JP2020031573A - Method for maintaining plasmid in microorganisms - Google Patents

Abstract

To provide a method for maintaining plasmid in microorganisms.SOLUTION: Decrease in an assimilation ability of sugar and compensation therefor with plasmid are combined, so that sugar functions as selection pressure, and plasmid can be maintained in microorganisms.SELECTED DRAWING: None

Description

  The present invention relates to a method for maintaining a plasmid in a microorganism and its use.

  In the field of biotechnology, a technique for imparting a desired phenotype to a microorganism by introducing a plasmid into the microorganism is widely used.

  In order to maintain the introduced plasmid in the microorganism, antibiotics are typically used as selective pressure. However, the use of antibiotics is problematic in terms of cost and the like.

  Therefore, a technique for maintaining a plasmid in a microorganism by selective pressure instead of an antibiotic is desired. Such techniques include, for example, a method of complementing a phenotypic defect such as auxotrophy with a gene on a plasmid (Patent Literatures 1 to 6), or a method using a medium containing only phosphorous acid as a phosphorus source. A method of culturing a microorganism into which a plasmid containing an acid dehydrogenase gene has been introduced (Patent Document 7) is known.

  It is also known that in bacteria such as Escherichia coli and Corynebacterium glutamicum, by disrupting genes encoding PTS (phosphotransferase system) and glycolytic enzymes, growth using sugar as a carbon source is known to be reduced ( Non-Patent Documents 1 to 6).

JP 6-086669 JP 7-067685 Special table 2004-517624 Special table 2004-524817 JP2006-067884 Special table 2008-509677 JP 2017-195907

Lovingshimer MR, et al., Construction of an inducible, pfkA and pfkB deficient strain of Escherichia coli for the expression and purification of phosphofructokinase from bacterial sources.Protein Expr Purif. 2006 Apr; 46 (2): 475-82. Siedler S, et al., Reductive whole-cell biotransformation with Corynebacterium glutamicum: improvement of NADPH generation from glucose by a cyclized pentose phosphate pathway using pfkA and gapA deletion mutants.Appl Microbiol Biotechnol. 2013 Jan; 97 (1): 143-52 . Fong SS, et al., Latent pathway activation and increased pathway capacity enable Escherichia coli adaptation to loss of key metabolic enzymes.J Biol Chem. 2006 Mar 24; 281 (12): 8024-33. Lindner SN, et al., Phosphotransferase system-independent glucose utilization in corynebacterium glutamicum by inositol permeases and glucokinases.Appl Environ Microbiol. 2011 Jun; 77 (11): 3571-81. Lindner SN, et al., Cg2091 encodes a polyphosphate / ATP-dependent glucokinase of Corynebacterium glutamicum.Appl Microbiol Biotechnol. 2010 Jun; 87 (2): 703-13. Moon MW, et al., Analyses of enzyme II gene mutants for sugar transport and heterologous expression of fructokinase gene in Corynebacterium glutamicum ATCC 13032.FEMS Microbiol Lett. 2005 Mar 15; 244 (2): 259-66.

  An object of the present invention is to provide a method for maintaining a plasmid in a microorganism.

  The present inventors have found that techniques using selective pressure instead of antibiotics as described above require strict control of the medium composition, and have been successfully used in culture systems using semi-synthetic or natural media, for example. Found it difficult to function. On the other hand, the present inventors have found that a sugar used as a carbon source can be used as selective pressure for maintaining a plasmid in a microorganism, and have completed the present invention.

That is, the present invention can be exemplified as follows.
[1]
A method of maintaining a plasmid in a microorganism, comprising:
Culturing a microorganism having a plasmid in a medium containing a sugar,
The microorganism has been modified such that the assimilation ability of the sugar is reduced as compared to an unmodified strain,
The method, wherein the plasmid comprises a first nucleotide sequence that complements the reduced assimilation ability.
[2]
A method for producing a target substance,
Culturing a microorganism having a target substance-producing ability and a plasmid in a medium containing a sugar to produce the target substance,
The microorganism has been modified such that the assimilation ability of the sugar is reduced as compared to an unmodified strain,
A method, wherein the plasmid comprises a first nucleotide sequence that complements the reduced assimilation ability and a second nucleotide sequence that is effective for producing the target substance.
[3]
The above method, wherein the medium further comprises a precursor of the target substance.
[4]
A method for producing a target substance,
Culturing a microorganism having the plasmid with the target substance-producing ability in a medium containing the sugar to produce cells having the plasmid; and converting the precursor of the target substance into the target substance using the cells. Including the step of
The microorganism has been modified such that the assimilation ability of the sugar is reduced as compared to an unmodified strain,
A method, wherein the plasmid comprises a first nucleotide sequence that complements the reduced assimilation ability and a second nucleotide sequence that is effective for producing the target substance.
[5]
The method further comprising recovering the target substance.
[6]
The above method, wherein the assimilation ability is reduced due to a decrease in the activity of the protein P1 involved in the assimilation of the sugar.
[7]
The above method, wherein the activity of the protein P1 is reduced by reducing the expression of a gene encoding the protein or by disrupting the gene.
[8]
The above method, wherein the activity of the protein P1 has been reduced by deletion of part or all of the amino acid sequence of the protein.
[9]
The above method, wherein the first base sequence is a gene encoding a protein P2 involved in the assimilation of the sugar.
[10]
The above method, wherein the proteins P1 and P2 are proteins selected from the sugar uptake system and the sugar metabolizing enzyme, respectively.
[11]
The above method, wherein the proteins P1 and P2 are each a protein selected from glycolytic enzymes.
[12]
The above method, wherein the proteins P1 and P2 are each 6-phosphofructokinase.
[13]
The method, wherein the 6-phosphofructokinase is a protein described in the following (a), (b), or (c), respectively:
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 38, 72, or 74;
(B) an amino acid sequence represented by SEQ ID NO: 38, 72 or 74, which comprises an amino acid sequence containing substitution, deletion, insertion and / or addition of 1 to 10 amino acid residues, and A protein having an actokinase activity;
(C) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 38, 72, or 74, and having 6-phosphofructokinase activity.
[14]
The above method, wherein the second nucleotide sequence is a gene selected from a gene encoding a protein capable of imparting or enhancing a target substance producing ability by increasing activity and a gene encoding the target substance.
[15]
The above method, wherein the protein capable of imparting or enhancing the target substance-producing ability by increasing the activity is a biosynthetic enzyme of the target substance.
[16]
The above method, wherein the second nucleotide sequence is a gene encoding aromatic carboxylate reductase.
[17]
The above method, wherein the target substance is selected from SAM-dependent metabolites, aldehydes, L-amino acids, nucleic acids, organic acids, γ-glutamyl peptides, sphingoids, proteins, and RNA.
[18]
The target substance is selected from vanillin, benzaldehyde, cinnamaldehyde, vanillic acid, melatonin, ergothioneine, mugineic acid, ferulic acid, polyamine, guaiacol, 4-vinylguaiacol, 4-ethylguaiacol, creatine, L-methionine; Method.
[19]
The precursor is selected from protocatechuic acid, protocatechualdehyde, vanillic acid, benzoic acid, cinnamic acid, L-tryptophan, L-histidine, L-phenylalanine, L-tyrosine, L-arginine, L-ornithine, glycine. , Said method.
[20]
The above method, wherein the target substance is vanillic acid.
[21]
The above method, wherein the target substance is vanillin, and the precursor is vanillic acid.
[22]
The above method, wherein the sugar is glucose or sucrose.
[23]
The above method, wherein the microorganism is a bacterium or a yeast.
[24]
The above method, wherein the microorganism is a coryneform bacterium or a bacterium of the family Enterobacteriaceae.
[25]
The above method, wherein the microorganism is a bacterium belonging to the genus Corynebacterium or a bacterium belonging to the genus Escherichia.
[26]
The above method, wherein the microorganism is Corynebacterium glutamicum or Escherichia coli.
[27]
The above method, wherein the medium is substantially free of antibiotics.

  According to the present invention, a plasmid can be maintained in a microorganism. Further, in one aspect, the present invention enables production of a target substance by maintaining a plasmid in a microorganism.

The figure which shows the construction procedure of plasmid pBS4S (DELTA) rnc. Agarose gel electrophoresis diagram (photo) for confirmation of rnc gene deletion. The figure which shows the construction procedure of plasmid pVC7-sacB. The figure which shows the construction procedure of plasmid pBS4S (DELTA) pfkA. Agarose gel electrophoresis diagram for confirming pfkA gene deletion (photograph). The figure which shows the construction procedure of plasmid pVC7-Pf1-Hv-iap. The figure which shows the construction procedure of plasmid pVC7-Pf1-Hv-iap-Pf1rev. The figure which shows the construction procedure of plasmid pVC7H2-Pf1-Hviap-Pf1rev-Pbl-pfkA, pVC7H2-Pf1-Hviap-Pf1rev-PmsrA-pfkA, and pVC7H2-Pf1-Hviap-Pf1rev-Plac-pfkA. Electropherogram (photograph) showing the results of dsRNA production in a culture system without antibiotics. The figure which shows the construction procedure of plasmid pPK4-Pbl-pfkA-dGFP and pPK4-Plac-pfkA-dGFP. FIG. 4 shows the results of GFP production in a culture system without antibiotics (photograph).

  Hereinafter, the present invention will be described in detail.

  In the methods described herein, the plasmid can be maintained in the microorganism using sugar as the selective pressure. That is, the method described herein is a method of maintaining a plasmid in a microorganism by using sugar as a selective pressure.

The phrase "plasmid is maintained in a microorganism" means that the plasmid is maintained in the microorganism during culture of the microorganism having the plasmid. That is, "the plasmid is maintained in the microorganism" may, in other words, mean that the microorganism grows (proliferates) while retaining the plasmid, and that the culturing produces bacterial cells having the plasmid. It may mean. Plasmid maintenance may be due, for example, to the ability of cells that retain the plasmid to grow preferentially over cells that have lost the plasmid. Maintaining the plasmid in the microorganism is also referred to as "plasmid stabilization". The degree of maintenance of the plasmid is not particularly limited as long as it is allowable according to various conditions such as the purpose of culture. "The plasmid is maintained in the microorganism" means, specifically, for example, when a microorganism having the plasmid is cultured, the ratio of the cells holding the plasmid in the cultured cells is 50% or more, It may mean 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, 99% or more, 99.5% or more, 99.7% or more, or 99.9% or more. The phrase "plasmid is maintained in a microorganism" specifically means that, for example, when a microorganism having a plasmid is cultured, the average copy number of the plasmid per cell in the cultured cells is determined before culture. 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more of the average number of copies of the plasmid per cell in the cells (ie, the cells at the time of inoculation) , 99% or more, 99.5% or more, 99.7% or more, or 99.9% or more.

  The phrase "plasmid is maintained in a microorganism using sugar as a selective pressure" means that the plasmid is maintained in the microorganism depending on the presence of the sugar. That is, "the plasmid is maintained in the microorganism with the sugar as the selective pressure" may specifically mean that the plasmid is maintained when the microorganism is cultured in the presence of the sugar. More specifically, "plasmid is maintained in a microorganism using sugar as a selective pressure" means, more specifically, that the plasmid is not maintained when the microorganism is cultured in the absence of sugar (i.e., "the plasmid is maintained in the microorganism. May not mean that the plasmid is maintained when the microorganism is cultured in the presence of sugar. More specifically, "plasmid is maintained in a microorganism using sugar as a selective pressure" means, more specifically, that when a microorganism is cultured in the absence of sugar, the plasmid is not maintained without using another selective pressure (ie, Does not satisfy the condition that “plasmid is maintained in microorganism”), but means that the plasmid is maintained without using any other selective pressure when the microorganism is cultured in the presence of sugar. Is also good.

  In the method described herein, specifically, the combination of reduced sugar assimilation and its complementation by a plasmid allows the sugar to function as a selective pressure, thereby maintaining the plasmid in the microorganism. Can be. That is, as the microorganism, a microorganism modified so as to reduce the assimilation ability of sugar can be used. As the plasmid, those having a base sequence (first base sequence) that complements the reduced ability to assimilate sugar can be used.

That is, the method described herein may be specifically the following method:
A method of maintaining a plasmid in a microorganism, comprising:
Culturing a microorganism having a plasmid in a medium containing a sugar,
The microorganism has been modified such that the assimilation ability of the sugar is reduced as compared to an unmodified strain,
The method, wherein the plasmid comprises a first nucleotide sequence that complements the reduced assimilation ability.

  In addition, when the microorganism has a target substance-producing ability, the target substance can be produced using the method described in the present specification. That is, when the microorganism has the target substance-producing ability, the target substance can be produced by culturing as described above or by utilizing the cells obtained by the culturing as described above. That is, one embodiment of the method described in this specification is a method for producing a target substance. When the target substance is produced for producing the target substance, a plasmid further having a base sequence (second base sequence) effective for producing the target substance can be used.

That is, one embodiment of the method for producing the target substance may be the following method:
A method for producing a target substance,
Culturing a microorganism having a target substance-producing ability and a plasmid in a medium containing a sugar to produce the target substance,
The microorganism has been modified such that the assimilation ability of the sugar is reduced as compared to an unmodified strain,
A method, wherein the plasmid comprises a first nucleotide sequence that complements the reduced assimilation ability and a second nucleotide sequence that is effective for producing the target substance.

Further, one embodiment of the method for producing the target substance may be the following method:
A method for producing a target substance,
Culturing a microorganism having the plasmid with the target substance-producing ability in a medium containing the sugar to produce cells having the plasmid, and
The microorganism has been modified such that the assimilation ability of the sugar is reduced as compared to an unmodified strain,
A method, wherein the plasmid comprises a first nucleotide sequence that complements the reduced assimilation ability and a second nucleotide sequence that is effective for producing the target substance.

  The above microorganism is also referred to as “the microorganism described in this specification”. The above plasmid is also referred to as “plasmid described in this specification”.

<1> Microorganism The microorganism described in the present specification is a microorganism having the plasmid described in the present specification, which is modified so as to reduce the ability to assimilate sugar.

  The microorganism described herein may further have any other property (for example, modification) as long as the plasmid can be maintained using sugar as a selective pressure. The microorganism may or may not have a target substance-producing ability, for example. Further, the microorganism may or may not have auxotrophy such as amino acid requirement. Also, the microorganism may or may not have, for example, another plasmid (ie, a plasmid other than the plasmids described herein).

  The microorganisms described in the present specification can be constructed by modifying the following microorganisms, such as reducing the ability to assimilate sugar and introducing the plasmids described in the present specification. That is, the microorganism described in the present specification may be a modified strain derived from the following microorganism. Modifications for constructing microorganisms can be performed in any order. For example, a microorganism may be modified to reduce its ability to assimilate sugar, and then the plasmid described herein may be introduced. The microorganism described in this specification or a parent strain for constructing the microorganism is also referred to as a “host”.

<1-1> Microorganism used as parent strain The microorganism used as a parent strain for constructing the microorganism described in this specification is not particularly limited. Microorganisms include bacteria and yeast.

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

  Examples of bacteria belonging to the Enterobacteriaceae family include the genus Escherichia, the genus Enterobacter, the genus Pantoea, the genus Klebsiella, the genus Serratia, the genus Erwinia, and the photolabdas. Bacteria belonging to the genus (Photorhabdus), the genus Providencia, the genus Salmonella, the genus Morganella, and the like. Specifically, 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 Enterobacteriaceae family can be used.

Examples of the bacterium belonging to the genus Escherichia include, but are not particularly limited to, bacteria classified into the genus Escherichia according to the classification known to experts in microbiology. Examples of bacteria belonging to the genus Escherichia include, for example, the book by 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 / Second Edition, American Society for Microbiology Press, Washington, DC) Described in the above. Examples of the bacterium belonging to the genus Escherichia include, for example, Escherichia coli. Specific examples of Escherichia coli include, for example, Escherichia coli K-12 strains such as W3110 strain (ATCC 27325) and MG1655 strain (ATCC 47076); Escherichia coli K5 strain (ATCC 23506); BL21 (DE3) strain Escherichia coli B strain; and derivatives thereof.

  Examples of the Enterobacter bacteria include, but are not particularly limited to, bacteria classified into the genus Enterobacter according to classifications known to experts in microbiology. Examples of the Enterobacter bacteria include, for example, 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, for example, those described in European Patent Application Publication No. EP0952221. Some Enterobacter agglomerans are classified as Pantoea agglomerans.

  Examples of the Pantoea bacteria include, but are not particularly limited to, bacteria classified into Pantoea by a classification known to experts in microbiology. Examples of the Pantoea bacteria include, for example, Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Specific examples of Pantoea ananatis include Pantoea ananatis LMG20103, AJ13355 (FERM BP-6614), AJ13356 (FERM BP-6615), AJ13601 (FERM BP-7207), and SC17 (FERM BP-7207). -11091), SC17 (0) strain (VKPM B-9246), and SC17sucA strain (FERM BP-8646). Some Enterobacter and Erbinia bacteria have been reclassified as Pantoea (Int. J. Syst. Bacteriol., 39, 337-345 (1989); Int. J. Syst. Bacteriol. , 43, 162-173 (1993)). For example, certain 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 that have thus been reclassified into the genus Pantoea.

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

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

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

Specific examples of 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 ammoniaphilum ATCC 15354

  Bacteria of the genus Corynebacterium, which were previously classified into the genus Brevibacterium, also include bacteria that are now integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1991)). included. Corynebacterium stationis, which was previously classified as Corynebacterium ammoniagenes, also includes bacteria that have been reclassified as Corynebacterium stationis by 16S rRNA nucleotide sequence analysis or the like (Int. J Syst. Evol. Microbiol., 60, 874-879 (2010)).

  The yeast may be a budding yeast or a fission yeast. The yeast may be a haploid yeast, a diploid or higher ploidy yeast. Examples of yeast include Saccharomyces such as Saccharomyces cerevisiae; Pichia ciferrii; ), Yeast belonging to the genus Candida such as Candida utilis, the genus Hansenula such as Hansenula polymorpha, the genus Schizosaccharomyces such as Schizosaccharomyces pombe. Is mentioned.

  These strains can be obtained, for example, from the American Type Culture Collection (address 12301 Parklawn Drive, Rockville, Maryland 20852 P.O. Box 1549, Manassas, VA 20108, United States of America). That is, a registration number corresponding to each strain is assigned, and the strain can be ordered using this registration number (see http://www.atcc.org/). The registration number corresponding to each strain is described in the catalog of the American Type Culture Collection. These strains can be obtained, for example, from a depository institution where the strains are deposited.

<1-2> Decrease in assimilation ability of sugar The microorganism described in the present specification has been modified so as to decrease assimilation ability of sugar. The microorganism is specifically modified so that the ability to assimilate sugar is reduced as compared to the unmodified strain. “Sugar assimilation ability” may mean the ability to grow using sugar. In microorganisms, the assimilation ability of one kind of sugar may be reduced, or the assimilation ability of two or more kinds of sugars may be decreased. In the microorganism, at least the assimilation ability of the sugar used as the selective pressure may be reduced.

  There is no particular limitation on the degree of decrease in sugar assimilation ability as long as the plasmid can be maintained using sugar as a selective pressure. The ability to assimilate the sugar may or may not be completely deficient. The decrease in sugar assimilation ability can be measured, for example, as a decrease in growth when a microorganism is cultured using only sugar as a carbon source. The term “sugar assimilation ability is reduced” is specifically defined as, for example, a modified strain when cultured in a liquid minimal medium containing only sugar as a carbon source (that is, as the sugar assimilation ability is reduced. The specific growth rate of the modified strain is 50% or less, 40% or less, 30% or less, and 20% or less of the non-modified strain (that is, the strain before being modified so as to reduce sugar assimilation ability). , 10% or less, 5% or less, 2% or less, or 1% or less. In addition, the term “sugar assimilation ability is reduced” specifically means that, for example, when cultured in a minimal medium containing only sugar as a carbon source (for example, a liquid minimal medium or a solid minimal medium), It may mean that the strain is able to grow (ie, grow), but the modified strain is unable to grow (ie, grow). In addition, since the reduced assimilation ability of sugar is complemented by the plasmid described in the present specification, whether or not the assimilation ability of sugar is reduced is determined in the absence of the plasmid. That is, the term "sugar assimilation ability" in the case of "sugar assimilation ability is reduced" means the assimilation ability of sugar in the absence of the plasmid described in this specification. In other words, the trait that “the ability to assimilate sugar is reduced” is the trait of the host in the absence of the plasmid described herein.

The method for reducing the ability to utilize sugar is not particularly limited as long as the ability to utilize sugar decreases to a desired degree. The ability to utilize sugar can be reduced, for example, by reducing the activity of a protein involved in the utilization of sugar. That is, the microorganism may be modified so that, for example, the activity of a protein involved in sugar utilization is reduced. The microorganism may be specifically modified so that the activity of a protein involved in assimilation of sugar is reduced as compared with an unmodified strain. The technique for reducing the activity of the protein will be described later. The activity of a protein involved in sugar utilization can be reduced, for example, by disrupting a gene encoding the protein. Proteins involved in sugar utilization are also referred to as “sugar utilization proteins”. Genes encoding proteins involved in sugar utilization are also referred to as “sugar utilization genes”. For the convenience of distinguishing a saccharide-assimilating protein whose activity is decreased in order to reduce the saccharide assimilation ability from a protein P2 (a saccharide-assimilating protein encoded by the first base sequence) described below, “protein P1” is used. Also called. In a microorganism, the activity of one kind of glycosimilating protein may be reduced, or the activity of two or more kinds of glycosimilating proteins may be reduced.

  Hereinafter, the sugar utilization gene and the sugar utilization protein will be described. In the following description, in addition to the description of the gene or protein that is modified to reduce the ability to utilize sugar, the description of the gene or protein that is used to complement the reduced ability to utilize sugar is described. Doubles. The protein whose activity is reduced in the microorganism is selected from proteins possessed by a non-modified strain (that is, a strain before being modified so as to reduce sugar assimilation ability). Examples of the protein possessed by the unmodified strain include a protein encoded by a gene present on the chromosome of the unmodified strain.

  Examples of the sugar assimilation gene and the sugar assimilation protein include those of various organisms such as the microorganisms exemplified above. That is, specific examples of the saccharide assimilating gene and the saccharide assimilating protein include bacteria of the family Enterobacteriaceae (eg, bacteria belonging to the genus Escherichia such as E. coli) and coryneform bacteria (eg, C. glutamicum). Corynebacterium genus). In addition, examples of the sugar utilization gene and the sugar utilization protein include those of organisms individually exemplified below. The nucleotide sequences of saccharide-utilizing genes derived from various organisms and the amino acid sequences of saccharide-utilizing proteins encoded by them can be obtained from public databases such as NCBI and technical documents such as patent documents.

  Examples of the sugar-assimilating protein include proteins constituting a sugar-assimilating pathway. That is, by lowering the activity of the protein constituting the sugar assimilation pathway, the sugar assimilation pathway can be weakened, and thus the sugar assimilation ability can be reduced. When the microorganism has two or more assimilation pathways for a certain sugar, in order to reduce the assimilation ability of the sugar, one of the assimilation pathways selected from those assimilation pathways is weakened. Alternatively, two or more (eg, all) assimilation routes selected from those assimilation routes may be weakened. When a certain utilization pathway is composed of two or more proteins, the activity of one protein selected from those proteins may be reduced in order to weaken the utilization pathway. The activity of two or more (eg all) proteins selected from the proteins may be reduced.

  Proteins constituting the sugar utilization pathway include sugar uptake systems and sugar metabolizing enzymes.

Examples of the sugar uptake system include PTS (phosphotransferase system). The “PTS (phosphotransferase system)” may mean a system having an activity of phosphorylating a sugar using PEP (phosphoenolpyruvate) as a phosphate donor and transporting the sugar into a cell. This activity is also called “PTS activity”. For example, glucose, sucrose, and fructose may be converted to glucose-6-phosphate, sucrose-6-phosphate, and fructose-1-phosphate, respectively, and transported into cells. PTS consists of three proteins: Enzyme I (EI), Histidine-phosp
It may be composed of horylatable protein (HPr) and Enzyme II (EII). That is, PTS includes EI, HPr, and EII. In PTS, specifically, a phosphate group derived from PEP may be sequentially transferred to EI, HPr, and EII, and EII may phosphorylate a sugar and transport it into cells. In order to reduce the activity of PTS, for example, the activity of one protein selected from EI, HPr, and EII may be reduced, and two or more proteins selected from EI, HPr, and EII (eg, The activity of all) proteins may be reduced.

  EI can be commonly used regardless of the type of sugar. EI includes PtsI protein encoded by the ptsI gene. The nucleotide sequence of the ptsI gene (NCgl1858) of C. glutamicum ATCC 13869 strain is shown in SEQ ID NO: 1, and the amino acid sequence of the PtsI protein encoded by the gene is shown in SEQ ID NO: 2. The nucleotide sequence of the ptsI gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 15, and the amino acid sequence of the PtsI protein encoded by the gene is shown in SEQ ID NO: 16.

  HPr can be commonly used regardless of the type of sugar. HPr includes PtsH protein encoded by the ptsH gene. The nucleotide sequence of the ptsH gene (NCgl1862) of C. glutamicum ATCC 13869 strain is shown in SEQ ID NO: 3, and the amino acid sequence of the PtsH protein encoded by the gene is shown in SEQ ID NO: 4. The nucleotide sequence of the ptsH gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 17, and the amino acid sequence of the PtsH protein encoded by the gene is shown in SEQ ID NO: 18.

EII includes EII specific to each sugar. Specific examples of EII include glucose-specific EII (EII ^Glc ), sucrose-specific EII (EII ^Scr ), and fructose-specific EII (EII ^Fru ). The EII may be composed of, for example, three components: EIIA, EIIB, and EIIC. These components may be encoded by the gene alone or in an appropriate combination. EIIA is also referred to as “Enzyme III (EIII)”. EII ^Glc includes PtsG and Crr proteins encoded by the ptsG and crr genes, respectively. EII ^Scrs include the PtsS, ScrA, and Crr proteins encoded by the ptsS, scrA, and crr genes, respectively. EII ^Fru includes PtsF, FruA, and FruB proteins encoded by the ptsF, fruA, and fruB genes, respectively.

For example, in the case of coryneform bacteria such as C. glutamicum, the ptsG gene is glucose-specific EIIBCA (EIIBCA ^Glc ), the ptsS gene is sucrose-specific EIIBCA (EIIBCA ^Scr ), and the ptsF gene is fructose-specific EIIBCA (EIIBCA ^Fru ). Can be respectively coded. The nucleotide sequence of the ptsG gene (NCgl1305) of C. glutamicum ATCC 13869 strain is shown in SEQ ID NO: 5, and the amino acid sequence of the PtsG protein encoded by the gene is shown in SEQ ID NO: 6. The nucleotide sequence of the ptsS gene (NCgl2553) of C. glutamicum ATCC 13869 strain is shown in SEQ ID NO: 7, and the amino acid sequence of the PtsS protein encoded by the gene is shown in SEQ ID NO: 8. The nucleotide sequence of the ptsF gene (NCgl1861) of C. glutamicum ATCC 13869 strain is shown in SEQ ID NO: 9, and the amino acid sequence of the PtsF protein encoded by the gene is shown in SEQ ID NO: 10.

For example, E. If bacteria Enterobacteriaceae such coli (Enterobacteriaceae), ptsG gene glucose-specific EIIBC (EIIBC ^Glc), crr gene glucose and specific EIIA both sucrose (EIIA ^{Glc, Scr} ), The scrA gene is sucrose-specific EIIBC (EIIBC ^Scr ), the fruA gene is fructose-specific EIIBC (EIIBC ^Fru ), the fruB gene is fructose-specific EIIA and HPr fusion protein (fused EIIA ^Fru / HPr), You can code each. The nucleotide sequence of the ptsG gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 19, and the amino acid sequence of the PtsG protein encoded by the gene is shown in SEQ ID NO: 20. The nucleotide sequence of the crr gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 21, and the amino acid sequence of the Crr protein encoded by the gene is shown in SEQ ID NO: 22. SEQ ID NO: 23 shows the nucleotide sequence of the scrA gene mounted on plasmid pUR400 found in Salmonella enterica, and SEQ ID NO: 24 shows the amino acid sequence of the ScrA protein encoded by the gene. The nucleotide sequence of the fruA gene of E. coli K-12 MG1655 is shown in SEQ ID NO: 25, and the amino acid sequence of the FruA protein encoded by the gene is shown in SEQ ID NO: 26. E.
The nucleotide sequence of the fruB gene of Escherichia coli K-12 MG1655 is shown in SEQ ID NO: 27, and the amino acid sequence of the FruB protein encoded by the gene is shown in SEQ ID NO: 28.

  In addition, the sugar uptake system also includes a non-PTS uptake system. The “non-PTS uptake system” may mean a system having an activity of transporting a sugar into a cell without phosphorylation. This activity is also referred to as “non-PTS uptake activity”. Examples of the non-PTS uptake system for sugar include a non-PTS uptake system for glucose and a non-PTS uptake system for sucrose.

  A non-PTS uptake system for glucose includes a GalP protein encoded by the galP gene. GalP protein is known as galactose permease. The nucleotide sequence of the galP gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 29, and the amino acid sequence of the GalP protein encoded by the gene is shown in SEQ ID NO: 30. Non-PTS uptake systems for glucose also include the IolT1 and IolT2 proteins encoded by the iolT1 and iolT2 genes, respectively. IolT1 and IolT2 proteins are known as myo-inositol transporters. The nucleotide sequence of the iolT1 gene (NCgl0178) of C. glutamicum ATCC 13032 is shown in SEQ ID NO: 11, and the amino acid sequence of the IolT1 protein encoded by the gene is shown in SEQ ID NO: 12. The nucleotide sequence of the iolT2 gene (NCgl2953) of C. glutamicum ATCC 13032 is shown in SEQ ID NO: 13, and the amino acid sequence of the IolT2 protein encoded by the gene is shown in SEQ ID NO: 14.

  The non-PTS uptake system of sucrose includes the CscB protein encoded by the cscB gene. CscB protein is known as sucrose permease. The nucleotide sequence of the cscB gene of E. coli EC3132 strain is shown in SEQ ID NO: 31, and the amino acid sequence of the CscB protein encoded by the gene (NCBI ACCESSION P30000) is shown in SEQ ID NO: 32.

Examples of sugar metabolizing enzymes include glycolytic enzymes. The glycolysis, include a series of metabolic pathway that converts sugar or phosphorylated saccharide to acetyl CoA and CO ₂ is. Specific examples of the glycolytic pathway include the Emden-Meierhof pathway (EM pathway), the Entner-Doudoroff pathway (ED pathway), and the pentose phosphate pathway (PP pathway). Specific examples of glycolytic enzymes include, for example, glucokinase, glucokinase, phosphoglucose isomerase, 6-phosphofructokinase, and fructose-1,6-bisphosphate aldolase. fructose 1,6-bisphosphate aldolase), triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase ( phosphoglycerate mutase, enolase, pyruvate kinase, pyruvate dehydrogenase, sucrose-6-phosphate hydrolase, 1-phosphofructokinase (1- phosphofructokinase) Invertase (invertase), and the like fructokinase (fructokinase) is. Glycolytic enzymes include, among others, 6-phosphofructokinase.

  "Glucokinase" may refer to a protein having the activity of catalyzing the reaction of phosphorylating glucose using PEP as a phosphate donor to produce glucose-6-phosphate (EC 2.7.1.2). ). This activity is also called "glucokinase activity". The gene encoding glucokinase is also called "glucokinase gene". The glucokinase includes a Glk protein encoded by the glk gene. The nucleotide sequence of the glk gene of C. glutamicum ATCC 13869 is shown in SEQ ID NO: 33, and the amino acid sequence of the Glk protein encoded by the gene is shown in SEQ ID NO: 34. The nucleotide sequence of the glk gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 67, and the amino acid sequence of the Glk protein encoded by the gene is shown in SEQ ID NO: 68.

  "Phosphoglucose isomerase" may mean a protein having the activity of catalyzing the reaction of isomerizing glucose-6-phosphate to produce fructose-6-phosphate (EC 5.3.1.9). . This activity is also called “phosphoglucose isomerase activity”. The gene encoding phosphoglucose isomerase is also referred to as “phosphoglucose isomerase gene”. Examples of phosphoglucose isomerase include a Pgi protein encoded by the pgi gene. The nucleotide sequence of the pgi gene of C. glutamicum ATCC 13869 is shown in SEQ ID NO: 35, and the amino acid sequence of the Pgi protein encoded by the gene is shown in SEQ ID NO: 36. The nucleotide sequence of the pgi gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 69, and the amino acid sequence of the Pgi protein encoded by the gene is shown in SEQ ID NO: 70.

