected in the reaction solution to the extent that it can be recovered. may mean a microorganism capable of accumulating in

Microorganisms having the ability to produce the target substance are, for example, 0.01 g/L or more, 0.05 g/L or more, or 0.09
It may be possible to accumulate the target substance in the medium or the reaction solution in an amount of g/L or more.

By "target substance" is meant vanillin or a vanillin precursor. By "vanillin precursor" may be meant a compound that can be converted to vanillin by microbial biotransformation. Vanillin precursors include protocatechuic acid, vanillic acid and protocatechualdehyde. Vanillin precursors include in particular vanillic acid. A microorganism may be capable of producing only one target substance, or may be capable of producing two or more target substances. Moreover, the microorganism may be able to produce the target substance from one type of target substance precursor, or may be capable of producing the target substance from two or more types of target substance precursors.

When the target substance is a compound that can take a salt form, 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 the target substance in free form, a salt thereof, or a mixture thereof. Salts include, for example, ammonium salts, sodium salts, and potassium salts. Salts of the target substance include in particular salts other than ammonium salts. As the salt of the target substance, one salt may be used, or two or more salts may be used in combination.

There are no particular restrictions on the microorganisms used to produce the target substance or the microorganisms used as parent strains for constructing it. Microorganisms include bacteria and yeast.

Examples of 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 Photolabdus. Bacteria belonging to genera such as Photorhabdus, Providencia, Salmonella, and Morganella. 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 family Enterobacteriaceae can be used.

Bacteria belonging to the genus Escherichia include, but are not limited to, bacteria classified into the genus Escherichia according to classifications known to microbiological experts. As Escherichia bacteria, for example, Neidhardt et al. (Backmann, BJ 1996. Derivations and Genotypes
of some mutant derivatives of Escherichia coli K-12, p. 2460-2488. Table 1. In FD Neidhardt (ed.), Escherichia coli and Salmonella Cellular and Molecular Biology
Physics/Second Edition, American Society for Microbiology Press, Washington, D.C.
). Escherichia bacteria include, for example, Escherichia coli. As Escherichia coli, specifically, for example, Escherichia coli K-12 strain such as W3110 strain (ATCC 27325) and MG1655 strain (ATCC 47076); Escherichia coli K5 strain (ATCC 23506); BL21 (DE3) strain Escherichia coli B strains such as E. coli; and derivatives thereof.

Bacteria belonging to the genus Enterobacter include, but are not limited to, bacteria classified into the genus Enterobacter according to the classification known to experts in microbiology. Examples of bacteria belonging to the genus Enterobacter include Enterobacter agglomerans and Enterobacter aerogenes. Specific examples of Enterobacter agglomerans include, for example, Enterobacter agglomerans ATCC12287 strain. Specific examples of Enterobacter aerogenes include, for example, 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. In addition, some Enterobacter agglomerans are classified as Pantoea agglomerans.

Bacteria belonging to the genus Pantoea include, but are not limited to, bacteria classified into the genus Pantoea according to classifications known to experts in microbiology. Examples of Pantoea bacteria include Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. As Pantoea ananatis, specifically, 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-11091
), SC17(0) strain (VKPM B-9246), and SC17sucA strain (FERM BP-8646). Some Enterobacter and Erwinia bacteria have been reclassified into the Pantoea genus (Int. J. Syst. Bacteriol., 39, 337-345 (1989); Int. J. Syst. Bacteriol. , 43, 162-173 (1993)). For example, some species of Enterobacter agglomerans have recently been reclassified into Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii, etc. based on 16S rRNA nucleotide sequence analysis (Int. J. Syst. Bacteriol ., 39, 337-345 (1989)). Bacteria of the genus Pantoea may also include bacteria reclassified into the genus Pantoea in this way.

Erwinia bacteria include Erwinia amylovora and Erwinia carotovora. Klebsiella bacteria include Klebsiella planticola.

Coryneform bacteria include bacteria belonging to genera such as the genus Corynebacterium, the genus Brevibacterium, and the genus Microbacterium.

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

Specific examples of coryneform bacteria 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 ammonophilum ATCC 15354

Bacteria of the genus Corynebacterium, which were formerly classified as the genus Brevibacterium, are now integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1991)). included. In addition, Corynebacterium stathionis includes bacteria that were previously classified as Corynebacterium ammoniagenes, but were reclassified as Corynebacterium stathionis based on 16S rRNA nucleotide sequence analysis (Int. J. Syst. Evol. Microbiol., 60, 874-879 (2010)).

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

These strains are listed, for example, in the American Type Culture Collection, address PO
Box 1549, Manassas, VA 20108, United States of America or atcc.org). In other words, a registration number corresponding to each strain is assigned, and this registration number can be used to receive distribution (see atcc.org/). The accession number corresponding to each strain can be found in the catalog of the American Type Culture Collection. In addition, these strains can be obtained, for example, from the depository to which each strain has been deposited.

Microorganisms may be those that inherently have the ability to produce the target substance, or those that have been modified to have the ability to produce the target substance. A microorganism capable of producing a target substance can be obtained, for example, by imparting the target substance-producing capability to the above-described microorganism, or by enhancing the target substance-producing capability of the above-described microorganism.

The method of imparting or enhancing the target substance-producing ability is not particularly limited. For example, a known method can be used as a method for imparting or enhancing the target substance-producing ability. Methods for imparting or enhancing the ability to produce a target substance include, for example, WO2018/079687, WO2018/079686, WO2018/079685, WO2018/079684, WO2018/079683, WO2017/073701, WO2018/079705, US2018-0334693A, and US2019- 0161776A.

Specific examples of methods for imparting or enhancing the target substance-producing ability are given below. Modifications for imparting or enhancing the ability to produce the target substance as exemplified below may be used alone or in combination as appropriate.

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

In addition, by increasing the activity of the target substance biosynthetic enzyme, the ability of the microorganism to produce the target substance can be improved. That is, a method for imparting or enhancing the target substance-producing ability includes a method for increasing the activity of the target substance biosynthetic enzyme. That is, the microorganism may be modified to increase the activity of the target substance biosynthetic enzyme. The activity of one target biosynthetic enzyme may be increased, or the activity of two or more target biosynthetic enzymes may be increased. Techniques for increasing the activity of proteins (such as enzymes) are described herein. Activity of a protein (such as an enzyme) can be increased, for example, by increasing 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, target substance biosynthetic enzymes include, for example, 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 shikimic acid 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 (3-dehydroquinate synthase), and 3-dehydroquinate dehydratase. 3-dehydroshikimate 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 protocatechualdehyde can be converted to vanillin by the action of ACAR or OMT, respectively. That is, target substance biosynthetic enzymes specifically include, for example, DAHP synthase, 3-dehydroquinate synthase, 3-dehydroquinate dehydratase, DHSD, OMT, and ACAR.

"3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthase (3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase; DAHP synthase)" is a combination of D-erythrose 4-phosphate and phosphoenol It may mean a protein having activity to catalyze a reaction converting pyruvate into D-arabino-hepturonate-7-phosphate (DAHP) and phosphate (EC 2.5.1.54 etc.). The same activity is also referred to as "DAHP synthase activity". A gene encoding DAHP synthase is also referred to as "DAHP synthase gene". DAHP synthase includes AroF, AroG and AroH proteins encoded by aroF, aroG and aroH genes, respectively. Among these, the AroG protein may function as the major DAHP synthase. DAHP synthases such as AroF, AroG and AroH proteins include those derived from various organisms such as Enterobacteriaceae bacteria and coryneform bacteria. As DAHP synthase, specifically, E. coli
AroF, AroG and AroH proteins of E. coli such as K-12 MG1655 strain.

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

"3-dehydroquinate dehydratase" may mean a protein having the activity of catalyzing the reaction of dehydrating 3-dehydroquinic acid to form 3-dehydroshikimic acid (EC 4.2.1.10 etc). The same activity is also called "3-dehydroquinate dehydratase activity". A gene encoding 3-dehydroquinate dehydratase is also referred to as a "3-dehydroquinate dehydratase gene". As a 3-dehydroquinate dehydratase, aroD
The gene-encoded AroD protein is included. Examples of 3-dehydroquinate dehydratase such as AroD protein include those derived from various organisms such as Enterobacteriaceae bacteria and coryneform bacteria. Specific examples of 3-dehydroquinate dehydratase include E. coli AroD protein such as E. coli K-12 MG1655 strain.

"3-dehydroshikimate dehydratase (DHSD)" may mean a protein having the activity of catalyzing the reaction of dehydrating 3-dehydroshikimate to form protocatechuic acid (EC 4.2. 1.118 etc.). The same activity is also referred to as "DHSD activity". A gene encoding DHSD is also referred to as a "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 such as Bacillus thuringiensis BMB171 strain, Neurospora crassa, and Podospora pauciseta.

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

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

OMTs of various organisms, such as Homo sapiens (Hs) OMT (GenBank Accession
No. NP_000745, NP_009294), Arabidopsis thaliana OMT (GenBank Accession No. NP_200227, NP_009294), Fragaria x ananassa OMT (GenBank Accession No. AAF28353), and other mammals, plants and microorganisms exemplified in WO2013/022881. Various OMTs can be mentioned. Four transcriptional variants and two OMT isoforms are known in the Homo sapiens OMT gene. OMTs further include OMTs of Bacteroidetes bacteria (ie, bacteria belonging to the Bacteroidetes phylum) (WO2018/079683). The phylum Bacteroidetes includes bacteria belonging to the genus Niastella, Terrimonas, Chitinophaga, etc. (International Journal of Systematic and Evolutionary Microbiology (2007), 57, 1828-1833). Niastella koreensis is mentioned as Niastella bacteria. OMTs further include mutant OMTs 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 protocatechualdehyde. It may mean a protein having catalytic activity (eg EC 1.2.99.6). This activity is also referred to as "ACAR activity". A gene encoding ACAR is also referred to as an "ACAR gene". ACAR may use both vanillic acid and protocatechuic acid as substrates, but is not limited to this. That is, ACAR may have the required substrate specificity depending on the type of biosynthetic pathway through which the target substance is produced. For example, when the method for producing a target substance includes conversion from vanillic acid to vanillin, ACAR using at least 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 protocatechualdehyde, ACAR using at least protocatechuic acid as a substrate can be used. Electron donors include NADH and NADPH.
Electron donors include in particular NADPH.

Examples of ACAR include those 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. ACARs also include ACARs of other bacteria of the genus Nocardia, such as Nocardia brasiliensis and Nocardia vulneris. Nocardia brasiliensis includes the Nocardia brasiliensis ATCC 700358 strain. ACAR further includes ACAR of bacteria of the genus Gordonia such as Gordonia effusa, ACAR of bacteria of the genus Novosphingobium such as Novosphingobium malaysiense, and ACAR of algae of the genus Coccomyxa such as Coccomyxa 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 phosphopantetheinylated (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 phosphopantetheinylation of proteins (also referred to as "phosphopantetheinylation enzyme"). That is, methods for imparting or enhancing the target substance-producing ability include methods for increasing the activity of phosphopantetheinylase. That is, the microorganism may be modified to have increased activity of a phosphopantetheinylase. Phosphopantetheinyltransferases include phosphopantetheinyl transferase (PPT).

A "phosphopantetheinyl transferase (PPT)" may refer to a protein having the activity of catalyzing the phosphopantetheinylation of ACAR in the presence of a phosphopantetheinyl group donor. This activity is also referred to as "PPT activity". A gene encoding PPT is also referred to as a "PPT gene". Phosphopantetheinyl group donors include coenzyme A (CoA). As a PPT, EntD encoded by the entD gene
A protein is mentioned. PPTs such as EntD proteins include those from various organisms. Specific examples of PPT include the EntD protein of E. coli 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 same gene is shown in SEQ ID NO: 4, respectively. As PPT, specifically, PPT of Nocardia brasiliensis, PPT of Nocardia farcinica IFM10152 (J. Biol. Chem. 2007, Vol. 282, No. 1, pp.478-485), PPT of C. glutamicum ( App Env.
Microbiol. 2009, Vol.75, No.9, pp.2765-2774). 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 ability to produce the target substance, a substance other than the target substance (for example, a substance generated as an intermediate during the production of the target substance, or a substance used as a precursor of the target substance) and methods for increasing the activity of the uptake system of That is, microorganisms may be modified to increase the activity of such uptake systems. A "substance uptake system" may mean a protein that has the function of taking up a substance from outside 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". Such uptake systems include the vanillic acid uptake system and the protocatechuic acid uptake system. A vanillic acid uptake system includes the VanK protein encoded by the vanK gene (MT Chaudhry, et al., Microbiology, 2007. 153:857-865). The protocatechuic acid uptake system includes the PcaK protein encoded by the pcaK gene (MT Chaudhry, et al., Microbiology, 2007. 153:857-865). Vanillic acid uptake systems such as VanK protein and vanillic acid uptake systems such as PcaK protein include those of various organisms such as coryneform bacteria. Specific examples of vanillic acid uptake systems include VanK proteins of C. glutamicum such as C. glutamicum ATCC 13032 and ATCC 13869 strains. The protocatechuic acid uptake system specifically includes the PcaK protein (NCgl1031 protein) of C. glutamicum 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, respectively.

