JP5790022B2 - Xylanase and gene encoding the same - Google Patents

Description

  The present invention relates to a xylanase, a gene encoding the same, and use thereof, and more specifically, a protein having a xylanase activity derived from a microorganism group present in bagasse compost, which is a bagasse high-decomposition environment, and the protein Related genes and their use.

  Cellulose is a high-molecular polysaccharide in which glucose is connected by β-1,4-glycoside bonds. Therefore, if this bond is hydrolyzed, glucose can be obtained from cellulose, and cellulose can be effectively used as a glucose supply source. However, cellulose is a hardly degradable substance, and the utilization of cellulosic biomass has not yet been put to practical use. Cellulase controls the reaction of efficiently hydrolyzing cellulose. Therefore, improving the characteristics of cellulase and producing cellulase efficiently can be said to form the basis of technologies related to effective utilization of cellulose resources. Conventionally, cellulase genes derived from microorganisms have been isolated. It has been broken.

  For example, Patent Document 1 describes that a cellulase gene is isolated from a Humicola insolens strain, the cellulase gene is introduced into a host microorganism, and expressed to obtain a target cellulase. Patent Document 2 describes a heat-resistant alkaline cellulase gene derived from the genus Bacillus.

  Cellulosic biomass is composed of hemicellulose and lignin mainly composed of xylan, and the removal of these is important for increasing the saccharification efficiency of cellulose by cellulase. In conventional cellulosic biomass saccharification methods, attempts have been made to remove lignin and hemicellulose by pretreatment with a chemical substance such as dilute sulfuric acid. There are problems such as “prone to occur” and “cannot use saccharides constituting hemicellulose”. On the other hand, in the natural world, cellulosic biomass is efficiently decomposed by the action of cellulase with lignin-degrading enzymes such as manganese peroxidase, lignin peroxidase, and laccase produced by microorganisms, and hemicellulose-degrading enzymes such as xylanase, glucanase, and pectinase. Has been.

  As one of the effective utilization methods of biomass other than cellulose saccharification, composting is known in which compost is produced by mixing and fermenting with livestock feces. In compost, nitrogen sources derived from urea in the excreta, proliferated microbial cells and decomposed plant proteins are important, and carbon sources such as cellulose are used as energy sources for microbial growth. Therefore, in the composting of biomass, microbial groups that decompose the biomass with high efficiency are accumulated, and these microbial groups are expected to have a degrading enzyme suitable for decomposing the biomass.

  However, 99% of the microorganisms present on the earth are said to be difficult-to-cultivate microorganisms that are difficult to cultivate. In conventional screening methods that use ordinary agar media, they grow on the used media. They are only screening for the enzymes and genes of interest from the few possible microbial species. Therefore, in recent years, in order to find useful enzymes from difficult-to-cultivate microorganisms, a DNA library is created by extracting total DNA directly from a sample such as environmental soil and cloning it into a vector. Screening is performed by a metagenomic technique (metagenomics) for screening.

JP-A-8-56663 JP 2000-210081

  An object of the present invention is to provide a xylanase useful for saccharification of biomass resources such as sugarcane bagasse useful as cellulosic biomass, a gene thereof, and a method for using them.

  In order to solve the above-mentioned problems, the inventor mixed sugarcane bagasse and cow feces, extracted total DNA from the fermented bagasse compost that had been fermented for about half a year, created a library of the DNA, and obtained xylanase activity. Screening was conducted to obtain a recombinant having xylanase activity, and the xylanase gene was identified. Furthermore, a novel recombinant protein having xylanase activity is produced using a transformant transformed with a recombinant vector containing these xylanase genes, and the recombinant xylanase is added to a commercially available biomass saccharifying enzyme. The present inventors have found that the saccharification rate is improved as compared with the cellulose saccharification rate using only a commercially available enzyme, and the present invention has been completed.