  "6-phosphofructokinase" refers to an activity of catalyzing a reaction of phosphorylating fructose-6-phosphate using ATP as a phosphate donor to produce fructose-1,6-bisphosphate. (EC 2.7.1.11). This activity is also referred to as “6-phosphofructokinase activity”. The gene encoding 6-phosphofructokinase is also referred to as “6-phosphofructokinase gene”. Examples of 6-phosphofructokinase include Pfk proteins (for example, PfkA and PfkB proteins) encoded by pfk genes (for example, pfkA and pfkB genes). The nucleotide sequence of the pfk gene (also referred to as the pfkA gene) of C. glutamicum ATCC 13869 is shown in SEQ ID NO: 37, and the amino acid sequence of the Pfk protein encoded by the gene is shown in SEQ ID NO: 38. The nucleotide sequence of the pfkA gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 71, and the amino acid sequence of the PfkA protein encoded by the gene is shown in SEQ ID NO: 72. The nucleotide sequence of the pfkB gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 73, and the amino acid sequence of the PfkB protein encoded by the gene is shown in SEQ ID NO: 74. In order to reduce the activity of 6-phosphofructokinase, for example, the activity of one or both of the PfkA protein and the PfkB protein may be reduced.

"Fructose 1,6-bisphosphate aldolase" decomposes fructose-1,6-bisphosphate to produce dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. May refer to a protein having the activity of catalyzing the reaction that takes place (EC 4.1.2.13). This activity is also referred to as “fructose 1,6-bisphosphate aldolase activity”. The gene encoding fructose 1,6-bisphosphate aldolase is referred to as “fructose
It is also called "1,6-bisphosphate aldolase gene". Examples of fructose 1,6-bisphosphate aldolase include the FbaA protein encoded by the fbaA gene. The nucleotide sequence of the fbaA gene of C. glutamicum ATCC 13869 is shown in SEQ ID NO: 39, and the amino acid sequence of the FbaA protein encoded by the gene is shown in SEQ ID NO: 40. The nucleotide sequence of the fbaA gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 75, and the amino acid sequence of the FbaA protein encoded by the gene is shown in SEQ ID NO: 76.

  "Triose phosphate isomerase" may mean a protein having the activity of catalyzing the reaction of isomerizing dihydroxyacetone phosphate to produce glyceraldehyde-3-phosphate (EC 5.3.1.1). ). This activity is also called “triose phosphate isomerase activity”. The gene encoding triose phosphate isomerase is also called "triose phosphate isomerase gene". The triose phosphate isomerase includes a TpiA protein encoded by the tpiA gene. The nucleotide sequence of the tpiA gene of C. glutamicum ATCC 13869 is shown in SEQ ID NO: 41, and the amino acid sequence of the TpiA protein encoded by the gene is shown in SEQ ID NO: 42. The nucleotide sequence of the tpiA gene of E. coli K-12 MG1655 is shown in SEQ ID NO: 77, and the amino acid sequence of the TpiA protein encoded by the gene is shown in SEQ ID NO: 78.

"Glyceraldehyde-3-phosphate dehydrogenase" refers to phosphorylation and oxidation of glyceraldehyde-3-phosphate in the presence of phosphate and an electron acceptor to give 1,3. A protein having an activity of catalyzing a reaction for producing bisphosphoglycerate (EC 1.2.1.12, EC 1.2.1.13, EC.1.2.1.59, etc.). This activity is also referred to as “glyceraldehyde-3-phosphate dehydrogenase activity”. The gene encoding glyceraldehyde-3-phosphate dehydrogenase is also referred to as “glyceraldehyde-3-phosphate dehydrogenase gene”. Examples of the electron acceptor include NAD ⁺ and NADP ⁺ . Glyceraldehyde-3-phosphate dehydrogenase may be, for example, one that can utilize at least one of these electron acceptors. Examples of glyceraldehyde-3-phosphate dehydrogenase include a GapA protein encoded by the gapA gene. The nucleotide sequence of the gapA gene of C. glutamicum ATCC 13869 is shown in SEQ ID NO: 43, and the amino acid sequence of the GapA protein encoded by the gene is shown in SEQ ID NO: 44. The nucleotide sequence of the gapA gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 79, and the amino acid sequence of the GapA protein encoded by the gene is shown in SEQ ID NO: 80.

  The term "phosphoglycerate kinase" may mean a protein having an activity of catalyzing a reaction to generate 3-phosphoglycerate and ATP from 1,3-bisphosphoglycerate and ADP (EC 2.7). .2.3). This activity is also called “phosphoglycerate kinase activity”. The gene encoding phosphoglycerate kinase is also referred to as “phosphoglycerate kinase gene”. Examples of phosphoglycerate kinase include Pgk protein encoded by pgk gene. The nucleotide sequence of the pgk gene of C. glutamicum ATCC 13869 is shown in SEQ ID NO: 45, and the amino acid sequence of the Pgk protein encoded by the gene is shown in SEQ ID NO: 46. The nucleotide sequence of the pgk gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 81, and the amino acid sequence of the Pgk protein encoded by the gene is shown in SEQ ID NO: 82.

  "Phosphoglycerate mutase" may mean a protein having the activity of catalyzing the reaction of isomerizing 3-phosphoglycerate to produce 2-phosphoglycerate (EC 5.4.2.11 or EC 5.4.2.12). This activity is also referred to as “phosphoglycerate mutase activity”. The gene encoding phosphoglycerate mutase is also referred to as “phosphoglycerate mutase gene”. Examples of the phosphoglycerate mutase include Gpm proteins (eg, GpmA proteins and GpmM proteins) encoded by gpm genes (eg, gpmA genes and gpmM genes). The nucleotide sequence of the gpmA gene of C. glutamicum ATCC 13869 is shown in SEQ ID NO: 47, and the amino acid sequence of the GpmA protein encoded by the gene is shown in SEQ ID NO: 48. The nucleotide sequence of the gpmM gene of C. glutamicum ATCC 13869 is shown in SEQ ID NO: 49, and the amino acid sequence of the GpmM protein encoded by the gene is shown in SEQ ID NO: 50. The nucleotide sequence of the gpmA gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 83, and the amino acid sequence of the GpmA protein encoded by the gene is shown in SEQ ID NO: 84. The nucleotide sequence of the gpmM gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 85, and the amino acid sequence of the GpmM protein encoded by the gene is shown in SEQ ID NO: 86. In order to reduce the activity of the phosphoglycerate mutase, for example, the activity of one or both of the GpmA protein and the GpmM protein may be reduced.

  "Enolase" may mean a protein having the activity of catalyzing the reaction of dehydrating 2-phosphoglycerate to produce PEP (EC 4.2.1.11). This activity is also called “enolase activity”. The gene encoding enolase is also called "enolase gene". The enolase includes an Eno protein encoded by the eno gene. The nucleotide sequence of the eno gene (NCgl0935) of C. glutamicum ATCC 13869 is shown in SEQ ID NO: 51, and the amino acid sequence of the Eno protein encoded by the gene is shown in SEQ ID NO: 52. The nucleotide sequence of the eno gene of E. coli K-12 MG1655 is shown in SEQ ID NO: 87, and the amino acid sequence of the Eno protein encoded by the gene is shown in SEQ ID NO: 88.

"Pyruvate kinase" may mean a protein having the activity of catalyzing the reaction of PEP and ADP to produce pyruvate and ATP (EC 2.7.1.40). This activity is also called “pyruvate kinase activity”. The gene encoding pyruvate kinase is called "pyru
It is also called “vate kinase gene”. Examples of the pyruvate kinase include a Pyk protein (for example, PykA protein and PykF protein) encoded by a pyk gene (for example, pykA gene and pykF gene). The nucleotide sequence of the pykA gene of C. glutamicum ATCC 13869 is shown in SEQ ID NO: 53, and the amino acid sequence of the PykA protein encoded by the gene is shown in SEQ ID NO: 54. The nucleotide sequence of the pykF gene of C. glutamicum ATCC 13869 is shown in SEQ ID NO: 55, and the amino acid sequence of the PykF protein encoded by the gene is shown in SEQ ID NO: 56. The nucleotide sequence of the pykA gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 89, and the amino acid sequence of the PykA protein encoded by the gene is shown in SEQ ID NO: 90. The nucleotide sequence of the pykF gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 91, and the amino acid sequence of the PykF protein encoded by the gene is shown in SEQ ID NO: 92. In order to reduce the activity of pyruvate kinase, for example, the activity of one or both of the PykA protein and the PykF protein may be reduced.

  "Pyruvate dehydrogenase" may refer to a protein having the activity of catalyzing the reaction of oxidatively decarboxylating pyruvate to produce acetyl-CoA. This activity is also referred to as "pyruvate dehydrogenase activity". The gene encoding pyruvate dehydrogenase is also referred to as "pyruvate dehydrogenase gene". Pyruvate dehydrogenase is composed of three subunits: pyruvate dehydrogenase / decarboxylase (E1; EC 1.2.4.1), dihydrolipoyl acetyltransferase (E2; EC 2.3.1.12), and dihydrolipo. It may be composed of dihydrolipoyl dehydrogenase (E3; EC 1.8.1.4). That is, examples of pyruvate dehydrogenase include E1, E2, and E3. In pyruvate dehydrogenase, specifically, E1 may be responsible for decarboxylation of pyruvate and transfer of an acetyl group to E2, E2 for production of acetyl-CoA, and E3 for production of NADH. In order to reduce the activity of pyruvate dehydrogenase, for example, the activity of one protein selected from E1, E2, E3 may be reduced, and two or more proteins selected from E1, E2, E3 ( For example, the activity of all) proteins may be reduced. E1 includes the AceE protein encoded by the aceE gene. E2 includes AceF protein encoded by the aceF gene. E3 includes Lpd protein encoded by the lpd gene. The nucleotide sequence of the aceE gene of C. glutamicum ATCC 13869 is shown in SEQ ID NO: 57, and the amino acid sequence of the AceE protein encoded by the gene is shown in SEQ ID NO: 58. The nucleotide sequence of the aceF gene of C. glutamicum ATCC 13869 is shown in SEQ ID NO: 59, and the amino acid sequence of the AceF protein encoded by the gene is shown in SEQ ID NO: 60. The nucleotide sequence of the lpd gene of C. glutamicum ATCC 13869 is shown in SEQ ID NO: 61, and the amino acid sequence of the Lpd protein encoded by the gene is shown in SEQ ID NO: 62. The nucleotide sequence of the aceE gene of E. coli K-12 MG1655 is shown in SEQ ID NO: 93, and the amino acid sequence of the AceE protein encoded by the gene is shown in SEQ ID NO: 94. The nucleotide sequence of the aceF gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 95, and the amino acid sequence of the AceF protein encoded by the gene is shown in SEQ ID NO: 96. The nucleotide sequence of the lpd gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 97, and the amino acid sequence of the Lpd protein encoded by the gene is shown in SEQ ID NO: 98.

"Sucrose-6-phosphate hydrolase" is a protein having an activity of catalyzing a reaction of hydrolyzing sucrose-6-phosphate to produce glucose-6-phosphate and fructose. (EC 3.2.1.B3). This activity is also referred to as "sucrose-6-phosphate hydrolase activity". The gene encoding sucrose-6-phosphate hydrolase is also referred to as "sucrose-6-phosphate hydrolase gene". The sucrose-6-phosphate hydrolase includes a ScrB protein encoded by the scrB gene. C.
SEQ ID NO: 63 shows the nucleotide sequence of the scrB gene (NCgl2554) of glutamicum ATCC 13869, and SEQ ID NO: 64 shows the amino acid sequence of the protein encoded by the gene. The nucleotide sequence of the scrB gene found in Salmonella enterica and carried on the plasmid pUR400 is shown in SEQ ID NO: 99, and the amino acid sequence of the ScrB protein encoded by the gene is shown in SEQ ID NO: 100. In addition, sucrose-6-phosphate hydrolase may further have an invertase activity.

  “1-phosphofructokinase” is an activity that catalyzes a reaction of phosphorylating fructose-1-phosphate using ATP as a phosphate donor to produce fructose-1,6-bisphosphate. (EC 2.7.1.56). This activity is also referred to as “1-phosphofructokinase activity”. The gene encoding 1-phosphofructokinase is also referred to as “1-phosphofructokinase gene”. 1-phosphofructokinase includes FruK protein encoded by fruK gene. The nucleotide sequence of the fruK gene (NCgl1860) of C. glutamicum ATCC 13869 is shown in SEQ ID NO: 65, and the amino acid sequence of the FruK protein encoded by the gene is shown in SEQ ID NO: 66. The nucleotide sequence of the fruK gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 101, and the amino acid sequence of the FruK protein encoded by the gene is shown in SEQ ID NO: 102.

  "Invertase" may mean a protein having the activity of catalyzing the reaction of hydrolyzing sucrose to produce glucose and fructose (EC 3.2.1.26). This activity is also called “invertase activity”. The gene encoding invertase is also called "invertase gene". Invertase includes a CscA protein encoded by the cscA gene. The nucleotide sequence of the cscA gene of E. coli EC3132 is shown in SEQ ID NO: 103, and the amino acid sequence of the CscA protein encoded by the gene (NCBI ACCESSION P40714) is shown in SEQ ID NO: 104. Invertase may further have sucrose-6-phosphate hydrolase activity.

  The term “fructokinase” may mean a protein having an activity of catalyzing a reaction of fructose phosphorylation using ATP as a phosphate donor to produce fructose-6-phosphate (EC 2.7. 1.4). This activity is also called “fructokinase activity”. The gene encoding fructokinase is also referred to as "fructokinase gene". The fructokinase includes a CscK protein encoded by the cscK gene. The nucleotide sequence of the cscK gene of E. coli EC3132 is shown in SEQ ID NO: 105, and the amino acid sequence of the CscK protein encoded by the gene (NCBI ACCESSION P40713) is shown in SEQ ID NO: 106.

  That is, the saccharide assimilation gene may be, for example, a gene having a known base sequence such as the base sequence exemplified above. In addition, the saccharide-utilizing protein may be, for example, a protein having a known amino acid sequence such as the amino acid sequence exemplified above. The expression “having a (amino acid or base) sequence” may mean “including an (amino acid or base) sequence” unless otherwise specified. May also be included.

  The sugar assimilation gene may be a variant of the above-described sugar assimilation gene (for example, a gene having a known nucleotide sequence such as the above-described nucleotide sequence) as long as the original function is maintained. Similarly, as long as the original function is maintained, the saccharide-utilizing protein may be a variant of the above-described saccharide-utilizing protein (for example, a protein having a known amino acid sequence such as the above-described amino acid sequence). Good. Note that a variant in which such an original function is maintained may be referred to as a “conservative variant”. Further, the gene specified by the gene name and the protein specified by the protein name are not limited to the genes and proteins exemplified above, respectively, and may include conservative variants thereof. That is, for example, the term “pfk gene” may include conservative variants thereof in addition to the pfk gene exemplified above (eg, a gene having the nucleotide sequence shown in SEQ ID NO: 37, 71, or 73). . Similarly, for example, the term “Pfk protein” encompasses the Pfk proteins exemplified above (eg, a protein having the amino acid sequence set forth in SEQ ID NO: 38, 72, or 74), as well as conservative variants thereof. Good. Conservative variants include, for example, homologs and artificial variants of the genes and proteins exemplified above.

  “Maintaining the original function” means that the gene or protein variant has a function (eg, activity or property) corresponding to the function (eg, activity or property) of the original gene or protein. I do. “Maintained original function” for a gene may mean that the variant of the gene encodes a protein that has maintained its original function. That is, “maintaining the original function” of a glycosylation gene may mean that the gene variant encodes the corresponding glycosylation protein. Further, “maintaining the original function” of a glycosimilating protein may mean that the protein variant has the activity of the corresponding glycosimilating protein. For example, "maintaining the original function" of the 6-phosphofructokinase gene may mean that the gene variant encodes 6-phosphofructokinase. Further, “maintaining the original function” of 6-phosphofructokinase may mean that the protein variant has 6-phosphofructokinase activity.

  The functions (eg, activities and properties) of the glycoproteins can be measured by, for example, a known method. That is, the activity of an enzyme such as a sugar metabolizing enzyme can be measured, for example, by incubating the enzyme with a corresponding substrate and measuring the production of the enzyme and the substrate-dependent corresponding product. Specifically, for example, 6-phosphofructokinase activity can be achieved by incubating the enzyme with the corresponding substrate (ie, fructose-6-phosphate) in the presence of ATP, and the enzyme and substrate-dependent corresponding product (ie, fructose). -1,6-bisphosphoric acid) can be measured. In addition, the activity of the sugar uptake system can be measured, for example, by incubating cells expressing the protein with sugar, and measuring the protein-dependent sugar uptake into the cells. The saccharide-utilizing protein may have at least one function (for example, activity or property) of the saccharide-utilizing protein measured under appropriate conditions. It should be noted that all other proteins described in the present specification need only have a corresponding function (eg, activity or property) measured under at least one appropriate condition.

  Hereinafter, conservative variants will be exemplified.

  A homologue of a glycosylation gene or a homologue of a glycosylation protein is, for example, a BLAST search or FASTA using a known base sequence such as the above-described base sequence or a known amino acid sequence such as the above-described amino acid sequence as a query sequence. It can be easily obtained from public databases by searching. In addition, the homologue of the sugar assimilation gene is, for example, by using a chromosome of an organism such as a coryneform bacterium as a template and performing PCR using an oligonucleotide prepared as a primer based on a known nucleotide sequence such as the above-described nucleotide sequence as a primer. Can be obtained.

  As long as the original function is maintained, one or several amino acids at one or several positions are substituted, deleted, or inserted in a known amino acid sequence such as the amino acid sequence described above, as long as the original function is maintained. And / or may encode a protein having an added amino acid sequence. For example, the encoded protein may be extended or shortened at its N-terminus and / or C-terminus. The “one or several” differs depending on the position and type of the amino acid residue in the three-dimensional structure of the protein, but specifically, for example, 1 to 50, 1 to 40, and 1 to 30 , 1-20, 1-10, 1-5, or 1-3.

The above substitutions, deletions, insertions, and / or additions of one or several amino acids are conservative mutations that maintain the original function of the protein. A typical conservative mutation is a conservative substitution. A conservative substitution is a polar amino acid between Phe, Trp and Tyr when the substitution site is an aromatic amino acid and between Leu, Ile and Val when the substitution site is a hydrophobic amino acid. In the case, between Gln and Asn, when it is a basic amino acid, between Lys, Arg, His, when it is an acidic amino acid, it is between Asp and Glu, when it is an amino acid having a hydroxyl group Is a mutation that substitutes for Ser and Thr. As the substitution considered as a conservative substitution, specifically, Ala to Ser or Thr substitution, Arg to Gln, His or Lys substitution, Asn to Glu, Gln, Lys, His or Asp substitution, Asp to Asn, Glu or Gln substitution, Cys to Ser or Ala substitution, Gln to Asn, Glu, Lys, His, Asp or Arg substitution, Glu to Gly, Asn, Gln, Lys or Asp Substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Lys to Asn, Glu, Gln, His or Arg substitution, Met to Ile, Leu, Val or Phe substitution, Phe to Trp, Tyr, Met, Ile or Leu substitution, Ser to Thr or Ala Substitution, substitution of Thr for Ser or Ala, substitution of Trp for Phe or Tyr, substitution of Tyr for His, Phe or Trp, and substitution of Val for Met, Ile or Leu. In addition, the substitution, deletion, insertion, or addition of amino acids as described above is caused by a naturally occurring mutation (mutant or variant) such as one based on individual differences or species differences of the organism from which the gene is derived. Is also included.

  Further, as long as the original function is maintained, the sugar assimilation gene is, for example, 50% or more, 65% or more, 80% or more, 90%, based on the entire known amino acid sequence such as the amino acid sequence exemplified above. The gene may encode a protein having an amino acid sequence having 95% or more, 97% or more, or 99% or more identity.

  Further, as long as the original function is maintained, a sugar assimilation gene can be prepared from a known nucleotide sequence such as the above-described nucleotide sequence, for example, the entire known nucleotide sequence such as the above-described nucleotide sequence or the like. It may be a gene, such as DNA, that hybridizes under stringent conditions with a complementary sequence to a part. "Stringent conditions" may mean conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. As an example, DNA with high identity, for example, DNA with 50% or more, 65% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more identity Hybridized, less identical DNA is not hybridized with each other, or the usual washing conditions for Southern hybridization at 60 ° C., 1 × SSC, 0.1% SDS, preferably 60 ° C., 0.1 × SSC, The conditions include washing once, preferably 2-3 times at a salt concentration and temperature corresponding to 0.1% SDS, more preferably 68 ° C., 0.1 × SSC, and 0.1% SDS.

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

  In addition, since the degeneracy of codons differs depending on the host, the sugar assimilation gene may be obtained by replacing any codon with an equivalent codon. That is, the sugar utilization gene may be a variant of the sugar utilization gene exemplified above due to the degeneracy of the genetic code. For example, the saccharide assimilation gene may be modified to have an optimal codon depending on the codon usage of the host used.

The "identity" between amino acid sequences is calculated by blastp using the default settings of Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Existing = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment). Means the identity between different amino acid sequences. The term “identity” between nucleotide sequences means the identity between nucleotide sequences calculated by blastn using Scoring Parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear) set by default. I do.

  In addition, the description regarding the conservative variants of the above-mentioned genes and proteins can be applied mutatis mutandis to any gene and protein other than the sugar-utilizing gene and the protein.

The sugar assimilating protein (protein P1) which reduces the activity in order to reduce the assimilation ability of sugar can be appropriately selected according to various conditions such as, for example, the type of sugar and the type of sugar assimilation pathway. For example, to reduce glucose assimilation through PTS, EI, HPr, EII ^Glc , phosphoglucose isomerase, 6-phosphofructokinase, fructose 1,6-bisphosphate aldolase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase Or one or more activities selected from phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, and pyruvate dehydrogenase. In addition, for example, in order to reduce the ability to assimilate sucrose through PTS, EI, HPr, EII ^Scr , phosphoglucose isomerase, 6-phosphofructokinase, fructose 1,6-bisphosphate aldolase, triose phosphate isomerase, glyceraldehyde-3- One or more activities selected from phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, pyruvate dehydrogenase, sucrose-6-phosphate hydrolase, and fructokinase may be reduced. In a microorganism, for example, the activity of one or more proteins selected from glycolytic enzymes may be reduced. In microorganisms, in particular, the activity of 6-phosphofructokinase may be reduced.

<1-3> Target substance producing ability The microorganism described in this specification may have a target substance producing ability.

  "Target substance production ability" means the ability to produce a target substance. That is, “a microorganism capable of producing a target substance” may mean a microorganism that can produce a target substance.

  The “microorganism having a target substance-producing ability” may mean a microorganism capable of producing a target substance by fermentation when the microorganism is used in a fermentation method. That is, the “microorganism capable of producing the target substance” may mean a microorganism capable of producing the target substance from sugar. The term "microorganism having a target substance-producing ability" is specifically used in a culture solution (for example, a culture medium) such that the target substance is produced and recovered when cultured in a medium (eg, a medium containing sugar). Medium, a cell surface layer, a cell body, or a combination thereof).

  The “microorganism having a target substance-producing ability” may mean a microorganism capable of producing a target substance by bioconversion when the microorganism is used in a bioconversion method. That is, “a microorganism having the ability to produce a target substance” may mean a microorganism that can produce a target substance from a precursor of the target substance. The term "microorganism capable of producing a target substance" specifically refers to a culture that can produce and recover the target substance when cultured in a medium (for example, a medium containing a sugar and a precursor of the target substance). A microorganism that can accumulate in a liquid (eg, in a medium, in a cell, or a combination thereof) may be referred to. In addition, the term "microorganism having a target substance-producing ability" specifically refers to an amount of a target substance produced and allowed to recover in a reaction solution (for example, , Liquid fraction, intracellular, or a combination thereof). “Liquid fraction” may mean the remainder of the reaction solution from which bacterial cells have been removed.

Microorganisms having the target substance-producing ability include, for example, a medium or reaction medium containing 0.01 g / L or more, 0.05 g / L or more, 0.1 g / L or more, 0.5 g / L or more, or 1.0 g / L or more of the target substance. It may be able to accumulate in the liquid.

  A microorganism may be capable of producing one type of target substance, or may be capable of producing two or more target substances. In addition, the microorganism may be capable of producing a target substance from one kind of target substance precursor, or may be capable of producing a target substance from two or more kinds of target substance precursors.

  The target substance is not particularly limited as long as it can be produced using a microorganism. Target substances include SAM-dependent metabolites, aldehydes, L-amino acids, nucleic acids, organic acids, γ-glutamyl peptides, sphingoids, proteins, and RNA.

  "SAM-dependent metabolite" may mean a metabolite that requires S-adenosylmethionine (SAM) for biosynthesis. SAM-dependent metabolites include vanillin, vanillic acid, melatonin, ergothioneine, ergothioneine, mugineic acid, ferulic acid, polyamine, guaiacol (Guaiacol), 4-vinylguaiacol, 4-ethylguaiacol, and creatine. Examples of the polyamine include spermidine and spermine.

  Aldehydes include aromatic aldehydes. Examples of the aromatic aldehyde include vanillin, benzaldehyde, and cinnamaldehyde.

  Examples of L-amino acids include basic amino acids such as L-lysine, L-ornithine, L-arginine, L-histidine, L-citrulline, L-isoleucine, L-alanine, L-valine, L-leucine, glycine and the like. Aliphatic amino acids, amino acids which are hydroxymonoaminocarboxylic acids such as L-threonine and L-serine, cyclic amino acids such as L-proline, aromatic amino acids such as L-phenylalanine, L-tyrosine and L-tryptophan; Examples include sulfur-containing amino acids such as cysteine, L-cystine and L-methionine; acidic amino acids such as L-glutamic acid and L-aspartic acid; and amino acids having an amide group in the side chain such as L-glutamine and L-asparagine. "Amino acid" may mean L-amino acid unless otherwise specified.

  Examples of the nucleic acid include purine-based substances. Purine-based substances include purine nucleosides and purine nucleotides. Purine nucleosides include inosine, guanosine, xanthosine, and adenosine. Purine nucleotides include 5'-phosphate esters of purine nucleosides. Examples of the 5′-phosphate ester of purine nucleoside include inosine acid (inosine-5′-phosphate ester; IMP), guanylic acid (guanosine-5′-phosphate ester; GMP), and xanthyl acid (xanthosin-5′-phosphate). Phosphate esters; XMP), and adenylic acid (adenosine-5'-phosphate esters; AMP).

Examples of the organic acid include a dicarboxylic acid. Examples of the dicarboxylic acids, since three carbon atoms eight dicarboxylic acids (C ₃ -C ₈ dicarboxylic acid). Specific examples of the dicarboxylic acid include α-ketoglutaric acid (α-KG; also known as 2-oxoglutaric acid), malic acid, fumaric acid, succinic acid, itaconic acid, malonic acid, adipic acid, glutaric acid, pimelic acid, and suberin. Acids.

Examples of the γ-glutamyl peptide include γ-glutamyl dipeptide and γ-glutamyl tripeptide. Examples of the γ-glutamyl dipeptide include γ-Glu-Val. γ
-Glutamyl tripeptide includes γ-Glu-Val-Gly (CAS 38837-70-6, also referred to as Gluvalicine).

  Sphingoids include sphingoid bases and sphingolipids. Examples of sphingoid bases include phytosphingosine (PHS). Examples of sphingolipids include phytoceramide (PHC).

  The protein includes any protein that can be expressed using a microorganism as a host. The protein may be a host-derived protein or a heterologous protein (heterologous protein). “Heterologous protein” refers to a protein that is exogenous to the host producing the protein (ie, the microorganisms described herein). The protein may be, for example, a naturally occurring protein, a modified protein thereof, or a protein having an artificially designed amino acid sequence. The protein may be, for example, a microorganism-derived protein, a plant-derived protein, an animal-derived protein, or a virus-derived protein. The protein may be a monomeric protein or a multimeric protein. The protein may be a secretory protein or a non-secretory protein. The “protein” also includes what is called a peptide, such as an oligopeptide or a polypeptide.

  Specific examples of proteins include enzymes, bioactive proteins, receptor proteins, antigen proteins, and other proteins.

  Enzymes include cellulase, transglutaminase, protein glutaminase, isomaltodextranase, protease, endopeptidase, exopeptidase, aminopeptidase, carboxypeptidase, collagenase, and chitinase.

  Examples of bioactive proteins include growth factors (growth factors), hormones, cytokines, and antibody-related molecules.

Examples of growth factors include epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and transforming growth factor (TGF). ), Nerve growth factor (NGF), Brain-derived neurotrophic factor (BDNF), Vesicular endothelial growth factor (VEGF), Granulocyte colony stimulating factor (Granulocyte- colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF), platelet-derived growth factor (PDGF), erythropoietin (EPO), Thrombopoietin (TPO), acidic fibroblast growth factor (aFGF or FGF1), basic fibroblast growth factor (basic fibrob) last growth factor; bFGF or FGF2), keratinocyte growth factor (keratinocyto growth)
factor; KGF-1 or FGF7, KGF-2 or FGF10), and hepatocyte growth factor (HGF).

  Hormones include insulin, glucagon, somatostatin, human growth hormone (hGH), parathyroid hormone (PTH), calcitonin (calcitonin), and exenatide (exenatide).

Cytokines include interleukin, interferon, tumor necrosis factor (Tumor
Necrosis Factor; TNF).

  The bioactive protein may be the whole protein or a part thereof. As a part of the protein, for example, a part having a physiological activity can be mentioned. Specific examples of the bioactive moiety include the bioactive peptide Teriparatide consisting of the N-terminal 34 amino acid residues of a mature form of parathyroid hormone (PTH).

The “antibody-related molecule” may mean a protein containing a single domain selected from domains constituting a complete antibody or a molecular species composed of a combination of two or more domains. The domains constituting a complete antibody include VH, CH1, CH2, and CH3, which are domains of the heavy chain, and VL and CL, which are domains of the light chain. The antibody-related molecule may be a monomeric protein or a multimeric protein as long as it contains the above-mentioned molecular species. When the antibody-related molecule is a multimeric protein, it may be a homomultimer composed of a single type of subunit or a heteromultimer composed of two or more types of subunits. Is also good. Specific examples of antibody-related molecules include a complete antibody, Fab, F (ab '), F (ab') ₂ , Fc, a dimer composed of a heavy chain (H chain) and a light chain (L chain), Fc Fusion proteins, heavy chains (H chains), light chains (L chains), single chain Fvs (scFv), sc (Fv) ₂ , disulfide-linked Fvs (sdFv), diabodies, VHH fragments (nanobody®) Can be More specifically, the antibody-related molecule includes trastuzumab.

  Receptor proteins include biologically active proteins and receptor proteins for other biologically active substances. Other physiologically active substances include neurotransmitters such as dopamine. The receptor protein may be an orphan receptor for which the corresponding ligand is not known.

  The antigen protein is not particularly limited as long as it can elicit an immune response. The antigen protein can be appropriately selected depending on, for example, the subject of an assumed immune response. The antigen protein can be used, for example, as a vaccine.

  Other proteins include Liver-type fatty acid-binding protein (LFABP), fluorescent protein, immunoglobulin binding protein, albumin, fibroin-like protein, and extracellular protein. Examples of the fluorescent protein include Green Fluorescent Protein (GFP). Examples of the immunoglobulin binding protein include Protein A, Protein G, and Protein L. Albumin includes human serum albumin. Fibroin-like proteins include those disclosed in WO2017 / 090665 and WO2017 / 171001. Extracellular proteins include fibronectin, vitronectin, collagen, osteopontin, laminin, and their partial sequences. The partial sequence includes laminin E8, which is an E8 fragment of laminin.

  The protein may be, for example, a protein having the amino acid sequence of a protein as described above. Further, the protein may be, for example, a protein having a variant sequence of the amino acid sequence of the protein as described above. As for the variant sequence, the description of the variant of glycoprotein can be applied mutatis mutandis.

Examples of the RNA include any RNA that can be expressed using a microorganism as a host. The RNA may be a host-derived RNA or a heterologous RNA (heterologous RNA). “Heterologous RNA” refers to RNA that is exogenous to a host that produces the RNA (ie, the microorganism described herein). The RNA may be, for example, a naturally-occurring RNA, a modified RNA thereof, or an RNA having an artificially designed base sequence. The RNA may be, for example, RNA from a microorganism, RNA from a plant, RNA from an animal, or RNA from a virus. RNA may be, for example, mRNA (messenger RNA), such as rRNA (ribosomal RNA), tRNA (transfer RNA), miRNA (micro RNA), siRNA (small interfering RNA), ribozyme (ribozyme), and RNA aptamer. It may be non-coding RNA. The mRNA may, for example, encode a protein having some function, or may encode a protein having no function. Examples of the protein encoded by the mRNA include the proteins exemplified as the target substance.

  The RNA may be, for example, an RNA having the above RNA base sequence. The RNA may be, for example, an RNA having a partial sequence of the above-described RNA base sequence. Further, the RNA may be, for example, an RNA having a complementary sequence of the above-described RNA base sequence or a partial sequence thereof. The RNA may be, for example, an RNA having a variant sequence of the above-described RNA base sequence, a partial sequence thereof, or a complementary sequence thereof. With respect to the variant sequence, the description of the variant of the sugar assimilation gene can be applied mutatis mutandis. Further, the RNA has, for example, a combination of two or more base sequences selected from the above-described RNA base sequences, their partial sequences, their complementary sequences, and their variant sequences. Is also good. Specific examples of the RNA include a partial sequence of mRNA of the apoptosis inhibitor of Citrus autum and a partial sequence of mRNA of subunits A and E constituting ATPase in the vacuole of Colorado potato beetle.