Methods for imparting or enhancing the target substance-producing ability include methods for reducing the activity of enzymes involved in the by-production of substances other than the target substance. Substances other than such target substances are also referred to as "by-products". Such enzymes are also referred to as "by-product producing enzymes". By-product-producing enzymes include, for example, enzymes involved in assimilation of target substances, and enzymes that catalyze reactions that branch from the biosynthetic pathway of target substances to produce substances other than target substances. Techniques for reducing the activity of proteins (such as enzymes) are described herein. The activity of a protein (enzyme, etc.) can be reduced, for example, by disrupting the gene encoding the protein. For example, it has been reported that in coryneform bacteria, vanillin is metabolized and utilized in the order of vanillin→vanillic acid→protocatechuic acid (Current Microbiology, 2005, Vol.51, p59-65). That is, by-product-producing enzymes specifically include enzymes that catalyze the conversion of vanillin to protocatechuic acid and enzymes that catalyze further metabolism of protocatechuic acid. Such enzymes include vanillate demethylase, protocatechuate 3,4-dioxygenase, and the reaction products of protocatechuate 3,4-dioxygenase, succinyl-CoA and acetyl-CoA. (Appl. Microbiol. Biotechnol., 2012, Vol.95, p77-89). 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, alcohol dehydrogenase (ADH) is specifically included as a byproduct-producing enzyme. Also, 3-dehydroshikimic acid, an intermediate in the vanillin biosynthetic pathway, can be converted to shikimic acid by the action of shikimate dehydrogenase. That is, as a byproduct-producing enzyme,
Specifically, shikimate dehydrogenase is also included.

By "vanillate demethylase" may be meant a protein that has the activity of catalyzing the reaction that demethylates vanillate to form protocatechuic acid. The same activity is also called "vanillate demethylase activity". A gene encoding vanillate demethylase is also referred to as "vanillate demethylase gene". Vanillate demethylases 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 vanillate demethylase activity is reduced, for example, both vanAB genes may be disrupted, or only one of them may be disrupted. Examples of vanillate demethylases such as VanAB protein include those derived from various organisms such as Enterobacteriaceae bacteria and coryneform bacteria. Specific examples of vanillate demethylase include C. glutam such as C. glutamicum ATCC 13032 strain and ATCC 13869 strain.
icum VanAB protein. The nucleotide sequence of the vanAB gene of C. glutamicum ATCC 13869 strain is shown in SEQ ID NOS: 7 and 9, and the amino acid sequence of the VanAB protein encoded by the same gene is 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 disrupted (eg, deleted) in order to reduce vanillate demethylase activity. In that case, the vanK gene may be introduced again into the microorganism. For example, when using extracellular vanillic acid, if the vanABK operons are disrupted (for example, deleted), it is preferable to reintroduce the vanK gene.

"protocatechuate 3,4-dioxygenase"
By may be meant a protein having the activity of catalyzing the reaction that oxidizes protocatechuic acid to form β-carboxyl cis,cis-muconic acid. The same activity is also called "protocatechuate 3,4-dioxygenase activity". A gene encoding protocatechuate 3,4-dioxygenase is also referred to as "protocatechuate 3,4-dioxygenase gene". Protocatechuate 3,4-dioxygenase includes PcaGH protein encoded by 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 protocatechuate 3,4-dioxygenase activity, for example, both pcaGH genes may be disrupted, or only one of them may be disrupted. Protocatechuate 3,4-dioxygenases such as PcaGH protein include those of various organisms such as Enterobacteriaceae bacteria and coryneform bacteria. Specific examples of the protocatechuate 3,4-dioxygenase include the PcaGH protein of C. glutamicum such as C. glutamicum ATCC 13032 strain and ATCC 13869 strain.

"Alcohol dehydrogenase (ADH)" may mean a protein that has the activity of catalyzing a reaction that reduces an aldehyde to form 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 referred to as "ADH activity". A gene encoding ADH is also referred to as an "ADH gene". Electron donors include NADH and NADPH.

ADHs include, in particular, those having activity to catalyze the reaction of reducing vanillin to form vanillyl alcohol in the presence of an electron donor. This activity is also particularly referred to as "vanillyl alcohol dehydrogenase activity". 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 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. Such ADHs include Enterobacteriaceae
and various organisms such as 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, for example, in coryneform bacteria such as C. glutamicum. Specifically, as ADH, E. coli
Examples include the YqhD protein of E. coli, such as K-12 MG1655 strain. Further, 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, the NCgl0313 gene, and the 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 these genes are shown in SEQ ID NOS: 12, 14, and 16. each 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 NCgl0324 protein, NCgl2709 protein, and NCgl0313 protein may be reduced. In particular, at least the activity of the NCgl0324 protein may be reduced.

"Shikimate dehydrogenase" may mean a protein having the activity of catalyzing the reaction of reducing 3-dehydroshikimate to form shikimate in the presence of an electron donor (EC 1.1. 1.25 etc.). This activity is also called "shikimate dehydrogenase activity". A gene encoding shikimate dehydrogenase is also referred to as a "shikimate dehydrogenase gene". Electron donors include NADH and NADPH. A shikimate dehydrogenase includes an AroE protein encoded by the aroE gene. Examples of shikimate dehydrogenase such as AroE protein include those of various organisms such as Enterobacteriaceae bacteria and coryneform bacteria. Specific examples of shikimate dehydrogenase include E. coli AroE protein such as E. coli K-12 MG1655 strain.

Methods for imparting or enhancing the ability to produce a target substance include methods for increasing the activity of L-cysteine biosynthetic enzymes (WO2018/079687).

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

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

Expression of the L-cysteine biosynthetic gene can be increased, for example, by altering (eg, increasing or decreasing) the activity of the expression regulator of the gene. Thus, L-cysteine biosynthetic gene expression can be increased, for example, by increasing the activity of positive expression regulators (eg, activators) of the gene. Expression of L-cysteine biosynthetic genes can also be increased, for example, by reducing the activity of negative expression regulators (eg, repressors) of the same genes. Such regulatory factors are also referred to as "regulatory proteins". A gene encoding such a regulatory factor is also referred to as a "regulatory gene".

Such activators include the CysR and SsuR proteins encoded by the cysR and ssuR genes, respectively. Increased activity of the CysR protein may result in increased expression of one or more of the cysIXHDNYZ, fpr2, and ssuR genes. Also, increased activity of the SsuR protein may increase expression of genes involved in the utilization of organosulfur compounds. Such activators include those of various organisms such as bacteria of the Enterobacteriaceae family and coryneform bacteria. Specific examples of such activators include C. glutamicum ATCC 13032 and ATCC 13869 strains.
glutamicum CysR and SsuR proteins. The activity of one or both CysR and SsuR proteins may be increased. For example, at least the activity of the CysR protein may be reduced. The activity of such activators can be increased, for example, by increasing expression of the gene encoding the activator.

Such repressors include the McbR protein encoded by the mcbR gene. Decreased activity of the McbR protein may increase expression of one or more of the cysR and ssuR genes, and thereby increase expression of one or more of the cysIXHDNYZ and fpr2 genes. can. 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 and ATCC 13869 strains. The activity of such repressors can be reduced, for example, by reducing 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 can be 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, "the activity of the L-cysteine biosynthetic enzyme is increased" means, for example, that the expression of one or more of the cysIXHDNYZ gene, fpr2 gene, cysR gene, and ssuR gene is increased. 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, the fpr2 gene, and the ssuR gene may be increased by increasing expression of the cysR gene.

Methods for imparting or enhancing the ability to produce a target substance include methods for reducing the activity of the NCgl2048 protein (WO2018/079686).

"NCgl2048 protein" may refer to a protein encoded by the NCgl2048 gene. NCgl2048 proteins include those of various organisms such as Enterobacteriaceae bacteria and coryneform bacteria. Specific examples of NCgl2048 proteins include NCgl2048 proteins of C. glutamicum such as C. glutamicum ATCC 13869 strain. Regarding conservative variants, "original function of NCgl2048 protein" refers to C. glutamicum ATCC
It may refer to the function of a protein having the amino acid sequence of the 13869 strain NCgl2048 protein, or it may refer to the property of increasing the production of a target substance by reducing the activity in a microorganism.

Methods for imparting or enhancing the ability to produce a target substance include a method for reducing enolase activity (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. (e.g. EC 4.2.1.11). This activity is also called "enolase activity". Enolase is also called "phosphopyruvate hydratase". A gene encoding enolase is also referred to as an "enolase gene". Enolase includes the Eno protein encoded by the eno gene. Examples of enolase such as Eno protein include those derived from various organisms such as Enterobacteriaceae bacteria and coryneform bacteria. Specific examples of enolase include Eno proteins of C. glutamicum such as C. glutamicum ATCC 13869 strain.

Methods for imparting or enhancing the ability to produce a target substance include methods for increasing the activity of S-adenosyl-L-homocysteine hydrolase (WO2018/079684). .

"S-adenosyl-L-homocysteine hydrolase (S-adenosyl-L-homocysteine hydrolase)" refers to the hydrolysis of S-adenosyl-L-homocysteine (S-adenosyl-L-homocysteine; SAH) to L- It may mean a protein having activity to catalyze a reaction that produces homocysteine and adenosine (EC 3.3.1.1, etc.). The same activity is also referred to as "S-adenosyl-L-homocysteine hydrolase activity". S-adenosyl-L-homocysteine hydrolase is also called "adenosylhomocysteinase". A gene encoding S-adenosyl-L-homocysteine hydrolase is also referred to as "S-adenosyl-L-homocysteine hydrolase gene". S-adenosyl-L-homocysteine hydrolase includes SahH protein encoded by sahH gene. Examples of S-adenosyl-L-homocysteine hydrolases such as SahH protein include those derived from various organisms such as yeast, bacteria belonging to the genus Streptomyces, and coryneform bacteria. As S-adenosyl-L-homocysteine hydrolase, specifically C. glutamicum ATCC 13032
and the SahH protein of C. glutamicum, such as strain ATCC 13869.

In addition, methods for imparting or enhancing the ability to produce a target substance include methods for reducing the activity of L-serine deaminase (US2018-0334693A).
.

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

In addition, as a method for imparting or enhancing the ability to produce the target substance, the activity of AICAR formyltransferase / IMP cyclohydrolase is reduced, or AICAR is modified so as to have the mutation described in US2018-0334693A. A method of modifying a gene encoding formyltransferase/IMP cyclohydrolase is included (US2018-0334693A).

"AICAR formyltransferase/IMP cyclohydrolase" may mean AICAR formyltransferase and/or IMP cyclohydrolase, ie one or both of AICAR formyltransferase and IMP cyclohydrolase. "AICAR formyltransferase" means 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide (5-amino-1-(5-phospho-D-ribosyl)imidazole -4-carboxamide; AICAR) and 10-formyltetrahydrofolic acid (10-formyltetrahydrofolate), 5-formamide-1- (
Catalyzes the conversion of 5-phospho-D-ribosyl)imidazole-4-carboxamide (5-formamido-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide; FAICAR) to tetrahydrofolate (EC 2.1.2.3, etc.). The same activity is also referred to as "AICAR formyltransferase activity". "IMP cyclohydrolase" may mean a protein that has the activity of catalyzing the reaction that dehydrates FAICAR to form IMP (EC 3.5.4.10, etc.). This activity is called “IMP cyclohydrolase
Also called "activation". A 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, i.e. a protein having both AICAR formyltransferase activity and IMP cyclohydrolase activity. may mean. AICAR formyltransferase/IMP cyclohydrolase includes the PurH protein, a bifunctional AICAR formyltransferase/IMP cyclohydrolase encoded by the purH gene. Examples of AICAR formyltransferase/IMP cyclohydrolase such as PurH protein include those of various organisms such as Enterobacteriaceae bacteria and coryneform bacteria. AICAR formyltransferase/IMP cyclohydrolase specifically includes the PurH protein of C. glutamicum such as C. glutamicum ATCC 13869 strain.