That is, the present invention is as follows.
(1) DNA encoding the following protein (A) or (B):
(A) a protein having an amino acid sequence of SEQ ID NO: 20, 22, or 25 or an amino acid sequence consisting of at least positions 56 to 455 of SEQ ID NO: 24 and having xylanase activity;
(B) an amino acid sequence of SEQ ID NO: 20, 22, or 25, or an amino acid sequence consisting of at least positions 56 to 455 of SEQ ID NO: 24, wherein one or several amino acids are substituted, deleted, inserted or added A protein having a sequence and having xylanase activity.
(2) The DNA, wherein the amino acid sequence consisting of at least positions 56 to 455 of SEQ ID NO: 24 is the amino acid sequence of SEQ ID NO: 24.
(3)
The DNA which is the DNA of the following (a) or (b):
(A) a DNA having a base sequence of SEQ ID NO: 19 or 21, or a base sequence consisting of at least positions 166 to 1368 of SEQ ID NO: 23;
(B) a probe having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 19 or 21, or a nucleotide sequence consisting of at least positions 166 to 1368 of SEQ ID NO: 23, or a probe that can be prepared from the complementary nucleotide sequence; A DNA that hybridizes under stringent conditions and encodes a protein having xylanase activity.
(4) The DNA, wherein the base sequence consisting of at least positions 166 to 1368 of SEQ ID NO: 23 is the base sequence of SEQ ID NO: 23.
(5) The following protein (A) or (B):
(A) a protein having an amino acid sequence of SEQ ID NO: 20, 22, or 25, or an amino acid sequence consisting of at least positions 56 to 455 of SEQ ID NO: 24 and having xylanase activity;
(B) an amino acid sequence of SEQ ID NO: 20, 22, or 25, or an amino acid sequence consisting of at least positions 56 to 455 of SEQ ID NO: 24, wherein one or several amino acids are substituted, deleted, inserted or added A protein having a sequence and having xylanase activity.
(6) The protein, wherein the amino acid sequence consisting of at least positions 56 to 455 of SEQ ID NO: 24 is the amino acid sequence of SEQ ID NO: 24.
(7) A fusion protein in which the protein is bound to a peptide selected from a marker peptide, a peptide tag, a secretory signal peptide, and a part thereof.
(8) DNA encoding the protein.
(9) A recombinant vector containing the DNA.
(10) A transformed cell containing the recombinant vector.
(11) A method for producing xylanase, comprising culturing the transformed cell in a medium and collecting a protein having xylanase activity from the obtained culture.
(12) The recombinant vector can express the fusion protein containing a secretion signal, and the transformed cell has the ability to secrete the fusion protein and cultures a recombinant protein having xylanase activity. A method for producing the recombinant protein, wherein the recombinant protein is collected from the supernatant.
(13) A method for producing a sugar solution, wherein the protein or fusion protein and cellulase are reacted with a biomass resource containing xylan to produce sugar.
(14) The method for producing a sugar solution, wherein the reaction is performed at 50 to 75 ° C.
(15) A method for producing a target substance, comprising culturing a microorganism producing the target substance in a medium containing the sugar solution produced by the above method or a fraction thereof as a carbon source, and collecting the target substance from the culture.
(16) The method of the target substance, wherein the microorganism is a bacterium selected from microorganisms belonging to Corynebacterium glutamicum and Escherichia coli.
(17) The method of the target substance, wherein the target substance is an L-amino acid.
(18) A method for decomposing biomass resources, wherein the protein or fusion protein is reacted with biomass resources containing xylan.
(19) The method for decomposing biomass resources, wherein the reaction is performed at 50 to 75 ° C.

Since the novel xylanase derived from bagasse compost provided by the present invention can improve the decomposition efficiency of biomass resources such as bagasse in combination with cellulase, it is useful for sugar production from biomass resources.
In particular, the preferred form has a high optimum reaction temperature and temperature stability, and is therefore useful for saccharification of bagasse requiring a long-time treatment at a high temperature.
The DNA of the present invention is useful for producing the xylanase.

The figure which shows the bagasse saccharification rate by xylanase in pH5. The vertical axis represents the relative saccharification rate of each xylanase-added reaction group relative to the xylanase-free group (NC). `` NC '' is 50 mM sodium acetate buffer (pH 5.0), `` P '' is YDK010 introduced with pPK4, `` X11-1 '' is an Xyn11-1 expression strain, `` X11-2 '' is an Xyn11-2 expression strain, `` X11 "-5" indicates that each culture supernatant of the Xyn11-5 expression strain was added to the reaction system. “X14” represents NS50014 (Xyn14), and “X30” represents NS50030 (Xyn30) (same for FIG. 2). The figure which shows the bagasse saccharification rate by xylanase in pH7. The figure which shows the optimal reaction pH of a novel xylanase and a commercially available xylanase. The vertical axis shows the specific activity at each pH when the sample showing the maximum amount of produced sugar is taken as 1. The figure which shows the optimal reaction temperature of a novel xylanase and a commercially available xylanase. The vertical axis shows the specific activity at each reaction temperature when the sample showing the maximum amount of produced sugar is taken as 1. The figure which shows the temperature stability in the short time (5 minutes) of a novel xylanase and a commercial xylanase. The vertical axis shows the specific activity at each heat treatment temperature when the average value of the sample to which the non-heat treated enzyme solution is added is 1. The commercial enzyme Xyn14 was examined at 35, 50, 70, and 80 ° C. The figure which shows the temperature stability in the long time (16 hours) of a novel xylanase and a commercially available xylanase. The vertical axis shows the specific activity at each heat treatment temperature when the average value of the sample to which the non-heat treated enzyme solution is added is 1.