  The RNA may be, for example, a single-stranded RNA (RNA consisting of one RNA strand) or a double-stranded RNA (RNA consisting of two RNA strands). The double-stranded RNA may be a double strand (homoduplex) composed of a single type of RNA molecule, or a double strand (heteroduplex) composed of two different RNA molecules. Is also good. Specific examples of the double-stranded RNA include a double-stranded RNA consisting of a certain RNA strand and its complementary strand. The RNA may be, for example, a double strand consisting of one RNA strand and one DNA strand. RNA may include both single-stranded and double-stranded regions. That is, for example, single-stranded RNA may partially form a double-stranded structure (eg, a stem-loop structure) in the molecule. In addition, for example, the double-stranded RNA may partially include a single-stranded structure.

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

  The microorganism described in the present specification may originally have a target substance-producing ability, or may be modified to have a target substance-producing ability. The microorganism having the target substance-producing ability can be obtained, for example, by imparting the target substance-producing ability to the above-mentioned microorganisms or by enhancing the target substance-producing ability of the microorganism as described above. When the plasmid described in the present specification has the second base sequence (a base sequence effective for production of a target substance), the ability to produce the target substance can be improved by introducing the plasmid. That is, the target substance-producing ability may be imparted or enhanced by introducing a plasmid. The microorganism may have acquired the target substance-producing ability by, for example, introduction of a plasmid or a combination of introduction of a plasmid and other modifications.

The method for imparting or enhancing the target substance-producing ability is not particularly limited. As a method for imparting or enhancing the target substance-producing ability, for example, a known method can be used. Methods for imparting or enhancing the ability to produce SAM-dependent metabolites are disclosed, for example, in WO2018 / 079687, WO2018 / 079686, WO2018 / 079685, WO2018 / 079684, WO2018 / 079683. Methods for imparting or enhancing the ability to produce aldehydes are disclosed, for example, in WO2017 / 073701, WO2018 / 079705, WO2017 / 122747. Methods for imparting or enhancing the ability to produce L-amino acids are disclosed, for example, in WO2015 / 060391 and WO2018 / 030507. Methods for imparting or enhancing the ability to produce nucleic acids are disclosed, for example, in WO2015 / 060391. Methods for imparting or enhancing the ability to produce organic acids are disclosed, for example, in WO 2016/104814. Methods for imparting or enhancing the ability to produce γ-glutamyl peptide are disclosed, for example, in WO2015 / 133547 and WO2017 / 039001. Methods for imparting or enhancing sphingoid productivity are disclosed, for example, in WO2017 / 033463 and WO2017 / 033464. Methods for imparting or enhancing the ability to produce proteins are disclosed, for example, in WO2018 / 074578 and WO2018 / 074579.

  Hereinafter, a specific example of a method for imparting or enhancing the target substance producing ability will be described. Any of the modifications exemplified below for imparting or enhancing the target substance-producing ability may be used alone or in an appropriate combination.

  The target substance can be produced by the action of an enzyme involved in the biosynthesis of the target substance. Such an enzyme is also referred to as “target substance biosynthetic enzyme”. Therefore, the microorganism may have the target substance biosynthetic enzyme. In other words, the microorganism may have a gene encoding the target substance biosynthetic enzyme. Such a gene is also called a “target substance biosynthesis gene”. The microorganism may originally have the target substance biosynthesis gene, or may have the target substance biosynthesis gene introduced therein. The method for introducing a gene will be described later.

  In addition, the ability of the microorganism to produce the target substance can be improved by increasing the activity of the target substance biosynthetic enzyme. That is, as a method for imparting or enhancing the target substance-producing ability, a method for increasing the activity of the target substance biosynthetic enzyme can be mentioned. That is, the microorganism may be modified so that the activity of the target substance biosynthetic enzyme is increased. In the microorganism, the activity of one type of target substance biosynthetic enzyme may be increased, or the activity of two or more types of target substance biosynthetic enzymes may be increased. A method for increasing the activity of a protein (enzyme or the like) will be described later. The activity of a protein (such as an enzyme) can be increased, for example, by increasing the expression of a gene encoding the protein.

  Examples of the target substance biosynthetic gene and the target substance biosynthetic enzyme include those of various organisms such as the microorganisms exemplified above. Specific examples of the target substance biosynthetic gene and the target substance biosynthetic enzyme include bacteria of the family Enterobacteriaceae (eg, Escherichia bacteria such as E. coli) and coryneform bacteria (eg, C. glutamicum). Corynebacterium genus). Examples of the target substance biosynthetic gene and target substance biosynthetic enzyme include those of organisms individually exemplified below. The nucleotide sequence of the biosynthetic gene of the target substance derived from various organisms and the amino acid sequence of the biosynthetic enzyme of the target substance encoded thereby can be obtained from public databases such as NCBI and technical documents such as patent documents. The same applies to genes and proteins other than the target substance biosynthetic gene and the target substance biosynthetic enzyme that are used for imparting or enhancing the target substance producing ability.

The target substance can be produced, for example, from sugar and / or a precursor of the target substance. Therefore, examples of the target substance biosynthetic enzyme include enzymes that catalyze the conversion of sugar and / or precursor to the target substance. For example, 3-dehydroshikimic acid can be produced by part of the shikimate pathway. A part of the shikimate pathway includes 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthase (DAHP synthase) and 3-dehydroquinate synthase. (3-dehydroquinate synthase) and 3-dehydroquinate dehydratase (3-dehydroquinate synthase).
quinate dehydratase). 3-dehydroshikimate can be converted to protocatechuic acid by the action of 3-dehydroshikimate dehydratase (DHSD). Protocatechuic acid is converted to vanillin by the action of O-methyltransferase (O-methyltransferase; OMT) or aromatic aldehyde oxidoreductase (also called aromatic carboxylic acid reductase (ACAR)), respectively. It can be converted to vanillic acid or protocatechualdehyde. Vanillic acid or protocatechualdehyde can be converted to vanillin by the action of ACAR or OMT, respectively. In addition, benzaldehyde and cinnamaldehyde can be produced from benzoic acid and cinnamic acid by the action of ACAR, respectively. That is, specific examples of the target substance biosynthetic enzyme include, for example, DAHP synthase, 3-dehydroquinate synthase, 3-dehydroquinate dehydratase, DHSD, OMT, and ACAR (WO2018 / 079687, WO2018 / 079686, WO2018 / 079685, WO2018 / 079684, WO2018 / 079683, WO2017 / 073701, WO2018 / 079705).

  "3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthase (DAHP synthase)" refers to D-erythrose 4-phosphate and phosphoenol It may mean a protein having the activity of catalyzing the reaction of converting pyruvate to D-arabino-heptulonic acid-7-phosphate (DAHP) and phosphate (EC 2.5.1.54). This activity is also referred to as “DAHP synthase activity”. The 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. Of these, the AroG protein can function as a major DAHP synthase. The nucleotide sequence of the aroG gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 107, and the amino acid sequence of the AroG protein encoded by the gene is shown in SEQ ID NO: 108.

  DAHP synthase activity can be determined, for example, by incubating the enzyme with a substrate (ie, D-erythrose 4-phosphate and phosphoenolpyruvate) and measuring the production of the enzyme and substrate-dependent product (ie, DAHP). Can be measured.

  "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). This activity is also called “3-dehydroquinate synthase activity”. The gene encoding 3-dehydroquinate synthase is also referred to as “3-dehydroquinate synthase gene”. Examples of 3-dehydroquinate synthase include AroB protein encoded by aroB gene. The nucleotide sequence of the aroB gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 109, and the amino acid sequence of the AroB protein encoded by the gene is shown in SEQ ID NO: 110.

  3-dehydroquinate synthase activity can be measured, for example, by incubating the enzyme with a substrate (ie, DAHP) and measuring the production of the enzyme and substrate-dependent product (ie, 3-dehydroquinic acid).

  "3-dehydroquinate dehydratase" may mean a protein having the activity of catalyzing the reaction of dehydrating 3-dehydroquinic acid to produce 3-dehydroshikimic acid (EC 4.2.1.10). ). This activity is also referred to as “3-dehydroquinate dehydratase activity”. The gene encoding 3-dehydroquinate dehydratase is also referred to as “3-dehydroquinate dehydratase gene”. Examples of 3-dehydroquinate dehydratase include AroD protein encoded by aroD gene. The nucleotide sequence of the aroD gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 111, and the amino acid sequence of AroD protein encoded by the gene is shown in SEQ ID NO: 112.

  3-dehydroquinate dehydratase activity can be measured, for example, by incubating the enzyme with a substrate (ie, 3-dehydroquinic acid) and measuring the production of the enzyme and substrate-dependent product (ie, 3-dehydroshikimic acid). .

  “3-dehydroshikimate dehydratase (DHSD)” may mean a protein having an activity of catalyzing a reaction of dehydrating 3-dehydroshikimate to produce protocatechuate (EC 4.2. 1.118). This activity is also referred to as “DHSD activity”. The gene encoding DHSD is also called “DHSD gene”. DHSD includes AsbF protein encoded by the asbF gene. Specific examples of DHSD include those of various organisms such as Bacillus thuringiensis, Neurospora crassa, and Podospora pauciseta. The nucleotide sequence of the asbF gene of Bacillus thuringiensis strain BMB171 is shown in SEQ ID NO: 113, and the amino acid sequence of the AsbF protein encoded by the gene is shown in SEQ ID NO: 114.

  DHSD activity can be measured, for example, by incubating the enzyme with a substrate (ie, 3-dehydroshikimic acid) and measuring the production of the enzyme and substrate-dependent product (ie, protocatechuic acid).

Enzymes in the shikimate pathway (DAHP synthase, 3-dehydroquinate synthase, 3-dehydroquinate
The expression of a gene encoding dehydratase is suppressed by the tyrosine repressor TyrR encoded by the tyrR gene. Thus, the activity of the shikimate pathway enzyme can also be increased by reducing the activity of the tyrosine repressor TyrR. E. coli K-12
The nucleotide sequence of the tyrR gene of MG1655 strain is shown in SEQ ID NO: 115, and the amino acid sequence of the TyrR protein encoded by the gene is shown in SEQ ID NO: 116.

  “O-methyltransferase (OMT)” may mean a protein having an activity of catalyzing a reaction of methylating a hydroxyl group of a substrate in the presence of a methyl group donor (EC 2.1.1.68, etc.). ). This activity is also called "OMT activity". The gene encoding OMT is also called "OMT gene". OMT may have the required substrate specificity depending on the type of biosynthetic pathway in which the target substance is produced in the method described herein. For example, when producing a target substance via conversion of protocatechuic acid to vanillic acid, OMT using at least protocatechuic acid as a substrate can be used. For example, when the target substance is produced through conversion of protocatechualdehyde to vanillin, OMT using at least protocatechualdehyde as a substrate can be used. That is, “O-methyltransferase (OMT)” specifically refers to methylation of protocatechuic acid and / or protocatechualdehyde in the presence of a methyl group donor to convert vanillic acid and / or vanillin. It may also mean a protein having the activity of catalyzing the resulting reaction (ie, methylation of the meta-hydroxyl group). The OMT may be, but is not limited to, typically both protocatechuic acid and protocatechualdehyde. Examples of the methyl group donor include S-adenosylmethionine (SAM).

As the OMT, OMT of various organisms, for example, OMT (GenBank Accession) of Homo sapiens (Hs)
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 / 022881A1. Various OMTs. Four transcription variants and two OMT isoforms are known in the OMT gene of Homo sapiens. The nucleotide sequences of these four transcription variants (transcript variant 1-4; GenBank Accession No. NM_000754.3, NM_001135161.1, NM_001135162.1, NM_007310.2) are shown in SEQ ID NOS: 117 to 120, and the long OMT isoform (MB- The amino acid sequence of COMT; GenBank Accession No. NP_000745.1) is shown in SEQ ID NO: 121, and the short OMT isoform (S-COMT;
The amino acid sequence of GenBank Accession No. NP_009294.1) is shown in SEQ ID NO: 122, respectively. SEQ ID NO: 122 corresponds to the amino acid sequence of SEQ ID NO: 121 lacking the N-terminal 50 amino acid residues. OMT further includes OMT of Bacteroidetes bacteria (that is, bacteria belonging to the Bacteroidetes phylum). Examples of bacteria belonging to the phylum Bacteroidetes include bacteria belonging to the genus Niastella, the genus Terrimonas, or the genus Chitinophaga (International Journal of Systematic and Evolutionary Microbiology (2007), 57, 1828-1833). Examples of the bacteria of the genus Niastella include Niastella koreensis. The nucleotide sequence of the OMT gene of Niastella koreensis is shown in SEQ ID NO: 123, and the amino acid sequence of the OMT encoded by the gene is shown in SEQ ID NO: 124.

  OMT can also catalyze, as a side reaction, a reaction that methylates protocatechuic acid and / or protocatechualdehyde to produce isovanillic acid and / or isovanillin (ie, methylation of the hydroxyl group at the para-position). OMT may selectively catalyze the methylation of the meta-hydroxyl group. "Selectively catalyze the methylation of the hydroxyl group at the meta position" means selectively producing vanillic acid from protocatechuic acid and / or selectively producing vanillin from protocatechualdehyde. May be. "Selectively producing vanillic acid from protocatechuic acid" means that when OMT is allowed to act on protocatechuic acid, for example, the molar ratio is 3 or more, 5 or more, 10 or more, or 15 times that of isovanillic acid. Above, 20 times or more, 25 times or more, or 30 times or more of vanillic acid may be generated. Further, "selectively producing vanillic acid from protocatechuic acid" means that when OMT is allowed to act on protocatechuic acid, for example, at a molar ratio of 60% or more based on the total amount of vanillic acid and isovanillic acid, It may mean producing 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of vanillic acid. This ratio, that is, the amount of vanillic acid with respect to the total amount of vanillic acid and isovanillic acid is also referred to as “VA / (VA + iVA) ratio”. Further, "selectively generate vanillin from protocatechualdehyde" means that when OMT is allowed to act on protocatechualdehyde, for example, the molar ratio is 3 times or more, 5 times or more, 10 times or more of isovanillin, It may mean producing 15 times or more, 20 times or more, 25 times or more, or 30 times or more of vanillin. Further, "selectively producing vanillin from protocatechualdehyde" means that, when OMT is allowed to act on protocatechualdehyde, for example, the molar ratio is 60% or more and 65% or more based on the total amount of vanillin and isovanillin. % Or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of producing vanillin. This ratio, that is, the amount of vanillin relative to the total amount of vanillin and isovanillin is also referred to as “Vn / (Vn + iVn) ratio”. An OMT that selectively catalyzes the methylation of a meta-hydroxyl group includes an OMT having a “specific mutation” described herein.

  An OMT having a “specific mutation” is also referred to as a “mutant OMT”. The gene encoding the mutant OMT is also referred to as “mutant OMT gene”.

  An OMT that does not have a “specific mutation” is also referred to as a “wild-type OMT”. The gene encoding the wild-type OMT is also referred to as “wild-type OMT gene”. Here, the term `` wild-type '' is a description for the convenience of distinguishing the `` wild-type '' OMT from the `` mutant '' OMT, and is not limited to those naturally obtained, Any OMT that does not have a "mutation" may be included. Examples of the wild-type OMT include the OMT exemplified above. In addition, all the conservative variants of the OMT exemplified above are included in the wild-type OMT unless they have a “specific mutation”.

The “specific mutation” includes a mutation of the mutant OMT described in WO2013 / 022881A1. That is, the “specific mutation” includes a mutation in which the leucine residue (L198) at position 198 of the wild-type OMT is replaced with an amino acid residue having a lower hydropathy index than the leucine residue, A glutamic acid residue at position 199 (E199) of type OMT is an amino acid residue having either a neutral or uncharged side chain at pH 7.4.
or positive side-chain charge at pH 7.4). The mutant OMT may have one of these mutations, or may have both.

"Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Lys, Met, Phe, Pro, Ser, Thr, Trp, and Tyr. The “amino acid residue having a lower hydropathy index than a leucine residue” includes, in particular, Ala, Arg, Asn, Asp, Glu, Gln, Gly, His,
Examples include amino acid residues selected from Lys, Met, Pro, Ser, Thr, Trp, and Tyr, and more particularly, Tyr.

As "an amino acid residue whose side chain is uncharged or positively charged at pH 7.4", Ala, Arg,
Asn, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val. The “amino acid residue whose side chain is uncharged or positively charged at pH 7.4” particularly includes Ala or Gln.

  “L198” and “E199” in any wild-type OMT refer to “an amino acid residue corresponding to the leucine residue at position 198 of the amino acid sequence shown in SEQ ID NO: 122” and “amino acid sequence shown in SEQ ID NO: 122, respectively”. An amino acid residue corresponding to the glutamic acid residue at position 199 "may be used. The positions of these amino acid residues indicate relative positions, and the absolute positions may be changed before and after due to amino acid deletion, insertion or addition. For example, in the amino acid sequence represented by SEQ ID NO: 122, when one amino acid residue is deleted or inserted at a position N-terminal to position X, the original amino acid residue at position X is N-terminal. And the amino acid residue at position X-1 or X + 1 is counted as "an amino acid residue corresponding to the amino acid residue at position X in the amino acid sequence shown in SEQ ID NO: 122". “L198” and “E199” are usually leucine residues and glutamic acid residues, respectively, but need not be. That is, the `` specific mutation '' also includes a mutation in which, when `` L198 '' and `` E199 '' are not a leucine residue and a glutamic acid residue, respectively, the amino acid residue is substituted with the amino acid residue after the above mutation. May be.

In the amino acid sequence of an arbitrary OMT, which amino acid residue is “L198” or “E199” can be determined by aligning the amino acid sequence of the arbitrary OMT with the amino acid sequence shown in SEQ ID NO: 122. The alignment can be performed using, for example, known gene analysis software. Specific software includes DNASIS manufactured by Hitachi Solutions and GENETYX manufactured by Genetics (Elizabeth C. Tyler et al., Computers and Biomedical Research, 24 (1), 72-96, 1991; Barton GJ et al.,
Journal of molecular biology, 198 (2), 327-37. 1987).

  “Specific mutations” further include mutations at the following amino acid residues (WO2018 / 079683): D21, L31, M36, S42, L67, Y90, P144. "Specific mutation" may be a mutation at one amino acid residue or a combination of mutations at two or more amino acid residues. That is, "specific mutations" may include, for example, mutations at one or more of the following amino acid residues: D21, L31, M36, S42, L67, Y90, P144.

In the above description for specifying an amino acid residue, a numeral indicates a position in the amino acid sequence shown in SEQ ID NO: 124, and a character to the left of the numeral indicates an amino acid residue at each position in the amino acid sequence shown in SEQ ID NO: 124 (that is, each position). Before modification) are shown. That is, for example, “D21” indicates a D (Asp) residue at position 21 in the amino acid sequence shown in SEQ ID NO: 124. In any wild-type OMT, each of these amino acid residues indicates “an amino acid residue corresponding to the amino acid residue in the amino acid sequence shown in SEQ ID NO: 124”. That is, for example, “D21” in any wild-type OMT indicates an amino acid residue corresponding to the D (Asp) residue at position 21 in the amino acid sequence shown in SEQ ID NO: 124.

  In each of the above mutations, the amino acid residue after modification may be any amino acid residue other than the amino acid residue before modification. As the amino acid residue after modification, specifically, K (Lys), R (Arg), H (His), A (Ala), V (Val), L (Leu), I (Ile), G ( Gly), S (Ser), T (Thr), P (Pro), F (Phe), W (Trp), Y (Tyr), C (Cys), M (Met), D (Asp), E ( Glu), N (Asn) and Q (Gln), other than the amino acid residue before modification. As the amino acid residues after modification, those effective for production of the target substance (for example, those that increase the VA / (VA + iVA) ratio and / or Vn / (Vn + iVn) ratio of OMT) are selected. Good.

  The “specific mutation” specifically includes the following mutations: D21Y, L31H, M36 (K, V), S42C, L67F, Y90 (A, C, G, S), P144 (E, G , S, V, Y). That is, mutations at the amino acid residues D21, L31, M36, S42, L67, Y90, and P144 are, for example, D21Y, L31H, M36 (K, V), S42C, L67F, Y90 (A, C, G, respectively) , S), and P144 (E, G, S, V, Y). A “specific mutation” may include, for example, one or more of the following mutations: D21Y, L31H, M36 (K, V), S42C, L67F, Y90 (A, C, G, S), P144 (E, G, S, V, Y).

  In the above notation for specifying a mutation, the numbers and the characters to the left thereof are the same as described above. In the above notation for specifying a mutation, the letter to the right of the number indicates the amino acid residue after modification at each position. That is, for example, “D21Y” indicates a mutation in which the D (Asp) residue at position 21 in the amino acid sequence shown in SEQ ID NO: 124 is substituted with a Y (Tyr) residue. For example, “M36 (K, V)” means that the M (Met) residue at position 36 in the amino acid sequence shown in SEQ ID NO: 124 is substituted with a K (Lys) residue or a V (Val) residue. Indicates a mutation. In any wild-type OMT, each of these mutations indicates “a mutation corresponding to the mutation in the amino acid sequence shown in SEQ ID NO: 124”. "The mutation corresponding to the mutation at the amino acid residue at position X in the amino acid sequence shown in SEQ ID NO: 124" is "mutation at the amino acid residue corresponding to the amino acid residue at position X in the amino acid sequence shown in SEQ ID NO: 124" Shall be replaced. That is, for example, in any wild-type OMT, “D21Y” means that an amino acid residue corresponding to the D (Asp) residue at position 21 in the amino acid sequence shown in SEQ ID NO: 124 is substituted with a Y (Tyr) residue. Mutation.

  The combination of mutations is not particularly limited. As a combination of mutations, specifically, D21Y / M36K / L67F, D21Y / M36K / L67F / Y90A, L31H / M36K / L67F / P144V, L31H / L67F / Y90A, M36K / S42C / L67F, M36K / L67F, M36K / L67F / Y90A, M36K / L67F / Y90A / P144E, M36K / L67F / Y90C, M36K / L67F / Y90C / P144V, M36K / L67F / Y90G, M36K / L67F / Y90S / P144G, M36K / L67F / P144S, M36K / L P144Y, M36K / Y90A / P144V, M36K / P144E, and M36V / L67F / P144S. That is, the “specific mutation” may include, for example, any combination of these.

  In the above description for specifying the combination, the numbers and the meaning of the characters on the left and right sides thereof are the same as described above. In the above notation for specifying a combination, a combination of two or more mutations separated by “/” indicates a double mutation or more than one multiple mutation. That is, for example, “M36K / P144E” indicates a double mutation of M36K and P144E.

  Descriptions of mutations in “L198” and “E199” (eg, descriptions for identifying absolute positions) include other “specific mutations” (eg, D21, L31, M36, S42, L67, Y90, and Mutation at the amino acid residue corresponding to P144). However, for these other “specific mutations”, the amino acid sequence shown in SEQ ID NO: 124 is used as a wild-type OMT reference sequence.

  The mutant OMT gene can be obtained, for example, by modifying a wild-type OMT gene so that the encoded OMT has a “specific mutation”. The wild-type OMT gene to be modified can be obtained, for example, by cloning from an organism having the wild-type OMT gene or by chemical synthesis. Alternatively, the mutant OMT gene can be obtained without using the wild-type OMT gene. The mutant OMT gene may be directly obtained, for example, by cloning from an organism having the mutant OMT gene or by chemical synthesis. The obtained mutant OMT gene may be used as it is or after further modification. The wild-type or mutant OMT gene from which the modification is made may or may not be derived from a host, such as a microorganism from which the microorganisms described herein are derived.

Gene modification can be performed by a known technique. For example, a target mutation can be introduced into a target site of DNA by a site-specific mutation method. As a site-specific mutation 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 a method using a phage (Kramer, W. and Frits, HJ, Meth. In Enzymol., 154, 350 (1987); Kunkel,
TA et al., Meth. In Enzymol., 154, 367 (1987)).

  OMT activity is determined, for example, by incubating an enzyme with a substrate (eg, protocatechuic acid or protocatechualdehyde) in the presence of SAM and measuring the production of the enzyme and a substrate-dependent product (eg, vanillic acid or vanillin). Can be measured (WO2013 / 022881A1). Also, by measuring the production of by-products (e.g., isovanillic acid or isovanillin) under similar conditions and comparing with the production of the product, it can be determined whether OMT selectively produces the product.

  “Aromatic aldehyde oxidoreductase (aromatic carboxylic acid reductase; ACAR)” refers to the reduction of aromatic carboxylic acid in the presence of an electron donor and ATP to the corresponding aromatic carboxylic acid reductase. A protein having an activity of catalyzing a reaction to produce an aromatic aldehyde may be used (such as EC 1.2.99.6). This activity is also called “ACAR activity”. The gene encoding ACAR is also called “ACAR gene”. ACAR may have the required substrate specificity depending on the type of biosynthetic pathway in which the target substance is produced in the method described herein. For example, when producing a target substance via conversion of vanillic acid to vanillin, ACAR using at least vanillic acid as a substrate can be used. In addition, for example, when the target substance is produced via conversion of protocatechuic acid to protocatechualdehyde, ACAR using at least protocatechuic acid as a substrate can be used. That is, "ACAR" specifically has an activity of catalyzing a reaction of reducing vanillic acid and / or protocatechuic acid in the presence of an electron donor and ATP to produce vanillin and / or protocatechualdehyde. It may mean protein. ACAR may be specific for both vanillic acid and protocatechuic acid, but is not so limited. For example, when producing benzaldehyde, ACAR using at least benzoic acid as a substrate can be used. That is, “ACAR” may specifically mean a protein having an activity of catalyzing a reaction of reducing benzoic acid to generate benzaldehyde in the presence of an electron donor and ATP. For example, when producing cinnamaldehyde, ACAR using at least cinnamic acid as a substrate can be used. That is, “ACAR” may specifically mean a protein having an activity of catalyzing a reaction of reducing cinnamic acid to generate cinnamaldehyde in the presence of an electron donor and ATP. Examples of the electron donor include NADH and NADPH. The ACAR may, for example, be able to utilize at least one of these electron donors.

ACAR includes Nocardia sp.NRRL 5646 strain, Actinomyces sp., Clostridium thermoace
ACAR of various organisms such as ticum, Aspergillus niger, Corynespora melonis, Coriolus sp., Neurospora sp., and the like (J. Biol. Chem. 2007, Vol. 282, No. 1, p478-485). Nocardia sp. NRRL 5646 strain is classified as Nocardia iowensis. ACAR further includes ACAR of other bacteria belonging to the genus Nocardia, such as Nocardia brasiliensis and Nocardia vulneris. The nucleotide sequence of the ACAR gene of Nocardia brasiliensis ATCC 700358 is shown in SEQ ID NO: 125, and the amino acid sequence of ACAR encoded by the gene is shown in SEQ ID NO: 126. Nocardia
The nucleotide sequence of an example of the variant ACAR gene of the brasiliensis ATCC 700358 strain is shown in SEQ ID NO: 127, and the amino acid sequence of ACAR encoded by the gene is shown in SEQ ID NO: 128. ACAR also includes ACAR of a microorganism belonging to the genus Gordonia such as Gordonia effusa, a bacterium belonging to the genus Novosphingobium such as Novosphingobium malaysiense, or a microorganism belonging to the genus Coccomyxa such as Coccomyxa subellipsoidea (WO2018 / 079705A1). The nucleotide sequence of the Gordonia effusa ACAR gene is shown in SEQ ID NO: 129, and the amino acid sequence of ACAR encoded by the gene is shown in SEQ ID NO: 130. SEQ ID NO: 131 shows the nucleotide sequence of the ACAR gene of Novosphingobium malaysiense, and SEQ ID NO: 132 shows the amino acid sequence of ACAR encoded by the gene. The nucleotide sequence of the ACAR gene of Coccomyxa subellipsoidea C-169 is shown in SEQ ID NO: 133, and the amino acid sequence of ACAR encoded by the gene is shown in SEQ ID NO: 134.

  ACAR activity can be measured, for example, by incubating the enzyme with a substrate (eg, vanillic acid or protocatechuic acid) in the presence of ATP and NADPH, and measuring enzyme and substrate-dependent oxidation of NADPH (J. Biol. Chem. 2007, Vol. 282, No. 1, p478-485 modified).

  ACAR can become an active enzyme by phosphopantethenylation (J. Biol. Chem. 2007, Vol. 282, No. 1, p478-485). Thus, ACAR activity can be increased by increasing the activity of an enzyme that catalyzes phosphopantethenylation of a protein (also referred to as “phosphopantethenylation enzyme”). That is, as a method for imparting or enhancing the target substance-producing ability, a method for increasing the activity of a phosphopantetheinylase is exemplified. That is, the microorganism may be modified such that the activity of the phosphopantetheinylating enzyme is increased. Examples of the phosphopantetheinylating enzyme include phosphopantetheinyl transferase (PPT).

The term “phosphopantetheinyl transferase (PPT)” may mean a protein having an activity of catalyzing a reaction of phosphopantetheinylation of ACAR in the presence of a phosphopantetheinyl group donor. This activity is also called “PPT activity”. The gene encoding PPT is also called “PPT gene”. Examples of the phosphopantetheinyl group donor include coenzyme A (CoA). PPT includes EntD protein encoded by the entD gene. Specific examples of the PPT include PPT of Nocardia brasiliensis, PPT of Nocardia farcinica IFM10152 (J. Biol. Chem. 2007, Vol. 282, No. 1, pp. 478-485), and PPT of Corynebacterium glutamicum (App. Env. Microbiol. 2009, Vol. 75, No. 9, pp. 2765-2774), and various organisms such as E. coli EntD protein. Corynebacterium
The nucleotide sequence of the PPT gene of glutamicum ATCC 13032 is shown in SEQ ID NO: 135, and the amino acid sequence of the PPT encoded by the gene is shown in SEQ ID NO: 136. The nucleotide sequence of the entD gene of E. coli K-12 MG1655 strain is shown in SEQ ID NO: 137, and the amino acid sequence of the EntD protein encoded by the gene is shown in SEQ ID NO: 138.

  The PPT activity can be measured, for example, by incubating the enzyme with ACAR in the presence of CoA and using the enhancement of ACAR activity as an index (J. Biol. Chem. 2007, Vol. 282, No. 1, pp. 478). -485).

Melatonin can be produced from L-tryptophan. That is, examples of the target substance biosynthetic enzyme include L-tryptophan biosynthetic enzyme and an enzyme that catalyzes the conversion of L-tryptophan to melatonin. Examples of L-tryptophan biosynthetic enzymes include 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (aroF, aroG, aroH), and 3-dehydroquinic acid. Synthase (3-dehydroquinate synthase; aroB), 3-dehydroquinate dehydratase (aroD), shikimate dehydrogenase (aroE), shikimate kinase (aroK, aroL), 5-enol pill Biosynthetic enzymes common to aromatic amino acids such as virshikimate-3-phosphate synthase (5-enolpyruvylshikimate-3-phosphate synthase; aroA) and chorismate synthase (aroC), and anthranilate synthase (anthranilate synthase) TrpED) and tryptophan synthase (trpAB). An example of a gene name encoding each enzyme is shown in parentheses after the enzyme name (the same applies hereinafter). L-tryptophan is tryptophan 5-hydroxylase (EC 1.14.16.4), 5-hydroxytryptophan decarboxylase (EC 4.1.1.28), aralkylamine-N-acetyltransferase (aralkylamine). By the action of N-acetyltransferase; AANAT; EC 2.3.1.87) and acetylserotonin O-methyltransferase (EC 2.1.1.4), hydroxytryptophan, hydroxytryptophan, serotonin, and N- It can be converted to N-acetylserotonin, and melatonin. That is, enzymes that catalyze the conversion of L-tryptophan to melatonin include these enzymes. In addition, acetylserotonin O-methyltransferase is an example of OMT which produces melatonin by methylating N-acetylserotonin using SAM as a methyl group donor.

Ergothioneine can be produced from L-histidine. That is, examples of the target substance biosynthetic enzyme include L-histidine biosynthetic enzyme and an enzyme that catalyzes the conversion of L-histidine to ergothioneine. Examples of L-histidine biosynthetic enzymes include ATP phosphoribosyltransferase (hisG), phosphoribosyl-AMP cyclohydrolase (hisI), phosphoribosyl-ATP pyrophosphohydrolase (hisI), and phosphoribosylformase. Imino-5-aminoimidazole carboxamide ribotide isomerase (phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase; hisA), amide transferase (amidotransferase; hisH), histidinol phosphate aminotransferase (hisC), histidinol Examples include phosphatase (histidinol phosphatase; hisB) and histidinol dehydrogenase (histD). L-histidine is sequentially converted into hercynine, hercynyl-γ-L-glutamyl-L-cysteine by the action of EgtB, EgtC, EgtD, and EgtE proteins encoded by egtB, egtC, egtD, and egtE genes, respectively. Sulfoxide (hercynyl-gamma-L-glutamyl-L-cysteine
sulfoxide), hercynyl-L-cysteine sulfoxide, and ergothioneine. Hercinin can be converted to hercinyl-L-cysteine sulfoxide by the action of the Egt1 protein encoded by the egt1 gene. That is, these enzymes are mentioned as enzymes that catalyze the conversion of L-histidine to ergothioneine. EgtD is a SAM-dependent histidine-N, N, N-methyltransferase (SAM) -dependent histidine N, which methylates histidine to generate hercinin using SAM as a methyl group donor. N, N-methyltransferase).