In addition, as a method for imparting or enhancing the ability to produce a target substance, the activity of D-3-phosphoglycerate dehydrogenase (3-PGDH) is increased, or feedback inhibition by L-serine is used. (US2018-0334693A) to modify the microorganism so that it has a gene encoding 3-PGDH that is resistant to .

"D-3-phosphoglycerate dehydrogenase (3-PGDH)" oxidizes 3-phosphoglyceric acid in the presence of an electron acceptor to form 3-phosphoglyceric acid. 3-phosphohydroxylpyruvic acid
) (EC 1.1.1.95 etc.)
. The same activity is also referred to as "3-PGDH activity". Electron acceptors include NAD ⁺ and NADP ⁺ . A gene encoding 3-PGDH is also referred to as "3-PGDH gene". 3-PGDH includes the SerA protein encoded by the serA gene. Examples of 3-PGDH such as SerA protein include those of various organisms such as Enterobacteriaceae bacteria and coryneform bacteria. 3-PGDH specifically includes the SerA protein of B. flavum such as B. flavum ATCC 14067 strain. 3-PGDH also specifically includes E. coli SerA protein such as E. coli K-12 MG1655 strain. 3-PGDHs that are resistant to feedback inhibition by L-serine include those disclosed in US2018-0334693A. Specific examples of 3-PGDH resistant to feedback inhibition by L-serine include the SerA* protein of B. flavum AJ13327 strain encoded by the serA* gene (US2018-0334693A).

Genes and proteins used for breeding microorganisms capable of producing target substances include, for example, the type of target substance, the type of biosynthetic pathway that produces the target substance, and the type and activity of the protein originally possessed by the microorganism. can be appropriately selected according to various conditions. For example, the activity of one or more of the OMT, ACAR, PPT, and protocatechuic acid uptake systems may be increased, particularly if vanillin is produced by bioconversion from protocatechuic acid. Also, for example, the activity of one or more of the ACAR, PPT, and vanillic acid uptake systems may be increased, particularly if vanillin is produced by bioconversion from vanillic acid. When vanillin is produced by bioconversion from vanillic acid, more particularly at least ACAR and/or PP
The activity of T may be increased. When producing vanillin by bioconversion from vanillic acid,
More particularly, the activity of at least ACAR and PPT may be increased. Also, the activity of OMT may be increased, particularly when vanillin is produced by bioconversion from protocatechualdehyde.

Genes and proteins used for breeding microorganisms capable of producing a target substance may each have, for example, known nucleotide sequences and amino acid sequences (including those exemplified above). In addition to those exemplified above, known base sequences and amino acid sequences include WO2018/079687, WO2018/079686, WO2018/079685, WO2018/079684, WO2018/079683, WO2017/073701, WO2018/079705, US2018-0334691A, and US2091A. -0161776A. The expression "a gene or protein has a base sequence or amino acid sequence", unless otherwise specified, may mean that the gene or protein contains the base sequence or amino acid sequence, and the gene or protein has the base sequence or amino acid sequence. A case consisting of an amino acid sequence may also be included.

Genes and proteins used for breeding microorganisms capable of producing a target substance may be conservative variants of genes and proteins having known base sequences and amino acid sequences, respectively. A "conservative variant" refers to a variant that maintains the original function. Conservative variants include, for example, homologues and artificial variants of genes and proteins having known nucleotide and amino acid sequences.

"Original function is maintained" means that a variant of a gene or protein has a function (e.g., activity or property) that corresponds to the function (e.g., activity or property) of the original gene or protein. . "Maintaining the original function" of a gene means that a variant of the gene encodes a protein that retains the original function. For example, "maintaining the original function" for an ACAR gene means that the variant of the gene encodes a protein that has ACAR activity. In addition, "original function is maintained" with respect to ACAR means that a protein variant has ACAR activity. The activity of each protein is measured by the method described in, for example, WO2018/079687, WO2018/079686, WO2018/079685, WO2018/079684, WO2018/079683, WO2017/073701, WO2018/079705, US2018-0334693A, or US2019-016177. can do.

Examples of conservative variants are given below.

Homologs of genes or proteins used for breeding microorganisms capable of producing a target substance can be easily obtained from public databases by, for example, BLAST searches or FASTA searches using known base sequences or amino acid sequences as query sequences. can. In addition, homologues of genes used for breeding microorganisms having the ability to produce a desired substance can be obtained by using, as primers, oligonucleotides prepared based on known nucleotide sequences using chromosomes of organisms such as coryneform bacteria as templates. It can be obtained by PCR.

Genes used for breeding microorganisms having the ability to produce the target substance have one or several amino acid substitutions at one or several positions in the known amino acid sequence, as long as the original function is maintained. , may encode proteins with deleted, inserted or added amino acid sequences. For example, the encoded protein may be lengthened or truncated at its N-terminus and/or C-terminus. The above-mentioned "one or several" varies depending on the position and type of the amino acid residue in the three-dimensional structure of the protein. It means preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5, particularly preferably 1 to 3.

Substitution, deletion, insertion, or addition of one or several amino acids described above is a conservative mutation that maintains normal protein function. A typical conservative mutation is a conservative substitution. Conservative substitution is between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid,
If the substitution site is a hydrophobic amino acid, between Leu, Ile, and Val; if it is a polar amino acid, between Gln and Asn; and if it is a basic amino acid, between Lys, Arg, and His. , and between Asp and Glu in the case of acidic amino acids, and between Ser and Thr in the case of amino acids having a hydroxyl group. Substitutions regarded as conservative substitutions specifically include substitutions of Ala to Ser or Thr, substitutions of Arg to Gln, His or Lys, substitutions of Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp 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 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 substitution, deletion, insertion, addition, or inversion of amino acids as described above may include naturally occurring mutations (mutants or variants), such as those based on individual differences in organisms from which genes are derived, and differences in species. It also includes those caused by

In addition, genes used for breeding microorganisms having the ability to produce the target substance may have, for example, 50% or more, 65% or more, 80% or more of the entire known amino acid sequence, as long as the original function is maintained. % or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, and particularly preferably 99% or more, encoding a protein having an amino acid sequence.

In addition, genes used for breeding microorganisms capable of producing the target substance are probes that can be prepared from known nucleotide sequences, as long as the original functions are maintained. It may be a DNA that hybridizes under stringent conditions with a complementary sequence to . “Stringent conditions” refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, DNAs with high identity, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 97% or more, particularly preferably 99% Conditions that hybridize DNAs with more than 10% identity but do not hybridize DNAs with lower identities, or normal Southern hybridization washing conditions of 60°C, 1×SSC, 0.1%
SDS, preferably 60 ° C., 0.1 × SSC, 0.1% SDS, more preferably at a salt concentration and temperature corresponding to 68 ° C., 0.1 × SSC, 0.1% SDS, wash once, preferably 2-3 times. can be mentioned.

As noted above, the probes used for the hybridization may be part of the complementary sequence of a gene. Such probes can be prepared by PCR using oligonucleotides prepared based on known nucleotide sequences as primers and a DNA fragment containing the above gene as a template. For example, a DNA fragment with a length of about 300 bp can be used as a probe. When using a DNA fragment with a length of about 300 bp as a probe, washing conditions for hybridization include 50° C., 2×SSC, and 0.1% SDS.

In addition, since the degeneracy of codons differs depending on the host, genes used for breeding microorganisms capable of producing the target substance may be those in which arbitrary codons are replaced with equivalent codons.

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

<1-2> Technique for Increasing Protein Activity Techniques for increasing protein activity will be described below.

"The activity of a protein is increased" may mean that the activity of the same protein is increased compared to an unmodified strain. "The activity of a protein is increased" may specifically mean that the activity per cell of the same protein is increased relative to the unmodified strain. "Activity of a protein per cell" may mean the average value of the activity of the same protein per cell. Unmodified strains are also referred to as "unmodified microorganisms" or "strains of unmodified microorganisms." As used herein, "unmodified strain" may refer to a control strain that has not been modified to increase the activity of the target protein. Unmodified strains include wild strains and parental strains. Unmodified strains specifically include type strains of each microbial species. In addition, specific examples of unmodified strains include the strains exemplified in the description of microorganisms. Thus, in one aspect, the activity of the protein may be increased compared to a reference strain (ie, the reference strain of the species to which the microorganism capable of producing the desired substance belongs). Also, in another embodiment, the activity of the protein may be increased compared to C. glutamicum ATCC 13869 strain. Also, in another embodiment, the activity of the protein may be increased compared to C. glutamicum ATCC 13032 strain. Also, in another embodiment, the activity of the protein may be increased compared to E. coli K-12 strain MG1655. In addition, "the activity of the protein is increased" is also referred to as "the activity of the protein is enhanced". More specifically, "the activity of the protein is increased" means that the number of molecules of the same protein per cell is increased compared to an unmodified strain, and/or It may mean that the function is increased. That is, "activity" in the case of "increase in protein activity" means not only the catalytic activity of the protein but also the transcription level (mRNA level) or translation level (protein level) of the gene encoding the protein. may "Number of molecules of protein per cell" may mean the average number of molecules of the same protein per cell. In addition, "increasing the activity of the protein" includes not only increasing the activity of the protein in a strain that originally has the activity of the target protein, but also increasing the activity of the same protein in a strain that does not originally have the activity of the target protein. Conferring activity is also included. In addition, 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 preferred target protein may be imparted.

The degree of increase in protein activity is not particularly limited as long as the protein activity is increased compared to the unmodified strain. The activity of the protein may be increased, for example, by 1.2-fold or more, 1.5-fold or more, 2-fold or more, or 3-fold or more over the unmodified strain. In addition, if the unmodified strain does not have the activity of the target protein, the protein may be produced by introducing the gene encoding the protein. It should be produced as much as possible.

Modifications that increase the activity of a protein can be achieved, for example, by increasing the expression of the gene encoding the same protein. “Increase in gene expression” may mean that the expression of the same gene is increased compared to unmodified strains such as wild strains and parent strains. “Increase in gene expression” may specifically mean that the expression level of the same gene per cell is increased compared to an unmodified strain. “Gene expression level per cell” may mean the average expression level of the gene per cell. “Increase in gene expression” more specifically means an increase in the transcription level (mRNA level) of the gene and/or an increase in the translation level (protein level) of the gene. you can In addition, "gene expression is increased" is also referred to as "gene expression is enhanced". Expression of the gene may be increased, for example, by 1.2-fold or more, 1.5-fold or more, 2-fold or more, or 3-fold or more over the unmodified strain. In addition, "increased gene expression" means not only increasing the expression level of the gene in a strain that originally expresses the target gene, but also in a strain that does not originally express the target gene, Expression of the same gene is also included. That is, "increasing the expression of a gene" may mean, for example, introducing the target gene into a strain that does not retain the target gene and expressing the same gene.

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

An increase in gene copy number can be achieved by introducing the same gene into the host chromosome. Introduction of genes into chromosomes can be performed, for example, by utilizing homologous recombination (Miller, JH Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Examples of gene introduction methods using homologous recombination include Red-driven integration (Datsenko, K. A, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97:6640-6645). (2000)), a method using a plasmid containing a temperature-sensitive replication origin, a method using a conjugative transferable plasmid, a method using a suicide vector that does not have a replication origin that functions in the host, A transduction method using phage can be mentioned. The gene may be introduced in only one copy, two copies or more. For example, multiple copies of a gene can be introduced into the chromosome by targeting homologous recombination to a sequence that exists in multiple copies on the chromosome. Sequences that have multiple copies on the chromosome include repetitive DNA sequences,
Examples include inverted repeats present at both ends of transposons. Alternatively, homologous recombination may be performed by targeting an appropriate sequence on the chromosome, such as a gene that is not required for the production of the target substance. A gene can also be randomly introduced onto a chromosome using a transposon or Mini-Mu (JP-A-2-109985, US Pat. No. 5,882,888, EP805867B1).

Confirmation that the target gene has been introduced onto the chromosome is performed by Southern hybridization using a probe having a sequence complementary to all or part of the gene, or by using a primer prepared based on the sequence of the gene. It can be confirmed by PCR or the like.