  The protein of the present invention is a protein having xylanase activity (hereinafter sometimes referred to as “xylanase”), and the DNA of the present invention is a DNA encoding the protein (hereinafter also referred to as “xylanase gene”). . The xylanase may be xylanase itself or a fusion protein in which another peptide is bound to xylanase. The xylanase gene may be a structural gene encoding xylanase or a fusion protein of xylanase and another peptide, and may be one in which an expression regulatory sequence such as a promoter or a terminator is bound to the structural gene.

Xylanase activity means the activity of decomposing hemicellulose by cleaving the β-1,4-glycosidic bond between xylose sugars that form the skeleton of the hemicellulose molecule. When xylanase is allowed to act on hemicellulose, xylan, which is the main component of the hemicellulose, is decomposed to produce xylooligosaccharides and xylose. In addition, when hemicellulose surrounding the cellulose fiber in the cellulosic biomass is decomposed by xylanase, the cellulose is easily decomposed by cellulase.
Hereinafter, the present invention will be described in detail.

<1> Xylanase and DNA encoding the same
The xylanase gene of the present invention has been identified as DNA encoding a protein having xylanase activity from bagasse compost by metagenomic cloning. The base sequences of these xylanase genes are shown in SEQ ID NOs: 19, 21, and 23. The amino acid sequences encoded by these xylanase genes are shown in SEQ ID NOs: 20, 22, and 24.

  The xylanase of the present invention includes a protein having the amino acid sequence of SEQ ID NO: 20, 22, or 24. These amino acid sequences were input to SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/), and signal peptide prediction was performed. As a result, in the amino acid sequence of SEQ ID NO: 24, the sequence at the N-terminal side relative to the 55th amino acid residue was predicted to be a signal peptide. Then, as shown in the Examples, a fusion protein in which a peptide consisting of positions 56 to 455 of SEQ ID NO: 24 and a Sec or Tat signal peptide are linked is expressed in Corynebacterium glutamicum. As a result, it showed xylanase activity. Therefore, the xylanase of the present invention includes a protein having an amino acid sequence consisting of at least positions 56 to 455 of SEQ ID NO: 24. When a fusion protein in which a peptide having the amino acid sequence of SEQ ID NO: 20 or 22 and a Sec or Tat signal peptide were linked was expressed in the same manner as described above, all showed xylanase activity.

  The amino acid sequences of SEQ ID NOs: 22 and 24 are highly homologous, and their common sequence is shown in SEQ ID NO: 25. Of the 410 amino acid residues of SEQ ID NO: 25, 336 are common amino acid residues. In addition, at positions 8 to 188 in SEQ ID NO: 25, 173 amino acids out of 181 amino acids (95.6%) are the same amino acids. A protein having the amino acid sequence of SEQ ID NO: 25 is also included in the xylanase of the present invention.

  The xylanase of the present invention is a conservative variant of a protein having an amino acid sequence of SEQ ID NO: 20, 22, or 25, or an amino acid sequence consisting of at least positions 56 to 455 of SEQ ID NO: 24, that is, a protein having the amino acid sequence described above. It may be a homolog or an artificially modified product. That is, it may be a protein having a sequence including substitution, deletion, insertion or addition of one or several amino acids at one or several positions in the above amino acid sequence. The first methionine (Met) contained in the amino acid sequence may be deleted.