Guaiacol can be produced from vanillic acid. Therefore, as for the target substance biosynthetic enzyme for guaiacol, the description of the target substance biosynthetic enzyme for vanillic acid described above can be applied mutatis mutandis. Vanillic acid can be converted to guaiacol by the action of vanillic acid decarboxylase (VDC). That is, the target substance biosynthetic enzyme includes VDC.

  Ferulic acid, 4-vinyl guaiacol, and 4-ethyl guaiacol can be produced from L-phenylalanine or L-tyrosine. That is, as the target substance biosynthetic enzyme, for example, L-phenylalanine biosynthetic enzyme, L-tyrosine biosynthetic enzyme, and L-phenylalanine or L-tyrosine to ferulic acid, 4-vinylguaiacol, or 4-ethylguaiacol Also included are enzymes that catalyze the conversion. Examples of the L-phenylalanine biosynthetic enzyme include a biosynthetic enzyme common to the aromatic amino acids exemplified above, chorismate mutase (pheA), prephenate dehydratase (pheA), and tyrosine aminotransferase (tyrosine amino transferase). TyrB). Chorismate mutase and prephenate dehydratase may be encoded in the pheA gene as bifunctional enzymes. Examples of the L-tyrosine biosynthetic enzyme include a biosynthetic enzyme common to the aromatic amino acids exemplified above, chorismate mutase (tyrA), prephenate dehydrogenase (tyrA), and tyrosine aminotransferase (tyrosine amino transferase). TyrB). Chorismate mutase and prephenate dehydrogenase may be encoded in the tyrA gene as bifunctional enzymes. L-phenylalanine is converted to cinnamic acid by the action of phenylalanine ammonia lyase (PAL; EC 4.3.1.24), and then to cinnamic acid 4-hydroxylase (C4H; EC 1.14). .13.11) can be converted to p-coumaric acid. In addition, L-tyrosine can be converted to p-coumaric acid by the action of tyrosine ammonia lyase (TAL; EC 4.3.1.23). p-Coumaric acid includes hydroxycinnamic acid 3-hydroxylase (C3H), O-methyltransferase (O-methyltransferase; OMT), ferulic acid decarboxylase (FDC), and vinyl. It can be converted into caffeic acid, ferulic acid, 4-vinyl guaiacol, and 4-ethyl guaiacol in turn by the action of phenol reductase (VPR). That is, enzymes that catalyze the conversion of L-phenylalanine or L-tyrosine to ferulic acid, 4-vinylguaiacol, or 4-ethylguaiacol include these enzymes. To produce ferulic acid, 4-vinyl guaiacol, or 4-ethyl guaiacol, an OMT utilizing at least caffeic acid can be used.

Polyamines can be produced from L-arginine or L-ornithine. That is, examples of the target substance biosynthetic enzyme include L-arginine biosynthetic enzyme, L-ornithine biosynthetic enzyme, and an enzyme that catalyzes the conversion of L-arginine or L-ornithine to polyamine. Examples of L-ornithine biosynthetic enzymes include N-acetylglutamate synthase (argA), N-acetylglutamate kinase (argB), and N-acetylglutamyl phosphate reductase. ArgC), acetylornithine transaminase (argD), and acetylornithine deacetylase (argE). Examples of L-arginine biosynthetic enzymes include L-ornithine biosynthetic enzymes exemplified above, carbamoyl phosphate synthetase (carAB), ornithine carbamoyl transferase (argF, argI), and argininosuccinate synthase (argininosuccinate). synthetase; argG) and argininosuccinate lyase (argH). L-arginine is converted to agmatine by the action of arginine decarboxylase (arginine decarboxylase; speA; EC 4.1.1.19) and then putrescine (agmatine ureohydrolase; speB; EC 3.5.3.11) by the action of agmatine ureohydrolase (EC 3.5.3.11). putrescine). In addition, L-ornithine can be converted to putrescine by the action of ornithine decarboxylase (ornithine decarboxylase; speC; EC 4.1.1.17). Putrescine can be converted to spermidine by the action of spermidine synthase (speE; EC 2.5.1.16) and then to spermine by the action of spermine synthase (EC 2.5.1.22). Agmatine is also converted to aminopropylagmatine by the action of agmatine / triamine aminopropyl transferase and then to spermidine by the action of aminopropylagmatine ureohydrolase. obtain. That is, the enzymes that catalyze the conversion of L-arginine or L-ornithine to polyamine include these enzymes. Note that spermidine synthase, spermine synthase, and agmatine / triamine aminopropyl transferase all transfer propylamine groups from decarboxylated S-adenosyl methionine (dcSAM), which can be generated by decarboxylation of SAM, to the corresponding substrate. Catalyze the reaction.

  Creatine can be produced from L-arginine and glycine. That is, examples of the target substance biosynthetic enzyme include L-arginine biosynthetic enzyme, glycine biosynthetic enzyme, and an enzyme that catalyzes the conversion of L-arginine and glycine to creatine. L-arginine and glycine can combine to produce guanidinoacetate and ornithine by the action of arginine: glycine amidinotransferase (AGAT; EC 2.1.4.1). Guanidinoacetic acid can be methylated with SAM as a methyl group donor to produce creatine by the action of guanidinoacetate-N-methyltransferase (guanidinoacetate N-methyltransferase; GAMT; EC 2.1.1.2). That is, enzymes that catalyze the conversion of L-arginine and glycine to creatine include these enzymes.

  Mugineic acid can be produced from SAM. That is, examples of the target substance biosynthetic enzyme include an enzyme that catalyzes the conversion of SAM to mugineic acid. One molecule of nicotianamine can be synthesized from three molecules of SAM by the action of nicotianamine synthase (nicotianamine synthase; EC 2.5.1.43). Nicotianamine is nicotianamine aminotransferase (EC 2.6.1.80), 3 "-deamino-3" -oxonicotianamine reductase (3 "-deamino-3" -oxonicotianamine reductase; EC 1.1.1.285), and 2'-deoxymugine. By the action of 2'-deoxymugineic-acid 2'-dioxygenase (EC 1.14.11.24), 3 "-deamino-3" -oxonicotianamine (3 "-deamino-3" -oxonicotianamine) ), 2'-deoxymugineic-acid, and mugineic acid. That is, enzymes that catalyze the conversion of SAM to mugineic acid include these enzymes.

  L-methionine may be generated from L-cysteine. That is, examples of the target substance biosynthetic enzyme include L-cysteine biosynthetic enzyme and an enzyme that catalyzes the conversion of L-cysteine to L-methionine. Examples of the L-cysteine biosynthetic enzyme include CysIXHDNYZ protein, Fpr2 protein, and CysK protein described herein. Examples of enzymes that catalyze the conversion of L-cysteine to L-methionine include cystathionine-gamma-synthase and cystathionine-β-lyase.

Also, as described above, benzaldehyde and cinnamaldehyde, for example, can be produced from benzoic acid and cinnamic acid, respectively, by the action of ACAR. That is, examples of the target substance biosynthetic enzyme include benzoic acid biosynthetic enzyme and cinnamic acid biosynthetic enzyme in addition to ACAR. Specifically, cinnamic acid is, for example, phenylalanine ammonia lyase (
It can be produced from L-phenylalanine by the action of phenylalanine ammonia lyase; PAL; EC 4.3.1.24). That is, examples of the cinnamic acid biosynthetic enzyme include L-phenylalanine biosynthetic enzyme and PAL.

  Benzaldehyde can also be produced, for example, from L-phenylalanine (WO2017 / 122747). That is, examples of the target substance biosynthetic enzyme include L-phenylalanine biosynthetic enzyme and an enzyme that catalyzes the conversion of L-phenylalanine to benzaldehyde. L-phenylalanine is an amino acid deaminase (AAD; EC 1.4.3.2), 4-hydroxymandelate synthase (HMAS; EC 1.13.11.46), (S) -mandelic acid dehydrogenase ((S ) -mandelate dehydrogenase; SMDH; EC 1.1.99.31), and phenylpyruvic acid, (S) -mandelic acid, benzoylformic acid, and benzoylformate decarboxylase; BFDC; It can be converted to benzaldehyde. That is, these enzymes are exemplified as enzymes that catalyze the conversion of L-phenylalanine to benzaldehyde.

  In addition, as a method for imparting or enhancing the target substance producing ability, a substance other than the target substance (for example, a substance generated as an intermediate during the production of the target substance or a substance used as a precursor of the target substance) A method for increasing the activity of an uptake system of is mentioned. That is, the microorganism may be modified such that the activity of such an uptake system is increased. The “substance uptake system” may mean a protein having a function of taking up a substance from outside a cell into a cell. This activity is also referred to as "substance uptake activity". A gene encoding such an uptake system is also referred to as an “uptake system gene”. Examples of such an uptake system include a vanillic acid uptake system and a protocatechuate acid uptake system. Examples of the vanillic acid uptake system include the VanK protein encoded by the vanK gene (MT Chaudhry, et al., Microbiology, 2007. 153: 857-865). The nucleotide sequence of the vanK gene (NCgl2302) of C. glutamicum ATCC 13869 is shown in SEQ ID NO: 139, and the amino acid sequence of the VanK protein encoded by the gene is shown in SEQ ID NO: 140. The protocatechuic acid uptake system includes a PcaK protein encoded by the pcaK gene (MT Chaudhry, et al., Microbiology, 2007. 153: 857-865). The nucleotide sequence of the pcaK gene (NCgl1031) of C. glutamicum ATCC 13869 is shown in SEQ ID NO: 141, and the amino acid sequence of the PcaK protein encoded by the gene is shown in SEQ ID NO: 142.

  The substance uptake activity can be measured, for example, by a known method (MT Chaudhry, et al., Microbiology, 2007. 153: 857-865).

Examples of a method for imparting or enhancing the target substance-producing ability include a method of reducing the activity of an enzyme involved in by-product of a substance other than the target substance. Such a substance other than the target substance is also referred to as “by-product”. Such enzymes are also referred to as "byproduct-forming enzymes." Examples of the by-product-forming enzyme include an enzyme involved in assimilation of the target substance and an enzyme that catalyzes a reaction that diverges from the biosynthetic pathway of the target substance to generate a substance other than the target substance. A method for reducing the activity of a protein (enzyme or the like) will be described later. The activity of a protein (such as an enzyme) can be reduced, for example, by disrupting a gene encoding the protein. For example, in coryneform bacteria, it has been reported that vanillin is metabolized and assimilated in the order of vanillin → vanillic acid → protocatechuic acid (Current Microbiology, 2005, Vol. 51, p59-65). That is, specific examples of by-product-forming enzymes include enzymes that catalyze the conversion of vanillin into protocatechuic acid and enzymes that catalyze the further metabolism of protocatechuic acid. Examples of such enzymes include vanillate demethylase, protocatechuate 3,4-dioxygenase, and the reaction product of protocatechuate 3,4-dioxygenase with succinyl-CoA and acetyl-CoA. Various enzymes that further degrade (Appl. Microbiol. Biotechnol., 2012, Vol. 95, p77-89). Also, vanillin can be converted to vanillyl alcohol by the action of alcohol dehydrogenase (ADH) (Kunjapur AM. Et al.
al., J. Am. Chem. Soc., 2014, Vol. 136, p11644-11654 .; Hansen EH. et al., App. Env. Microbiol., 2009, Vol.75, p2765-2774.). That is, ADH is specifically mentioned as a by-product-forming enzyme. Also, 3-dehydroshikimic acid, which is an intermediate in the vanillin biosynthetic pathway, can be converted to shikimic acid by the action of shikimate dehydrogenase. That is, as the by-product-forming enzyme, specifically, shikimate dehydrogenase can be mentioned.

  “Vanillate demethylase” may refer to a protein that has the activity of catalyzing the reaction of demethylating vanillic acid to produce protocatechuic acid. This activity is also referred to as "vanillic acid demethylase activity". The gene encoding vanillic acid demethylase is also referred to as "vanillic acid demethylase gene". Vanillic acid demethylase includes VanAB protein encoded by 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 the vanillic acid demethylase activity is reduced, for example, both vanAB genes may be destroyed, or only one of them may be destroyed. The nucleotide sequences of the vanAB gene of C. glutamicum ATCC 13869 are shown in SEQ ID NOs: 143 and 145, and the amino acid sequences of the VanAB protein encoded by the gene are shown in SEQ ID NOs: 144 and 146, respectively. The vanAB gene usually constitutes the vanK gene and the vanABK operon. Thus, the vanABK operon may be destroyed (eg, deleted) collectively in order to reduce vanillic acid demethylase activity. In that case, the vanK gene may be introduced again into the host. For example, when vanillic acid existing outside the cells is used, and vanABK operon is destroyed (eg, deleted) as a whole, it is preferable to introduce the vanK gene again.

  Vanillate demethylase activity can be measured, for example, by incubating the enzyme with a substrate (ie, vanillic acid) and measuring the production of the enzyme and substrate-dependent product (ie, protocatechuic acid) (J Bacteriol, 2001, Vol.183, p3276-3281).

  “Protocatechuate 3,4-dioxygenase” may mean a protein having an activity of catalyzing a reaction of oxidizing protocatechuic acid to generate β-carboxyl cis, cis-muconic acid. This activity is also referred to as "protocatechuate 3,4-dioxygenase activity". The gene encoding protocatechuate 3,4-dioxygenase is also referred to as "protocatechuate 3,4-dioxygenase gene". Examples of protocatechuate 3,4-dioxygenase include a PcaGH protein encoded by the pcaGH gene (Appl. Microbiol. Biotechnol., 2012, Vol. 95, p77-89). The pcaG and pcaH genes encode the α and β subunits of protocatechuate 3,4-dioxygenase, respectively. When the protocatechuic acid 3,4-dioxygenase activity is reduced, for example, both pcaGH genes may be destroyed, or only one of them may be destroyed. The nucleotide sequences of the pcaGH gene of C. glutamicum ATCC 13032 are shown in SEQ ID NOS: 147 and 149, and the amino acid sequence of the PcaGH protein encoded by the gene is shown in SEQ ID NOs: 148 and 150, respectively.

  Protocatechuate 3,4-dioxygenase activity can be measured, for example, by incubating the enzyme with a substrate (ie, protocatechuic acid) and measuring enzyme and substrate-dependent oxygen consumption (Meth. Enz., 1970, Vol.17A, p526-529).

“Alcohol dehydrogenase (ADH)” may mean a protein having an activity of catalyzing a reaction of reducing an aldehyde in the presence of an electron donor to produce an alcohol (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”. The gene encoding ADH is also referred to as "ADH gene". Examples of the aldehyde serving as a substrate of ADH include the aldehydes exemplified as the target substance, for example, aromatic aldehydes such as vanillin, benzaldehyde, and cinnamaldehyde. That is, the combination of aldehyde and alcohol referred to in the description of `` ADH activity '' includes aromatic aldehydes such as a combination of vanillin and vanillyl alcohol, a combination of benzaldehyde and benzyl alcohol, and a combination of cinnamaldehyde and cinnamyl alcohol. Combinations of aromatic alcohols are included. ADH using aromatic aldehyde, vanillin, benzaldehyde and cinnamaldehyde as substrates is called "aromatic alcohol dehydrogenase", "vanillyl alcohol dehydrogenase" and "benzyl alcohol dehydrogenase", respectively. dehydrogenase) and "cinnamyl alcohol dehydrogenase". In addition, ADH activity using aromatic aldehyde, vanillin, benzaldehyde, and cinnamaldehyde as substrates is referred to as "aromatic alcohol dehydrogenase activity", "vanillyl alcohol dehydrogenase activity", and "benzyl alcohol dehydrogenase activity", respectively. Also called "alcohol dehydrogenase (benzyl alcohol dehydrogenase) activity" and "cinnamyl alcohol dehydrogenase activity". ADH may use one aldehyde as a substrate or two or more aldehydes as a substrate. Examples of the electron donor include NADH and NADPH. ADH may, for example, utilize at least one of these electron donors.

  Examples of ADH include the yqhD gene, the NCgl0324 gene, the NCgl0313 gene, the NCgl2709 gene, the NCgl0219 gene, and the NCgl2382 gene, respectively. The yqhD gene and NCgl0324 gene both encode vanillyl alcohol dehydrogenase. The yqhD gene can be found, for example, in bacteria of the family Enterobacteriaceae, such as E. coli. The NCgl0324 gene, NCgl0313 gene, NCgl2709 gene, NCgl0219 gene, NCgl2382 gene can be found in coryneform bacteria such as C. glutamicum. SEQ ID NO: 151 shows the nucleotide sequence of the yqhD gene of E. coli K-12 MG1655, and SEQ ID NO: 152 shows the amino acid sequence of the YqhD protein encoded by the gene. The nucleotide sequences of the NCgl0324, NCgl0313, and NCgl2709 genes of C. glutamicum ATCC 13869 are shown in SEQ ID NOs: 153, 155, and 157, and the amino acid sequences of the proteins encoded by the genes are shown in SEQ ID NOs: 154, 156, and 158, respectively. The nucleotide sequences of the NCgl0219 gene and NCgl2382 gene of C. glutamicum ATCC 13032 are shown in SEQ ID NOs: 159 and 161 and the amino acid sequences of the proteins encoded by the genes are shown in SEQ ID NOs: 160 and 162, respectively.

In microorganisms, 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, for example all, ADH selected from the NCgl0324 protein, NCgl2709 protein, and NCgl0313 protein may be reduced. Further, at least the activity of one or both of the NCgl0324 protein and the NCgl2709 protein may be reduced. That is, for example, the activity of at least the NCgl0324 protein may be reduced, and the activity of the NCgl2709 protein may be further reduced. Alternatively, at least the activity of the NCgl2709 protein may be reduced, and the activity of the NCgl0324 protein may be further reduced. The combination of ADH and the target substance is not particularly limited as long as the production of the target substance is increased by reducing the activity of ADH in coryneform bacteria. For example, at least the activity of ADH using an aldehyde produced as a target substance as a substrate may be reduced. That is, for example, for the production of aromatic aldehydes such as vanillin, benzaldehyde and cinnamaldehyde, respectively, aromatic alcohol deh such as vanillyl alcohol dehydrogenase, benzyl alcohol dehydrogenase, cinnamyl alcohol dehydrogenase, etc.
The activity of ydrogenase may be reduced. For example, when producing vanillin, the activity of the YqhD protein may be reduced. For example, when producing vanillin, the activity of one or both of the NCgl0324 protein and the NCgl0313 protein may be reduced, or at least the activity of the NCgl0324 protein may be reduced. Further, for example, when producing benzaldehyde, the activity of one or both of NCgl0324 protein and NCgl2709 protein may be reduced. For example, when producing cinnamaldehyde, the activity of one or both of NCgl0324 protein and NCgl2709 protein may be reduced. The YqhD protein may have vanillyl alcohol dehydrogenase activity. NCgl0324 protein has vanillyl alcohol dehydrogenase activity, benzyl alcohol dehydrogenase activity, and cinnamyl
It may have all of the alcohol dehydrogenase activity. The NCgl2709 protein may have both benzyl alcohol dehydrogenase activity and cinnamyl alcohol dehydrogenase activity.

  ADH activity can be measured, for example, by incubating the enzyme with a substrate (eg, an aldehyde such as vanillin) in the presence of NADPH or NADH, and measuring enzyme and substrate-dependent oxidation of NADPH or NADH.

  By "shikimate dehydrogenase" may be meant a protein having the activity of catalyzing the reaction of reducing 3-dehydroshikimate in the presence of an electron donor to produce shikimate (EC 1.1. 1.25). This activity is also called “shikimate dehydrogenase activity”. The gene encoding shikimate dehydrogenase is also called "shikimate dehydrogenase gene". Examples of the electron donor include NADH and NADPH. The shikimate dehydrogenase may, for example, be one that can utilize at least one of these electron donors. The shikimate dehydrogenase includes AroE protein encoded by the aroE gene. The nucleotide sequence of the aroE gene of E. coli K-12 MG1655 is shown in SEQ ID NO: 163, and the amino acid sequence of the AroE protein encoded by the gene is shown in SEQ ID NO: 164.

  Shikimate dehydrogenase activity can be measured, for example, by incubating the enzyme with a substrate (ie, 3-dehydroshikimic acid) in the presence of NADH or NADPH and measuring enzyme and substrate-dependent oxidation of NADH or NADPH.

  Examples of a method for imparting or enhancing the ability to produce a target substance such as a SAM-dependent metabolite or L-methionine include a method for increasing the activity of L-cysteine biosynthetic enzyme (WO2018 / 079687).

“L-cysteine biosynthetic enzyme” may mean a protein involved in L-cysteine biosynthesis. The gene encoding the L-cysteine biosynthetic enzyme is also referred to as “L-cysteine biosynthesis gene”. L-cysteine biosynthetic enzymes include proteins involved in sulfur utilization. Proteins involved in sulfur utilization include CysIXHDNYZ protein and Fpr2 protein encoded by the cysIXHDNYZ gene and fpr2 gene, respectively. CysIXHDNYZ protein is particularly involved in the reduction of inorganic sulfur compounds such as sulfates and sulfites. The Fpr2 protein may particularly be involved in electron transport for sulfite reduction. The L-cysteine biosynthetic enzyme also includes O-acetylserine (thiol) -lyase. O-acetylserine (thiol) -lyase also includes CysK protein encoded by the cysK gene. C. glutamicum ATCC 13869 strain cysIXHDNYZ gene, fpr2 gene, and cysK gene (NCgl2473) have the nucleotide sequences of SEQ ID NOs: 165, 167, 169, 171, 173, 175, 177, 179, 181; Are shown in SEQ ID NOs: 166, 168, 170, 172, 174, 176, 178, 180, and 182, respectively. The activity of one L-cysteine biosynthetic enzyme may be increased, and the activity of two or more L-cysteine biosynthetic enzymes may be increased. For example, the activity of one or more of the CysIXHDNYZ protein, the Fpr2 protein, and the CysK protein may be increased, and the activity of one or more of the CysIXHDNYZ protein and the Fpr2 protein may be increased.

  The activity of L-cysteine biosynthetic enzyme can be determined, for example, by increasing the expression of a gene encoding L-cysteine biosynthetic enzyme (that is, an L-cysteine biosynthetic gene such as cysIXHDNYZ gene, fpr2 gene, cysK gene, etc.) Can be increased.

  The expression of the L-cysteine biosynthesis gene can be increased, for example, by modifying (eg, increasing or decreasing) the activity of an expression regulator of the gene. That is, the expression of the L-cysteine biosynthesis gene can be increased, for example, by increasing the activity of a positive expression regulator (for example, an activator) of the gene. In addition, the expression of the L-cysteine biosynthesis gene can be increased, for example, by decreasing the activity of a negative expression regulator (for example, a repressor) of the gene. 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”.

  The activator as described above includes CysR protein and SsuR protein encoded by the cysR gene and ssuR gene, respectively. Increased activity of the CysR protein may increase expression of one or more of the cysIXHDNYZ gene, fpr2 gene, and ssuR gene. In addition, an increase in the activity of the SsuR protein may increase the expression of genes involved in the use of organic sulfur compounds. The nucleotide sequences of the cysR gene (NCgl0120) and ssuR gene of C. glutamicum ATCC 13869 are shown in SEQ ID NOs: 183 and 185, and the amino acid sequences of the proteins encoded by the gene are shown in SEQ ID NOs: 184 and 186, respectively. The activity of one or both of the CysR and SsuR proteins may be increased. For example, at least the activity of the CysR protein may be reduced. The activity of such an activator can be increased, for example, by increasing the expression of a gene encoding the activator.

  The repressor as described above includes 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 I can do it. The activity of such a repressor 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 L-cysteine biosynthetic enzyme is increased, for example, by increasing the expression of one or more of the cysIXHDNYZ gene, fpr2 gene, cysR gene, and ssuR gene. Can be. That is, "the activity of L-cysteine biosynthetic enzyme is increased" means that, for example, 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, fpr2 gene, and ssuR gene may be increased by increasing the expression of the cysR gene.

  Examples of a method for imparting or enhancing the ability to produce a target substance such as a SAM-dependent metabolite or L-methionine include a method of reducing the activity of NCgl2048 protein (WO2018 / 079686).

“NCgl2048 protein” may mean a protein encoded by the NCgl2048 gene. The nucleotide sequence of the NCgl2048 gene of C. glutamicum ATCC 13869 is shown in SEQ ID NO: 187, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 188. With respect to the conservative variant, the "original function" of the NCgl2048 protein may mean the function of the protein having the amino acid sequence of SEQ ID NO: 188, and the production of the target substance is increased by reducing the activity in the microorganism. It may mean a property.

  Examples of a method for imparting or enhancing the ability to produce a target substance such as a SAM-dependent metabolite or L-methionine include a method of reducing enolase activity (WO2018 / 079684). The enolase is as described above. When the activity of enolase is decreased and increased in order to decrease and supplement the ability to assimilate sugar, the activity of enolase after complementation is changed to a non-modified strain (ie, The strain may be limited to be lower than the strain before the sugar is assimilated.

  As a method for imparting or enhancing the ability to produce a target substance such as a SAM-dependent metabolite or L-methionine, the activity of S-adenosyl-L-homocysteine hydrolase is used. There is a method of increasing (WO2018 / 079684).

  "S-adenosyl-L-homocysteine hydrolase" refers to S-adenosyl-L-homocysteine hydrolase, which hydrolyzes S-adenosyl-L-homocysteine (SAH) to L-. It may mean a protein having the activity of catalyzing the reaction to produce homocysteine and adenosine (EC 3.3.1.1). This activity is also referred to as “S-adenosyl-L-homocysteine hydrolase activity”. S-adenosyl-L-homocysteine hydrolase is also referred to as "adenosylhomocysteinase". The gene encoding S-adenosyl-L-homocysteine hydrolase is also referred to as “S-adenosyl-L-homocysteine hydrolase gene”. S-adenosyl-L-homocysteine hydrolase includes a SahH protein encoded by the sahH gene. Specific examples of S-adenosyl-L-homocysteine hydrolase include those of various organisms such as yeast, bacteria of the genus Streptomyces, and coryneform bacteria. The nucleotide sequence of the sahH gene (NCgl0719) of C. glutamicum ATCC 13869 is shown in SEQ ID NO: 189, and the amino acid sequence of the SahH protein encoded by the gene is shown in SEQ ID NO: 190.

S-adenosyl-L-homocysteine hydrolase activity can be determined, for example, by incubating the enzyme with a substrate (eg, S-adenosyl-L-homocysteine) and DTNB (5,5′-Dithiobis (2-nitrobenzoic acid)), For example, it can be measured by measuring homocysteine-dependent TNB (5-Mercapto-2-nitrobenzoic acid) production based on the absorbance at 412 nm (J
Mol Microbiol Biotechnol 2008, 15: 277-286).

  The protein whose activity is to be modified can be appropriately selected depending on, for example, the type of the target substance, the type of the biosynthetic pathway in which the target substance is produced, and various conditions such as the type and activity of the protein that the microorganism originally has. For example, if vanillin is produced by bioconversion from protocatechuic acid, the activity of one or more of the OMT, ACAR, PPT, and protocatechuic acid uptake systems, among others, may be increased. Also, for example, if vanillin is produced by bioconversion from vanillic acid, one or more of the activities of the ACAR, PPT, and vanillic acid uptake systems may be particularly increased. In addition, when vanillin is produced by bioconversion from protocatechualdehyde, the activity of OMT may be particularly increased.

When the target substance is an expression product of a gene (for example, a protein or RNA), the microorganism described in the present specification has a target substance-producing ability based on having at least a gene encoding the target substance. The gene encoding the target substance is also called “target substance gene”. That is, the microorganism may have the target substance gene. The microorganism may have a target protein-producing ability, for example, by having a target substance gene, or a combination of having the target substance gene and other properties. Examples of a method for imparting or enhancing the target substance-producing ability include a method of introducing a target substance gene into a microorganism. The method for introducing a gene will be described later.

  For example, the protein may be secreted and produced extracellularly (for example, in the medium or on the surface of the cell), or may be accumulated in the cell. For example, a protein can be secreted and produced by using a signal peptide. The gene encoding the target protein (target protein gene) only needs to be retained in the host so that it can be expressed. Specifically, for example, a protein expression unit containing a promoter sequence that functions in the host in the 5 ′ to 3 ′ direction, a base sequence that encodes a signal peptide that functions in the host, and a base sequence that encodes the target protein is used as a host. And expressing the target protein, it is possible to secrete and produce the target protein. The base sequence encoding the target protein may be linked downstream of the base sequence encoding the signal peptide so that the target protein is expressed as a fusion protein with the signal peptide. That is, the target protein gene includes a gene encoding such a fusion protein. For secretory production of a protein, for example, a coryneform bacterium can be used as a host. Coryneform bacteria used for secretory production of proteins include, for example, strains in which the activity of cell surface proteins is reduced. Examples of such a strain include C. glutamicum YDK010 strain (WO2004 / 029254), which is a cell surface protein PS2 deficient strain of C. glutamicum AJ12036 (FERM BP-734), and a modified strain thereof. In addition, as a method for imparting or enhancing the secretory productivity of a coryneform bacterium, a method for decreasing the activity of a cell surface protein (WO2013 / 065869, WO2013 / 065772, WO2013 / 118544, WO2013 / 062029), penicillin A method for decreasing the activity of a binding protein (WO2013 / 065869), a method for increasing the expression of a gene encoding a metallopeptidase (WO2013 / 065772), and Method for modifying (WO2013 / 118544), method for modifying a host to have a mutant phoS gene (WO2016 / 171224), method for decreasing the activity of RegX3 protein (WO2018 / 074578), method for decreasing the activity of the HrrSA system (WO2018 / 074579), Inserting an amino acid sequence containing Gln-Glu-Thr between a signal peptide and a target protein to express the target protein The method (WO2013 / 062029), a method of enhancing the secretion system of proteins such as Tat-based secretion system (Japanese Patent No. 4730302) and the like.

  RNA may, for example, accumulate in the cells. The gene encoding the target RNA (target RNA gene) may be held in a host so that it can be expressed. Specifically, for example, an expression unit for RNA containing a promoter sequence that functions in the host in the 5 ′ to 3 ′ direction and a base sequence (target RNA gene) encoding the target RNA is retained in the host, and the target RNA is transferred to the host. By expression, the desired RNA can be produced. For production of RNA, for example, a coryneform bacterium can be used as a host. The transcription mode of the target RNA gene is not particularly limited as long as the target RNA is obtained. The target RNA gene may be transcribed in one direction (ie, using only one strand of a double-stranded strand as a template), or may be transcribed bidirectionally (ie, using each strand of a double-stranded strand as a template). Good. To bidirectionally transcribe the target RNA gene, the promoter is placed in the opposite direction with respect to the gene (that is, the promoter located on the 5 ′ side of the gene in each of the double-stranded strands). It can be carried out by transferring. That is, the RNA expression unit may contain such two promoters. In that case, both promoters may or may not be the same. Transcription of the target RNA gene in one direction typically yields single-stranded RNA. By bidirectionally transcribing the target RNA gene, a double-stranded RNA is typically obtained. Alternatively, double-stranded RNA can be obtained by transcribing each strand of the double-stranded RNA from an individual expression unit.

As a method for imparting or enhancing the ability to produce RNA, ribonuclease III (
(ribonuclease III; RNaseIII).

  “Ribonuclease III (RNase III)” refers to a protein having an activity of catalyzing a reaction of cleaving a specific RNA such as a double-stranded RNA (EC 3.1.26.3). This activity is also referred to as “RNase III activity”. The gene encoding RNaseIII is also referred to as "RNaseIII gene". Examples of RNaseIII include Rnc protein encoded by rnc gene. The nucleotide sequence of the rnc gene of C. glutamicum ATCC 13869 is shown in SEQ ID NO: 191, and the amino acid sequence of the Rnc protein encoded by the gene is shown in SEQ ID NO: 192.

Ribonuclease III activity can be measured by incubating the enzyme with its substrate RNA (eg, double-stranded RNA) and measuring enzyme-dependent RNA cleavage. Specifically, the activity of ribonuclease III is usually measured as follows (Methods Enzymol. 2001; 342: 143-58.). One method is to purify a crude extract from an enzyme (eg, a cell (microbial cell) or partially purify the same with respect to a synthetic substrate (double-stranded state) of poly (A-U) labeled with ³ H. Enzyme), react at 35 ° C, treat the reaction mixture with trichloroacetic acid, and determine the degree of decrease in radioactivity (radioactivity) in the precipitate fraction (containing high-molecular-weight nucleic acids) with the reaction time. It is a method of measuring. That is, the activity of ribonuclease III can be calculated using the degree of decrease in radioactivity as an index of cleavage of the substrate. Another method is to use a double-stranded RNA radiolabeled with ³² P as a substrate and convert it into a reaction solution containing an enzyme (30 mM Tris-HCl (pH 8.0), 250 mM potassium glutamate or 160 mM NaCl). , 5 mM spermidine, 0.1 mM EDTA, 0.1 mM DTT), incubated at 37 ° C. for 5 minutes, added 10 mM MgCl ₂ to a final concentration of 10 mM to start the RNA degradation reaction, and after appropriate reaction, The reaction is stopped by adding an equal volume of an EDTA-electrophoresis marker dye mixture (concentration such that the final concentration of EDTA is 20 mM or more). Next, the sample after the reaction was subjected to electrophoresis using a 15% (w / v) denaturing polyacrylamide gel in a TBE buffer (89 mM Tris / Tris-borate, 2 mM EDTA) containing 7 M urea, The activity of ribonuclease III can be detected by running the gel on a radiological image analyzer and analyzing the degraded RNA fragments.