An increase in gene copy number can also be achieved by introducing a vector containing the same gene into a host. For example, a DNA fragment containing a target gene is ligated to a vector that functions in a host to construct an expression vector for the same gene, and the host is transformed with the expression vector to increase the copy number of the same gene. can. A DNA fragment containing a target gene can be obtained, for example, by PCR using the genomic DNA of a microorganism containing the target gene as a template. As a vector, a vector capable of autonomous replication in host cells can be used. A vector may be a multi-copy vector. The vector may also have a marker such as an antibiotic resistance gene for selection of transformants. The vector may also have a promoter and terminator for expressing the inserted gene. A vector can be, for example, a bacterial plasmid-derived vector, a yeast plasmid-derived vector, a bacteriophage-derived vector, a cosmid, or a phagemid, or the like. Specific examples of vectors capable of autonomous replication in Enterobacteriaceae bacteria such as Escherichia coli include, for example, pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, pSTV29 (all available from Takara Bio Inc.), pACYC184, pMW219 (Nippon Gene), pTrc99A (
Pharmacia), pPROK vector (Clontech), pKK233-2 (Clontech), pET vector (Novagen), pQE vector (Qiagen), pCold TF DNA (TaKa
Ra), pACYC-based vector, broad host range vector RSF1010. Specific examples of vectors capable of autonomous replication in coryneform bacteria include, for example, pHM1519 (Agric. Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, 2901- 2903 (1984)); improved plasmids with drug resistance genes; pCRY30 (JP-A-3-210184); pCRY21, pCRY2KE
, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX (JP-A-2-72876, US Pat. No. 5,185,262)
pCRY2 and pCRY3 (JP-A-1-191686); pAJ655, pAJ611, and pAJ1844 (JP-A-58-192900); pCG1 (JP-A-57-134500); pCG2 (JP-A-58-35197); pCG4 and pCG11 (JP 57-183799); pPK4 (U.S. Pat. No. 6,090,597); pVK4 (JP No. 9-322774); pVK7 (JP 10-215883); pVK9 (WO2007/046389); pVS7 (WO2013/069634) and pVC7 (JP-A-9-070291). Further, as a vector capable of autonomous replication in coryneform bacteria, specifically,
Examples include variants of pVC7 such as pVC7H2 (WO2018/179834).

When introducing a gene, it is sufficient that the gene can 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. A "promoter that functions in the host" may mean a promoter that has promoter activity in the host. The promoter may be a host-derived promoter or a heterologous promoter. The promoter may be the intrinsic promoter of the gene to be introduced, or the promoter of another gene. The promoter may be, for example, a stronger promoter as described herein.

A terminator for terminating transcription 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 heterologous terminator. The terminator may be a terminator specific to the gene to be introduced, or may be a terminator of another gene. Specific examples of terminators include T7 terminator, T4 terminator, fd phage terminator, tet terminator, and trpA terminator.

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

Also, when introducing two or more genes, each gene may be retained in the host so as to be expressible. For example, each gene may be all maintained on a single expression vector, or all may be maintained on a chromosome. In addition, each gene may be separately maintained on a plurality of expression vectors, or may be separately maintained on a single or multiple expression vectors and on the chromosome. Alternatively, two or more genes may be introduced to form an operon. When "two or more genes are introduced," e.g., when introducing genes encoding two or more proteins (e.g., enzymes) respectively, a single protein complex (e.g., enzyme conjugate) is When introducing genes encoding each of the constituent two or more subunits, or combinations thereof may be introduced.

The introduced gene is not particularly limited as long as it encodes a protein that functions in the host. The introduced gene may be a host-derived gene or a heterologous gene. The gene to be introduced can be obtained by PCR, for example, using primers designed based on the base sequence of the gene and genomic DNA of an organism having the gene or a plasmid carrying the gene as a template. Also, the introduced gene may be totally synthesized based on the base sequence of the same gene (Gene, 60(1), 115-127 (1987)). The obtained gene can be used as it is or after being modified as appropriate. That is, by modifying the gene, its variants can be obtained. Gene modification can be performed by a known technique. For example, site-directed mutagenesis can be used to introduce a desired mutation at a desired site in DNA. Thus, for example, by site-directed mutagenesis, the coding region of a gene can be altered such that the encoded protein contains substitutions, deletions, insertions, and/or additions of amino acid residues at specific sites. . As the site-directed mutagenesis method, a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and methods using phage (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 ( 1987)). Alternatively, all variants of the gene may be synthesized.

When a protein functions as a complex composed of multiple subunits, all or only some of the subunits may be modified as long as the activity of the protein is increased as a result. That is, for example, when increasing protein activity by increasing gene expression, the expression of all of the genes encoding these subunits may be enhanced, or only a portion of the expression may be enhanced. good. It is usually preferred to enhance expression of all of the genes encoding those subunits. Each subunit constituting the complex may be derived from one organism or from two or more different organisms, as long as the complex has the function of the target protein. That is, for example, genes encoding a plurality of subunits and derived from the same organism may be introduced into the host, or genes derived from different organisms 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. Improving gene transcription efficiency and translation efficiency can be achieved, for example, by modifying an expression control sequence. "Expression control sequence" may be a generic term for sites that affect gene expression. Expression control sequences include, for example, promoters, Shine-Dalgarno (SD) sequences (also called ribosome binding sites (RBS)), and spacer regions between the RBS and the initiation codon. The expression regulatory sequence can be determined using a promoter search vector or gene analysis software such as GENETYX. These expression control sequences can be modified, for example, by a method using a temperature-sensitive vector or by the Red-driven integration method (WO2005/010175).

Improving the transcription efficiency of a gene can be achieved, for example, by replacing the promoter of the gene on the chromosome with a stronger promoter. A "stronger promoter" may refer to a promoter that improves transcription of a gene over the naturally occurring wild-type promoter. Examples of stronger promoters include known high expression promoters such as T7 promoter, trp promoter, lac promoter, thr promoter, tac promoter, trc promoter, tet promoter, araBAD promoter, rpoH promoter, msrA promoter, Bifidobacterium-derived Pm1 promoters, PR promoters, and PL promoters. In addition, stronger promoters that can be used in coryneform bacteria include, for example, an artificially modified P54-6 promoter (Appl. Microbiol. Biotechnol., 53, 674-679 (2000)), coryneform bacteria pta, aceA, aceB, adh, amyE promoters, which can be induced by acetic acid, ethanol, pyruvic acid, etc., cspB, SOD, tuf (EF-Tu) promoters, which are strong promoters with high expression levels in coryneform bacteria (Journal of Biotechnology 104 (2003) 311-323, Appl Environ Microbiol. 2005 Dec;71(12):8587-96.), P2 promoter (US2018-0334693A), P3 promoter (US2018-0334693A), F1 promoter (WO2018/179834) , lac promoter, tac promoter and trc promoter. As a stronger promoter, a conventional promoter with high activity may be obtained by using various reporter genes. For example, -35 in the promoter region, -
Promoter activity can be increased by bringing the 10 region closer to the consensus sequence (WO 00/18935). Examples of highly active promoters 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 Goldstein et al.

Improving the translation efficiency of a gene can be achieved, for example, by replacing the Shine-Dalgarno (SD) sequence (also called ribosome binding site (RBS)) of the gene on the chromosome with a stronger SD sequence. A "stronger SD sequence" may refer to an SD sequence that improves translation of mRNA over the native wild-type SD sequence. Stronger SD sequences include, for example, the RBS of gene 10 from phage T7 (Olins PO et al, Gene,
1988, 73, 227-235). Furthermore, substitutions, insertions, or deletions of a few nucleotides in the spacer region between the RBS and the initiation codon, especially in the sequence immediately upstream of the initiation codon (5'-UTR), may affect mRNA stability and translation efficiency. These are known to have a significant effect, and their modification can also improve the efficiency of gene translation.

Improving the efficiency of gene translation can also be achieved, for example, by modifying codons. For example, gene translation efficiency can be improved by replacing rare codons present in a gene with synonymous codons that are used more frequently. That is, the introduced gene may be modified so as to have optimal codons according to the codon usage of the host to be used, for example. Codon substitution can be performed, for example, by site-directed mutagenesis in which a desired mutation is introduced into a desired site of DNA. Alternatively, a gene fragment in which codons are replaced may be totally synthesized. The frequency of codon usage in various organisms can be found in "Codon Usage Database"(http://www.kazusa.or.jp/codon; Nakamura, Y. et al, Nucl. Acids Res., 28, 292 (2000)). disclosed in

Gene expression can also be increased by amplifying a regulator that increases gene expression, or by deleting or weakening a regulator that decreases gene expression.

The techniques for increasing gene expression as described above may be used alone or in any combination.

Modifications to increase the activity of a protein can also be achieved, for example, by enhancing the specific activity of the protein. Enhancement of specific activity may also include desensitization to feedback inhibition. That is, if a protein is subject to feedback inhibition by a metabolite, the activity of the protein can be increased by mutating the gene or protein in the host such that the feedback inhibition is desensitized. Unless otherwise specified, "desensitization of feedback inhibition" may include cases where feedback inhibition is completely eliminated and cases where feedback inhibition is reduced. In addition, "feedback inhibition is desensitized" (that is, feedback inhibition is reduced or eliminated) is also referred to as "tolerant to feedback inhibition". Proteins with enhanced specific activity can be obtained by searching various organisms, for example. Alternatively, a highly active protein may be obtained by introducing a mutation into a conventional protein. The mutations introduced may be, for example, substitutions, deletions, insertions and/or additions of one or several amino acids at one or several positions of the protein. Mutations can be introduced, for example, by site-directed mutagenesis as described above. In addition, mutation may be introduced by, for example, mutagenesis. Mutation treatments include X-ray irradiation, ultraviolet irradiation, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS). ) and other mutating agents. We also treated the DNA directly with hydroxylamine in vitro,
Random mutations may be induced. Enhancement of specific activity may be used alone, or may be used in combination with any of the methods for enhancing gene expression as described above.

The transformation method is not particularly limited, and conventionally known methods can be used. For example, treatment of 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; , 1977. Gene 1: 153-167) can be used. Alternatively, cells of DNA recipients, such as those known for Bacillus subtilis, actinomycetes, and yeast,
A method for introducing recombinant DNA into DNA recipient bacteria in the form of protoplasts or spheroplasts that readily incorporate recombinant DNA (Chang, S. and Choen, SN, 1979. Mol. Gen. Genet. 168: 111-115 Bibb, MJ, Ward, JM and Hopwood, OA 1978. Nature 274: 398-400; Hinnen, A., Hicks, JB and Fink, GR 1978. Proc. Natl. Acad. can also be applied. Alternatively, an electric pulse method (JP-A-2-207791), which has been reported for coryneform bacteria, can also be used.

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

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

An increase in the transcription level of a gene can be confirmed by comparing the amount of mRNA transcribed from the same gene with that of a wild strain or an unmodified strain such as 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 Press, Cold Spring Harbor (USA), 2001). The amount of mRNA (e.g., the number of molecules per cell) is, for example, 1.2 times or more that of the unmodified strain, 1.5
It may be increased by a factor of 2 or more, 2 times or more, or 3 times or more.

Confirmation of increased protein abundance can be performed by Western blot using antibodies (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, by 1.2-fold or more, 1.5-fold or more, 2-fold or more, or 3-fold or more over the unmodified strain.

The techniques for increasing protein activity described above can be used to enhance the activity of any protein or to enhance the expression of any gene.

<1-3> Technique for Reducing Protein Activity Techniques for reducing protein activity will be described below.

"The activity of a protein is reduced" may mean that the activity of the same protein is reduced compared to an unmodified strain. "The activity of a protein is reduced" may specifically mean that the activity of the same protein per cell is reduced compared to an unmodified strain. "Activity of a protein per cell" may mean the average value of the activity of the same protein per cell. Unmodified strains are also referred to as "unmodified microorganisms" or "strains of unmodified microorganisms." As used herein, "unmodified strain" may refer to a control strain that has not been modified to reduce the activity of the target protein. Unmodified strains include wild strains and parental strains. Unmodified strains specifically include type strains of each microbial species. In addition, specific examples of unmodified strains include the strains exemplified in the description of microorganisms. That is, in one aspect, the activity of the protein may be reduced compared to the reference strain (ie, the reference strain of the species to which the microorganism capable of producing the desired substance belongs). Also, in another embodiment, the activity of the protein may be reduced compared to C. glutamicum ATCC 13869 strain. Also, in another embodiment, the activity of the protein may be reduced compared to C. glutamicum ATCC 13032 strain. Also, in another embodiment, the activity of the protein may be reduced compared to E. coli K-12 strain MG1655. In addition, "the activity of the protein is decreased" may include the case where the activity of the same protein is completely lost. "The activity of the protein is reduced" means, more specifically, that the number of molecules of the same protein per cell is reduced as compared with an unmodified strain, and/or It may mean that the function is degraded. That is, the "activity" in the case of "the activity of the protein is decreased" is not limited to the catalytic activity of the protein, but means the transcription level (mRNA level) or translation level (protein level) of the gene encoding the protein. may "Number of molecules of protein per cell" may mean the average number of molecules of the same protein per cell. It should be noted that "the number of protein molecules 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 same protein is completely lost. The degree of reduction in protein activity is not particularly limited as long as the protein activity is reduced compared to the unmodified strain. The activity of the protein may be reduced, eg, to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain.