  “One or several” amino acid residues vary depending on the position of the protein in the three-dimensional structure of the protein and the type of amino acid residue, but specifically, preferably 1-20, more preferably 1-10, Preferably it means 1-5. A typical conservative mutation is a conservative substitution. 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 this case, between Gln and Asn, when it is a basic amino acid, between Lys, Arg, and His, when it is an acidic amino acid, between Asp and Glu, when it is an amino acid having a hydroxyl group Is a mutation that substitutes between Ser and Thr. Specifically, substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp 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, Substitution from Lys to Asn, Glu, Gln, His or Arg, substitution from Met to Ile, Leu, Val or Phe, substitution from Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala Substitution, substitution from Thr to Ser or Ala, substitution from Trp to Phe or Tyr, substitution from Tyr to His, Phe or Trp, and substitution from Val to Met, Ile or Leu.

Further, the xylanase having a conservative mutation as described above is preferably 80% or more, preferably 80% or more of the amino acid sequence of SEQ ID NO: 20, 22, or 24 or the amino acid sequence consisting of at least positions 56 to 455 of SEQ ID NO: 24. May be a protein having homology of 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more, and having xylanase activity.
In the present specification, “homology” may refer to “identity”.

In a preferred form, the xylanase of the present invention has the following properties.
(1) Optimum reaction pH: 5.5 to 7.5
The optimum reaction pH of Xyn11-1, Xyn11-2, and Xyn11-5 described below is around 7, and around 7.0-7.5, respectively. It is around 5.5 to 7.5.
(2) Optimal reaction temperature: 65 ° C to 80 ° C
The optimum reaction temperatures of Xyn11-1, Xyn11-2, and Xyn11-5 are around 65 ° C, 70 ° C, and 75-80 ° C, respectively.
(3) Temperature stability: stable for about 16 hours at 50 ° C
Xyn11-1, Xyn11-2, and Xyn11-5 maintain an activity of 80% or more after holding at 50 ° C. for 16 hours. In particular, Xyn11-2 and Xyn11-5 maintain about 60% activity after 16 hours of holding at 65 ° C and 70 ° C, respectively.

  The xylanase of the present invention may be a fusion protein with another peptide. Examples of other peptides include marker peptides (marker proteins), peptide tags, and secretory signal peptides. There may be only one peptide linked to xylanase, or two or more peptides. In the case where the xylanase is a fusion protein with a signal peptide, after expression in a secretable host cell, the signal peptide can be cleaved and only the xylanase moiety can be secreted outside the cell.

  The marker peptide is not particularly limited as long as it is a peptide that can function as a marker, and specific examples thereof include alkaline phosphatase, antibody Fc region, HRP, and GFP. In addition, the peptide tag is not particularly limited, and conventionally known peptide tags such as Myc tag, His tag, FLAG tag, and GST tag can be specifically exemplified. In addition, the secretion signal peptide is not particularly limited as long as it can function in a host that expresses the xylanase gene. For example, when a coryneform bacterium is used as a host, Sec and Tat secretion from coryneform bacterium is used. The secretory signal peptide of the pathway (see WO01 / 23591, WO2005 / 103278). The fusion protein can be prepared by a conventional method.

  For example, the His tag is useful for purification of a protein having xylanase activity utilizing the affinity between Ni-NTA and His tag, and for producing a culture supernatant containing a protein having a high concentration of xylanase activity.

The DNA of the present invention encodes the above xylanase. Specifically, DNA encoding the amino acid sequence of SEQ ID NO: 20, 22, or 25, or an amino acid sequence consisting of at least positions 56 to 455 of SEQ ID NO: 24 is included. It may also be a DNA encoding a conservative variant of a protein having these amino acid sequences.
More specifically, DNA having the base sequence of SEQ ID NO: 19 or 21 or the base sequence consisting of at least positions 166 to 1368 of SEQ ID NO: 23 can be mentioned. Examples of the base sequence consisting of at least positions 166 to 1368 of SEQ ID NO: 23 include the base sequence of SEQ ID NO: 23. The DNA of the present invention is not limited to these, and may be one in which a codon encoding each amino acid in the coding region is replaced with another equivalent codon encoding the same amino acid.

  The DNA of the present invention includes a probe having a base sequence complementary to the base sequence of SEQ ID NO: 19 or 21, or a base sequence consisting of at least positions 166 to 1368 of SEQ ID NO: 23, or the complementary base Also included is a DNA that hybridizes with a probe that can be prepared from the sequence under stringent conditions and encodes a protein having xylanase activity. Stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, highly homologous DNAs, for example, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97%, particularly preferably 99% or more DNAs having homology. Conditions that hybridize and DNAs with lower homology do not hybridize, such as hybridization at 42 ° C and washing treatment at 42 ° C with a buffer containing 1x SSC, 0.1% SDS More preferred examples include hybridization at 65 ° C. and washing treatment at 65 ° C. with a buffer containing 0.1 × SSC and 0.1% SDS. In addition, there are various factors other than the above temperature conditions as factors affecting the stringency of hybridization, and those skilled in the art will be able to combine various components as appropriate to achieve the same stringency of hybridization as exemplified above. Stringency can be realized.