  The gene and protein used for breeding a microorganism having the target substance-producing ability may have, for example, a known base sequence and amino acid sequence such as the above-described base sequence and amino acid sequence, respectively. In addition, the genes and proteins used for breeding microorganisms having the target substance-producing ability include the genes and proteins exemplified above (for example, having the known base sequences and amino acid sequences such as the exemplified base sequences and amino acid sequences, respectively). (Genes and proteins). Specifically, for example, a gene used for breeding a microorganism having an ability to produce a target substance may have the amino acid sequence exemplified above as long as the original function (ie, enzyme activity, transporter activity, etc.) is maintained. May be a gene encoding a protein having an amino acid sequence in which one or several amino acids at one or several positions are substituted, deleted, inserted, and / or added. Further, for example, a gene used for breeding a microorganism having a target substance-producing ability, as long as the original function is maintained, relative to the entire known amino acid sequence such as the amino acid sequence exemplified above, for example, 50% The gene may be a gene encoding a protein having an amino acid sequence having 65% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more identity. As for the conservative variants of genes and proteins, the description on the conservative variants of glycated genes and glycated proteins can be applied mutatis mutandis.

<1-4> Plasmid The microorganism described in this specification has the plasmid described in this specification.

The plasmids described herein are maintained in a microorganism according to the methods described herein. Therefore, a plasmid that can be autonomously replicated in a microorganism can be used as the plasmid.

  The plasmid described in the present specification has a base sequence (also referred to as “first base sequence”) that complements the reduced ability to assimilate sugar.

  The plasmids described herein may further have any other properties so long as the sugar is maintained at the selective pressure. The plasmid may or may not have, for example, a base sequence (also referred to as “second base sequence”) that is effective for producing the target substance. Further, the plasmid may or may not have a marker gene such as an antibiotic resistance gene, for example.

  Regarding the plasmid described in the present specification, the description relating to the vector used for gene transfer in the “technique for increasing the activity of a protein” can be applied mutatis mutandis, except that it has the above properties.

  The plasmid described in the present specification can be constructed, for example, by introducing a base sequence such as a first base sequence or a second base sequence into a vector described later. Further, the plasmid described in the present specification can also be directly constructed by, for example, total synthesis.

<1-4-1> First base sequence The first base sequence is a base sequence that complements the reduced ability to utilize sugar. Complementation is also called “complementation” or “recovery”. “The reduced assimilation ability of sugar is complemented” means that the assimilation ability of reduced sugar is improved. That is, the microorganism described in the present specification has a reduced sugar assimilation ability by having the plasmid described in the present specification. Specifically, the microorganism described in the present specification has an improved ability to assimilate reduced sugars as compared with the case where the microorganism has no plasmid. The first base sequence may complement the decrease in the assimilation ability of one kind of sugar, or may complement the decrease in the assimilation ability of two or more kinds of sugars. The first base sequence may at least complement the decrease in the ability to utilize sugar.

  The degree of complementation of the reduced sugar assimilation ability is not particularly limited as long as the sugar can be used as a selective pressure to maintain the plasmid. The ability of the sugar to be assimilated after complementation by the plasmid may or may not be the same as that of the non-modified strain (that is, the strain before being modified so that the sugar assimilating ability is reduced). The ability of the sugar to be assimilated after complementation by the plasmid may be lower or higher than that of an unmodified strain (that is, a strain before being modified so that the sugar assimilation ability is reduced). The complementation of the reduced sugar assimilation ability can be measured, for example, as an improvement in growth when a microorganism is cultured using sugar as a sole carbon source. “Complementing the reduced ability to assimilate sugars” specifically means, for example, a modified strain having a plasmid when cultured in a liquid minimal medium containing sugar as a sole carbon source (ie, assimilation of sugars). The specific growth rate of the strain modified so as to decrease its ability and the plasmid introduced therein) is 50% or more of that of the non-modified strain (that is, the strain before being modified so as to reduce the assimilation ability of sugar), It may mean 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, 99% or more, 99.5% or more, 99.7% or more, or 99.9% or more. In addition, "complementing the reduced ability to assimilate sugar" specifically means, for example, a modified strain having a plasmid when cultured in a liquid minimal medium containing only sugar as a carbon source (that is, a modified strain of sugar). The specific growth rate of the strain that has been modified so as to reduce the assimilation ability and into which the plasmid has been introduced, has been modified so that the modified strain without the plasmid (that is, the ability to assimilate the sugar is reduced). , A strain into which no plasmid has been introduced), 2 times or more, 3 times or more, 5 times or more, 10 times or more, 20 times or more, 50 times or more, or 100 times or more. In addition, the phrase “the reduced ability to assimilate sugar is complemented” specifically means, for example, when cultured in a minimal medium containing only sugar as a carbon source (for example, a liquid minimal medium or a solid minimal medium). Alternatively, it may mean that the modified strain having the plasmid can grow (ie, grow), but the modified strain having no plasmid cannot grow (ie, grow).

  The first base sequence is not particularly limited as long as it can complement the reduced assimilation ability of the saccharide to a desired extent. The reduced assimilation ability of sugar can be complemented by, for example, increasing the activity of a protein (sugar assimilating protein) involved in sugar assimilation. The activity of a sugar-utilizing protein can be increased, for example, by increasing the copy number of a gene (sugar-utilizing gene) encoding the protein. The copy number of a sugar assimilation gene can be increased, for example, by introducing a plasmid carrying the gene into a microorganism. That is, as the first base sequence, a sugar assimilation gene can be mentioned. That is, by introducing a plasmid having a sugar assimilation gene as the first base sequence into a microorganism, the activity of a sugar assimilating protein can be increased, thereby complementing the reduced sugar assimilating ability. it can. For the convenience of distinguishing the saccharide-assimilating protein encoded by the first base sequence from the above-mentioned protein P1 (sugar-assimilating protein that reduces the activity for decreasing the ability to utilize sugar), “protein P2” is used. Also called. The use of the first base sequence may increase the activity of one type of saccharide-utilizing protein, or may increase the activity of two or more types of saccharide-utilizing proteins. That is, as the first base sequence, one kind of sugar assimilating gene (specifically, a gene encoding one kind of sugar assimilating protein) may be used. A gene (specifically, a gene encoding two or more glycoproteins) may be used. As the first base sequence, one copy of a certain gene may be used, or two or more copies may be used.

  The sugar assimilation gene and the sugar assimilation protein are as described above. That is, examples of the sugar-assimilating protein include proteins constituting the sugar-assimilating pathway as exemplified above. That is, by increasing the activity of the protein constituting the sugar assimilation pathway, the sugar assimilation pathway can be strengthened, and thus the sugar assimilation ability can be complemented. When two or more assimilation pathways exist for a sugar, in order to supplement the assimilation ability of the sugar, one assimilation pathway selected from those assimilation pathways may be strengthened. Often, two or more (eg, all) assimilation routes selected from those assimilation routes may be strengthened. When a certain utilization pathway is composed of two or more proteins, the activity of one protein selected from those proteins may be increased in order to enhance the utilization pathway. The activity of two or more (eg all) proteins selected from the proteins may be increased.

  The sugar assimilation gene used as the first base sequence is, for example, a kind of sugar, a kind of sugar assimilation pathway, or a sugar assimilating protein (protein P1) whose activity has been reduced to reduce the ability to assimilate sugar. Can be selected as appropriate according to various conditions such as the type of.

The sugar assimilating protein (protein P2) encoded by the first base sequence is, for example, a protein of the same type as the sugar assimilating protein (protein P1) whose activity has been reduced in order to reduce the ability to assimilate sugar. Good. “A protein of the same type as a certain protein” may mean a protein having the same function as the certain protein. Identical functions include functions that take on (eg, catalyze) the same step in the sugar assimilation pathway. That is, for example, when the activity of a protein that carries out a certain step in the sugar assimilation pathway is reduced in order to reduce the sugar assimilation ability, a gene encoding the protein that carries out the same step is used as the first base sequence. May be used. Specifically, for example, EI, HPr, EII ^Glc , EII ^Scr , EII ^Fru , non-PTS uptake system of glucose, non-PTS uptake system of sucrose, glucokinase, phosphoglucose isomerase, 6 -phosphofructokinase, fructose 1,6-bisphosphate aldolase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, pyruvate dehydrogenase, sucrose-6-phosphate hydrolase, 1-phosphofructokinase, invertase, and And / or when the activity of fructokinase is reduced, EI and H
Pr, EII ^Glc , EII ^Scr , EII ^Fru , non-PTS uptake system for glucose, non-PTS uptake system for sucrose, glucokinase, phosphoglucose isomerase, 6-phosphofructokinase, fructose 1,6-bisphosphate aldolase, triose phosphate isomerase, glyceraldehyde-3- A gene encoding phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, pyruvate dehydrogenase, sucrose-6-phosphate hydrolase, 1-phosphofructokinase, invertase, and / or fructokinase may be used as the first base sequence. More specifically, for example, when the activity of 6-phosphofructokinase is reduced in order to reduce the ability to utilize sugar, the 6-phosphofructokinase gene may be used as the first base sequence. When the activity of two or more proteins is reduced in order to reduce the ability to assimilate sugar, if the activity of two or more proteins is reduced, as long as the reduced ability to assimilate sugar can be complemented to a desired extent, one of the proteins selected from those proteins may The activity of a protein of the same type as the protein of the species may be increased, and the activity of a protein of the same type as each of two or more (for example, all) proteins selected from the proteins may be increased. When the activity of two or more proteins is reduced in order to reduce the ability to utilize sugar, typically, the activity of all of those proteins may be increased, respectively.

The sugar-assimilating protein (protein P2) encoded by the first base sequence is, for example, a protein not homologous to the sugar-assimilating protein (protein P1) whose activity has been reduced in order to reduce the ability to assimilate sugar. It may be. That is, for example, when two or more assimilation pathways exist for a certain sugar, at least one assimilation pathway selected from those assimilation pathways is weakened and another assimilation pathway selected from those assimilation pathways is reduced. May be strengthened. That is, a gene encoding such a protein constituting another assimilation pathway may be used as the first base sequence. Specifically, for example, in a microorganism that assimilates glucose via PTS, the activity of EI, HPr, and / or EII ^Glc is reduced, and a gene encoding a non-PTS uptake system of glucose is used as the first base sequence. It may be used. The assimilation pathway to be strengthened here may or may not be a pathway originally possessed by a non-modified strain (that is, a strain before being modified so as to reduce sugar assimilation ability). .

  The saccharide-assimilating protein (protein P2) encoded by the first base sequence is, for example, the same species as the saccharide-assimilating protein (protein P1) whose activity has been reduced in order to reduce the ability to assimilate sugar. A protein that is not the same species as the protein may be used in combination.

  As the sugar assimilation gene used as the first base sequence, a gene encoding a protein that functions in a host can be selected. The sugar assimilation gene used as the first base sequence may be, for example, a host-derived gene, a heterologous gene, or a variant thereof. For example, when the activity of 6-phosphofructokinase is reduced in order to reduce the ability to assimilate sugar, a 6-phosphofructokinase gene derived from a host may be used as the first base sequence, or a 6-phosphofructokinase gene derived from a different species may be used. Or a variant thereof, the 6-phosphofructokinase gene, may be used.

  The sugar assimilation gene may be mounted on the plasmid so that it can be expressed in the host into which the plasmid has been introduced. Specifically, the sugar assimilation gene may be mounted on a plasmid so as to be expressed under the control of a promoter that functions in the host. For expression of a gene by a plasmid (including, for example, gene acquisition and expression-possible configuration), the description of gene introduction in “Method of increasing protein activity” can be applied mutatis mutandis.

<1-4-2> Second base sequence The second base sequence is a base sequence effective for production of a target substance. An effective base sequence for the production of the target substance includes a base sequence that increases the production of the target substance. That is, when the plasmid has the second nucleotide sequence, the ability of the microorganism to produce the target substance can be improved by introducing the plasmid into the microorganism. That is, it is possible to increase the production of the target substance by the microorganism. it can. When the plasmid has the second base sequence, the ability of the microorganism to maintain the target substance can be improved by maintaining the plasmid in the microorganism as compared with the case where the plasmid is not maintained. The production of the target substance by the microorganism can be increased. The increase in the production of the target substance includes an increase in the production amount of the target substance, an increase in the production rate of the target substance, and an increase in the yield of the target substance. Examples of the increase in the production amount of the target substance include an increase in the production amount of the target substance per cell weight, and an increase in the production amount of the target substance per culture medium amount. The second base sequence may improve the ability to produce one kind of target substance, or may enhance the ability to produce two or more kinds of target substances.

  The second base sequence is not particularly limited as long as it is effective for producing the target substance. The second base sequence may or may not be essential for the production of the target substance. That is, the microorganism described in the present specification may have a target substance-producing ability based on having a plasmid having at least the second base sequence.

  Examples of the second base sequence include a gene encoding a protein whose increase in activity is effective in producing a target substance. That is, examples of the second base sequence include a gene encoding a protein capable of imparting or enhancing the target substance-producing ability by increasing the activity. That is, by introducing a plasmid having such a gene as the second base sequence into a microorganism, the activity of such a protein can be increased, and thereby the ability to produce the target substance can be imparted or enhanced. . Such genes and proteins are as described above. That is, examples of such a protein include a target substance biosynthetic enzyme and others described above. The use of the second base sequence may increase the activity of one protein, or may increase the activity of two or more proteins. That is, as the second base sequence, one kind of gene (specifically, a gene encoding one kind of protein) may be used, or two or more kinds of genes (specifically, two kinds of genes). Or a gene encoding a higher protein).

  When the target substance is a gene expression product (eg, protein or RNA), the second base sequence also includes a gene encoding the target substance (target substance gene). Such genes are as described above. From the second base sequence, one kind of target substance may be expressed, or two or more kinds of target substances may be expressed. That is, as the second base sequence, one kind of gene (specifically, a gene encoding one kind of target substance) may be used, or two or more kinds of genes (specifically, two Or a gene encoding a species or more of the target substance).

  Further, as the second base sequence, a gene encoding a protein whose increase in activity is effective in producing the target substance and a gene encoding the target substance may be used in combination. As the second base sequence, one copy of a certain gene may be used, or two or more copies may be used.

  The gene as described above may be carried on a plasmid so that it can be expressed in a host into which the plasmid has been introduced. Specifically, the gene may be mounted on a plasmid so that the gene is expressed under the control of a promoter that functions in the host. For expression of a gene by a plasmid (including, for example, gene acquisition and expression-possible configuration), the description of gene introduction in “Method of increasing protein activity” can be applied mutatis mutandis.

<1-4-3> Introduction of plasmid The method of introducing a plasmid into a microorganism is not particularly limited. For the method of introducing a plasmid into a microorganism, the description of transformation in “Method of increasing protein activity” can be applied mutatis mutandis.

<1-5> Technique for increasing protein activity Hereinafter, a technique for increasing the protein activity will be described.

  “Increased protein activity” may mean that the activity of the protein is increased as compared to the unmodified strain. “Increased activity of a protein” may specifically mean that the activity per cell of the protein is increased relative to an unmodified strain. The “unmodified strain” here may mean a control strain that has not been modified so that the activity of the target protein is increased. Unmodified strains include wild strains and parent strains. The non-modified strain specifically includes a reference strain (type strain) of each microorganism species. In addition, specific examples of the unmodified strain include the strains exemplified in the description of the microorganism. That is, in one embodiment, the activity of the protein may be increased as compared to a reference strain (ie, a reference strain of the species to which the microorganisms described herein belong). In another embodiment, the activity of the protein may be increased as compared to the C. glutamicum ATCC 13869 strain. Also, in another embodiment, the activity of the protein may be increased as compared to the C. glutamicum ATCC 13032 strain. In another embodiment, the activity of the protein may be increased as compared to the E. coli K-12 MG1655 strain. In addition, “the protein activity is increased” is also referred to as “the protein activity is enhanced”. "Increased protein activity" refers more specifically to an increase in the number of molecules per cell of the protein compared to the unmodified strain, and / or It may mean that the function is increasing. In other words, “activity” in the case of “enhancement of protein activity” means not only the catalytic activity of the protein but also the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein. You may. In addition, “increase in protein activity” means not only that the activity of the target protein is increased in a strain that originally has the activity of the target protein, but also that the activity of the protein is increased in a strain that does not originally have the activity of the target protein. Giving activity is also included. In addition, as long as the activity of the protein increases as a result, the activity of the target protein originally possessed by the host may be reduced or eliminated, and then a suitable target protein activity may be imparted.

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

  The modification that increases the activity of the protein can be achieved, for example, by increasing the expression of a gene encoding the protein. “Elevated gene expression” may mean that the expression of the gene is increased as compared to an unmodified strain such as a wild-type strain or a parent strain. “Elevated gene expression” may specifically mean that the expression level of the gene per cell is increased as compared to an unmodified strain. More specifically, “the expression of a gene is increased” means that the transcription amount (mRNA amount) of a gene is increased and / or the translation amount (protein amount) of a gene is increased. May be. In addition, "the expression of a gene is increased" is also referred to as "the expression of a gene is enhanced". Gene expression may be increased, for example, by a factor of 1.2 or more, 1.5 or more, 2 or more, or 3 or more times the unmodified strain. In addition, "increase in gene expression" means not only increasing the expression level of the target gene in a strain in which the target gene is originally expressed, but also in a strain in which the target gene is not originally expressed, Expression of the same gene is also included. That is, “the expression of a gene is increased” may mean, for example, introducing the gene into a strain that does not have the target gene and expressing the gene.

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

  Increasing the copy number of a gene can be achieved by introducing the gene into the host chromosome. Introduction of a gene into a chromosome can be performed using, for example, homologous recombination (Miller, JH Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). As a gene transfer method using homologous recombination, for example, a Red-driven integration method (Datsenko, KA, 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 plasmid capable of conjugative transfer, a method using a suicide vector having no replication origin that functions in a host, A transduction method using a phage can be used. Only one copy of the gene may be introduced, or two or more copies may be introduced. For example, multiple copies of a gene can be introduced into a chromosome by performing homologous recombination targeting a sequence having multiple copies on the chromosome. Sequences in which many copies exist on a chromosome include repetitive DNA sequences and inverted repeats present at both ends of a transposon. Alternatively, homologous recombination may be performed by targeting an appropriate sequence on a chromosome such as a gene unnecessary for production of the target substance. The gene can also be randomly introduced into the chromosome using a transposon or Mini-Mu (Japanese Patent Laid-Open No. 2-109985, US Pat. No. 5,882,888, EP805867B1).

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

The copy number of a gene can also be increased by introducing a vector containing the gene into a host. For example, by linking a DNA fragment containing a target gene with a vector that functions in a host to construct an expression vector for the gene, and transforming the host with the expression vector, it is possible to increase the copy number of the gene. it can. The DNA fragment containing the target gene can be obtained, for example, by PCR using genomic DNA of a microorganism having the target gene as a template. As the vector, a vector that can autonomously replicate in a host cell can be used. The vector may be a multicopy vector. The copy number of the vector is, for example, 5 copies / cell or more, 10 copies / cell or more, 20 copies / cell or more, 30 copies / cell or more, 50 copies / cell or more, 70 copies / cell or more, 100 copies / cell or more, It may be 150 copies / cell or more, 200 copies / cell or more, 300 copies / cell or more, 500 copies / cell or more, or 1000 copies / cell or more, 2000 copies / cell or less, 1500 copies / cell or less, 1000 copies. / Cell or less, 500 copies / cell or less, or 300 copies / cell or less, or a combination thereof that is not inconsistent. In addition, in order to select a transformant, the vector may have a marker such as an antibiotic resistance gene. Further, the vector may be provided with a promoter or a terminator for expressing the inserted gene. The vector may be, for example, a bacterial plasmid-derived vector, a yeast plasmid-derived vector, a bacteriophage-derived vector, a cosmid, or a phagemid. 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-based vector (Clontech), pKK233-2 (Clontech), pET-based vector (Novagen), pQE-based vector (Qiagen), pCold TF DNA ( TaKaRa), a pACYC-based vector, and a broad host range vector RSF1010. As a vector capable of autonomously replicating in a coryneform bacterium, specifically, for example, pHM1519 (Agr
ic. Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, 2901-2903 (1984)); a plasmid having improved drug resistance gene; PCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX (JP-A-2-72876, U.S. Pat. No. 5,185,262); pCRY2 and pCRY3 (JP-A-1-91686); pAJ655, pAJ611, and pAJ1844 PCG1 (JP-A-57-134500); pCG2 (JP-A-58-35197); pCG4 and pCG11 (JP-A-57-183799); pPK4 (US Pat. No. 6,090,597); PVK7 (WO2007 / 046389); pVS7 (WO2013 / 069634); pVC7 (Japanese Patent Application Laid-Open No. 9-070291). In addition, specific examples of the vector that can autonomously replicate in coryneform bacteria include, for example, pVC7H2, which is a variant of pVC7.

  When introducing a gene, the gene only needs to be expressible by the host. Specifically, the gene may be maintained so that it is expressed under the control of a promoter that functions in the host. “Promoter that functions in a 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 an intrinsic promoter of the gene to be introduced or a promoter of another gene. As a promoter, for example, a stronger promoter as described herein may be utilized.

  A terminator for terminating transcription can be arranged 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-derived terminator. The terminator may be a specific terminator of the gene to be introduced, or may be a terminator of another gene. Specific examples of the terminator 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 in, for example, "Basic Lectures on Microbiology 8 Genetic Engineering, Kyoritsu Shuppan, 1987", and these can be used.

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

The gene to be introduced is not particularly limited as long as it encodes a protein that functions in the host. The gene to be introduced may be a host-derived gene or a heterologous gene. The gene to be introduced can be obtained by, for example, PCR using primers designed based on the nucleotide sequence of the gene and using genomic DNA of an organism having the gene or a plasmid or the like carrying the gene as a template. The gene to be introduced may be totally synthesized, for example, based on the nucleotide sequence of the gene (Gene, 60 (1), 115-127 (1987)). The obtained gene can be used as it is or after being appropriately modified. That is, the variant can be obtained by modifying the gene. Gene modification can be performed by a known technique. For example, a target mutation can be introduced into a target site of DNA by a site-specific mutation method. That is, for example, the coding region of a gene can be modified by site-directed mutagenesis such that the encoded protein contains amino acid residue substitutions, deletions, insertions, and / or additions at specific sites. . As a site-specific mutation 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 a method using phage (Kramer, W. and Frits, HJ, Meth. In Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. In Enzymol., 154, 367 ( 1987)). Alternatively, gene variants may be totally synthesized.

  When the protein functions as a complex composed of a plurality of subunits, all of these subunits may be modified or only a part thereof may be modified as long as the activity of the protein increases. That is, for example, when increasing the activity of a protein by increasing the expression of a gene, the expression of all of the genes encoding each of those subunits may be enhanced, or only a part of the expression may be enhanced. Good. Usually, it is preferred to enhance the expression of all of the genes encoding those subunits. Each subunit constituting the complex may be derived from one organism, or may be derived from two or more different organisms, as long as the complex has the function of the target protein. That is, for example, genes derived from the same organism that encode a plurality of subunits 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 the expression of a gene can be achieved by improving the translation efficiency of the gene. Improvement in transcription efficiency and translation efficiency of a gene can be achieved, for example, by modifying an expression control sequence. “Expression control sequence” may be a general term for sites that affect gene expression. Expression control sequences include, for example, a promoter, a Shine-Dalgarno (SD) sequence (also referred to as a ribosome binding site (RBS)), and a spacer region between the RBS and the initiation codon. The expression control sequence can be determined using a gene search software such as a promoter search vector or GENETYX. These expression control sequences can be modified by, for example, a method using a temperature-sensitive vector or a 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 mean a promoter that enhances gene transcription over a naturally occurring wild-type promoter. Examples of stronger promoters include, for example, 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, Pm1 derived from Bifidobacterium Promoters, PR promoters, and PL promoters. Examples of stronger promoters usable in coryneform bacteria include, for example, artificially modified P54-6 promoter (Appl. Microbiol. Biotechnol., 53, 674-679 (2000)) and coryneform bacteria. Pta, aceA, aceB, adh, and amyE promoters that can be induced with acetic acid, ethanol, pyruvate, etc., and cspB, SOD, and 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 (WO2018 / 079684), P3 promoter (WO2018 / 079684), lac promoter, tac promoter, trc Promoter and F1 promoter. As a stronger promoter, a highly active type of a conventional promoter may be obtained by using various reporter genes. For example, by bringing the -35 and -10 regions within the promoter region closer to the consensus sequence, the activity of the promoter can be increased (WO 00/18935). Various tac-like promoters (Katashkina JI et
al. Russian Federation Patent application 2006134574). Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al.'S article (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)).

The translation efficiency of a gene can be improved by, for example, replacing the Shine-Dalgarno (SD) sequence (also referred to as a 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 enhances translation of mRNA over a native SD sequence that originally exists. More potent SD sequences include, for example, the RBS of gene 10 from phage T7 (Olins PO et al, Gene,
1988, 73, 227-235). Furthermore, substitution, insertion or deletion of several nucleotides in the spacer region between the RBS and the start codon, particularly in the sequence immediately upstream of the start codon (5'-UTR), may affect the stability and translation efficiency of mRNA. It is known to have a great effect, and it is possible to improve the translation efficiency of the gene by modifying them.

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

  In addition, the expression of a gene can be increased by amplifying a regulator that increases the expression of the gene, or by deleting or weakening a regulator that decreases the expression of the gene.

  The above-described methods for increasing the expression of a gene may be used alone or in any combination.

Further, the modification that increases the activity of the protein can also be achieved, for example, by enhancing the specific activity of the protein. Increasing specific activity may also include desensitization to feedback inhibition. That is, if the protein is subject to feedback inhibition by metabolites, the activity of the protein can be increased by mutating the gene or protein in the host such that the feedback inhibition is desensitized. Unless otherwise specified, “feedback inhibition desensitization” may include a case where feedback inhibition is completely canceled and a case where feedback inhibition is reduced. Further, "feedback inhibition is desensitized" (that is, feedback inhibition is reduced or released) is also referred to as "resistance to feedback inhibition". For example, a protein with enhanced specific activity can be obtained by searching various organisms. Alternatively, a highly active form may be obtained by introducing a mutation into a native protein. The introduced mutation may be, for example, a substitution, deletion, insertion and / or addition of one or several amino acids at one or several positions of the protein. The mutation can be introduced, for example, by the site-specific mutation method described above. The mutation may be introduced, for example, by a mutation treatment. Mutation treatments included X-ray irradiation, ultraviolet irradiation, and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS). ) And the like. In addition, DNA is directly treated with hydroxylamine in vitro,
Random mutations may be induced. The enhancement of the specific activity may be used alone or in any combination with the above-described technique for enhancing the expression of a gene.

  The transformation method is not particularly limited, and a conventionally known method can be used. For example, methods for increasing the permeability of DNA by treating recipient cells with calcium chloride, as reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol. 1970, 53, 159-162) and methods for preparing competent cells from cells at the growth stage and introducing DNA as reported for Bacillus subtilis (Duncan, CH, Wilson, GA and Young, FE). , 1997. Gene 1: 153-167) can be used. Alternatively, as is known for Bacillus subtilis, actinomycetes, and yeast, cells of the DNA recipient are transformed into protoplasts or spheroplasts that readily take up the recombinant DNA and the recombinant DNA is transformed into a DNA recipient. Method of introduction (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. Sci. USA 75: 1929-1933). Alternatively, an electric pulse method (JP-A-2-207791) as reported for coryneform bacteria can be used.

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

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

  The increase in the transcription level of the gene can be confirmed by comparing the amount of mRNA transcribed from the gene with a wild-type strain or an unmodified strain such as a parent strain. Methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR, microarray, and RNA-seq (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 may be increased, for example, by a factor of at least 1.2, at least 1.5, at least 2, or at least 3 times that of the unmodified strain.

  The increase in the amount of protein can be confirmed by Western blot using an antibody (Molecular Cloning (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 a factor of at least 1.2, at least 1.5, at least 2, or at least 3 times that of the unmodified strain.

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

<1-6> Technique for reducing protein activity A technique for reducing protein activity will be described below.

The expression “the activity of a protein is reduced” may mean that the activity of the protein is reduced as compared to an unmodified strain. The phrase “the activity of the protein is reduced” may specifically mean that the activity per cell of the protein is reduced as compared with the unmodified strain. The “unmodified strain” here may mean a control strain that has not been modified so that the activity of the target protein is reduced. Unmodified strains include wild strains and parent strains. The non-modified strain specifically includes a reference strain (type strain) of each microorganism species. In addition, specific examples of the unmodified strain include the strains exemplified in the description of the microorganism. That is, in one embodiment, the activity of the protein may be reduced as compared to a reference strain (ie, a reference strain of the species to which the microorganisms described herein belong). Also, in another embodiment, the activity of the protein may be reduced compared to the C. glutamicum ATCC 13869 strain. In another embodiment, the activity of the protein may be reduced as compared to the C. glutamicum ATCC 13032 strain. In another embodiment, the activity of the protein may be reduced as compared to the E. coli K-12 MG1655 strain. The phrase “the activity of the protein is reduced” may include the case where the activity of the protein is completely lost. More specifically, “the activity of the protein is reduced” means that the number of molecules per cell of the protein is lower than that of the unmodified strain, and / or It may mean that the function is reduced. In other words, “activity” in the case of “protein activity is reduced” means not only the catalytic activity of the protein but also the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein. You may. The phrase “the number of molecules of the protein per cell is reduced” may include the case where the protein is not present at all. Further, the phrase “function per molecule of the protein is reduced” may include a case where the function per molecule of the protein is completely lost. The degree of decrease in the activity of the protein is not particularly limited as long as the activity of the protein is reduced as compared with the unmodified strain. The activity of the protein may be reduced, for example, to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain.

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

The decrease in gene expression may be due to, for example, a decrease in transcription efficiency, a decrease in translation efficiency, or a combination thereof. The decrease in the expression of a gene can be attained by, for example, modifying an expression control sequence such as a gene promoter, a Shine-Dalgarno (SD) sequence (also referred to as a ribosome binding site (RBS)), or a spacer region between the RBS and the initiation codon. Can be achieved by When modifying the expression control sequence, the expression control sequence is modified by one or more bases, two or more bases, or three or more bases. Reduction of the transcription efficiency of a gene can be achieved, for example, by replacing the promoter of the gene on the chromosome with a weaker promoter. “Weaker promoter” may mean a promoter whose transcription of a gene is weaker than the naturally occurring wild-type promoter. Weaker promoters include, for example, inducible promoters. Examples of weaker promoters include the P4 promoter (WO2018 / 079684) and the P8 promoter (WO2018 / 079684). That is, an inducible promoter can function as a weaker promoter under non-inducing conditions (eg, in the absence of an inducer). Further, part or all of the expression control sequence may be deleted. Further, the reduction of gene expression can also be achieved, for example, by manipulating factors involved in expression control. Factors involved in the regulation of expression include small molecules (such as inducers and inhibitors), proteins (such as transcription factors), and nucleic acids (such as siRNA) that are involved in the regulation of transcription and translation. Further, the decrease in gene expression can also be achieved by, for example, introducing a mutation into the coding region of the gene so that the expression of the gene decreases. 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. Also, for example, disruption of a gene as described herein can reduce the expression of the gene itself.

  Further, the modification that reduces the activity of the protein can be achieved, for example, by disrupting the gene encoding the protein. “Gene disruption” may mean that the gene is modified so that it does not produce a normally functioning protein. "Does not produce a protein that functions normally" means that no protein is produced from the gene, or a protein is produced from the gene that has reduced or lost function per molecule (eg, activity or property). May also be included.

  Gene disruption can be achieved, for example, by deleting (deleting) a gene on a chromosome. “Gene deletion” may mean deletion of part or all of the coding region of a gene. Furthermore, the entire gene may be deleted, including the sequence before and after the coding region of the gene on the chromosome. As long as a reduction in the activity of the protein can be achieved, the region to be deleted is the N-terminal region (ie, the region encoding the N-terminal side of the protein), the internal region, the C-terminal region (ie, the region encoding the C-terminal side of the protein) And so on. 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 length of the gene coding region. Alternatively, it may be a region having a length of 95% or more. In addition, it is preferable that the sequences before and after the region to be deleted do not have the same reading frame. Reading frame mismatches can cause a frameshift downstream of the region to be deleted.