A modification that reduces the activity of a protein can be achieved, for example, by reducing the expression of the gene encoding the same protein. “Gene expression is reduced” may mean that the expression of the same gene is reduced compared to unmodified strains such as wild strains and parent strains. "Reduced expression of a gene" may specifically mean that the expression level of the same gene per cell is reduced compared to an unmodified strain. “Gene expression level per cell” may mean the average expression level of the same gene per cell. More specifically, the term "gene expression is decreased" means that the transcription level (mRNA level) of the gene is decreased and/or the translation level (protein level) of the gene is decreased. you can "The expression of a gene is decreased" may include the case where the same gene is not expressed at all. In addition, "the expression of a gene is decreased" is also referred to as "the expression of a gene is attenuated". Expression of the gene may be reduced to, eg, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain.

A decrease in gene expression may be due to, for example, a decrease in transcription efficiency, a decrease in translation efficiency, or a combination thereof. Reduction of gene expression is achieved, for example, by altering expression regulatory sequences such as gene promoters, Shine-Dalgarno (SD) sequences (also referred to as ribosome binding sites (RBS)), and spacer regions between RBS and start codons. can be achieved by When modifying the expression control sequence, the expression control sequence is modified at 1 or more bases, 2 or more bases, or 3 or more bases. A decrease in transcription efficiency of a gene can be achieved, for example, by replacing the promoter of the gene on the chromosome with a weaker promoter. A “weaker promoter” may refer to a promoter whose gene transcription is weaker than the naturally occurring wild-type promoter. Weaker promoters include, for example, inducible promoters. Weaker promoters also 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-inducing conditions (eg, in the absence of an inducer). Also, part or all of the expression control sequence may be deleted. In addition, reduction of gene expression can also be achieved, for example, by manipulating factors involved in expression control. Factors involved in expression control include small molecules (inducing substances, inhibitors, etc.), proteins (transcription factors, etc.), nucleic acids (siRNA, etc.), etc., involved in transcription and translation control. In addition, the reduction of gene expression can also be achieved, for example, by introducing a mutation that reduces gene expression into the coding region of the gene. For example, expression of a gene can be reduced by replacing codons in the coding region of the gene with synonymous codons that are used less frequently in the host. Gene expression itself can also be reduced, for example, by disruption of the gene as described herein.

Also, modification that reduces the activity of a protein can be achieved, for example, by disrupting the gene encoding the same protein. "Gene is disrupted" may mean that the same gene is altered so that it does not produce a protein that functions normally. "Do not produce a protein that functions normally" includes cases where no protein is produced from the same gene, or cases where the same gene produces a protein whose function per molecule (e.g., activity or property) is reduced or lost. may also be included.

Gene disruption can be achieved, for example, by deleting (deleting) the gene on the chromosome. A "gene deletion" may refer to a deletion of part or all of the coding region of a gene. Furthermore, the entire gene may be deleted, including sequences before and after the coding region of the gene on the chromosome. As long as the protein activity can be reduced, the regions to be deleted include the N-terminal region (that is, the region that encodes the N-terminal side of the protein), the internal region, and the C-terminal region (that is, the region that encodes the C-terminal side of the protein). It may be any region such as Generally, the longer the region to be deleted, the more reliably 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 entire coding region of the gene, Or it may be a region with a length of 95% or more. Moreover, it is preferable that the sequences before and after the region to be deleted do not have the same reading frame. A reading frame mismatch can result in a frameshift downstream of the deleted region.

In addition, gene disruption includes, for example, introducing an amino acid substitution (missense mutation) into the coding region of the gene on the chromosome, introducing a stop codon (nonsense mutation), or adding or deleting 1 to 2 bases ( (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)).

Gene disruption can also be achieved, for example, by inserting other nucleotide sequences 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 reliably the gene can be inactivated. Moreover, it is preferable that the sequences before and after the insertion site do not have the same reading frame. A reading frame mismatch can result in a frameshift downstream of the insertion site. Other nucleotide sequences are not particularly limited as long as they reduce or eliminate the activity of the encoded protein, and examples thereof include marker genes such as antibiotic resistance genes and genes useful for the production of target substances.

Disruption of the gene may in particular be carried out such that the amino acid sequence of the encoded protein is deleted (deleted). In other words, modifications that reduce the activity of a protein are, for example, deletion of the amino acid sequence of the protein (a region of part or all of the amino acid sequence). This can be achieved by modifying the gene to encode a protein that lacks a partial or complete region. In addition, "deletion of the amino acid sequence of the protein" may mean deletion of part or all of the region of the amino acid sequence of the protein. In addition, "deletion of the amino acid sequence of a protein" may mean that the original amino acid sequence does not exist in the protein, and may include cases in which the original amino acid sequence is changed to another amino acid sequence. That is, for example, a region changed to a different amino acid sequence by frameshifting may be considered a deleted region. Deletion of an amino acid sequence typically shortens the overall length of the protein, but in some cases the overall length of the protein remains unchanged or is lengthened. For example, deletion of part or all of the coding region of a gene can result in deletion of the region encoded by the deleted region in the amino acid sequence of the encoded protein. In addition, for example, by introducing a stop codon into the coding region of a gene, it is possible to delete the region encoded by the region downstream from the introduction site in the amino acid sequence of the encoded protein. Also, for example, by frameshifting in the coding region of a gene, the region encoded by the frameshift site can be deleted. Regarding the position and length of the region to be deleted in amino acid sequence deletion, the description of the position and length of the region to be deleted in gene deletion can be applied mutatis mutandis.

Modifying a gene on a chromosome as described above involves, for example, preparing a disrupted gene that is modified so as not to produce a protein that functions normally, and transforming a host with recombinant DNA containing the disrupted gene. This can be achieved by causing homologous recombination between the disrupted gene and the wild-type gene on the chromosome to replace the wild-type gene on the chromosome with the disrupted gene. In this case, the manipulation is facilitated if the recombinant DNA contains a marker gene according to the traits such as auxotrophy of the host. Disruption-type genes include genes with partially or entirely deleted coding regions, genes with missense mutations, genes with nonsense mutations, genes with frameshift mutations, transposons, and marker genes. Includes inserted genes. 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 replacement using such homologous recombination has already been established, and a method called "Red-driven integration" (Datsenko, K. A, and Wanner, BL Proc. Natl. Acad. Sci. US A. 97:6640-6645 (2000)), Red-driven integration method and λ phage-derived excision system (Cho, EH, Gumport, RI, Gardner, JFJ Bacteriol. 184: 5200-5203 (2002) ) (see WO2005/010175), a method using linear DNA, a method using a plasmid containing a temperature-sensitive replication origin, a method using a plasmid capable of conjugative transfer, replication that functions in the host There is a method using a suicide vector that does not have an origin (US Pat. No. 6,303,383, JP-A-05-007491).

Further, modifications that reduce protein activity may be performed, for example, by mutagenesis. Mutation treatments include X-ray irradiation, ultraviolet irradiation, and N-methyl-N'
-treatment with mutating agents such as nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

When a protein functions as a complex composed of multiple subunits, all or only some of these subunits 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 disrupted, or only some of them may be disrupted. In addition, when a protein has multiple isozymes, the activities of all or only some of these isozymes 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 disrupted, or only some of them may be disrupted.

The techniques for reducing protein activity as described above may be used alone or in any combination.

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

Decreased protein activity can also be confirmed by confirming decreased expression of the gene encoding the same protein. Decreased gene expression can be confirmed by confirming that the amount of transcription of the gene has decreased or by confirming that the amount of protein expressed from the gene has decreased.

Decrease in gene transcription level can be confirmed by comparing the amount of mRNA transcribed from the same gene with that of an unmodified 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 Press, Cold Spring Harbor (USA), 2001)). amount of mRNA (e.g.
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 of decreased protein levels can be performed by Western blotting using antibodies (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, eg, to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain.

Disruption of a gene can be confirmed by determining the base sequence, restriction enzyme map, full length, or the like of part or all of the gene, depending on the means used for the disruption.

The techniques for reducing protein activity described above can be used to reduce the activity of any protein or to reduce the expression of any gene.

<2> Method for producing target substance The target substance can be produced using 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. A process of producing a target substance 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 called "fermentation method". Moreover, the process of manufacturing a target substance by fermentation using microorganisms is also called a "fermentation process." That is, the target substance manufacturing process may include, for example, a fermentation process. Also, the target substance manufacturing process may be carried out by, for example, a fermentation process.

A fermentation process can be implemented by culturing microorganisms. Specifically, in the fermentation process, the target substance can be produced from the carbon source. That is, the fermentation step may be, for example, a step of culturing a 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 of producing a target substance, including 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 microorganisms.

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

Carbon sources are not particularly limited as long as they can be assimilated by microorganisms and produce the target substance. Specific examples of carbon sources include sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, blackstrap molasses, starch hydrolysates, biomass hydrolysates, acetic acid, citric acid, and succinic acid. , organic acids such as gluconic acid, alcohols such as ethanol, glycerol and crude glycerol, and fatty acids. In addition, as a carbon source, in particular, a plant-derived raw material can be used. Plants include, for example, corn, rice, wheat, soybeans, sugarcane, beets, and cotton. Plant-derived raw materials include, for example, organs such as roots, stems, trunks, branches, leaves, flowers, seeds, plants containing them, and decomposition products of these plant organs. The form of utilization of the plant-derived raw material is not particularly limited, and for example, it can be used in any form such as unprocessed product, squeezed juice, pulverized product, refined product, and the like. In addition, a pentose such as xylose, a hexose such as glucose, or a mixture thereof can be obtained and used, for example, from plant biomass. Specifically, these sugars 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. In addition, 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 hexose. good. Alternatively, xylose may be supplied by conversion from the hexose, for example, by making the microorganism possess a conversion pathway from hexose such as glucose to xylose. 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 can be produced. The concentration of the carbon source in the medium may be, for example, as high as possible within a range in which the production of the desired substance is not inhibited. The initial concentration of carbon source in the medium may be, for example, 5-30 w/v%, or 10-20 w/v%. Moreover, a carbon source may be added to the medium as appropriate. For example, a carbon source may be added to the medium as the fermentation progresses and the carbon source is depleted or depleted. 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 state of carbon source depletion does not continue. may be preferred.

Specific examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate; organic nitrogen sources such as peptone, yeast extract, meat extract and soy protein hydrolyzate; ammonia; and urea. Ammonia gas or ammonia water used for pH adjustment may be used as the 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 phosphate source include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate. As the phosphoric acid source, one phosphoric acid source may be used, or two or more phosphoric acid sources may be used in combination.

Specific examples of sulfur sources 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 sulfur source may be used, or two or more sulfur sources may be used in combination.

Specific examples of other organic and inorganic components include 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. Acids, nicotinamide, vitamins such as vitamin B12; amino acids; nucleic acids; As other various organic components and inorganic components, one component may be used, or two or more components may be used in combination.

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

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

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

Cultivation can be performed by batch culture, fed-batch culture, continuous culture, or a combination thereof. In addition, the medium at the start of culture is also referred to as "starting medium". A medium added to a culture system (for example, a fermenter) in fed-batch culture or continuous culture is also referred to as a "fed-batch medium". In addition, addition of a feed medium to a culture system in fed-batch culture or continuous culture is also referred to as "fed-batch". When culture is performed separately for seed culture and main culture, the culture forms of seed culture and main culture may or may not be the same. For example, both the seed culture and the main culture may be performed by batch culture, or the seed culture may be performed by batch culture, and the main culture may be performed by fed-batch culture or continuous culture.