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

  The DNA of the present invention is based on the DNA sequence information disclosed in the present specification, for example, bagasse compost produced by mixing sugarcane bagasse with various animal feces, or cultivated land or bagasse compost fertilized with bagasse compost. It can be prepared from the total DNA of soil such as a storage place by a known method such as PCR or hybridization. In addition, it can also be prepared by chemical synthesis based on the DNA information.

  In addition, the xylanase gene may include naturally occurring mutations (mutant or variant) such as cases based on individual differences of microorganisms or differences in species. A xylanase gene having such a mutation is also included in the DNA of the present invention. include. In addition, the gene encoding a conservative variant of the above xylanase is, for example, xylanase so that the amino acid residue at a specific site of the encoded protein includes substitution, deletion, insertion or addition by site-directed mutagenesis. It can be obtained by modifying the base sequence of the gene. Such a gene can also be obtained by using a known mutation means such as a commercially available mutagenesis kit.

  A DNA encoding a fusion protein of xylanase and another peptide can be constructed by ligating DNA encoding xylanase and DNA encoding another peptide. By expressing the obtained DNA, a fusion protein can be produced.

<2> Recombinant vector The recombinant vector of the present invention is a recombinant vector containing a xylanase gene. The xylanase gene may be DNA encoding only xylanase, or may be DNA encoding the fusion protein. The recombinant vector is not particularly limited as long as it contains the xylanase gene, and can be constructed by inserting the xylanase gene into any vector.

The vector can be appropriately selected depending on the host to be used. Examples of the host include Escherichia bacteria such as Escherichia coli, and coryneform bacteria. Examples of coryneform bacterium vectors include plasmids capable of autonomous replication in coryneform bacteria, such as plasmid pCRY30 described in JP-A-3-210184; JP-A-2-72876 and US Pat. No. 5,185,262. Plasmids pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX; plasmids pCRY2 and pCRY3 described in JP-A-1-91686; pAM330 described in JP-A-58-67679; JP-A-58-77895 PHM1519 described in; pAJ655, pAJ611 and pAJ1844 described in JP-A-58-192900; pCG1 described in JP-A-57-134500; pCG2 described in JP-A-58-35197; Examples include pCG4 and pCG11 described in JP-A-57-183799.
Examples of vectors that can be expressed in Escherichia coli include pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, and pMW218.

  Although it does not restrict | limit especially as a vector, What contains the expression system which can express a xylanase in a host cell is preferable. Examples of such expression systems include, for example, expression systems derived from chromosomes, episomes and viruses, and more specifically derived from bacterial plasmids, yeast plasmids, bacteriophages, transposons and combinations thereof. Vectors such as those derived from plasmids such as cosmids and phagemids and bacteriophage genetic elements can be mentioned. Expression systems may contain control sequences that not only cause expression but also regulate expression.

  The expression system may also be a secretory expression system that includes a secretion signal. The secretory expression system of coryneform bacteria includes the general secretion pathway (Sec system) in which proteins are folded extracellularly (see WO01 / 23591) and the twin-arginine translocation pathway (in which proteins are secreted by folding in cells) Tat type) (see WO2005 / 103278).

<3> Transformed Cell The transformed cell of the present invention contains the DNA or recombinant vector of the present invention. A transformed cell means a cell obtained by introducing the DNA or recombinant vector of the present invention into a host cell, and the method of introduction is not limited.

  Host cells include Escherichia coli bacteria such as Escherichia coli, coryneform bacteria such as Corynebacterium glutamicum, actinomycetes such as Streptomyces bacteria, Bacillus bacteria such as Bacillus subtilis, Streptococcus bacteria, Examples include prokaryotic cells such as phylococcus bacteria, fungal cells such as yeast and Aspergillus, insect cells such as Drosophila S2, Spodoptera Sf9, and silkworm cultured cells, and plant cells.