  Disruption of a gene can be performed, for example, by 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 another base sequence into the coding region of the gene on the chromosome. The insertion site may be in any region of the gene, but the longer the inserted base sequence, the more reliably the gene can be inactivated. Further, it is preferable that the sequences before and after the insertion site do not match in the reading frame. Reading frame mismatches can cause a frameshift downstream of the insertion site. The other nucleotide sequence is not particularly limited as long as it reduces or eliminates the activity of the encoded protein, and examples thereof include a marker gene such as an antibiotic resistance gene and a gene useful for producing a target substance.

Disruption of the gene may be carried out, in particular, such that the amino acid sequence of the encoded protein is deleted (deleted). In other words, the modification that reduces the activity of the protein is carried out, for example, by deleting the amino acid sequence of the protein (part or all of the amino acid sequence), specifically, by deleting the amino acid sequence (a part of the amino acid sequence). (Or part or all of the region) can be achieved by modifying a gene so as to encode a protein lacking the same. In addition, "deletion of the amino acid sequence of a protein" may mean deletion of a part or the whole region of the amino acid sequence of a protein. Further, “deletion of the amino acid sequence of the protein” may mean that the original amino acid sequence is not present in the protein, and may include a case where the original amino acid sequence is changed to another amino acid sequence. That is, for example, a region that has been changed to another amino acid sequence by a frameshift may be regarded as a deleted region. Deletion of an amino acid sequence typically reduces the full length of the protein, but may not change or extend the full length of the protein. For example, by deleting part or all of the coding region of a gene, the region encoded by the deleted region can be deleted in the amino acid sequence of the encoded protein. Also, for example, by introducing a stop codon into the coding region of a gene, a region encoded by a region downstream from the introduced site can be deleted in the amino acid sequence of the encoded protein. Further, for example, the region encoded by the frameshift site can be deleted by a frameshift in the coding region of the gene. Regarding the position and length of the region to be deleted in the deletion of the amino acid sequence, the description of the position and length of the region to be deleted in the deletion of the gene can be applied mutatis mutandis.

  Modifying a gene on a chromosome as described above involves, for example, preparing a disrupted gene modified so as not to produce a normally functioning protein, and transforming a host with a recombinant DNA containing the disrupted gene. By causing homologous recombination between the disrupted gene and the wild-type gene on the chromosome, it can be achieved by replacing the wild-type gene on the chromosome with the disrupted gene. At this time, if the marker DNA is included in the recombinant DNA according to the trait such as auxotrophy of the host, the operation is easy. Examples of the disrupted gene include a gene in which part or all of the coding region of the gene has been deleted, a gene in which a missense mutation has been introduced, a gene in which a nonsense mutation has been introduced, a gene in which a frameshift mutation has been introduced, a transposon or a marker gene. Inserted genes. Even if produced, the protein encoded by the disrupted gene has a different three-dimensional structure from the wild-type protein, and its function is reduced or lost. Such gene disruption by gene replacement using 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 excision system derived from λ phage (Cho, EH, Gumport, RI, Gardner, JFJ Bacteriol. 184: 5200-5203 (2002)). ), A method using a plasmid containing a temperature-sensitive replication origin, a method using a plasmid capable of conjugative transfer, a replication functioning in a host, and the like (see WO2005 / 010175). There is a method using a suicide vector having no origin (US Pat. No. 6,303,383, Japanese Patent Laid-Open No. 05-007491).

  Further, the modification that reduces the activity of the protein may be performed by, for example, a mutation treatment. Mutation treatments included X-ray irradiation, UV irradiation, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS). )).

  When a protein functions as a complex composed of a plurality of subunits, all of these subunits may be modified or only a part thereof may be modified as long as the activity of the protein is reduced. That is, for example, all of the genes encoding the respective subunits may be destroyed, or only a part thereof may be destroyed. When a protein has a plurality of isozymes, all the activities of the isozymes may be reduced or only a part of the activities may be reduced as long as the activity of the protein is reduced. That is, for example, all of the genes encoding the isozymes may be destroyed, or only some of them may be destroyed.

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

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

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

  Confirmation that the transcription amount of the gene has decreased can be performed by comparing the amount of mRNA transcribed from the gene with that of the non-modified strain. Methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR, microarray, RNA-seq, and the like (Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The amount of mRNA may be reduced, for example, to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain.

  The decrease in the amount of protein can be confirmed by Western blot using an antibody (Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The amount of protein (eg, number of molecules per cell) may be reduced, for example, to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of the unmodified strain.

  The disruption of a gene can be confirmed by determining the base sequence, restriction map, full length, etc. of a part or all of the gene according to the means used for the disruption.

  The above-described technique for reducing the activity of a protein can be used for reducing the activity of any protein or the expression of any gene.

<2> Method for Maintaining Plasmid The method described in this specification is a method for maintaining a plasmid in a microorganism using sugar as a selective pressure. The method described herein may specifically be a method of maintaining a plasmid in a microorganism, comprising culturing the microorganism described herein in a medium containing a sugar. This step is also referred to as “culturing step”.

  The method described herein can be applied to culture for any purpose. The purpose of the culture is not particularly limited. The purpose of the culture includes subculture of microorganisms, production of microbial cells, and production of a target substance.

  The medium to be used is not particularly limited as long as it contains sugar, can grow microorganisms, and maintains the plasmid. When culturing is performed for a specific purpose, the medium to be used can be set so as to achieve the purpose. As the medium, for example, a liquid medium or a solid medium can be used. As the medium, specifically, for example, a normal medium used for culturing microorganisms such as bacteria and yeasts can be used as it is or appropriately modified except that it contains sugar. The medium may contain, in addition to sugar, medium components such as a carbon source, a nitrogen source, a phosphate source, a sulfur source, and other various organic and inorganic components as necessary. The type and concentration of the medium component may be appropriately set according to various conditions such as the type of microorganism used and the purpose of culture.

  The sugar used as the selection pressure is not particularly limited. Sugars include glucose, fructose, sucrose, lactose, galactose, xylose, and arabinose. Sugars include glucose and sucrose, among others. Sugars more particularly include glucose. As the selection pressure, one kind of sugar may be used, or two or more kinds of sugars may be used in combination.

Sugar is used as a carbon source. Sugar may or may not be used as the sole carbon source. That is, in addition to the sugar, another carbon source may be used in combination. Other carbon sources are not particularly limited as long as the microorganism can assimilate. Other carbon sources include
Organic acids such as acetic acid, fumaric acid, citric acid and succinic acid; alcohols such as glycerol and ethanol; and fatty acids. Other carbon sources also include sugars not employed as selective pressure. It should be noted that when simply described as “sugar”, it means a sugar adopted as the selective pressure. As the other carbon source, one type of carbon source may be used, or two or more types of carbon sources may be used in combination. When another carbon source is used in combination, the ratio of the sugar to the total carbon source is not particularly limited as long as the plasmid can be maintained. The ratio of sugar to total carbon source is, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, 99% or more, 99.5% or more by weight. , 99.7% or more, or 99.9% or more. For example, the ratio of the sugar to the total carbon source in the initial medium may be set in the above-described range. Further, for example, the ratio of the sugar to the total carbon source in the feed medium may be set in the above-described range.

  As the sugar, a purified product purified to a desired degree may or may not be used. As the sugar, for example, a mixture containing the sugar may be used. For example, examples of the mixture containing sugar include molasses, starch hydrolyzate, and biomass hydrolyzate. The mixture may be, for example, a mixture containing sugar and another carbon source. That is, the sugar and the other carbon source may be used in combination, for example, in the form of such a mixture.

The sugar may be contained in the medium during the entire period of the culture, or may be contained in the medium only during a part of the culture. That is, “culturing a microorganism in a medium containing sugar” does not require that the sugar be contained in the medium during the entire culture period. The period during which the sugar is contained in the medium is not particularly limited as long as the plasmid can be maintained. The period in which the sugar is contained in the medium is, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, 99% or more of the entire culture period. %, 99.5% or more, 99.7% or more, or 99.9% or more. For example, the sugar may or may not be included in the medium from the start of the culture. To ensure the maintenance of the plasmid, the sugar may usually be included in the medium from the start of the culture. If the sugar is not contained in the medium at the start of the culture, the sugar is added to the medium after the start of the culture. The timing of the addition can be appropriately set according to various conditions such as the culture time. The sugar may be contained in the medium, for example, at least until the microorganism grows to a desired degree. The sugar may be specifically contained in the medium, for example, at least until the end of the logarithmic growth phase. When the target substance is produced by culturing, the saccharide may be contained in the medium, for example, at least until the target substance is produced to a desired degree. In any case, sugar may be appropriately added to the medium. For example, sugar may be added to the medium according to the decrease or depletion of sugar as the culture proceeds. The means for adding sugar to the medium is not particularly limited. For example, sugar can be added to a culture medium by feeding the medium with a feed medium containing the sugar. During the period when the sugar is not contained in the medium, for example, other carbon sources may be contained in the medium. That is, the cultivation may be performed so that the carbon source is not depleted or the state in which the carbon source is depleted does not continue. The concentration of the sugar in the medium is not particularly limited as long as the plasmid can be maintained. The concentration of the sugar in the medium is, for example, 1 g / L or more, 2 g / L or more, 5 g / L or more, 10 g / L or more, 20 g / L or more, 50 g / L or more, or 100 g / L or more. L or more, 300 g / L or less, 200 g / L or less, 100 g / L or less, 50 g / L or less, or 20 g / L or less. There may be. The concentration of the sugar in the medium may specifically be, for example, 50 to 300 g / L, or 100 to 200 g / L. The sugar may or may not be contained in the medium at the concentrations exemplified above throughout the culture. For example, the sugar may be contained in the medium at the concentration exemplified above at the start of the culture, or may be added to the medium at the concentration exemplified above after the start of the culture. In addition, the sugar may be contained in the medium at the above-mentioned concentration during the above-mentioned period, for example. The sugar concentration may be set in the above-described range, for example, when sugar is used solely as a carbon source. In addition, the concentration of the sugar may be set in the above-described range, for example, when the sugar is used in combination with another carbon source. When the main culture is performed after the preculture, the saccharide may be contained in the medium at least during the main culture, that is, during the entire main culture or a part of the main culture. That is, the sugar may or may not be contained in the medium during the pre-culture period. In such a case, the description about culturing (for example, “culturing period (culturing period)” or “start of culturing”) can be read as that for main culture.

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

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

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

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

  When using an auxotrophic mutant that requires nutrients such as amino acids for growth, the medium may preferably contain such nutrients.

  The medium may contain appropriate components depending on various conditions such as the purpose of the culture. The culture medium may contain, for example, components used for production of the target substance. Specific examples of such a component include a methyl group donor (for example, SAM) and a precursor thereof (for example, methionine). Further, as such a component, specifically, for example, a precursor of a target substance is also exemplified.

In the methods described herein, the selection pressure of the sugar allows the plasmid to be maintained without other selection pressures. However, this does not preclude the use of other selection pressures. That is, the medium may or may not contain, for example, components other than sugar that function as selective pressure. Components that function as selective pressure other than sugar include antibiotics. As a component that functions as a selective pressure other than sugar, specifically, when the plasmid described in the present specification has an antibiotic resistance gene, an antibiotic which becomes more resistant by the gene is exemplified. Usually, the medium may be substantially free of components that function as selective pressures other than sugar. The medium may be substantially free of components that function as selective pressure other than sugar, for example, during the entire period of the culture. "The medium does not substantially contain a component that functions as a selection pressure other than sugar" means, for example, the concentration of the component in the medium that cannot maintain the plasmid when the component is used as the selection pressure alone. It may mean that. "The medium does not substantially contain a component that functions as a selective pressure other than sugar" specifically means, for example, that the concentration of the component in the medium is 1/10 of the concentration usually used for maintaining the plasmid. Hereinafter, it may mean 1/100 or less, 1/1000 or less, or 1/10000 or less. Further, “the medium does not substantially contain a component that functions as a selective pressure other than sugar” specifically means, for example, that the concentration of the component in the medium is 10 mg / L or less, 1 mg / L or less, 0 mg / L or less. It may mean 0.1 mg / L or less, 0.01 mg / L or less, or 0.001 mg / L or less. "The medium does not substantially contain a component that functions as a selective pressure other than sugar" includes the case where the medium does not contain the component at all. Regardless of whether or not another selection pressure is used, the saccharide may be used in such a manner that the plasmid can be maintained when the selection pressure is used alone (for example, the concentration or the period). When the microorganism has another plasmid (that is, a plasmid other than the plasmid described in the present specification), a component that functions as a selective pressure for maintaining the plasmid may be used.

  The culture conditions are not particularly limited as long as the microorganism can grow and the plasmid is maintained. When culturing is performed for a specific purpose, the culturing conditions can be set so that the purpose is achieved. The cultivation can be performed, for example, under ordinary 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 and the purpose of culture.

  The cultivation can be performed using an appropriate culture vessel. Examples of the culture container include a petri dish, a multiwell plate, a microtube, a test tube, a flask, and a jar fermenter.

  At the time of culturing, first, a microorganism can be inoculated into a medium. The microorganism may be inoculated into the medium from a preserved product such as dried cells or glycerol stock, or may be inoculated into the medium after preliminary culture. Such preliminary culture is also referred to as “pre-culture”. The culture after the pre-culture is also referred to as “main culture”. In such a case, “culturing” in the method described in this specification shall mean main culture. The culture conditions for the preculture and the main culture may or may not be the same. For example, when the main culture is performed in a liquid medium, a microorganism pre-cultured in a solid medium such as an agar medium may be inoculated into the liquid medium, and the microorganism pre-cultured in the liquid medium may be used as a liquid for main culture. The medium may be inoculated. The inoculation amount of the microorganism is not particularly limited. For example, a pre-culture solution having an OD660 of 4 to 100 may be inoculated at the start of the culture at 0.1% to 100% by mass or 1% to 50% by mass with respect to the main culture medium.

  The culture can be performed by batch culture, fed-batch culture, continuous culture, or a combination thereof. Fed-batch culture or continuous culture can be particularly performed using a liquid medium. The culture medium at the start of the culture is also referred to as “initial culture 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”. The addition of a feed medium to a culture system in fed-batch culture or continuous culture is also referred to as “fed-batch”. When the main culture is performed after the preculture, the culture forms of the preculture and the main culture may or may not be the same. For example, both the preculture and the main culture may be performed by batch culture, the preculture may be performed by batch culture, and the main culture may be performed by fed-batch culture or continuous culture.

Various components such as sugar may be contained in the starting medium, the feed medium, or both. That is, in the course of the culture, various components such as sugar may be added to the medium alone or in any combination. Any of these components may be added once or multiple times, or may be added continuously. The type of components contained in the initial medium may or may not be the same as the type of components contained in the feed medium. Further, the concentration of each component contained in the initial medium may or may not be the same as the concentration of each component contained in the feed medium. Alternatively, two or more feed media having different types and / or different concentrations of the components may be used. For example, when a plurality of feedings are performed intermittently, the type and / or concentration of the components contained in each feeding medium may or may not be the same.

  The culture may be performed, for example, under aerobic, microaerobic, or anaerobic conditions. The cultivation may in particular be carried out under aerobic conditions. “Aerobic conditions” may mean conditions under which the concentration of dissolved oxygen in a medium is 0.33 ppm or more, or 1.5 ppm or more. Specifically, the oxygen concentration may be controlled, for example, to about 1 to 50% or about 5% with respect to the saturated oxygen concentration. The culture can be performed, for example, by aeration culture or shaking culture. The pH of the medium may be, for example, pH 3-10, or pH 4.0-9.5. During the culture, the pH of the medium can be adjusted as needed. The pH of the medium is adjusted using various alkaline or acidic substances such as ammonia gas, aqueous ammonia, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, and magnesium hydroxide. can do. 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. The cultivation may be continued, for example, until the carbon source (especially sugar) in the medium is consumed, or until the activity of the microorganism is lost. The culturing may be continued, for example, until the microorganisms grow to a desired degree. When the target substance is produced by culturing, the culturing may be continued, for example, until the target substance is produced to a desired degree.

  The concentration of each component present in the medium can be determined by a known method used for detecting or identifying a compound. Such techniques include, for example, HPLC, UPLC, LC / MS, GC / MS, NMR. These methods can be used alone or in an appropriate combination. These techniques can also be used to confirm the production of the target substance in the method for producing the target substance described below.

  By carrying out the culturing step in this way, the microorganism grows while retaining the plasmid. In other words, by carrying out the culturing step in this way, cells having the plasmid are produced. That is, the culturing step may be a step of culturing the microorganism described in the present specification in a medium containing a sugar to produce cells having a plasmid. This step is also referred to as a "cell formation step". That is, a microbial cell having a plasmid can also be produced using the method described in the present specification. That is, one embodiment of the method described in the present specification may be a method for producing a microbial cell having a plasmid. One embodiment of the method described in the present specification may specifically be a method for producing a microbial cell having a plasmid, which includes a cell generation step. The generated cells can be collected as appropriate. That is, the method described in the present specification may further include a step of collecting cells.

  The methods described herein may or may not be performed alone. That is, when performing a plurality of cultures, the method described herein may be applied to, for example, any of the plurality of cultures. In that case, the method described in the present specification may or may not be applied to the remaining cultures of the multiple cultures. In other words, the methods described herein may be applied, for example, to one or more of a plurality of cultures. Specifically, for example, when the preculture is performed before the culture (main culture) in the method described in the present specification, the method described in the present specification may be applied to the preculture, You don't have to. Where the methods described herein are applied to one or more of the multiple cultures, embodiments of the methods described herein may be set independently for each culture. Can be. In culturing without applying the methods described herein, the plasmid may be maintained, for example, by using other selection pressures.

<3> Method for producing target substance When the microorganism described in the present specification has a target substance-producing ability, the target substance can be produced using the method described in the present specification. That is, one embodiment of the method described in this specification is a method for producing a target substance.

  Specifically, the target substance is produced by culturing according to the method for maintaining the plasmid described above, or by utilizing cells obtained by culturing according to the method for maintaining the plasmid described above. be able to. That is, one embodiment of the method for producing a target substance may be a method for producing the target substance, which includes a step of culturing a microorganism in a medium containing saccharide to produce the target substance. One embodiment of the method for producing a target substance includes a step of culturing a microorganism in a medium containing a sugar to generate cells having a plasmid, and a step of generating the target substance using the cells. Alternatively, a method for producing a target substance may be used. The target substance can be produced, for example, from sugar and / or a precursor of the target substance. That is, in the method for producing the target substance, for example, the sugar may be used for both maintaining the plasmid and producing the target substance. In the method for producing the target substance, for example, the sugar may be used for maintaining the plasmid, and the precursor of the target substance may be used for producing the target substance. In any case, by maintaining the plasmid in the microorganism during culture, the plasmid (specifically, the second base sequence carried on the plasmid) may contribute to the production of the target substance.

<3-1> Fermentation method The target substance can be produced, for example, by fermentation using the microorganism described in this specification. That is, one embodiment of the method for producing the target substance may be a method for producing the target substance by fermentation using a microorganism. This embodiment is also called “fermentation method”. The step of producing a target substance by fermentation using a microorganism is also referred to as a “fermentation step”. That is, the fermentation method may be a method for producing a target substance, including a fermentation step.

  The fermentation step can be performed by culturing the microorganism described herein according to the method for maintaining a plasmid described above. Specifically, in the fermentation step, the target substance can be produced from sugar. That is, the fermentation step may be, for example, a step of culturing a microorganism in a medium containing sugar to produce a target substance. The fermentation step may be, in particular, a step of culturing a microorganism in a medium containing sugar to produce a target substance from sugar. That is, the fermentation method includes, for example, a step of culturing a microorganism in a medium containing sugar to produce a target substance, particularly, a step of culturing the microorganism in a medium containing sugar to produce the target substance from sugar. It may be a method for producing a target substance. The fermentation step is, specifically, a step of culturing a microorganism in a medium containing sugar and producing and accumulating a target substance in a culture solution (for example, in the medium, the cell surface, the cells, or a combination thereof). There may be. In other words, the fermentation step may be, for example, a step of producing a target substance from sugar using a microorganism.

  Regarding the culture in the fermentation method, except for the production of the target substance in the fermentation method, the description of the culture in the above-described method for maintaining the plasmid (for example, the description of the culture medium and culture conditions) can be applied mutatis mutandis. That is, the medium to be used is not particularly limited as long as it contains a saccharide, a microorganism can grow while retaining the plasmid, and the target substance is produced. The culture conditions are not particularly limited as long as the microorganism can grow while retaining the plasmid and the target substance is produced. The culture in the fermentation method may be performed using, for example, a liquid medium. Further, the culture in the fermentation method may be performed, for example, under aerobic conditions. When the main culture is performed after the preculture, the target substance may be produced at least during the main culture.

  By culturing the microorganism under such conditions, a target substance is produced, that is, a culture solution containing the target substance is obtained. Specifically, the target substance may be accumulated in, for example, a culture medium, a cell surface layer, a cell body, or a combination thereof.

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

  The generated target substance can be appropriately collected. That is, the fermentation method may further include a step of collecting the target substance. This step is also called a “recovery step”. The recovery step may be a step of recovering the target substance from the culture solution, specifically, from the culture medium, the cell surface, the cells, or a combination thereof. The target substance can be recovered by a known method used for separation and purification of a compound. Examples of such a method include an ion exchange resin method, a membrane treatment method, a precipitation method, an extraction method, a distillation method, and a crystallization method. The target substance can be specifically recovered, for example, by extraction with an organic solvent such as ethyl acetate or by steam distillation. The method of collecting the target substance can be appropriately selected according to various conditions such as the type of the target substance. These methods can be used alone or in an appropriate combination.

  When the target substance precipitates in the medium, it can be recovered by, for example, centrifugation or filtration. Further, the target substance precipitated in the medium may be isolated together after crystallization of the target substance dissolved in the medium.

  When the target substance accumulates in the cells, for example, the cells are crushed by ultrasonic waves or the like, and the supernatant obtained by removing the cells by centrifugation or the like is used to separate and purify the compound as described above. The target substance can be recovered by the technique used.

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

<3-2> Bioconversion method The target substance can also be produced by, for example, bioconversion using the microorganism described in this specification. That is, another aspect of the method for producing the target substance may be a method for producing the target substance by bioconversion using a microorganism. This embodiment is also referred to as “bioconversion method”. The step of producing a target substance by bioconversion using a microorganism is also referred to as a “bioconversion step”. That is, the bioconversion method may be a method for producing a target substance including a bioconversion step.

  Specifically, in the bioconversion step, the target substance can be produced from a precursor of the target substance. That is, the bioconversion step may be a step of generating the target substance from a precursor of the target substance using a microorganism. That is, the bioconversion method may be a method for producing a target substance, including a step of generating the target substance from a precursor of the target substance using a microorganism. More specifically, in the bioconversion step, the target substance can be produced by using a microorganism to convert a precursor of the target substance into the target substance. That is, the bioconversion step may be a step of converting a precursor of the target substance into the target substance using a microorganism. That is, the bioconversion method may be a method for producing a target substance, including a step of converting a precursor of the target substance into the target substance using a microorganism.

The precursor of the target substance is simply referred to as “precursor”. Precursors include substances for which SAM is required for conversion to the target substance. Specifically, the precursor is an intermediate in the biosynthetic pathway of the target substance (for example, the one mentioned in connection with the description of the target substance biosynthetic enzyme), and SAM is required for conversion to the target substance. Things. As the precursor, more specifically, for example, protocatechuic acid, protocatechualdehyde, vanillic acid, benzoic acid, cinnamic acid, L-tryptophan, L-histidine, L-phenylalanine, L-tyrosine, L-tyrosine
Arginine, L-ornithine, glycine. Protocatechuic acid may be used, for example, as a precursor to produce vanillin, vanillic acid, or guaiacol. Both protocatechualdehyde and vanillic acid may be used, for example, as precursors for producing vanillin. Benzoic acid and L-phenylalanine may be used, for example, as precursors for producing benzaldehyde. Both cinnamic acid and L-phenylalanine may be used, for example, as precursors for producing cinnamaldehyde. L-tryptophan may be used, for example, as a precursor for producing melatonin. L-histidine may be used, for example, as a precursor for producing ergothioneine. Both L-phenylalanine and L-tyrosine may be used, for example, as precursors for producing ferulic acid, 4-vinylguaiacol, or 4-ethylguaiacol. Both L-arginine and L-ornithine may be used, for example, as precursors for producing polyamines. Both L-arginine and glycine may be used, for example, as precursors for producing creatine. As the precursor, one kind of precursor may be used, or two or more kinds of precursors may be used in combination. When the precursor is a compound that can take the form of a salt, the precursor may be used as a free form, may be used as a salt, or may be used as a mixture thereof. That is, the “precursor” may mean a free precursor, a salt thereof, or a mixture thereof, unless otherwise specified. Examples of the salt include a sulfate, a hydrochloride, a carbonate, an ammonium salt, a sodium salt, and a potassium salt. As the salt of the precursor, one kind of salt may be used, or two or more kinds of salts may be used in combination.

As the precursor, a commercially available product may be used, or a precursor that is appropriately manufactured and obtained may be used. That is, the bioconversion method may further include a step of producing a precursor. The method for producing the precursor is not particularly limited, and for example, a known method can be used. The precursor can be produced, for example, by a chemical synthesis method, an enzymatic method, a bioconversion method, a fermentation method, an extraction method, or a combination thereof. That is, for example, the precursor of the target substance is prepared by utilizing an enzyme that catalyzes a conversion reaction from the further precursor to the precursor of the target substance (also referred to as “precursor-forming enzyme”). Can be manufactured from the body. In addition, for example, the precursor of the target substance can be produced from a carbon source or from such a further precursor using a microorganism capable of producing a precursor. A “microorganism capable of producing a precursor” may mean a microorganism capable of producing a precursor of a target substance from a carbon source or from such a further precursor. For example, as a method for producing protocatechuic acid by an enzymatic method or a bioconversion method, a method for converting paracresol to protocatechuic acid using Pseudomonas putida KS-0180 (JP-A-7-75589), NADH Method for converting parahydroxybenzoic acid to protocatechuic acid using dependent parahydroxybenzoic acid hydroxylase (Japanese Patent Application Laid-Open No. 5-244941), a method comprising introducing a gene involved in a reaction to generate protocatechuic acid from terephthalic acid A method for producing protocatechuic acid by culturing the transformant in a medium to which terephthalic acid has been added (Japanese Patent Application Laid-Open No. 2007-104942), a method having protocatechuic acid-producing ability and having reduced or defective protocatechuic acid 5-position oxidase activity A method of producing protocatechuic acid from its precursor using an isolated microorganism (Japanese Patent Application Laid-Open No. 2010-207094). . Further, as a method for producing protocatechuic acid by fermentation, a method for producing protocatechuic acid using bacteria belonging to the genus Brevibacterium using acetic acid as a carbon source (Japanese Patent Application Laid-Open No. 50-89592), -A method for producing protocatechuic acid using glucose as a carbon source using a bacterium belonging to the genus Escherichia or Klebsiella into which a gene encoding dihydroshikimate dehydrogenase has been introduced (U.S. Pat. No. 5,272,073) Is mentioned. In addition, vanillic acid can be obtained using protocatechuic acid as a precursor by an enzymatic method using OMT, a bioconversion method using a microorganism having OMT (J. Am. CHm. Soc., 1998, Vol. 120), or ferulic acid. acid as a precursor, Pseudomonas sp. bioconversion method using AV ₁₀ strain (J. App. Microbiol., 2013 , Vol.116, p903-910) it can be by, for manufacturing. In addition, protocatechuic aldehyde is obtained by using protocatechuic acid as a precursor.
It can be produced by an enzymatic method using AR or a bioconversion method using a microorganism having ACAR. The produced precursor can be used for a bioconversion method as it is or after appropriately subjecting it to a treatment such as concentration, dilution, drying, dissolution, fractionation, extraction, and purification. That is, as the precursor, for example, a purified product purified to a desired degree may be used, or a material containing the precursor may be used. The material containing the precursor is not particularly limited as long as the microorganism can use the precursor. Specific examples of the precursor-containing material include, for example, a culture solution obtained by culturing a microorganism having a precursor-producing ability, a culture supernatant separated from the culture solution, and a concentrate thereof (for example, concentrated Liquid) and processed products such as dried products.

  In one aspect, the biotransformation step can be performed by culturing the microorganisms described herein according to the method for maintaining a plasmid described above. This aspect is also referred to as “first aspect of the bioconversion method”. That is, the bioconversion step may be, for example, a step of producing a target substance by culturing a microorganism in a medium containing a sugar and a precursor of the target substance. The bioconversion step may be, in particular, a step of culturing a microorganism in a medium containing a precursor of a sugar and a target substance to produce the target substance from the precursor. That is, the first embodiment of the bioconversion method is, for example, a step of producing a target substance by culturing a microorganism in a medium containing a precursor of the sugar and the target substance, in particular, containing a precursor of the sugar and the target substance. The method for producing a target substance may include a step of culturing a microorganism in a medium to produce the target substance from a precursor. The biotransformation step is, specifically, a step of culturing a microorganism in a medium containing a precursor of the target substance, and producing and accumulating the target substance in a culture solution (for example, in the medium, inside the cells, or a combination thereof). It may be.

  Regarding the culture in the first embodiment of the bioconversion method, in the same embodiment, the culture in the above-described method for maintaining the plasmid except that the medium further contains the precursor of the target substance and the target substance is produced (For example, description of culture medium and culture conditions) can be applied mutatis mutandis. That is, the medium to be used is not particularly limited as long as it contains a precursor of the sugar and the target substance, the microorganism can grow while retaining the plasmid, and the target substance is produced. The culture conditions are not particularly limited as long as the microorganism can grow while retaining the plasmid and the target substance is produced. The culturing in the first embodiment of the bioconversion method may be performed, for example, using a liquid medium. The culture in the first embodiment of the bioconversion method may be performed, for example, under aerobic conditions. When the main culture is performed after the preculture, the target substance may be produced at least during the main culture.

The precursor may be contained in the medium during the entire period of the culture, or may be contained in the medium only during a part of the culture. That is, "culturing a microorganism in a medium containing a precursor" does not require that the precursor be contained in the medium during the entire period of the culture. The period during which the precursor is contained in the medium is not particularly limited as long as the target substance is produced. The period in which the precursor is contained in the medium is, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more of the entire culture period. The period may be 99% or more, 99.5% or more, 99.7% or more, or 99.9% or more. For example, the precursor may or may not be contained in the medium from the start of the culture. If the precursor is not contained in the medium at the start of the culture, the precursor is added to the medium after the start of the culture. The timing of the addition can be appropriately set according to various conditions such as the culture time. For example, the precursor may be added to the medium after the microorganism has sufficiently grown. The precursor may be contained in the medium, for example, at least until the target substance is produced to a desired degree. In any case, a precursor may be appropriately added to the medium. For example, the precursor may be added to the medium according to the decrease or depletion of the precursor accompanying the production of the target substance. The means for adding the precursor to the medium is not particularly limited. For example, the precursor can be added to the medium by feeding the medium with the feed medium containing the precursor. Also, for example, by co-culturing the microorganism described in the present specification and a microorganism having a precursor producing ability, a precursor is produced in the medium by the microorganism having the precursor producing ability, and thus the precursor is transformed into the medium. It can also be added. The “component” in the case of “adding a certain component to the medium” may include those produced or regenerated in the medium. These addition means may be used alone or in an appropriate combination. The concentration of the precursor in the medium is not particularly limited as long as the microorganism can use the precursor as a raw material of the target substance. The precursor concentration in the medium is converted into the weight of the free form, 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 throughout the culture. For example, the precursor may be contained in the medium at the concentration exemplified above at the start of the culture, or may be added to the medium at the concentration exemplified above after the start of the culture. Further, for example, the precursor may be contained in the medium at the above-described concentration during the above-described period. When the main culture is performed after the preculture, the target substance may be produced at least during the main culture. Therefore, it is sufficient that the precursor is contained in the medium at least during the main culture, that is, during the entire main culture or a part of the main culture. That is, the precursor may or may not be contained in the medium during the seed culture. In such a case, the description about culturing (for example, “culturing period (culturing period)” or “start of culturing”) can be read as that for main culture.

  In another embodiment, the bioconversion step can be performed by utilizing the cells obtained by culturing the microorganism described herein according to the method for maintaining a plasmid described above. This embodiment is also referred to as “the second embodiment of the bioconversion method”. That is, the bioconversion step may be, for example, a step of converting a precursor of the target substance into the target substance using such a cell. In other words, the bioconversion step may be a step of producing a target substance from a precursor of the target substance by utilizing such cells. In other words, the bioconversion step may be a step in which such cells are allowed to act on a precursor of the target substance to generate the target substance. That is, the second embodiment of the bioconversion method includes, for example, a step of culturing a microorganism in a medium containing a sugar to produce cells having a plasmid, and using the cells to produce a precursor of a target substance. It may be a method for producing a target substance, including a step of converting the substance into a substance. In the bioconversion step, specifically, such cells are allowed to act on a precursor of a target substance in a reaction solution, and the target substance is reacted in the reaction liquid (for example, in a liquid fraction, in a cell, or a combination thereof). ). The bioconversion process performed using such cells is also referred to as a “conversion reaction”.