Various components, such as carbon sources, may be contained in the starting medium, the feed medium, or both. That is, in the process of culturing, various components such as a carbon source may be added to the medium singly or in any combination. Any of these components may be added once or multiple times, or may be added continuously. The types of components contained in the starting medium may or may not be the same as the types of components contained in the feed medium. Also, the concentration of each component contained in the starting medium may or may not be the same as the concentration of each component contained in the feed medium. Also, two or more feed media containing different types and/or concentrations of components may be used. For example, when feeding is performed intermittently a plurality of times, the types and/or concentrations of components contained in each feeding medium may or may not be the same.

Culturing may be performed, for example, under aerobic conditions. “Aerobic conditions” may mean conditions under which the dissolved oxygen concentration in the medium is 0.33 ppm or higher, or 1.5 ppm or higher. Specifically, the oxygen concentration may be controlled to, for example, 1 to 50% or about 5% of the saturated oxygen concentration. Cultivation can be performed, for example, by aerobic culture or shaking culture. The pH of the medium can be, for example, pH 3-10, or pH 4.0-9.5. During cultivation, the pH of the medium can be adjusted as needed. The pH of the medium can be adjusted with various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, and magnesium hydroxide. can be adjusted using The pH of the medium may be adjusted with substances other than ammonia, such as ammonia gas and aqueous ammonia, among others. The culture temperature may be, for example, 20-45°C, or 25-37°C. The culture period may be, for example, 10 hours to 120 hours. Cultivation may be continued, for example, until the carbon source in the medium is consumed, or until the activity of the microorganisms ceases.

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

Production of the target substance can be confirmed by known methods used for compound detection or identification. Such techniques include, for example, HPLC, UPLC, LC/MS, GC/MS, NMR. These techniques can be used alone or in combination as appropriate. These techniques can also be used to determine the concentrations of various components present in the medium.

The produced target substance can be collected 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 from the medium. The target substance can be recovered by a known technique used for separation and purification of compounds. Such techniques include, for example, ion exchange resin methods, membrane treatment methods, precipitation methods, extraction methods, distillation methods, and crystallization methods. Specifically, the target substance can be recovered by extraction with an organic solvent such as ethyl acetate, or by steam distillation. These techniques can be used alone or in combination as appropriate.

In addition, when the target substance precipitates in the medium, it can be recovered by, for example, centrifugation or filtration. In addition, the target substance precipitated in the medium may be isolated together with the target substance dissolved in the medium after crystallization.

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

<2-2> Biotransformation method The target substance can also be produced by, for example, biotransformation 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 biotransformation using microorganisms. This aspect is also called "bioconversion method". In addition, a process of producing a target substance by biotransformation using microorganisms is also referred to as a "biological transformation process". That is, the target substance production process may include, for example, a biotransformation process. Also, the target substance production process may be carried out by, for example, a biotransformation process.

Specifically, in the biotransformation process, a target substance can be produced from a precursor of the target substance. More specifically, in the bioconversion step, the target substance can be produced by converting a precursor of the target substance into the target substance using microorganisms. That is, the bioconversion step may be a step of converting a precursor of a target substance into the target substance using microorganisms.

A precursor of a target substance is also simply referred to as a “precursor”. Precursors include intermediates in the target substance biosynthetic pathway (eg, those mentioned in connection with the description of target substance biosynthetic enzymes). Vanillin precursors are as described above. That is, vanillin precursors include protocatechuic acid, vanillic acid, and protocatechualdehyde. Vanillin precursors include in particular vanillic acid. Vanillic acid precursors include protocatechuic acid. Protocatechuic aldehyde precursors include protocatechuic acid. As the precursor, one type of precursor may be used, or two or more types of precursors may be used in combination. When the precursor is a compound that can take a salt form, the precursor may be used as a free form, as a salt, or as a mixture thereof. That is, "precursor" may mean a precursor in free form, or a salt thereof, or a mixture thereof, unless otherwise specified. Salts include, for example, ammonium salts, sodium salts, and potassium salts. Precursor salts include, among others, salts other than ammonium salts. As the salt of the precursor, 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 an appropriately manufactured product may be used. A method for producing the precursor is not particularly limited, and for example, a known method can be used. Precursors can be produced, for example, by chemical synthetic methods, enzymatic methods, biotransformation methods, fermentation methods, extraction methods, or combinations thereof. That is, for example, a precursor of a target substance is converted into a further precursor by utilizing an enzyme (also referred to as a "precursor-generating enzyme") that catalyzes a conversion reaction from the further precursor to the precursor of the target substance. It can be manufactured from the body. Also, for example, a precursor of a target substance can be produced from a carbon source or from such a further precursor using a microorganism having precursor-producing ability. A "precursor-producing microorganism" may mean a microorganism capable of producing a precursor. A "precursor-producing microorganism" may specifically mean a microorganism capable of producing a precursor of a target substance from a carbon source and/or from such a further precursor. . For microorganisms capable of producing precursors, the description of microorganisms capable of producing the target substance can be applied mutatis mutandis. In addition, the description of the production of the target substance using a microorganism capable of producing the target substance can be applied mutatis mutandis to the production of the precursor using the microorganism having the precursor-producing capability. For example, as a method for producing protocatechuic acid by an enzymatic method or a bioconversion method, a method of converting paracresol into protocatechuic acid using Pseudomonas putida KS-0180 (JP-A-7-75589), NADH A method for converting parahydroxybenzoic acid to protocatechuic acid using dependent parahydroxybenzoic acid hydroxylase (Japanese Patent Laid-Open No. 5-244941), a trait introduced with a gene involved in the reaction to produce protocatechuic acid from terephthalic acid A method for producing protocatechuic acid by culturing a transformant in a medium supplemented with terephthalic acid (Japanese Patent Application Laid-Open No. 2007-104942), having protocatechuic acid-producing ability and having reduced or absent protocatechuic acid 5-oxidase activity and a method for producing protocatechuic acid from its precursor using a microorganism produced by the method (Japanese Patent Application Laid-Open No. 2010-207094). Methods for producing protocatechuic acid by fermentation include a method of producing protocatechuic acid using acetic acid as a carbon source using bacteria belonging to the genus Brevibacterium (JP-A-50-89592); - A method for producing protocatechuic acid using glucose as a carbon source using bacteria belonging to the genus Escherichia or Klebsiella into which a gene encoding dihydroshikimate dehydrogenase has been introduced (US Pat. No. 5,272,073) is mentioned. In addition, protocatechualdehyde can be produced from protocatechuic acid as a precursor by an enzymatic method using ACAR or a biotransformation method using a microorganism having ACAR. In addition, vanillic acid can be produced from protocatechuic acid as a precursor by an enzymatic method using OMT or a bioconversion method using a microorganism having OMT (J. Am. CHm. Soc., 1998, Vol. 120). also,
Vanillic acid can be produced using ferulic acid as a precursor by a biotransformation method using Pseudomonas sp. AV ₁₀ strain (J. App. Microbiol., 2013, Vol.116, p903-910). The precursor may have been produced as described above. For example, vanillin precursors such as vanillic acid may be produced using a microorganism capable of producing vanillin precursors. The biotransformation method may further comprise a step of producing a precursor. The bioconversion method may specifically include the steps of producing precursors as described above. For example, when the target substance is vanillin, the bioconversion method may particularly include a step of producing a vanillin precursor such as vanillic acid using a microorganism capable of producing a vanillin precursor. The produced precursor can be used for the biotransformation method as it is, or after being appropriately subjected to treatments such as concentration, dilution, drying, dissolution, fractionation, sterilization, extraction, and purification. That is, as the precursor, for example, a purified product refined 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. Materials containing precursors are not particularly limited as long as the precursors can be used by microorganisms. Specific examples of materials containing precursors include culture media or reaction solutions containing precursors, supernatants separated from the culture media or reaction solutions, concentrates thereof (e.g., concentrates), dilutions ( For example, diluents) and processed materials such as dried materials can be mentioned.

In one aspect, the bioconversion step can be performed by culturing a microorganism capable of producing the target substance. This aspect is also called "the 1st aspect of a bioconversion method." That is, the bioconversion step may be, for example, a step of culturing microorganisms in a medium containing a precursor of the target substance and converting the precursor into the target substance. Specifically, the biotransformation step may be a step of culturing microorganisms in a medium containing a precursor of the target substance and producing and accumulating the target substance in the medium.

The medium to be used is not particularly limited as long as it contains the precursor of the target substance, allows the microorganisms to grow, and produces the target substance. Culture conditions are not particularly limited as long as the microorganisms can grow and the target substance can be produced. Regarding the culture in the first aspect of the bioconversion method, in the same aspect, the description of the culture in the fermentation method (for example, the description of the medium and culture conditions) except that the medium contains the precursor of the target substance. Applicable mutatis mutandis.

The precursor may be contained in the medium during the entire period of culture, or may be contained in the medium only during a part of the period of culture. That is, "cultivating a microorganism in a medium containing a precursor" does not require that the precursor be contained in the medium during the entire culture period. For example, precursors may or may not be contained in the medium from the start of culture. If the precursors are not contained in the medium at the start of the culture, the precursors are 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 grown sufficiently. In either case, precursors may be added to the medium as appropriate. For example, precursors may be added to the medium according to the decrease or depletion of precursors associated with production of the target substance. There are no particular restrictions on the means for adding precursors to the medium. For example, precursors can be added to the medium by feeding a feed medium containing the precursors to the medium. Alternatively, for example, by co-cultivating a microorganism capable of producing a target substance and a microorganism capable of producing a precursor, the microorganism capable of producing a precursor is caused to produce a precursor in the medium, thereby transferring the precursor to the medium. It can also be added. A "component" when referring to "adding a component to a medium" may include those that are produced or regenerated in the medium. These adding means may be used alone or in combination as appropriate. The precursor concentration 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, in terms of the weight of the free form, 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 exemplified above during the entire period of culture. For example, the precursor may be contained in the medium at the concentration exemplified above at the start of culture, or may be added to the medium so as to have the concentration exemplified above after the start of culture. When culture is performed separately for seed culture and main culture, the target substance may be produced at least during the period of main culture. Therefore, the precursor should be contained in the medium at least during the main culture, that is, during the entire main culture or part of the main culture. That is, the precursor may or may not be contained in the medium during seed culture. In such a case, descriptions about culture (eg, “culturing period (culturing period)” and “start of culture”) can be read as those about main culture.

In another embodiment, the bioconversion step can be carried out by using cells of a microorganism capable of producing the target substance. This aspect is also called "the second aspect of the bioconversion method". That is, the bioconversion step may be, for example, a step of converting a precursor of the target substance in the reaction solution into the target substance using cells of microorganisms. Specifically, the biotransformation step may be a step of reacting the precursor of the target substance in the reaction solution with the cells of microorganisms to produce and accumulate the target substance in the reaction solution. A biotransformation process carried out using such cells is also referred to as a "transformation reaction."

Microorganism cells are obtained by culturing microorganisms. The culture method for obtaining the microbial cells is not particularly limited as long as the microorganisms can grow. The precursor may or may not be contained in the medium during the culture for obtaining the cells. In addition, the target substance may or may not be produced in the medium during the culture for obtaining the cells. For the culture for obtaining the cells in the second aspect of the bioconversion method, the description of the culture in the fermentation method (for example, the description of the medium and culture conditions) can be applied mutatis mutandis.

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

The technique for recovering the cells from the culture solution is not particularly limited, and for example, a known technique can be used. Such techniques include, for example, natural sedimentation, centrifugation, and filtration. A flocculant may also be utilized. These techniques may be used alone or in combination as appropriate. The recovered cells can be appropriately washed using an appropriate medium. In addition, the collected cells can be appropriately resuspended using an appropriate medium. Examples of media that can be used for washing and suspension include aqueous media (aqueous solvents) such as water and aqueous buffers.

The treatment of the cells includes, for example, dilution, concentration, immobilization to a carrier such as acrylamide or carrageenan, freeze-thaw treatment, and treatment to increase membrane permeability. The permeability of the membrane can be enhanced using, for example, surfactants or organic solvents. These treatments may be used alone or in combination as appropriate.

Microbial 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 microbial cells have the ability to assimilate the carbon source and produce or regenerate the substances necessary for the production of the target substance. 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 a suitable reaction liquid. Specifically, the conversion reaction can be carried out by allowing the cells and precursors to coexist in an appropriate reaction solution. The conversion reaction may be performed batchwise or columnwise. In the case of a batch system, for example, the conversion reaction can be carried out by mixing microbial cells and precursors in a reaction solution in a reaction vessel. The conversion reaction may be carried out by standing, or may be carried out by stirring or shaking. In the case of a column system, for example, the conversion reaction can be carried out by passing a precursor-containing reaction solution through a column filled with immobilized cells. Examples of the reaction solution include aqueous media (aqueous solvents) such as water and aqueous buffer solutions.