  Host cells include various microbial cells, particularly including Escherichia coli, coryneform bacteria, and actinomycetes. Escherichia coli includes strains commonly used for cloning and expression of heterologous proteins, such as HB101, MC1061, JM109, CJ236, MV1184, and derivatives thereof. Coryneform bacteria are aerobic Gram-positive rods and include bacteria that were previously classified as genus Brevibacterium but are now integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1981)), and also includes Brevibacterium, which is very closely related to Corynebacterium. The advantage of using coryneform bacteria is that there are very few proteins that are inherently secreted outside the fungus compared to fungi, yeast and Bacillus bacteria that have been considered suitable for protein secretion. When protein is secreted, its purification process can be simplified and omitted, and it grows easily on a simple medium containing sugar, ammonia, inorganic salts, etc. It includes being excellent.

Examples of such coryneform bacteria include the following.
Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium carnae Corynebacterium glutamicum Corynebacterium lylium Corynebacterium merasecola Corynebacterium thermoaminogenes Coryne Bacterium Herculis Brevibacterium divaricatam Brevibacterium flavum Brevibacterium immariophyllum Brevibacterium lactofermentum Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalis Corynebacterium・ Ammonia Genes Brevibacterium album Brevibacterium cerinum Microbacteria Ammonia film

Specifically, the following strains can be exemplified.
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium alkanolyticum ATCC21511
Corynebacterium carnae ATCC15991
Corynebacterium glutamicum ATCC13020, ATCC13032, ATCC13060, ATCC13869, FERM BP-734
Corynebacterium lilium ATCC15990
Corynebacterium melasecola ATCC17965
Corynebacterium efficiens AJ12340 (FERM BP-1539)
Corynebacterium herculis ATCC13868
Brevibacterium divaricatam ATCC14020
Brevibacterium flavum ATCC13826, ATCC14067, AJ12418 (FERM BP-2205)
Brevibacterium immariophilum ATCC14068
Brevibacterium lactofermentum ATCC13869
Brevibacterium rose ATCC13825
Brevibacterium saccharolyticum ATCC14066
Brevibacterium thiogenitalis ATCC19240
Corynebacterium ammoniagenes ATCC6871ATCC6872
Brevibacterium album ATCC15111
Brevibacterium cerinum ATCC15112
Microbacterium ammonia philus ATCC15354

  In particular, Corynebacterium glutamicum AJ12036 (see WO 02/081694) isolated as a streptomycin (Sm) -resistant mutant strain from wild-type Corynebacterium glutamicum ATCC 13869 has a protein that is higher than that of its parent strain (wild-type strain). It is predicted that there will be a mutation in the functional gene involved in secretion of the protein, and the protein secretion production ability is extremely high, about 2 to 3 times as the accumulated amount under the optimum culture conditions, and therefore it is suitable as a host fungus.

  Furthermore, if a strain modified so as not to produce cell surface proteins from such a strain is used as a host, purification of the target protein secreted in the medium is facilitated, which is particularly preferable. Such modification can be performed by introducing a mutation into a cell surface protein on the chromosome or its expression regulatory region by a mutation or gene recombination method. Examples of coryneform bacteria modified so as not to produce cell surface protein include Corynebacterium glutamicum YDK010, which is a cell surface protein (PS2) disruption strain of AJ12036 (see WO 01/23491).

  Further, xylanase may be produced in the medium by utilizing the protein secretory ability of Bacillus subtilis, yeast, koji mold, actinomycetes and the like.

  The recombinant expression vector can be introduced into the host cell by a conventional and conventionally used method. Examples include a competent cell method, a protoplast method, an electroporation method, a microinjection method, a liposome fusion method, and the like. Specific examples of the method for introduction into coryneform bacteria include, for example, protoplast method (Gene, 39, 281-286 (1985)), electroporation method (Bio / Technology, 7, 1067-1070) (1989)). Etc. can be used, but is not limited thereto.

<3> Method for Producing Xylanase Xylanase can be produced by culturing such transformed cells in a medium and collecting a protein having xylanase activity from the resulting culture.

  Medium for cultivating transformants is known. For example, E. coli culture is supplemented with nutrient medium such as LB medium or minimal medium such as M9 medium with carbon source, nitrogen source, vitamin source, etc. A culture medium can be used. Depending on the host, the transformant is usually cultured at 16 to 42 ° C., preferably 25 to 37 ° C. for 5 to 168 hours, preferably 8 to 72 hours. Depending on the host, either shaking culture or stationary culture is possible, but stirring may be performed as necessary, and aeration may be performed. When a coryneform bacterium is selected as an expression host, for example, a CM2G medium can be used as the medium. In addition, culture conditions for L-amino acid production of coryneform bacteria and other conditions described in the protein production method using Sec and Tat signal peptides can be used (see WO01 / 23591 and WO2005 / 103278). . When an inducible promoter is used for xylanase expression, a promoter inducer can be added to the medium for cultivation.