  Regarding the culture in the second embodiment of the bioconversion method, the description about the culture in the above-described method for maintaining the plasmid (for example, the description about the culture medium and culture conditions) can be applied mutatis mutandis. The culturing in the second embodiment of the bioconversion method may be performed, for example, using a liquid medium. The culturing in the second embodiment of the bioconversion method may be performed, for example, under aerobic conditions. In the culture in the second aspect of the bioconversion method, the precursor may or may not be contained in the medium. In the culture in the second aspect of the bioconversion method, the target substance may or may not be produced.

  The cells may be used in the conversion reaction while being 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. The cells may be used in the conversion reaction after appropriate treatment. That is, examples of the cells include a culture solution containing the cells, cells recovered from the culture solution, and processed products thereof. In other words, the cells include cells contained in the culture solution of the microorganism, cells collected from the culture solution, and cells contained in a processed product thereof. Examples of the processed product include those obtained by subjecting bacterial cells to processing. Specific examples of the processed product include a product obtained by subjecting a culture solution containing bacterial cells to a process, and a product obtained by subjecting bacterial cells recovered from the culture solution to a process. The cells of these embodiments may be used in appropriate combination.

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

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

  The cells used in the conversion reaction are not particularly limited as long as they have the ability to produce the target substance and the plasmid described in this specification. The cells preferably maintain their metabolic activity. "Maintained metabolic activity" may mean that the cells have the ability to assimilate a carbon source (particularly sugar) to produce or regenerate a substance required for production of the target substance. . Examples of such a substance include an electron donor such as ATP, NADH and NADP, and a methyl group donor such as SAM. The cells may or may not have the ability to grow.

  The conversion reaction can be performed in a suitable reaction solution. The conversion reaction can be specifically carried out by allowing the cells and the precursor to coexist in an appropriate reaction solution. The conversion reaction may be performed in a batch system or a column system. In the case of a batch system, for example, a conversion reaction can be carried out by mixing microbial cells and a precursor in a reaction solution in a reaction vessel. The conversion reaction may be carried out while standing, or may be carried out with stirring or shaking. In the case of a column type, for example, the conversion reaction can be performed by passing a reaction solution containing a precursor through a column filled with immobilized cells. Examples of the reaction solution include an aqueous medium (aqueous solvent) such as water or an aqueous buffer.

  The reaction solution may contain components other than the precursor, if necessary, in addition to the precursor. 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 (especially sugars), and phosphate sources. And other various medium components. That is, for example, a medium containing a precursor may be used as a reaction solution. The reaction solution may or may not contain a sugar, for example. That is, as for the reaction solution in the second embodiment of the bioconversion method, the description of the medium in the first embodiment of the bioconversion method, except that the reaction solution may or may not contain sugar. Can be applied mutatis mutandis. The type and concentration of the components contained in the reaction solution may be appropriately set according to various conditions such as the type of the precursor used and the mode of the bacterial cell used.

The conditions of the conversion reaction (dissolved oxygen concentration, reaction solution pH, reaction temperature, reaction time, concentration of various components, etc.) are not particularly limited as long as the target substance is produced. The conversion reaction can be performed, for example, under normal conditions used for substance conversion using microbial cells such as quiescent cells. The conditions for the conversion reaction may be appropriately set according to various conditions such as the type of microorganism used. The conversion reaction may be performed, for example, under aerobic conditions. “Aerobic conditions” may mean conditions under which the concentration of dissolved oxygen in a reaction solution is 0.33 ppm or more, or 1.5 ppm or more. Specifically, the oxygen concentration may be controlled to, for example, about 1 to 50% or about 5% with respect to the saturated oxygen concentration. The pH of the reaction solution may be, for example, usually 6.0 to 10.0, or 6.5 to 9.0. The reaction temperature may be, for example, usually 15 to 50 ° C, 15 to 45 ° C, or 20 to 40 ° C. The reaction time may be, for example, from 5 minutes to 200 hours. In the case of the column method, the flow rate of the reaction solution may be, for example, a speed such that the reaction time falls within the range of the reaction time exemplified above. Further, the conversion reaction can be performed under culture conditions such as ordinary conditions used for culture of microorganisms such as bacteria and yeast. In the conversion reaction, the cells may or may not grow. That is, as for the conversion reaction in the second embodiment of the bioconversion method, the description of the culture in the first embodiment of the bioconversion method applies mutatis mutandis, except that the cells may or may not grow in the second embodiment. it can. In such a case, the culture conditions for obtaining the cells and the conditions for the conversion reaction may or may not be the same. The concentration of the precursor in the reaction solution is converted to the weight of the free form, 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 15 g / L or more, 200 g / L or less, 100 g / L or less, 50 g / L or less, or 20 g / L or less, or a combination thereof. Good. The concentration of bacterial 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 alone or in any combination to the reaction solution. For example, the precursor may be added to the reaction solution in accordance with the decrease or depletion of the precursor due to generation of the target substance. These components may be added one or more times, or may be added continuously.

Means for adding various components such as a precursor to the reaction solution is not particularly limited. Any of these components can be added to the reaction solution by directly adding it to the reaction solution. In addition, for example, by co-culturing the microorganism described in this specification and a microorganism having a precursor producing ability, a precursor is generated in a reaction solution by the microorganism having the precursor producing ability, thereby reacting the precursor. It can also be added to the liquid. Further, for example, components such as ATP, an electron donor, and a methyl group donor may all be generated or regenerated in a reaction solution, or may be generated or regenerated in the cells of a microorganism, It may be generated or reproduced by interconjugation. For example, when the metabolic activity is maintained in the microbial cells, use a carbon source (especially sugar) to generate or regenerate components such as ATP, an electron donor, and a methyl group donor in the microbial cells. Can be. For example, specifically, a microorganism may have enhanced ability to produce or regenerate a SAM, and SAM produced or regenerated by the microorganism may be used in a conversion reaction. SAM generation or regeneration can be further enhanced by combination with other approaches to generating or reproducing SAM. As a method of producing or regenerating ATP, for example, a method of supplying ATP from a carbon source (especially sugar) using a bacterium belonging to 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)), polyphosphate and polyphosphate kinase A method of regenerating ATP using (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 a component generated or regenerated in the reaction solution.

  The reaction conditions may be uniform from the start to the end of the conversion reaction, or may change during the conversion reaction. The phrase "reaction conditions change in the course of the conversion reaction" is not limited to time-dependent changes in reaction conditions, but may also include spatial changes in reaction conditions. "The reaction conditions vary spatially" means that, for example, when a conversion reaction is carried out by a column method, the reaction conditions such as the reaction temperature and the cell density vary depending on the position on the flow path. You may.

By performing the bioconversion process in this way, a target substance is generated, that is, a culture solution or a reaction solution containing the target substance is obtained. The target substance may specifically accumulate, for example, in a medium, in a bacterial cell, or a combination thereof. In addition, the target substance may specifically accumulate in, for example, a liquid fraction of the reaction solution, cells, or a combination thereof. Confirmation of the 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 bioconversion method further comprises a collection step (for example, a culture solution (specifically, a medium, a cell, or a combination thereof)) or a reaction solution (specifically, a liquid fraction, a cell, or a combination thereof). )) And recovering the target substance from)). The target substance to be collected may contain, in addition to the target substance, other components such as a microbial cell, a medium component, a reaction solution component, water, and a metabolic by-product of a microorganism. The purity of the recovered target substance is, for example, 30% (w / w) or more, 50% (w / w) or more, 70% (w / w) or more, 80% (w / w) or more, 90% (w / w) or more. w / w) or more, or 95% (w / w) or more.

<3-3> Method for producing vanillin and other target substances When the target substance is produced using the method described herein (that is, by a fermentation method or a bioconversion method), the produced target substance is further added. It can be converted to other target substances. That is, the present invention provides a step of producing a first target substance using the method described in the present specification (that is, by a fermentation method or a bioconversion method), and a step of converting the produced first target substance to a second target substance. Provided is a method for producing a second target substance, comprising a step of converting the target substance into a target substance.

  For example, if vanillic acid is produced utilizing the methods described herein (ie, by fermentation or bioconversion methods), the produced vanillic acid can be further converted to vanillin. That is, the present invention provides a method for producing vanillic acid using the method described herein (ie, by fermentation or bioconversion), and the step of converting the produced vanillic acid to vanillin. And a method for producing the same. This method is also called "vanillin production method".

  Vanillic acid produced by using a microorganism can be used for conversion to vanillin as it is or after being appropriately subjected to processes such as concentration, dilution, drying, dissolution, fractionation, extraction, and purification. That is, as vanillic acid, for example, a purified product purified to a desired degree may be used, or a material containing vanillic acid may be used. The material containing vanillic acid is not particularly limited as long as a component that catalyzes the conversion (for example, a microorganism or an enzyme) can use vanillic acid. As the material containing vanillic acid, specifically, for example, a culture solution or a reaction solution containing vanillic acid, a supernatant separated from the culture solution or the reaction solution, a concentrate thereof (for example, a concentrate) or a dried solution thereof And other processed materials.

  The method for converting vanillic acid to vanillin is not particularly limited.

  Vanillic acid can be converted to vanillin, for example, by a bioconversion method using a microorganism having ACAR. The microorganism having ACAR may or may not be modified so that the ability to assimilate sugars is reduced. Moreover, the microorganism having ACAR may or may not have the plasmid described herein. With respect to a microorganism having ACAR, the microorganism has ACAR and may or may not be modified so as to have a reduced ability to assimilate sugar, and may have the plasmid described in the present specification. The description of the microorganism described in the present specification can be applied mutatis mutandis except that it may or may not be necessary. A microorganism having an ACAR may be modified to increase the activity of one or more of the ACAR, PPT, and vanillic acid uptake systems. As for the bioconversion method for converting vanillic acid into vanillin using a microorganism having ACAR, the description on the bioconversion method for producing a target substance using a microorganism can be applied mutatis mutandis. The culture of the microorganism having ACAR may or may not be performed according to the method for maintaining the plasmid described above.

  Vanillic acid can be converted to vanillin by, for example, an enzymatic method using ACAR.

ACAR can be produced by expressing the ACAR gene in a host having the ACAR gene. ACAR can be produced using a cell-free protein synthesis system.

  The host having the ACAR gene is also referred to as “the host having ACAR”. The host having the ACAR gene may have the ACAR gene in nature, or may have been modified to have the ACAR gene. Hosts that inherently have an ACAR gene include organisms from which the above-described ACARs are derived. The host modified to have the ACAR gene includes a host into which the ACAR gene has been introduced. Further, a host originally having the ACAR gene may be modified so that the expression of the ACAR gene is increased. The host used for the expression of the ACAR gene is not particularly limited as long as a functional ACAR can be expressed. Examples of the host include microorganisms such as bacteria and yeast (fungi), plant cells, insect cells, and animal cells.

  The ACAR gene can be expressed by culturing a host having the ACAR gene. The culture method is not particularly limited as long as the host having the ACAR gene can grow and express ACAR. As for the culture of the host having the ACAR gene, the description of the culture in the fermentation method can be applied mutatis mutandis. If necessary, the expression of the ACAR gene can be induced. By culturing, a culture solution containing ACAR is obtained. ACARs can accumulate in host cells and / or media.

  ACAR present in the host cell or medium may be used as it is in the enzyme reaction, or may be purified therefrom and used in the enzyme reaction. Purification can be performed to the desired degree. That is, as ACAR, purified ACAR may be used, or a fraction containing ACAR may be used. Such a fraction is not particularly limited as long as ACAR is contained so as to act on vanillic acid. Examples of such a fraction include a culture solution of a host having an ACAR gene (that is, a host having an ACAR), cells recovered from the culture, and a processed product of the cells (eg, a crushed cell, a cell lysate). Products, cell extracts, immobilized cells immobilized with acrylamide, carrageenan, etc.), culture supernatants recovered from the culture, partially purified products thereof (ie, crude products), and combinations thereof. These fractions can be used alone or in combination with purified ACAR.

  The enzymatic reaction can be performed by allowing ACAR to act on vanillic acid. The enzyme reaction conditions are not particularly limited as long as vanillin is produced. The enzymatic reaction can be carried out, for example, under ordinary conditions used for substance conversion using enzymes or microbial cells (for example, quiescent cells). For the enzyme reaction in the vanillin production method, for example, the description of the conversion reaction in the second embodiment of the bioconversion method can be applied mutatis mutandis.

  By carrying out the conversion in this manner, a reaction solution containing vanillin is obtained. Confirmation of the production of vanillin and recovery of vanillin can be carried out in the same manner as in the fermentation method described above. That is, the method for producing vanillin may further include a step of recovering vanillin from the reaction solution. The recovered vanillin may contain, in addition to vanillin, other components such as microbial cells, medium components, reaction solution components, ACAR, moisture, and metabolic by-products of microorganisms. The purity of the recovered vanillin is, for example, 30% (w / w) or more, 50% (w / w) or more, 70% (w / w) or more, 80% (w / w) or more, 90% (w / w). / w) or more, or 95% (w / w) or more.

Vanillic acid can also be converted to guaiacol by, for example, a bioconversion method using a microorganism having VDC or an enzymatic method using VDC. Ferulic acid can be converted to 4-vinylguaiacol by, for example, a bioconversion method using a microorganism having FDC or an enzymatic method using FDC. 4-vinylguaiacol can be converted to 4-ethylguaiacol by, for example, a bioconversion method using a microorganism having VPR or an enzymatic method using VPR. Ferulic acid can also be converted to 4-ethylguaiacol by a combination of these methods. Specifically, ferulic acid can be used, for example, by utilizing FDC or a microorganism having the same and VPR or a microorganism having the same simultaneously or individually, in combination, or by using a microorganism having both FDC and VPR. By utilizing, it can be converted to 4-ethylguaiacol. The above description on the method for producing vanillin can be applied mutatis mutandis to the method for producing other target substances.

  Hereinafter, the present invention will be described more specifically with reference to examples.

<1> Acquisition of Corynebacterium glutamicum ribonuclease III gene-deficient strain
A disrupted strain of the ribonuclease III (RNaseIII) homolog gene (hereinafter also referred to as the rnc gene) of C. glutamicum 2256 strain (ATCC 13869 strain; hereinafter also referred to as 2256 strain) was constructed as follows.

  First, based on the homology of the amino acid sequence with the known RNaseIII, the region present in REGION: 2115207..2115950 in the genome sequence information of C. glutamicum 2256 (Accession No. AP017557) in the gene database (GenBank) Was estimated to be the rnc gene. Therefore, as information necessary for deletion of the gene, the ORF (open reading frame) region and DNA base sequence information of about 1,000 bases (1 kb) each of its upstream region and downstream region are stored in a gene database (GenBank). Obtained from.

  Next, genomic DNA was obtained from the cells of the 2256 strain using the DNeasy Blood & Tissue Kit (manufactured by QIAGEN), and using this as a template, the primers of SEQ ID NOs: 193 and 194 were used to obtain about 1 kb upstream of the rnc gene. And a DNA fragment of about 1 kb downstream of the rnc gene was amplified by PCR using PrimeSTAR GXL DNA Polymerase (TAKARA BIO) using the primers of SEQ ID NOs: 195 and 196, respectively. In addition, PCR conditions followed the protocol recommended by the manufacturer. Subsequently, these DNA fragments were ligated to a plasmid pBS4S (WO2005 / 113745 and WO2005 / 113744; which has no replication ability in C. glutamicum) carrying the sacB gene by the following procedure. Specifically, PCR amplification was performed with PrimeSTAR GXL DNA Polymerase using pBS4S as a template and primers of SEQ ID NOs: 197 and 198 to obtain an amplified fragment of pBS4S. Next, both the upstream and downstream DNA fragments of the rnc gene obtained above were mixed with the amplified fragment of pBS4S, and the three fragments were ligated using an In-Fusion HD Cloning Kit (Clontech). (FIG. 1). Then, using the reaction solution, competent cells of Escherichia coli JM109 strain (manufactured by TAKARA BIO) were transformed, and applied to an LB agar medium containing 25 μg / ml of kanamycin. After culturing overnight at 37 ° C., a single colony was separated from the colony that appeared on the agar medium, and a transformant which became kanamycin resistant was obtained. Plasmids were extracted from the resulting transformants by a conventional method, and plasmids containing DNA fragments in the upstream and downstream regions of the rnc gene were identified by structural analysis. This was named pBS4SΔrnc (FIG. 1).

Since this plasmid cannot replicate autonomously in coryneform bacteria, the frequency of this plasmid is extremely low when introduced into coryneform bacteria, but the plasmid is integrated into the chromosome by genetic homologous recombination and exhibits kanamycin resistance. Transformants appear. Therefore, the 2256 strain was transformed with the above plasmid pBS4Δrnc at a high concentration by the electric pulse method, and CM-Dex agar medium containing 25 μg / ml of kanamycin (glucose 5 g / L, polypeptone 10 g / L, yeast extract 10 g / L) _{, KH 2 PO 4 1g / L} , MgSO 4 · 7H 2 O 0.4g / L, FeSO 4 · 7H 2 O 0.01g / L, MnSO 4 · 7H 2 O 0.01g / L, urea 3 g / L, soybean hydrolyzate 1.2 g / L, pH 7.5 (adjusted by KOH), agar 20 g / L) and cultured at 30 ° C. overnight. As a result, several colonies appeared. The strain grown on this medium undergoes homologous recombination between the DNA sequence fragment of the plasmid near the rnc gene (upstream or downstream) and the region of the 2256 strain genome near the same gene. As a result, a strain in which the kanamycin resistance gene and sacB gene derived from the plasmid are inserted into the same genome, that is, a so-called single recombinant strain is obtained.

Next, these single recombinants were cultured overnight at 30 ° C. in CM-Dex liquid medium without kanamycin (same composition as CM-Dex agar medium except that no agar was contained), and After dilution, Kanamycin-free Dex-S10 agar medium containing 10% (w / v) sucrose (sucrose 10 g / L, polypeptone 10 g / L, yeast extract 10 g / L, KH ₂ PO ₄ 1 g / L, MgSO _{4 · 7H 2 O 0.4g / L} , FeSO 4 · 7H 2 O 0.01g / L, MnSO 4 · 4H 2 O 0.01g / L, urea 3 g / L, soybean hydrolyzate 1.2 g / L, biotin 10 [mu] g / L , PH 7.5 (adjusted with KOH), agar 20 g / L) and cultured at 30 ° C overnight. As a result, several colonies appeared. These were considered to be sucrose-insensitive strains due to the loss of the sacB gene by the second homologous recombination. The strains thus obtained include those in which the rnc gene has been replaced with a defective type and those in which the rnc gene has returned to the wild type. Therefore, the emerged colonies were subjected to a colony PCR reaction using KOD FX NEO (manufactured by TOYOBO) to select rnc gene-deficient strains. Using the primers of SEQ ID NOs: 199 and 200, the length of the rnc gene region of those strains was analyzed by PCR amplification. As a result, it was found that the genomic DNA of 2256 strains (wild type) was used as a template in PCR amplification. Some DNA fragments were short in length. Therefore, one of them was selected as a rnc gene-deficient strain and named 2256Δrnc strain. The length of the PCR-amplified DNA fragment in the case of the wild-type rnc gene is about 3 kbp, while the length of the PCR-amplified DNA fragment in the case of the rnc gene deletion is about 2 kbp (FIG. 2).

<2> Curing of endogenous plasmid
The 2256 strain has pAM330 as an endogenous plasmid (Yamaguchi, Ryuji, et al. "Determination of the complete nucleotide sequence of Brevibacterium lactofermentum plasmid pAM330 and analysis of its genetic information." Agricultural and biological chemistry 50.11 (1986): 2771- 2778.). pVC7-sacB was prepared by mounting the sacB gene on pVC7 (JP-A-1997-070291), a composite plasmid of pAM330 and pHSG399 (manufactured by TAKARA BIO), a general vector for Escherichia coli. Specifically, PCR amplification was performed with PrimeSTAR GXL DNA Polymerase using the pBS4S plasmid as a template and primers of SEQ ID NOs: 201 and 202 to obtain an amplified fragment of the sacB gene. In addition, PCR amplification was performed with KOD FX NEO (manufactured by TOYOBO) using the pVC7 plasmid as a template and the primers of SEQ ID NOs: 203 and 204 to obtain an amplified fragment of the pVC7 vector. The amplified fragments thus obtained were mixed and ligated to each other using an In-Fusion HD Cloning Kit (Clontech). Next, using this reaction solution, competent cells of Escherichia coli JM109 strain (manufactured by TAKARA BIO) were transformed, spread on an LB agar medium containing 25 μg / ml of chloramphenicol, and incubated at 37 ° C. Cultured overnight. Thereafter, a single colony was separated from the emerged colony. Plasmid DNA was extracted from the thus obtained transformant by a conventional method, and the target plasmid was confirmed by decoding the DNA sequence, and this was named pVC7-sacB (FIG. 3). Introduced pVC7-sacB to the 2256 strain and 2256Δrnc strain by the electric pulse method, applied to a CM-Dex agar medium containing 5 μg / ml of chloramphenicol, and cultured at 30 ° C. overnight, followed by 2256 / pVC7-sacB strain and 2256Δrnc. A plurality of transformants of the / pVC7-sacB strain were obtained. Subsequently, 2256ΔpAM330 / pVC7-sacB strain and 2256ΔrncΔpAM330 / pVC7-sacB strain from which the endogenous plasmid pAM330 had been removed were obtained. Furthermore, by applying these strains to Dex-S10 agar medium and culturing at 30 ° C. overnight, pVC7-sacB was dropped off and became sucrose-insensitive, 2256ΔpAM330 strain (hereinafter, also referred to as 2256LΔrnc strain) and 2256ΔrncΔpAM330 strain (hereinafter, also referred to as 2256L strain) was obtained.

<3> Construction of Corynebacterium glutamicum pfkA gene-deficient strain
The pfkA gene-disrupted strains of C. glutamicum 2256ΔrncΔpAM330 strain and C. glutamicum 2256ΔpAM330 strain were constructed as follows.

First, in the gene database (GenBank), the region present in REGION: 1421394..1422434 in the genome sequence information of C. glutamicum 2256 (Accession No. AP017557) is the pfkA gene, which is necessary for deletion of that gene. As information, the ORF (open reading frame) region, and DNA base sequence information of about 1,000 bases (1 kb) each of the upstream region and the downstream region thereof were obtained from the gene database (GenBank).

  Next, genomic DNA was obtained from the cells of the 2256 strain using a DNeasy Blood & Tissue Kit (manufactured by QIAGEN), and using this DNA as a template, primers of SEQ ID NOS: 205 and 206 were used to obtain about 1 kb upstream of the pfkA gene A DNA fragment of about 1 kb downstream of the pfkA gene was obtained by PCR amplification using PrimeSTAR Max DNA Polymerase (TAKARA BIO) using the primers of SEQ ID NOs: 207 and 208, respectively. In addition, PCR conditions followed the protocol recommended by the manufacturer. Subsequently, these DNA fragments were ligated to a plasmid pBS4S (WO2005 / 113745 and WO2005 / 113744; which has no replication ability in C. glutamicum) carrying the sacB gene by the following procedure. Specifically, after pBS4S was cleaved with restriction enzymes BamHI and PstI, both the upstream and downstream DNA fragments of the pfkA gene obtained above were mixed with the amplified fragment of pBS4S, and the mixture was mixed with an In-Fusion HD Cloning Kit (Clontech). Was used to ligate the three fragments (FIG. 4). Then, using the reaction solution, competent cells of Escherichia coli JM109 strain (manufactured by TAKARA BIO) were transformed, and applied to LB agar medium containing 50 μg / ml of kanamycin. After culturing overnight at 37 ° C., a single colony was separated from the colony that appeared on the agar medium, and a transformant which became kanamycin resistant was obtained. Plasmids were extracted from the resulting transformants by a conventional method, and plasmids containing DNA fragments in the upstream and downstream regions of the pfkA gene were identified by structural analysis. This was named pBS4SΔpfkA (FIG. 4).

Since this plasmid cannot replicate autonomously in coryneform bacteria, the frequency of this plasmid is extremely low when introduced into coryneform bacteria, but the plasmid is integrated into the chromosome by genetic homologous recombination and exhibits kanamycin resistance. Transformants appear. Therefore, the 2256LΔrnc strain and the 2256L strain were transformed using the above-mentioned plasmid pBS4SΔpfkA at a high concentration by the electric pulse method, and a CM-Dex agar medium containing 25 μg / ml of kanamycin (glucose 5 g / L, polypeptone 10 g / L, yeast extract) was used. _{10g / L, KH 2 PO 4} 1g / L, MgSO 4 · 7H 2 O 0.4g / L, FeSO 4 · 7H 2 O 0.01g / L, MnSO 4 · 7H 2 O 0.01g / L, urea 3 g / L, The soybean hydrolyzate was applied to 1.2 g / L, pH 7.5 (adjusted with KOH), agar 20 g / L) and cultured at 30 ° C. overnight. As a result, several colonies appeared. The strain grown on this medium undergoes homologous recombination between the DNA sequence fragment of the plasmid near the pfkA gene (upstream or downstream) and the region near the same gene on the genome of the 2256 strain. As a result, a strain in which the kanamycin resistance gene and the sacB gene derived from the plasmid are inserted into the same genome, that is, a so-called single recombinant strain is obtained.

Next, these single recombinants were cultured overnight at 30 ° C. in CM-Dex liquid medium without kanamycin (same composition as CM-Dex agar medium except that no agar was contained), and After dilution, Kanamycin-free Dex-S10 agar medium containing 10% (w / v) sucrose (sucrose 10 g / L, polypeptone 10 g / L, yeast extract 10 g / L, KH ₂ PO ₄ 1 g / L, MgSO _{4 · 7H 2 O 0.4g / L} , FeSO 4 · 7H 2 O 0.01g / L, MnSO 4 · 4H 2 O 0.01g / L, urea 3 g / L, soybean hydrolyzate 1.2 g / L, biotin 10 [mu] g / L , PH 7.5 (adjusted with KOH), agar 20 g / L) and cultured at 30 ° C overnight. As a result, several colonies appeared. These were considered to be sucrose-insensitive strains due to the loss of the sacB gene by the second homologous recombination. The strains thus obtained include those in which the pfkA gene has been replaced by a defective type and those in which the pfkA gene has returned to the wild type. Therefore, the emerged colonies were subjected to a colony PCR reaction using KOD FX NEO (manufactured by TOYOBO) to select pfkA gene-deficient strains. The length of the gene region of these strains was analyzed by PCR amplification using the primers of SEQ ID NOs: 209 and 210, and as a result, the DNA in the PCR amplification was smaller than that obtained by using genomic DNA of 2256 strain (wild type) as a template. Some fragments were short in length. Therefore, one of them was selected as a pfkA gene-deficient strain, and named 2256LΔrncΔpfkA strain and 2256LΔpfkA strain. The length of the PCR-amplified DNA fragment in the case of the wild-type pfkA gene is about 3 kbp, while the length of the PCR-amplified DNA fragment in the case of the deletion of the pfkA gene is about 2 kbp (see FIG. 5).

<4> Construction of plasmid for co-expression of pfkA gene and dsRNA <4-1> Construction of plasmid pVC7-Pf1-Hv-iap-Pf1rev for bidirectional transcription A DNA fragment of Hv-iap (SEQ ID NO: 211), which is a partial sequence of cDNA, was obtained by artificial gene synthesis based on the information described in WO2010 / 140675. The DNA fragment of SEQ ID NO: 212 containing the F1 promoter was obtained by artificial gene synthesis. The F1 promoter is a promoter sequence from bacteriophage BFK20 that infects coryneform bacteria (Koptides, M., et al., (1992).
Characterization of bacteriophage BFK20 from Brevibacterium flavum. Microbiology, 138 (7), 1387-1391.). The nucleotide sequence of the F1 promoter is shown in SEQ ID NO: 213. Then, a plasmid containing a DNA sequence in which an Hv-iap sequence was ligated immediately after the F1 promoter was constructed as follows (6). First, PCR amplification was performed using KOD FX NEO (manufactured by TOYOBO) using pVC7 as a template and primers of SEQ ID NOs: 214 and 215 to obtain a DNA fragment of the pVC7 vector region. Further, PCR amplification was performed using PrimeSTAR HS (manufactured by TAKARA BIO) using the DNA fragment of SEQ ID NO: 212 as a template and primers of SEQ ID NOs: 216 and 217 to obtain a DNA fragment containing the F1 promoter sequence. In addition, PCR amplification was performed with PrimeSTAR HS (manufactured by TAKARA BIO) using the DNA fragment of SEQ ID NO: 211 as a template and primers of SEQ ID NOs: 218 and 219 to obtain a DNA fragment of the Hv-iap sequence. Then, these three types of fragments were mixed, and the DNA fragments were ligated with an In-Fusion HD Cloning Kit (manufactured by Clontech). Next, using this reaction solution, competent cells of Escherichia coli JM109 strain (manufactured by TAKARA BIO) were transformed to obtain a resistant strain of chloramphenicol 25 μg / ml, and from the resulting transformant, Plasmid DNA was extracted by a conventional method. The DNA sequence was decoded to confirm that it was the target plasmid, and one of them was named pVC7-Pf1-Hv-iap (FIG. 6).

  Using pVC7 as a template and primers of SEQ ID NOs: 214 and 220, PCR amplification was performed using KOD FX NEO (manufactured by TOYOBO) to obtain a pVC7 vector DNA fragment. In addition, PCR amplification was performed with PrimeSTAR HS (manufactured by TAKARA BIO) using the DNA fragment of SEQ ID NO: 212 as a template and the primers of SEQ ID NOs: 221 and 222 to obtain an amplified fragment of the F1 promoter sequence. Both DNA fragments thus obtained were mixed and ligated with an In-Fusion HD Cloning Kit (Clontech). Next, using this reaction solution, competent cells of Escherichia coli JM109 strain (manufactured by TAKARA BIO) were transformed, spread on an LB agar medium containing 25 μg / ml of chloramphenicol, and incubated at 37 ° C. Cultured overnight. Thereafter, a single colony was separated from the emerged colonies, and plasmid DNA was extracted from the obtained transformant by a conventional method. The plasmid was confirmed to be the target plasmid by DNA sequencing, and was named pVC7-Pf1rev (FIG. 7).

  Next, in order to introduce the restriction enzyme sites KpnI site and XhoI site downstream of the F1 promoter of pVC7-Pf1rev, the KOD-Plus-Mutagenesis Kit (using pVC7-Pf1rev as a template and primers of SEQ ID NOs: 214 and 223) was used. After performing inverse PCR using TOYOBO), the amplified DNA fragment is circularized by performing DpnI treatment, phosphorylation reaction, and self-ligation reaction, and introduced into competent cells of Escherichia coli JM109 strain (TAKARA BIO). did. The cells were spread on an LB agar medium containing 25 μg / ml of chloramphenicol and cultured at 37 ° C. overnight. Thereafter, a single colony was separated from the emerged colonies, and plasmid DNA was extracted from the obtained transformant by a conventional method. It was confirmed that the plasmid was the target plasmid by DNA sequencing, and was named pVC7-KpnI-XhoI-Pf1rev (FIG. 7).

Subsequently, using pVC7-Pf1-Hv-iap as a template and primers of SEQ ID NOs: 224 and 225, PCR was performed using PrimeSTAR HS (manufactured by TAKARA BIO) to obtain a KpnI restriction enzyme site-F1 promoter region-Hv-iap A DNA fragment having a sequence of the region-XhoI restriction enzyme site was obtained. The DNA fragment and each of pVC7-KpnI-XhoI-Pf1rev were digested with restriction enzymes KpnI and XhoI, and then purified using MinElute PCR Purification Kit (manufactured by Qiagen). By performing a ligation reaction using Ligation high Ver. 2 (manufactured by TOYOBO), both DNA fragments were ligated. Next, using this reaction solution, competent cells (manufactured by TAKARA BIO) of Escherichia coli JM109 strain were transformed to obtain a resistant strain of chloramphenicol at 25 μg / ml. Plasmid DNA was extracted from the obtained transformant by a conventional method, and the plasmid was confirmed to be the target plasmid by DNA sequencing, and named pVC7-Pf1-Hv-iap-Pf1rev (FIG. 7). According to the plasmid, dsRNA derived from Hv-iap is transcribed.

<4-2> Construction of high copy number plasmid
High copy number version plasmids of pVC7 and pVC7-Pf1-Hv-iap-Pf1rev were constructed by the following procedure. Specifically, using pVC7 or pVC7-Pf1-Hv-iap-Pf1rev as a template, primers of SEQ ID NOs: 226 and 227 were used to perform inverse PCR using a KOD-Plus-Mutagenesis Kit (TOYOBO). Thereafter, each DNA fragment was circularized by DpnI treatment, phosphorylation reaction, and self-ligation reaction, and a competent cell of Escherichia coli JM109 strain (TAKARA BIO) was transformed. The cells were spread on an LB agar medium containing chloramphenicol (25 μg / ml), cultured at 37 ° C. overnight, and a single colony was separated from the appeared colony to obtain a transformant. Plasmid DNA was extracted from the obtained transformant by a conventional method, and the desired plasmid was confirmed by DNA sequencing, and named pVC7H2 and pVC7H2-Pf1-Hv-iap-Pf1rev, respectively. pVC7H2 has a C1172T mutation that increases the copy number of the plasmid. In the C1172T mutation, of the 6679 bp of the entire nucleotide sequence of pVC7, the 1172th base (the second base A from the 5 'end of the cleavage recognition site of the restriction enzyme HindIII is +1) is a cytosine (C) to thymine ( It is a mutation that is replaced by T). pVC7H2-Pf1-Hv-iap-Pf1rev also has a C1172T mutation in the pVC7 part.