In addition to the precursor, the reaction liquid may contain components other than the precursor, if necessary. Components other than precursors include ATP, electron donors such as NADH and NADPH, methyl group donors such as SAM, metal ions, buffers, surfactants, organic solvents, carbon sources, phosphate sources, and various other media. ingredients. That is, for example, a medium containing precursors may be used as the reaction solution. That is, the description of the medium in the first aspect of the bioconversion method can be applied mutatis mutandis to the reaction solution in the second aspect of the bioconversion method. The types and concentrations 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 bacterial cells to be used.

The conditions of the conversion reaction (dissolved oxygen concentration, pH of the reaction solution, reaction temperature, reaction time, concentrations of various components, etc.) are not particularly limited as long as the target substance is produced. The conversion reaction can be carried out, for example, under normal conditions used for material conversion using microbial cells such as stationary cells. The conditions for the conversion reaction may be appropriately set according to various conditions such as the type of microorganisms used. The conversion reaction may, for example, be carried out under aerobic conditions. “Aerobic conditions” may mean conditions under which the dissolved oxygen concentration in the reaction solution is 0.33 ppm or higher, or 1.5 ppm or higher. Specifically, the oxygen concentration is, for example,
It may be controlled to 1 to 50% or about 5% of the saturated oxygen concentration. The pH of the reaction solution may be, for example, generally 6.0-10.0, or 6.5-9.0. The reaction temperature may be, for example, generally 15-50°C, 15-45°C, or 20-40°C. The reaction time can 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, such a rate that the reaction time is within the range of the reaction times exemplified above. Moreover, the conversion reaction can also be carried out under culture conditions such as, for example, normal conditions used for culturing microorganisms such as bacteria and yeast. In the conversion reaction, the cells may or may not grow. That is, for the conversion reaction in the second aspect of the bioconversion method, the description of the culture in the first aspect of the bioconversion method applies mutatis mutandis, except that the cells may or may not grow in the same aspect. can. In such a case, the culture conditions for obtaining the cells and the conversion reaction conditions 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, or 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 any 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 course of the conversion reaction, cells, precursors, and other components may be added singly or in any combination to the reaction solution. For example, the precursor may be added to the reaction liquid according to the decrease or depletion of the precursor accompanying the production of the target substance. These ingredients may be added once or multiple times, or may be added continuously.

There are no particular restrictions on the means for adding various components such as precursors to the reaction solution. Any of these components can be added to the reaction solution by adding them directly to the reaction solution. Alternatively, for example, by co-cultivating a microorganism capable of producing a target substance and a microorganism capable of producing a precursor, the microorganism capable of producing a precursor is caused to produce a precursor in the reaction solution, thereby reacting the precursor. It can also be added to the liquid. In addition, for example, components such as ATP, electron donors, methyl group donors,
Any of them may be produced or regenerated in a reaction solution, may be produced or regenerated within the cells of a microorganism, or may be produced or regenerated by conjugation between different bacteria. For example, when metabolic activity is maintained in the cells of microorganisms, components such as ATP, electron donors, and methyl group donors can be produced or regenerated in the cells of microorganisms using carbon sources. For example, specifically, a microorganism may have an enhanced ability to produce or regenerate SAM, and SAM produced or regenerated by the same microorganism may be used in conversion reactions. Generation or regeneration of SAM can be further enhanced by combination with other techniques to generate or regenerate SAM. Methods for producing or regenerating ATP include, for example, a method of supplying ATP from a carbon source using bacteria of the genus Corynebacterium (Hori, H et al., Appl. Microbiol. Biotechnol. 48(6): 693-698 (1997)), a method for regenerating ATP using yeast cells and glucose (Yamamoto, S et
al., Biosci. Biotechnol. Biochem. 69(4): 784-789 (2005)), A method of regenerating ATP using phosphoenolpyruvate and pyruvate kinase (C. Aug'e and Ch. Gautheron, Tetrahedron Lett. 29:789-790 (1988)), a method for regenerating ATP using polyphosphate and polyphosphate kinase (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 include those that are produced or regenerated in the reaction solution.

Also, the reaction conditions may be uniform from the beginning to the end of the conversion reaction, or may change during the course of the conversion reaction. "The reaction conditions change during the conversion reaction" is not limited to temporal changes in the reaction conditions, and may include spatial changes in the reaction conditions. "The reaction conditions change spatially" means that, for example, when the conversion reaction is carried out in a column system, the reaction conditions such as reaction temperature and cell density vary depending on the position on the channel. You can

By carrying out the biotransformation step in this way, a culture solution (specifically, a medium) or a reaction solution containing the target substance can be obtained. Both confirmation of production of the target substance and recovery of the target substance can be carried out in the same manner as in the fermentation method described above. That is, the biotransformation method may further include a recovery step (for example, a step of recovering the target substance from the culture solution (specifically, medium) or reaction solution). In addition to the target substance, the recovered target substance may contain other components such as microbial cells, medium components, reaction liquid components, moisture, and metabolic by-products of the microorganism. The purity of the recovered target substance is, for example, 30% (w/w) or higher, 50% (w/w) or higher, 70% (w/w) or higher, 80% (w/w) or higher, 90% ( w/w) or higher, or 95% (w/w) or higher.

<2-3> Reduction of Ammonia Concentration In the method described in this specification (that is, the method for producing vanillin), the production of vanillin is carried out under conditions in which the ammonia concentration is reduced. That is, in the method described herein, the vanillin production step is carried out under conditions with reduced ammonia concentration.

Vanillin production is improved by producing vanillin under conditions with a reduced ammonia concentration. Specifically, by producing vanillin under conditions in which the ammonia concentration is reduced, vanillin production is improved compared to producing vanillin under conditions in which the ammonia concentration is not reduced. "Conditions under which the ammonia concentration is reduced" are also referred to as "ammonia reduction conditions". "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 the microorganisms used in the vanillin manufacturing process, by improving the vanillin production per cell of the microorganisms used in the vanillin manufacturing process, or by a combination thereof.

By "ammonia concentration" is meant the ammonia concentration in a medium or reaction solution (ie, in the case of vanillin production, the medium or reaction solution in which vanillin production is carried out). "Ammonia" is a generic term for ammonia molecules ( _NH3 ) and ammonium ions ₍ NH4 ⁺ ). That is _, the "ammonia concentration" specifically refers to the concentration and 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 the production of vanillin (that is, the ammonia concentration under 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.

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

The ammonia concentration (that is, the ammonia concentration under ammonia-reducing conditions) during production of vanillin is, for example, 0 mM or higher, 0.1 mM or higher, 0.3 mM or higher, 0.5 mM or higher, 0.7 mM or higher, 1 mM or higher. 5 mM or higher, 2 mM or higher, 2.5 mM or higher, 3 mM or higher, 3.5 mM or higher, 4 mM or higher, 4.5 mM or higher, 5 mM or higher, 5.5 mM or higher, 6 mM or higher, 6.5 mM or higher, 7 mM or higher, 7.5 mM or higher , 8 mM or more, 8.5 mM or more, 9 mM or more, 9.5 mM or more, or 10 mM or more; It may be 20 mM or less, or 10 mM or less, or any compatible combination thereof. Specifically, the ammonia concentration during the production of vanillin (that is, the ammonia concentration under ammonia-reducing conditions) may be, for example, 1 to 700 mM, 1 to 400 mM, or 1 to 100 mM.

The ammonia concentration may be reduced during the entire period of the vanillin manufacturing process, or may be reduced only during a part of the vanillin manufacturing 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 period of the fermentation process, or may be reduced only during a part of the period of the fermentation process. good. Further, for example, when the production of vanillin is carried out by bioconversion using microorganisms, the ammonia concentration may be reduced during the entire period of the bioconversion process, and is reduced only during a part of the bioconversion process. may be That is, "the production of vanillin or the process of producing vanillin is carried out under conditions with a reduced ammonia concentration" or "the production of vanillin or the process of producing vanillin is carried out in a medium or reaction solution with a reduced ammonia concentration" means that ammonia It is sufficient if the concentration is reduced during a part of the vanillin production process (e.g., fermentation process or bioconversion process), and the ammonia concentration is reduced during the entire vanillin production process (e.g., fermentation process or bioconversion process). not required to be

The ammonia concentration may be reduced to the concentration exemplified above during the entire period of the vanillin production process, or may be reduced to the concentration exemplified above 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 concentrations exemplified above during the entire period of the fermentation process, and only during a part of the period of the fermentation process. It may be reduced to the concentrations exemplified above. Further, for example, when the production of vanillin is carried out by bioconversion using microorganisms, the ammonia concentration may be reduced to the concentrations exemplified above during the entire period of the bioconversion process, and part of the bioconversion process It may be reduced to the concentration exemplified above only during the period. That is, "the production of vanillin or the process of producing vanillin is carried out under conditions in which 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 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 manufacturing process (e.g., fermentation process or bioconversion process), and the ammonia concentration is within the vanillin manufacturing process (e.g., fermentation process or bioconversion process). ) is not required to be within the range of concentration for the entire period.

"Part of the period" is not particularly limited as long as vanillin production is improved. 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. "Part of the 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 (e.g., fermentation process or bioconversion process) , 97% or greater, or 99% or greater. In addition, 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 hours or more, 70 hours or more, 100 hours or more, or 150 hours or more.

In addition, the ammonia concentration during the production of vanillin (that is, the ammonia concentration under 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 conditions in which 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 reaction solution having a certain ammonia concentration." By may mean that the average ammonia concentration is within the range of concentrations throughout the entire period of the vanillin manufacturing process. "Average ammonia concentration throughout the entire period of the vanillin manufacturing process" is not particularly limited as long as it reflects the change in ammonia concentration throughout the period of the vanillin manufacturing process, but for example, the entire period of the vanillin manufacturing process may mean the average ammonia concentration measured every 60 minutes, every 30 minutes, every 20 minutes, or every 10 minutes through

A means for reducing the ammonia concentration is not particularly limited. The ammonia concentration is, for example, to reduce the initial concentration of ammonia (i.e., the ammonia concentration in the medium or reaction solution at the start of the vanillin production process), the feed amount of ammonia (i.e., the medium or reaction after the start of the vanillin production process the amount of ammonia supplied to the liquid), or a combination thereof.

The initial concentration of ammonia in the production of vanillin (that is, the initial concentration of ammonia under reduced ammonia conditions) is, for example, 0 mM or higher, 0.1 mM or higher, 0.3 mM or higher, 0.5 mM or higher, 0.7 mM or higher, 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, 7.5 mM or higher, 8 mM or higher, 8.5 mM or higher, 9 mM or higher, 9.5 mM or higher, or 10 mM or higher, 700 mM or lower, 600 mM or lower, 500 mM or lower, 400 mM or lower, 300 mM or lower, 200 mM or lower, 100 mM or lower , 50 mM or less, 20 mM or less, or 10 mM or less, or any compatible combination thereof. Specifically, the initial concentration of ammonia during the production of vanillin (ie, the initial concentration of ammonia under ammonia-reducing conditions) 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 reduced ammonia conditions) is, for example, 300% or less, 250% or less, 200% or less, 150% or less 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 a molar ratio to the amount of carbon source used. 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 reduced ammonia conditions) is, for example, 300% or less, 250% or less, 200% or less as a 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 (i.e., the content of the component in the medium or reaction solution at the start of the vanillin manufacturing process) and the feed amount of the component (i.e., vanillin manufacturing amount of the component supplied to the medium or reaction solution after the start of the process). "Feed amount of a certain component" may particularly mean the fed amount of that component during the period from the start of the vanillin manufacturing process until sufficient vanillin is produced. "Sufficiently produced vanillin" 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 more. In addition, "sufficiently produced vanillin" means, for example, that the amount of vanillin produced 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 manufacturing process. may mean reaching an amount of

Ammonia concentration may be reduced, particularly by reducing the amount of ammonia used with the use of vanillin precursors. For example, when the vanillin precursor is used in the form of a composition that can contain ammonia, by reducing the amount of ammonia contained in the composition, the use of the vanillin precursor (specifically, the composition use) can reduce the amount of ammonia used. That is, by reducing the amount of ammonia contained in such a composition, the introduction of ammonia into the medium or reaction solution accompanying the use of the vanillin precursor (specifically, the use of the composition) is reduced. It is possible to reduce the amount of ammonia used. Such compositions include vanillin precursors produced using microorganisms. Specific examples of such compositions include materials containing vanillin precursors produced using microorganisms. Materials containing vanillin precursors are as described above. Specific examples of materials containing vanillin precursors include culture media or reaction solutions containing vanillin precursors, supernatants separated from the culture media or reaction solutions, concentrates thereof (e.g., concentrates), and dilutions. processed products such as products (for example, diluted solutions) and dried products. The content of vanillin precursors in such compositions is, for example, 1% (w/w) or more, 3% (w/w) or more, 5% (w/w) or more, 7% (w/w) 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 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% (w/w) below, 2
It may be 0% (w/w) or less, or 10% (w/w) or less, or any compatible combination thereof. The content of the vanillin precursor in such a composition is, for example, 1% (w/w) or more, 3% (w/w) or more as the amount of vanillin precursor relative to the total amount of the composition excluding water. , 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% (w/w) or less 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 combination that does not contradict.