  The method for collecting xylanase from the culture is not particularly limited, and a known method can be used. When xylanase is secreted and produced, the culture supernatant of the culture can be used as it is.

  To purify or isolate xylanase from the culture supernatant, ammonium sulfate precipitation, solvent precipitation such as ethanol, dialysis, ultrafiltration, acid extraction, and various chromatography such as gel filtration chromatography, anion or cation exchange chromatography, Examples include known methods including phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography, preferably high performance liquid chromatography, or a combination thereof. it can. Examples of the carrier used for the affinity chromatography include a carrier in which an antibody against xylanase is bound, and a carrier in which a peptide tag is attached to xylanase, and a substance having affinity for the peptide tag. . In addition, SDS polyacrylamide electrophoresis, isoelectric focusing, and the like can be used. The purification method of xylanase can also be applied during peptide synthesis.

  When xylanase is accumulated in cells, transformed cells can be disrupted, and xylanase can be purified or isolated from the centrifuged supernatant of the disrupted product in the same manner as described above. For example, after culturing the transformant, the cells collected by centrifugation are suspended in a cell disruption buffer (20-100 mM Tris-HCl (pH 8.0), 5 mM EDTA), and ultrasonic disruption is performed in Branson MODEL. -Using the Sonifier 250 microchip, the cells can be disrupted by performing output control 7, duty cycle 50% for about 10 minutes. This crushing solution can be centrifuged at 12000 rpm for 10 minutes to obtain a supernatant. The precipitate after centrifugation can be further purified after solubilization with guanidinium hydrochloride or urea, if necessary.

  The enzyme activity of the obtained xylanase can be confirmed by a saccharification test using biomass containing xylan, for example, bagasse. The activity of the purified enzyme can be measured by the method described in JP-A 2007-54050, Japanese Patent No. 2759209, Bailey et al. J. Biotech. 23 (1992) 257-270.

<4> Method for Producing Sugar Liquid Biomass resources can be decomposed by reacting xylanase with biomass resources containing xylan. A sugar solution can be obtained by reacting xylanase and cellulase with a biomass resource containing xylan to produce sugar. The xylanase of the present invention is a kind of hemicellulase, and in addition to the xylanase of the present invention, another xylanase or hemicellulase may be used.

  Cellulase is an enzyme involved in the hydrolysis of (1 → 4) -β-glycosidic bonds. It is an endoglucanase (EC3.2.1.4) that cleaves amorphous cellulose from the inside of the molecule. There is an exoglucanase that releases cellobiose (cellobiohydrolase) (EC 3.2.1.91) and β-glucosidase (EC 3.2.1.21) that produces glucose from the ends of cellobiose and cellooligosaccharide. Commercially available cellulases are sold as a mixture of cellulases having the aforementioned activity, and enzymes derived from Aspergillus niger and Trichoderma reesei are often used. Novozymes Cellic Ctec, Celluclast (Novozymes A / S), Novozyme 188 (Novozymes A / S), and Genencor Accellerase are representative commercial saccharifying enzymes with excellent cost performance. As other commercially available enzymes, there may be mentioned Meisserase from Meiji Seika Co., Ltd. Cellulase activity can be measured with reference to Irwin et al. Biotechnol Bioeng. 42 (1993) 1002-1013.

  Hemicellulase is a general term for enzymes involved in hydrolysis of glycosidic bonds contained in hemicellulose. Hemicellulose is a polysaccharide other than cellulose and pectin among the polysaccharides constituting the cell wall of land plant cells. The hemicellulose component contains a large amount of xylan, and the main component of hemicellulase is endo-1,4. -β-xylanase (EC 3.2.1.8), β-1,4-xylosidase (EC 3.2.1.37), etc., but other glucoside bond hydrolases are also included. Commercially available hemicellulases include Cellic Htec from Novozymes. They can also be obtained from Genencor and Amano Enzyme.