<4-3> Introduction of pfkA gene expression cassette DNA fragments of SEQ ID NOs: 228 to 230 containing three types of pfkA gene expression cassettes having different promoter sequences were obtained by artificial gene synthesis. The DNA fragment of SEQ ID NO: 228 (also referred to as Pbl-pfkA) is a bl promoter (with reference to the promoter region of the xfp gene of Bifidobacterium longum strain (accession number AY518215))-pfkA gene (derived from ATCC 13869 strain) -T7 terminator Contains an array. The DNA fragment of SEQ ID NO: 229 (also referred to as PmsrA-pfkA) contains a promoter derived from msrA gene (derived from ATCC 13869 strain) -pfkA gene (derived from ATCC 13869 strain) -T7 terminator sequence. The DNA fragment of SEQ ID NO: 230 (also referred to as Plac-pfkA) contains a lac promoter (with reference to the sequence on the pHSG299 (Takara Bio) plasmid) -pfkA gene (derived from ATCC 13869 strain) -T7 terminator sequence.

Construction of a plasmid containing a DNA sequence in which the pfkA expression cassette sequence and the pVC7H2-Pf1-Hviap-Pf1rev plasmid were linked was carried out as follows (FIG. 8). First, using Pbl-pfkA (SEQ ID NO: 228) as a template and primers of SEQ ID NOs: 231 and 232, and using PmsrA-pfkA (SEQ ID NO: 229) as a template, primers of SEQ ID NOs: 231 and 233 to obtain Plac-pfkA ( Using SEQ ID NO: 230) as a template and primers of SEQ ID NOs: 231 and 234, PCR amplification was performed with PrimeSTAR Max DNA Polymerase (TAKARA BIO) to obtain DNA fragments of three types of pfkA expression cassettes having different promoter sequences. In addition, using pVC7H2-Pf1-Hviap-Pf1rev as a template and primers of SEQ ID NOs: 235 and 236, PCR amplification was performed with PrimeSTAR Max DNA Polymerase (TAKARA BIO) to obtain a vector DNA fragment. Each DNA fragment was purified using a MinElute PCR Purification Kit (manufactured by Qiagen), digested with a restriction enzyme DpnI, and then purified again using the MinElute PCR Purification Kit. The purified vector DNA fragments were mixed with each of the three purified DNA fragments of the pfk gene expression cassette, and the DNA fragments were ligated by performing a ligation reaction using an In-Fusion HD Cloning Kit (Clontech). Next, using this reaction solution, competent cells (manufactured by TAKARA BIO) of the Escherichia coli JM109 strain were transformed to obtain a resistant strain of chloramphenicol at 25 μg / ml. Plasmid DNA was extracted from the obtained transformant by a conventional method. Decoding these DNA sequences and confirming that the plasmids are of interest, one of each is pVC7H2-Pf1-Hviap-Pf1rev-Pbl-pfkA, pVC7H2-Pf1
-Hviap-Pf1rev-PmsrA-pfkA and pVC7H2-Pf1-Hviap-Pf1rev-Plac-pfkA (FIG. 8).

<5> Production of target RNA
C. glutamicum 2256L Δrnc ΔpfkA strain, pVC7H2, pVC7H2-Pf1-Hviap-Pf1rev, pVC7H2-Pf1-Hviap-Pf1rev-Pbl-pfkA, pVC7H2-Pf1-Hviap-Pf1rev-PmsrA-pfkA and pVC1H2-Pp1-fp-A Each plasmid of -pfkA was introduced by an electric pulse method, spread on a CM-Dex agar medium containing 5 μg / ml of chloramphenicol, and cultured at 30 ° C. overnight. Then, each transformant was obtained.

  Colonies of each transformant obtained above were spread on a CM-Dex agar medium (containing 5 μg / ml of chloramphenicol) and cultured at 30 ° C. for about 16 hours. Next, a part of the cells was transferred to a test tube culture, and cultured with shaking at 30 ° C. for 20 hours in a CM-Dex liquid medium (2 ml) containing no antibiotic. Next, 200 μl of the culture solution was treated with RNAprotect Bacteria Reagent, and the supernatant was removed. Next, 225 μl of 15 mg-lysozyme (manufactured by Sigma) / ml-TE buffer was added thereto, and the mixture was reacted at room temperature for 30 minutes. After reacting for 30 minutes, RNA extraction was performed using TRIzol LS (manufactured by Thermo Fisher Scientific), which was dissolved in 50 μl of RNase-free water to prepare a total RNA solution. The obtained total RNA solution was subjected to total RNA analysis using Novex TBE Gels, 6% (manufactured by Thermo Fisher Scientific). That is, 1 μl of each total RNA solution was applied to a gel lane, and polyacrylamide gel electrophoresis (PAGE) was performed under non-denaturing conditions.

  As a result, compared with the Hviap-dsRNA band extracted from the pVC7H2-Pf1-Hviap-Pf1rev transformant not carrying the pfkA gene, pVC7H2-Pf1-Hviap-Pf1rev-Pbl-pfkA transformation carrying the pfkA gene Transformant, pVC7H2-Pf1-Hviap-Pf1rev-PmsrA-pfkA transformant, Hviap-dsRNA band extracted from pVC7H2-Pf1-Hviap-Pf1rev-Plac-pfkA transformant was clearly observed, and Hviap-dsRNA was abundant. It was found that it had accumulated (FIG. 9). This indicates that it is effective to utilize the pfkA gene deficiency and plasmid complementation to produce the target RNA in an antibiotic-free culture system.

<6> Construction of plasmid for co-expression of pfkA gene and GFP <6-1> Construction of pPK4-dGFP As an example of protein expression, a green fluorescent protein dasher GFP (also called dGFP, manufactured by ATUM) was used. First, construction of a plasmid containing a DNA sequence in which the cspB gene promoter sequence, the pfkA gene sequence and the pPK4 plasmid were linked was carried out as follows (FIG. 10). Specifically, using the genomic DNA sequence of the 2256 strain as a template, the cspB gene promoter sequence using the primers of SEQ ID NOs: 237 and 238, and using the dasher GFP expression vector as a template, the primers of SEQ ID NOs: 239 and 240, Using primers of SEQ ID NOs: 241 and 242 as templates, PCR amplification was performed with PrimeSTAR Max DNA Polymerase (TAKARA BIO) to obtain three types of DNA fragments. Each DNA fragment is purified using the MinElute PCR Purification Kit (Qiagen), mixed with three types of purified DNA fragments, and subjected to a ligation reaction using the In-Fusion HD Cloning Kit (Clontech) to perform DNA ligation. The fragments were ligated. Next, using this reaction solution, competent cells of Escherichia coli JM109 strain (manufactured by TAKARA BIO) were transformed to obtain a resistant strain of kanamycin 50 μg / ml. Plasmid DNA was extracted from the obtained transformant by a conventional method. The DNA sequence was decoded to confirm that the plasmid was the target plasmid, and one of them was named pPK4-dGFP (FIG. 10).

<6-2> Introduction of pfkA gene expression cassette
Using Pbl-pfkA (SEQ ID NO: 228) as a template and primers of SEQ ID NOs: 243 and 244, and using Plac-pfkA (SEQ ID NO: 230) as a template and primers of SEQ ID NOs: 243 and 245, PrimeSTAR Max DNA Polymerase (TAKARA BIO) to obtain DNA fragments of two pfkA expression cassettes having different promoter sequences. In addition, PCR amplification was performed with PrimeSTAR Max DNA Polymerase (TAKARA BIO) using the pPK4-dGFP plasmid as a template and primers of SEQ ID NOs: 242 and 246 to obtain a vector DNA fragment. Each DNA fragment was purified using a MinElute PCR Purification Kit (manufactured by Qiagen), digested with a restriction enzyme DpnI, and then purified again using the MinElute PCR Purification Kit. The purified vector DNA fragments were mixed with each of the two purified DNA fragments of the pfk gene expression cassette, and a ligation reaction was performed with an In-Fusion HD Cloning Kit (manufactured by Clontech) to link the DNA fragments. Next, using this reaction solution, competent cells of Escherichia coli JM109 strain (manufactured by TAKARA BIO) were transformed to obtain a resistant strain of kanamycin 50 μg / ml. Plasmid DNA was extracted from the obtained transformant by a conventional method. These DNA sequences were decoded and confirmed to be the target plasmids, one of which was named pPK4-Pbl-pfkA-dGFP and pPK4-Plac-pfkA-dGFP (FIG. 10).

<7> Production of target protein
C. glutamicum 2256L ΔpfkA strain, pPK4-dGFP, pPK4-Pbl-pfkA-dGFP, and each plasmid of pPK4-Plac-pfkA-dGFP introduced by an electric pulse method, CM-Dex agar medium containing kanamycin 25μg / ml It was spread on the plate and cultured at 30 ° C. overnight. Then, each transformant was obtained.

  The colonies of each transformant obtained above were spread on a CM-Dex agar medium (containing kanamycin 25 μg / ml) and cultured at 30 ° C. for about 16 hours. Next, a part of the cells was transferred to a test tube culture, and shake-cultured at 30 ° C. for 18 hours in a CM-Dex liquid medium (2 ml) containing no antibiotic. Next, 500 μl of the culture solution was collected, centrifuged (14,400 × g, 2 min, 4 ° C.), the supernatant was removed, and the cells were collected. Wash once with ice-cold 20 mM Tris-HCl (pH 8.0), suspend with 800 μl 20 mM Tris-HCl (pH 8.0), and multi-bead shocker with 0.1 mm glass beads (Yasui Kikai, 2,500 rpm , (ON: OFF = 30s: 30s) x 10 cycles, 4 ° C). The crushed liquid was centrifuged (5,000 xg, 5 min, 4 ° C), and the supernatant fraction was used as a crude extract. The total protein concentration of the crude extract was determined by Pierce BCA Protein Assay Kit (Thermo Fisher), and BSA included in this kit was used as a standard substance. The prepared crude extract was irradiated with a transmission-type LED light source CyanoView (manufactured by ATTO, excitation wavelength: 505 ± 25 nm), and a fluorescent image shielded by an orange cover (manufactured by ATTO, product number 2006122) was taken. In addition, 50 μl of the prepared crude extract was loaded on a white 96-well plate for fluorescence measurement (manufactured by Nunc, FluoroNunc Maxisorp plate), and fluorescence measurement was performed using a microplate reader SpectraMax Me2 (Molecular Device). 20 mM Tris-HCl (pH 8.0) was used as a blank. The plate reader measurement conditions were an excitation wavelength of 485 nm and a detection wavelength of 538 nm (cutoff 530 nm). As a result, compared with the extract from the pPK4-dGFP transformant not carrying the pfkA gene, the pPK4-Plac-pfkA-dGFP transformant and the pPK4-Pbl-pfkA-dGFP transformant carrying the pfkA gene It was found that the green fluorescence intensity of the extract from was high and the expression level of dGFP was high (FIG. 11). This indicates that it is effective to utilize pfkA gene deficiency and plasmid complementation to produce the target protein in an antibiotic-free culture system.

<Description of Sequence Listing>
SEQ ID NO: 1: Nucleotide sequence of the ptsI gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 2: Amino acid sequence of the PtsI protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 3: Base of the ptsH gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 4: Amino acid sequence of PtsH protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 5: Base sequence of ptsG gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 6: PtsG protein of Corynebacterium glutamicum 2256 (ATCC 13869) Amino acid sequence SEQ ID NO: 7: Base sequence of ptsS gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 8: Amino acid sequence of PtsS protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 9: ptsF gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 10: amino acid sequence of PtsF protein of Corynebacterium glutamicum 2256 (ATCC 13869) No. 11: Nucleotide sequence of iolT1 gene of Corynebacterium glutamicum ATCC 13032 SEQ ID NO: 12: Amino acid sequence of IolT1 protein of Corynebacterium glutamicum ATCC 13032 SEQ ID NO: 13: Nucleotide sequence of iolT2 gene of Corynebacterium glutamicum ATCC 13032 SEQ ID NO: 14: Corynebacterium glutamicum ATCC 13032 Amino acid sequence of IolT2 protein SEQ ID NO: 15: Nucleotide sequence of ptsI gene of Escherichia coli MG1655 SEQ ID NO: 16: Amino acid sequence of PtsI protein of Escherichia coli MG1655 SEQ ID NO: 17: Nucleotide sequence of ptsH gene of Escherichia coli MG1655 SEQ ID NO: 18: Escherichia coli Amino acid sequence of MG1655 PtsH protein SEQ ID NO: 19: Nucleotide sequence of ptsG gene of Escherichia coli MG1655 SEQ ID NO: 20: Amino acid sequence of PtsG protein of Escherichia coli MG1655 SEQ ID NO: 21: Nucleotide sequence of crr gene of Escherichia coli MG1655 SEQ ID NO: 22: Escherichia coli MG1655 Crr protein SEQ ID NO: 23: Nucleotide sequence of the ScrA gene of Salmonella enterica SEQ ID NO: 24: Amino acid sequence of the ScrA protein of Salmonella enterica SEQ ID NO: 25: Nucleotide sequence of the fruA gene of Escherichia coli MG1655 SEQ ID NO: 26: FruA protein of Escherichia coli MG1655 SEQ ID NO: 27: Nucleotide sequence of fruB gene of Escherichia coli MG1655 SEQ ID NO: 28: Amino acid sequence of FruB protein of Escherichia coli MG1655 SEQ ID NO: 29: Nucleotide sequence of galP gene of Escherichia coli MG1655 SEQ ID NO: 30: Escherichia coli MG1655 Amino acid sequence of GalP protein SEQ ID NO: 31: Nucleotide sequence of cscB gene of Escherichia coli EC3132 SEQ ID NO: 32: Amino acid sequence of CscB protein of Escherichia coli EC3132 SEQ ID NO: 33: Nucleotide sequence of Corynebacterium glutamicum 2256 (ATCC 13869) 34: Glk tamper of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 35: Nucleotide sequence of pgi gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 36: Amino acid sequence of Pgi protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 37: Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 38: Amino acid sequence of Pfk protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 39: Nucleotide sequence of fbaA gene of Corynebacterium glutamicum 2256 (ATCC 13869) 40: Corynebacterium glutamicum 2256 (ATCC 13869) ) FbaA protein amino acid sequence SEQ ID NO: 41: Corynebacterium glutamicum 2256 (ATCC 13869) tpiA gene nucleotide sequence SEQ ID NO: 42: Corynebacterium glutamicum 2256 (ATCC 13869) TpiA protein amino acid sequence SEQ ID NO: 43: Corynebacterium glutamicum 2256 (ATCC 13869) base sequence of gapA gene SEQ ID NO: 44: Corynebacterium glutamicum 2256 (ATCC 13869) Amino acid sequence of GapA protein of SEQ ID NO: 45: Nucleotide sequence of pgk gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 46: Amino acid sequence of Pgk protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 47: Corynebacterium glutamicum 2256 (ATCC 13869) ) Nucleotide sequence of the gpmA gene SEQ ID NO: 48: amino acid sequence of the GpmA protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 49: nucleotide sequence of the gpmM gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 50: Corynebacterium glutamicum 2256 (ATCC 13869) GpmM protein amino acid sequence SEQ ID NO: 51: Corynebacterium glutamicum 2256 (ATCC 13869) eno gene base sequence SEQ ID NO: 52: Corynebacterium glutamicum 2256 (ATCC 13869) Eno protein amino acid sequence SEQ ID NO: 53: Corynebacterium glutamicum 2256 (ATCC 13869) ATCC 13869) pykA gene nucleotide sequence SEQ ID NO: 54: Corynebacterium glutamicum 2 256 (ATCC 13869) PykA protein amino acid sequence SEQ ID NO: 55: Corynebacterium glutamicum 2256 (ATCC 13869) pykF gene nucleotide sequence SEQ ID NO: 56: Corynebacterium glutamicum 2256 (ATCC 13869) PykF protein amino acid sequence SEQ ID NO: 57: Corynebacterium Nucleotide sequence of aceE gene of glutamicum 2256 (ATCC 13869) SEQ ID NO: 58: Amino acid sequence of AceE protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 59: Nucleotide sequence of aceF gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 60 Amino acid sequence of AceF protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 61: Nucleotide sequence of lpd gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 62: Amino acid sequence of Lpd protein of Corynebacterium glutamicum 2256 (ATCC 13869) : Corynebacterium glutamicum 2256 (ATCC 13869) scrB gene nucleotide sequence SEQ ID NO: 64: Corynebact erium glutamicum 2256 (ATCC 13869) ScrB protein amino acid sequence SEQ ID NO: 65: Corynebacterium glutamicum 2256 (ATCC 13869) fruK gene nucleotide sequence SEQ ID NO: 66: Corynebacterium glutamicum 2256 (ATCC 13869) FruK protein amino acid sequence SEQ ID NO: 67 : Escherichia coli MG1655 glk gene SEQ ID NO: 68: Escherichia coli MG1655 Glk protein amino acid sequence SEQ ID NO: 69: Escherichia coli MG1655 pgi gene nucleotide sequence SEQ ID NO: 70: Escherichia coli MG1655 Pgi protein amino acid sequence No. 71: Nucleotide sequence of pfkA gene of Escherichia coli MG1655 SEQ ID NO: 72: Amino acid sequence of PfkA protein of Escherichia coli MG1655 SEQ ID NO: 73: Nucleotide sequence of pfkB gene of Escherichia coli MG1655 SEQ ID NO: 74: Amino acid of PfkB protein of Escherichia coli MG1655 SEQ ID NO: 75: Nucleotide sequence of fbaA gene of Escherichia coli MG1655 SEQ ID NO: 76: Amino acid sequence of FbaA protein of Escherichia coli MG1655 SEQ ID NO: 77: Nucleotide sequence of tpiA gene of Escherichia coli MG1655 SEQ ID NO: 78: Amino acid sequence of TpiA protein of Escherichia coli MG1655 SEQ ID NO: 79: GapA gene of Escherichia coli MG1655 Nucleotide sequence SEQ ID NO: 80: amino acid sequence of GapA protein of Escherichia coli MG1655 SEQ ID NO: 81: nucleotide sequence of pgk gene of Escherichia coli MG1655 SEQ ID NO: 82: amino acid sequence of Pgk protein of Escherichia coli MG1655 SEQ ID NO: 83: gpmA of Escherichia coli MG1655 SEQ ID NO: 84: Amino acid sequence of GpmA protein of Escherichia coli MG1655 SEQ ID NO: 85: Nucleotide sequence of gpmM gene of Escherichia coli MG1655 SEQ ID NO: 86: Amino acid sequence of GpmM protein of Escherichia coli MG1655 SEQ ID NO: 87: Escherichia coli MG1655 Nucleotide sequence of the eno gene of Escherichi amino acid sequence SEQ ID NO: 89 of the Eno protein of Escherichia coli MG1655 SEQ ID NO: 90: amino acid sequence of the PykA protein of Escherichia coli MG1655 SEQ ID NO: 91: nucleotide sequence of the pykF gene of Escherichia coli MG1655 92: amino acid sequence of PykF protein of Escherichia coli MG1655 SEQ ID NO: 93: nucleotide sequence of aceE gene of Escherichia coli MG1655 SEQ ID NO: 94: amino acid sequence of AceE protein of Escherichia coli MG1655 SEQ ID NO: 95: nucleotide sequence of aceF gene of Escherichia coli MG1655 SEQ ID NO: 96: Amino acid sequence of AceF protein of Escherichia coli MG1655 SEQ ID NO: 97: Nucleotide sequence of lpd gene of Escherichia coli MG1655 SEQ ID NO: 98: Amino acid sequence of Lpd protein of Escherichia coli MG1655 SEQ ID NO: 99: Base of scrB gene of Salmonella enterica SEQ ID NO: 100: ScrB protein of Salmonella enterica Amino acid sequence SEQ ID NO: 101: nucleotide sequence of fruK gene of Escherichia coli MG1655 SEQ ID NO: 102: amino acid sequence of FruK protein of Escherichia coli MG1655 SEQ ID NO: 103: nucleotide sequence of cscA gene of Escherichia coli EC3132 SEQ ID NO: 104: CscA of Escherichia coli EC3132 Amino acid sequence of protein SEQ ID NO: 105: Nucleotide sequence of cscK gene of Escherichia coli EC3132 SEQ ID NO: 106: Amino acid sequence of CscK protein of Escherichia coli EC3132 SEQ ID NO: 107: Nucleotide sequence of aroG gene of Escherichia coli MG1655 SEQ ID NO: 108: Escherichia coli MG1655 Amino acid sequence of AroG protein of SEQ ID NO: 109: Nucleotide sequence of aroB gene of Escherichia coli MG1655 SEQ ID NO: 110: Amino acid sequence of AroB protein of Escherichia coli MG1655 SEQ ID NO: 111: Nucleotide sequence of aroD gene of Escherichia coli MG1655 SEQ ID NO: 112: Escherichia AroD tan of coli MG1655 Amino acid sequence SEQ ID NO: 113: base sequence of the asbF gene of Bacillus thuringiensis BMB171 SEQ ID NO: 114: amino acid sequence of the AsbF protein of Bacillus thuringiensis BMB171 SEQ ID NO: 115: base sequence of the tyrR gene of Escherichia coli MG1655 116: Escherichia coli Amino acid sequence SEQ ID NOS: 117 to 120 of TyrR protein of MG1655: Nucleotide sequence SEQ ID NO: 121 of transcription variant 1-4 of HMT gene of Homo sapiens: Amino acid sequence SEQ ID NO: 122 of OMT isoform of Homo sapiens (MB-COMT) SEQ ID NO: 122: Homo Amino acid sequence of sapiens OMT isoform (S-COMT) SEQ ID NO: 123: Nucleotide sequence of OMT gene of Niastella koreensis SEQ ID NO: 124: Amino acid sequence of OMT protein of Niastella koreensis SEQ ID NO: 125: Nucleotide sequence of ACAR gene of Nocardia brasiliensis No. 126: Amino acid sequence of ACAR protein of Nocardia brasiliensis SEQ ID NO: 127: Nucleotide sequence of the ACAR gene of Nocardia brasiliensis SEQ ID NO: 128: Amino acid sequence of the ACAR protein of Nocardia brasiliensis SEQ ID NO: 129: Nucleotide sequence of the ACAR gene of Gordonia effusa SEQ ID NO: 130: Amino acid sequence of the ACAR protein of Gordonia effusa 131: nucleotide sequence of the ACAR gene of Novosphingobium malaysiense SEQ ID NO: 132: amino acid sequence of the ACAR protein of Novosphingobium malaysiense SEQ ID NO: 133: nucleotide sequence of the ACAR gene of Coccomyxa subellipsoidea 134: amino acid sequence of the ACAR protein of Coccomyxa subellipsoidea 135: Nucleotide sequence of entD gene of Escherichia coli MG1655 SEQ ID NO: 136: Amino acid sequence of EntD protein of Escherichia coli MG1655 SEQ ID NO: 137: Nucleotide sequence of PPT gene of Corynebacterium glutamicum ATCC 13032 SEQ ID NO: 138: PPT protein of Corynebacterium glutamicum ATCC 13032 Quality amino acid sequence SEQ ID NO: 139: nucleotide sequence of vanK gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 140: amino acid sequence of VanK protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 141: Corynebacterium glutamicum 2256 (ATCC 13869) Nucleotide sequence of pcaK gene SEQ ID NO: 142: Amino acid sequence of PcaK protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 143: Nucleotide sequence of vanA gene of Corynebacterium glutamicum 2256 (ATCC 13869) 144: Corynebacterium glutamicum 2256 (ATCC 13869) Amino acid sequence of VanA protein of SEQ ID NO: 145: Nucleotide sequence of vanB gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 146: Amino acid sequence of VanB protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 147: pcaG of Corynebacterium glutamicum ATCC 13032 Nucleotide sequence of the gene SEQ ID NO: 148: Corynebacterium gl Amino acid sequence of PcaG protein of utamicum ATCC 13032 SEQ ID NO: 149: nucleotide sequence of pcaH gene of Corynebacterium glutamicum ATCC 13032 SEQ ID NO: 150: amino acid sequence of PcaH protein of Corynebacterium glutamicum ATCC 13032 SEQ ID NO: 151: nucleotide sequence of yqhD gene of Escherichia coli MG1655 SEQ ID NO: 152: Amino acid sequence of YqhD protein of Escherichia coli MG1655 SEQ ID NO: 153: Nucleotide sequence of NCgl0324 gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 154: Amino acid sequence of NCgl0324 protein of Corynebacterium glutamicum 2256 (ATCC 13869) : Nucleotide sequence of NCgl0313 gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 156: amino acid sequence of NCgl0313 protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 157: nucleotide sequence of NCgl2709 gene of Corynebacterium glutamicum 2256 (ATCC 13869) 158: Corynebacte amino acid sequence of NCgl2709 protein of rium glutamicum 2256 (ATCC 13869) SEQ ID NO: 159: nucleotide sequence of NCgl0219 gene of Corynebacterium glutamicum ATCC 13032 SEQ ID NO: 160: amino acid sequence of NCgl0219 protein of Corynebacterium glutamicum ATCC 13032 SEQ ID NO: 161: Corynebacterium glutamicum ATCC 13032 Nucleotide sequence of the NCgl2382 gene SEQ ID NO: 162: Corynebacterium glutamicum Amino acid sequence of the NCgl2382 protein of ATCC 13032 SEQ ID NO: 163: Nucleotide sequence of the aroE gene of Escherichia coli MG1655 SEQ ID NO: 164: Amino acid sequence of the AroE protein of Escherichia coli MG1655 SEQ ID NO: 165: Corynebacterium Nucleotide sequence of cysI gene of glutamicum 2256 (ATCC 13869) SEQ ID NO: 166: Amino acid sequence of CysI protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 167: Nucleotide sequence of cysX gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 168: Corynebacte amino acid sequence of CysX protein of rium glutamicum 2256 (ATCC 13869) SEQ ID NO: 169: nucleotide sequence of cysH gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 170: amino acid sequence of CysH protein of Corynebacterium glutamicum 2256 (ATCC 13869) : Nucleotide sequence number of cysD gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 172: Amino acid sequence number of CysD protein of Corynebacterium glutamicum 2256 (ATCC 13869) 173: Nucleotide sequence number of cysN gene of Corynebacterium glutamicum 2256 (ATCC 13869) 174: amino acid sequence of CysN protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 175: nucleotide sequence of cysY gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 176: amino acid sequence of CysY protein of Corynebacterium glutamicum 2256 (ATCC 13869) No. 177: cysZ gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 178: Amino acid sequence of CysZ protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 179: Nucleotide sequence of fpr2 gene of Corynebacterium glutamicum 2256 (ATCC 13869) 180: Fpr2 of Corynebacterium glutamicum 2256 (ATCC 13869) Amino acid sequence of protein SEQ ID NO: 181: Nucleotide sequence of cysK gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 182: Amino acid sequence of CysK protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 183: Corynebacterium glutamicum 2256 (ATCC 13869) Nucleotide sequence of cysR gene SEQ ID NO: 184: Amino acid sequence of CysR protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 185: Nucleotide sequence of ssuR gene of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 186: Corynebacterium glutamicum 2256 (ATCC 13869) Amino acid sequence of SsuR protein of SEQ ID NO: 187: Corynebact base sequence of NCgl2048 gene of erium glutamicum 2256 (ATCC 13869) SEQ ID NO: 188: amino acid sequence of NCgl2048 protein of Corynebacterium glutamicum 2256 (ATCC 13869) SEQ ID NO: 189: base sequence of sahH gene of Corynebacterium glutamicum 2256 (ATCC 13869) : Corynebacterium glutamicum 2256 (ATCC 13869) SahH protein amino acid sequence SEQ ID NO: 191: Corynebacterium glutamicum 2256 (ATCC 13869) rnc gene nucleotide sequence SEQ ID NO: 192: Corynebacterium glutamicum 2256 (ATCC 13869) Rnc protein amino acid sequence SEQ ID NO: 193 to 210: Primer SEQ ID NO: 211: Hv-iap base sequence SEQ ID NO: 212: Base sequence of DNA fragment containing F1 promoter SEQ ID NO: 213: F1 promoter base sequence SEQ ID NOs: 214 to 227: Primer SEQ ID NOs: 228 to 230: nucleotide sequence SEQ ID NOS: 231 to 246 of pfkA gene expression unit :Primer

Claims (27)

A method of maintaining a plasmid in a microorganism, comprising:
Culturing a microorganism having a plasmid in a medium containing a sugar,
The microorganism has been modified such that the assimilation ability of the sugar is reduced as compared to an unmodified strain,
The method, wherein the plasmid comprises a first nucleotide sequence that complements the reduced assimilation ability. A method for producing a target substance,
Culturing a microorganism having a target substance-producing ability and a plasmid in a medium containing a sugar to produce the target substance,
The microorganism has been modified such that the assimilation ability of the sugar is reduced as compared to an unmodified strain,
A method, wherein the plasmid comprises a first nucleotide sequence that complements the reduced assimilation ability and a second nucleotide sequence that is effective for producing the target substance.   The method according to claim 2, wherein the medium further contains a precursor of the target substance. A method for producing a target substance,
Culturing a microorganism having the plasmid with the target substance-producing ability in a medium containing the sugar to produce cells having the plasmid; and converting the precursor of the target substance into the target substance using the cells. Including the step of
The microorganism has been modified such that the assimilation ability of the sugar is reduced as compared to an unmodified strain,
A method, wherein the plasmid comprises a first nucleotide sequence that complements the reduced assimilation ability and a second nucleotide sequence that is effective for producing the target substance.   The method according to claim 2, further comprising recovering the target substance.   The method according to any one of claims 1 to 5, wherein the assimilation ability is reduced due to a decrease in the activity of protein P1 involved in the assimilation of the sugar.   The method according to claim 6, wherein the activity of the protein P1 is reduced by reducing the expression of a gene encoding the protein or by disrupting the gene.   The method according to claim 6 or 7, wherein the activity of the protein P1 is reduced by deletion of a part or all of the amino acid sequence of the protein.   The method according to any one of claims 1 to 8, wherein the first base sequence is a gene encoding a protein P2 involved in assimilation of the sugar.   The method according to claim 9, wherein the proteins P1 and P2 are proteins selected from the sugar uptake system and the sugar metabolizing enzyme, respectively.   The method according to claim 9 or 10, wherein the proteins P1 and P2 are proteins selected from glycolytic enzymes, respectively.   The method according to any one of claims 9 to 11, wherein the proteins P1 and P2 are each 6-phosphofructokinase. The method according to claim 12, wherein the 6-phosphofructokinase is a protein according to the following (a), (b), or (c), respectively:
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 38, 72, or 74;
(B) an amino acid sequence represented by SEQ ID NO: 38, 72 or 74, which comprises an amino acid sequence containing substitution, deletion, insertion and / or addition of 1 to 10 amino acid residues, and A protein having an actokinase activity;
(C) a protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 38, 72, or 74, and having 6-phosphofructokinase activity.   The method according to any one of claims 2 to 13, wherein the second base sequence is a gene selected from a gene encoding a protein capable of imparting or enhancing a target substance producing ability by increasing activity and a gene encoding the target substance. Item 2. The method according to item 1.   The method according to claim 14, wherein the protein capable of imparting or enhancing the target substance-producing ability by increasing the activity is a biosynthetic enzyme of the target substance.   The method according to any one of claims 2 to 15, wherein the second base sequence is a gene encoding an aromatic carboxylate reductase.   17. The target substance according to claim 2, wherein the target substance is selected from SAM-dependent metabolites, aldehydes, L-amino acids, nucleic acids, organic acids, γ-glutamyl peptides, sphingoids, proteins, and RNA. the method of.   The target substance is selected from vanillin, benzaldehyde, cinnamaldehyde, vanillic acid, melatonin, ergothioneine, mugineic acid, ferulic acid, polyamine, guaiacol, 4-vinylguaiacol, 4-ethylguaiacol, creatine, L-methionine. Item 18. The method according to any one of Items 2 to 17.   The precursor is selected from protocatechuic acid, protocatechualdehyde, vanillic acid, benzoic acid, cinnamic acid, L-tryptophan, L-histidine, L-phenylalanine, L-tyrosine, L-arginine, L-ornithine, glycine. The method according to any one of claims 3 to 18.   The method according to any one of claims 2 to 19, wherein the target substance is vanillic acid.   The method according to any one of claims 3 to 19, wherein the target substance is vanillin, and the precursor is vanillic acid.   The method according to any one of claims 1 to 21, wherein the sugar is glucose or sucrose.   The method according to any one of claims 1 to 22, wherein the microorganism is a bacterium or a yeast.   The method according to any one of claims 1 to 23, wherein the microorganism is a coryneform bacterium or a bacterium of the family Enterobacteriaceae.   The method according to any one of claims 1 to 24, wherein the microorganism 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 25, wherein the microorganism is Corynebacterium glutamicum or Escherichia coli.   27. The method of any one of claims 1-26, wherein the medium is substantially free of antibiotics.