The vanillin precursor may be produced in a form with a reduced ammonia content. The vanillin precursor may in particular be produced in the form of a composition with a reduced content of ammonia. As for the method for producing the vanillin precursor in a form with a reduced ammonia content, the method described herein (that is, the method for producing vanillin) can be applied mutatis mutandis. That is, the production of the vanillin precursor may be carried out under conditions with a reduced ammonia concentration. "Ammonia concentration" means the concentration of ammonia in a medium or reaction solution (that is, in the case of production of vanillin precursors, the medium or reaction solution in which production of vanillin precursors is carried out). By producing the vanillin precursor under conditions where the ammonia concentration is reduced, it is possible to produce the vanillin precursor in a form in which the content of ammonia is reduced, thereby reducing the amount of ammonia used in the use of the vanillin precursor. can be reduced. In other words, the ammonia concentration during production of vanillin may be reduced by at least producing the vanillin precursor under conditions in which the ammonia concentration is reduced. For example, the amount of ammonia that can be used as a nitrogen source during the production of vanillin precursors may be reduced. Also, for example, the amount of ammonia that may be used for pH adjustment during the production of vanillin precursors may be reduced. pH adjustment includes neutralization of protocatechuic acid and/or vanillic acid. Specifically, for example, when protocatechuic acid is used as a raw material to produce vanillic acid, the amount of ammonia that can be used to neutralize protocatechuic acid and/or vanillic acid may be reduced. In particular, when protocatechuic acid is used as a raw material to produce vanillic acid, the amount of ammonia that can be used to neutralize protocatechuic acid may be reduced. Specifically, for example, when protocatechuic acid is used as a raw material to produce protocatechualdehyde, the amount of ammonia that can be used to neutralize 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, can be partially or completely controlled by a pH adjusting substance other than ammonia (e.g., sodium hydroxide or potassium hydroxide). It can be reduced by using the above-exemplified medium pH adjusting substances 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 such that some or all of the ammonia that can be used for pH adjustment is replaced by other pH adjusting substances (e.g., It can be reduced by substituting with a substance other than ammonia, such as sodium hydroxide or potassium hydroxide, for adjusting the pH of the culture medium. Ammonia that can be used for pH adjustment includes ammonia gas and ammonia water.

The content of ammonia in the above composition (for example, a material containing a vanillin precursor) is not particularly limited as long as the ammonia concentration can be reduced during the production of vanillin. The content of ammonia in the above composition (for example, a material containing a vanillin precursor) is, for example, 300% or less, 250% or less, 200% or less, as a molar ratio to the content of 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.

The present invention will now be described in more detail with reference to non-limiting examples.

Example: Vanillin production from vanillic acid under ammonia-limited conditions (1) Analysis conditions Vanillin was quantified by HPLC analysis. Analysis conditions are as shown below.
Mobile phase A: 0.1% phosphoric acid Mobile phase B: Acetonitrile Flow rate: 1.0 ml/min
Column temperature: 40℃
Detection: UV 210 nm
Column: Cadenza CD-C18, 4.6 x 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 using BF-7 (Oji Scientific Instruments). Analysis conditions are as shown below.
Mobile phase: BF buffer pH 7.0 (Oji Scientific Instruments) with α-ketoglutarate at a final concentration of 1 mM, NADH
was added to a final concentration of 1 mM Flow rate: 1 mL/min
Time: 90 seconds
Temperature: 37°C
Electrode: Glutamic acid electrode, ammonia electrode (Oji Scientific Instruments)
Remarks: Since the ammonia electrode also measures glutamic acid, the ammonia concentration was calculated by subtracting the glutamic acid concentration after measuring the glutamic acid concentration with the glutamic acid electrode.

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

(2) Construction of vanillin-producing strain Plasmid pVK9::Ptuf*-Ge_ACAR-entD (Example <3> of WO2018/079705) was transferred to Corynebacterium glutamicum FKFC14 (C. glutamicum 2256ΔvanABKΔNCgl0324ΔNCgl0313ΔNCgl2709; Example <2> of WO2018/079705). It was introduced by the electroporation method. FKFC14 is a strain deficient in vanillate demethylase and vanillic acid uptake system genes (vanABK) and alcohol dehydrogenase genes (NCgl0324, NCgl0313, NCgl2709) constructed using C. glutamicum 2256 (ATCC 13869) as the parent strain. Plasmid pVK9::Ptuf*-Ge_ACAR-entD is a co-expression plasmid of Gordonia effusa-derived aromatic carboxylic acid reductase (ACAR) and E. coli-derived phosphopantetheinyl transferase (PPT). Cells were plated on a CM-Dex SGFC plate (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, _FeSO4.7H2O 0.01 g/L, _MnSO4.7H2O 0.01 g/L, disodium succinate _{6H2O 2} g/L, sodium gluconate ₄ g/L, urea 3 g /L, biotin 10 μg/L, hydrolyzed soybean hydrolyzate (as total nitrogen) 1.2 g/L, adjusted to pH 7.5 with NaOH, kanamycin 25 mg/L, Agar 15 g/L) at 31.5°C did. The obtained colony was purified on a CM-Dex SGFC plate and designated as 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, and 0.9 mL of the culture medium was diluted to 0.6 mL.
50% glycerol and stored at -80°C as a glycerol stock.

(3) Vanillin production from vanillic acid
FKFC14/pVK9::Ptuf*-Ge_ACAR-entD was converted to CM-Dex-Km (glucose 5 g/L, polypeptone 10 g/L, yeast extract ₁₀ g/L, _{KH2PO4 1} g/L, MgSO4 7H _2O 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 μg/L, soybean hydrochloric acid hydrolyzate (total nitrogen as) 1.2 g/L, adjusted to pH 7.5 with KOH, kanamycin 25 mg/L, Agar 20 g/L) and cultured on a plate at 20°C for 3 days. CM-Dex-Glc10 medium containing 25 mg/L kanamycin (glucose 10 g/L, polypeptone 10 g/L, yeast extract 10 g/L, KH ₂ PO ₄ 1 g/L, MgSO ₄
・_7H2O 0.4 g/L, urea 3 g/L, _FeSO4・_7H2O 0.01 g/L, _MnSO4・_7H2O 0.01 g/L, biotin 10 μg/L, soybean hydrolyzate ( (as total nitrogen) 1.2 g/L, adjusted to pH 7.5 with KOH) 50 mL
and cultured with shaking at 31.5°C for 18 hours using a Sakaguchi flask. 25 mL of the resulting culture medium was centrifuged, the supernatant was removed, and the suspension was suspended in a 0.85% NaCl solution to an OD 600 nm of 15 to obtain a cell suspension. Add 1 mL of the cell suspension to 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, _{KH2PO4 1} g/L, MgSO4・_{7H2O 0.4} g/L, _FeSO4・_7H2O 0.01 g/L, _MnSO4
- _7H2O 0.01 g/L, biotin 10 µg/L, adjusted to pH 7.4 with KOH, kanamycin 25 mg/L), and cultured at 30°C for 48 hours. 8 μL of the culture medium at the start of culture was mixed with 1 mL of ultrapure water, and the ammonia concentration was measured. In addition, 10 μL of the culture medium after 48 hours of culture was mixed with 1 mL of culture termination solution (1% phosphoric acid, 50% ethanol), and the filtered filtrate was subjected to HPLC analysis. In addition, 20 μL of the culture medium after 48 hours of culture was mixed with 1 mL of ultrapure water, and the OD was measured. Table 1 shows the amount of NH ₄ Cl and (NH ₄ ) ₂ SO ₄ added, the ammonia concentration at the start of the culture, the OD after 48 hours of culture, and the amount of vanillin produced.

Improvements in growth and vanillin production were observed as the amount of ammonium salt added decreased. In particular, when the NH ₄ ⁺ addition concentration was 50-600 mM, an increase in vanillin production per OD was observed as compared to 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 kind of ammonium salt, it is considered that the improvement of growth and the improvement of vanillin production are due to the reduction of ammonia concentration.

<Description of Sequence Listing>
SEQ ID NO: 1: nucleotide sequence of ACAR gene of Gordonia effusa SEQ ID NO: 2: amino acid sequence of ACAR protein of Gordonia effusa SEQ ID NO: 3: nucleotide sequence of entD gene of Escherichia coli MG1655 SEQ ID NO: 4: amino acid sequence of EntD protein of Escherichia coli MG1655 SEQ ID NO: 5: base sequence of vanK gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 6: amino acid sequence of VanK protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 7: base of vanA gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 8: amino acid sequence of VanA protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 9: base sequence of vanB gene of Corynebacterium glutamicum 2256 (ATCC 13869) Amino acid sequence SEQ ID NO: 11: base sequence of NCgl0324 gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 12: amino acid sequence of NCgl0324 protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 13: NCgl0313 gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 14: amino acid sequence of NCgl0313 protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 15: nucleotide sequence of NCgl2709 gene of Corynebacterium glutamicum 2256 (ATCC 13869) amino acid sequence of protein

Claims (22)

A method for producing vanillin, comprising:
including producing vanillin using a microorganism capable of producing vanillin,
The method, wherein the production is carried out under conditions with reduced ammonia concentration. 2. The method according to claim 1, wherein the ammonia concentration during the production is 700 mM or less. 3. The method according to claim 1, wherein the concentration of ammonia during the production is 400 mM or less. The method according to any one of claims 1 to 3, wherein the ammonia concentration during the production is 100 mM or less. 5. The method of any one of claims 1-4, wherein said producing comprises converting a vanillin precursor into said vanillin utilizing said microorganism. 6. The method of claim 5, wherein said converting comprises culturing said microorganism in a medium containing said precursor to produce and accumulate vanillin in said medium. 6. The method according to claim 5, wherein said conversion includes allowing cells of said microorganism to act on said precursor in a reaction solution to produce and accumulate vanillin in said 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 collected from the culture solution, processed products thereof, or a combination thereof. A method according to any one of claims 5 to 8, wherein said precursor is vanillic acid. The method according to any one of claims 5 to 9, wherein said precursor is produced using a microorganism capable of producing said precursor. The method according to any one of claims 5 to 10, further comprising, prior to the production of vanillin, producing the precursor using a microorganism capable of producing the precursor. 12. A method according to claim 10 or 11, wherein the preparation of the precursor is carried out under conditions with reduced ammonia concentration. The precursor is used in the form of a material containing the precursor,
13. Any one of claims 10 to 12, wherein the material is a culture medium or reaction solution containing the precursor, a supernatant separated from the culture medium or reaction solution, a processed product thereof, or a combination thereof. The method described in . 14. The method of claim 13, wherein the content of ammonia in said material is less than or equal to 300% as a molar ratio to the content of said precursor in said material. 5. The method according to any one of claims 1 to 4, wherein the production of vanillin comprises culturing a microorganism capable of producing vanillin in a medium containing a carbon source to produce and accumulate vanillin in the medium. described method. 16. The method of any one of claims 1-15, further comprising recovering vanillin. The method according to any one of claims 1 to 16, wherein said microorganism capable of producing vanillin is a bacterium or yeast. 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 the Enterobacteriaceae family. 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. The method according to any one of claims 1 to 19, wherein the microorganism capable of producing vanillin is Corynebacterium glutamicum or Escherichia coli. 21. Any one of claims 1 to 20, wherein the microorganism capable of producing vanillin has been modified such that the activity of aromatic carboxylic acid reductase and/or phosphopantetheinyltransferase is increased compared to an unmodified strain. or the method according to item 1. 22. The microorganism according to any one of claims 1 to 21, wherein the microorganism capable of producing vanillin has been modified such that vanillate demethylase and/or alcohol dehydrogenase activity is reduced compared to an unmodified strain. the method of.