  Biomass resources include cellulosic and / or lignocellulosic biomass containing hemicellulose components that are produced by plants and algae. Examples of such biomass include bagasse, wood, bran, wheat straw, rice straw, and rice straw. These include potato, soybean meal, soybean okara, coffee cake, rice cake and the like. In the present invention, it is desirable to use bagasse. Bagasse is obtained as a residue after squeezing sugarcane.

As a method for decomposing or saccharifying biomass resources, a known method can be used. For example, the biomass resource to be used may be a dried product or a wet product, but is preferably pulverized to a size of 100 to 1000 μm in advance in order to increase the processing efficiency. This pulverization can be performed using a machine such as a ball mill, a vibration mill, a cutter mill, or a hammer mill. The pulverized biomass resource can be suspended in an aqueous medium, the xylanase and cellulase of the present invention can be added, and heated with stirring to decompose or saccharify the biomass resource. The xylanase and the cellulase may be added to the biomass resource simultaneously, or the xylanase may be added to the bioresource and reacted, and then the cellulase may be added and reacted.
In the above method, the pH and temperature of the reaction solution may be in a range where xylanase and cellulase are not inactivated. However, as described above, the xylanase of the present invention has a high optimum reaction temperature and excellent temperature stability. Therefore, the reaction is preferably performed at the optimum reaction temperature or a temperature in the vicinity thereof. For example, as long as the reaction is generally carried out at normal pressure, the temperature is 5-95 ° C, preferably 25-80 ° C, more preferably 50-75 ° C, 50-70 ° C, 50-65 ° C, or 50-55 ° C. The pH may range from 1-11, preferably 4-9. For example, when Cellic Ctec of Novozymes is used as the cellulase, the conditions are 35 to 55 ° C. and pH 4.5 to 6.0. Examples of the enzyme amount include 0.1 to 0.5% (w / w TS, total solid). The reaction time can be appropriately set depending on the amount of enzyme and the like.
The enzyme amount of the xylanase of the present invention is not particularly limited, and may be appropriately set by conducting a preliminary experiment.

  Xylanase and cellulase may act on biomass resources simultaneously, or cellulase may act after xylanase acts. In addition to the xylanase of the present invention, another xylanase or hemicellulase may be used. Any one or more of xylanase, cellulase and hemicellulase of the present invention may be added during the reaction.

When biomass resources are decomposed as described above, glucose is mainly produced, but sugars such as xylose, mannose, and arabinose are also produced. The obtained sugar solution may be further isomerized or decomposed by a chemical reaction or an enzymatic reaction depending on the application.
The obtained sugar solution can be used as it is, or can be used as a dried product after removing moisture. Moreover, you may fractionate and use the component in a sugar liquid suitably. The fraction includes a crude product and a purified product. An example of the purified product is glucose.

<5> Method for producing target substance The sugar liquid obtained by the above method or a fraction thereof can be used as a carbon source in the production of the target substance by, for example, fermentation. Examples of the target substance include alcohols such as methanol, ethanol, propanol, isopropanol, butanol, and butanediol. Other examples include acetic acid, lactic acid, propionic acid, 3-hydroxypropionic acid, succinic acid, citric acid, amino acid, and nucleic acid.

  In particular, L-lysine, L-ornithine, L-arginine, L-histidine, L-citrulline, L-isoleucine, L-alanine, L-valine, L-leucine, L-glycine, L-threonine L-serine, L-proline, L-phenylalanine, L-tyrosine, L-tryptophan, L-cysteine, L-cystine, L-methionine, L-glutamic acid, L-aspartic acid, L-glutamine and L-asparagine. Can be mentioned.

  The L-amino acid of the present invention also includes L-amino acid derivatives obtained using the above amino acids as starting materials, and examples include GABA, p-hydroxy-D-phenylglycine, DOPA, succinic acid, malic acid, and pyruvic acid. .

Examples of nucleic acids include purine nucleosides and purine nucleotides. Purine nucleosides include inosine, xanthosine, guanosine, adenosine, and the like, and purine nucleotides include 5'-phosphate esters of purine nucleosides, such as inosinic acid (inosine-5'-phosphate, hereinafter also referred to as "IMP"). Xanthylic acid (xanthosine-5′-phosphoric acid; hereinafter also referred to as “XMP”), guanylic acid (guanosine-5′-monophosphoric acid; hereinafter also referred to as “GMP”), adenylic acid (adenosine-5′-monophosphoric acid). (Hereinafter also referred to as “AMP”). The nucleic acid of the present invention also includes nucleic acid derivatives obtained using the above nucleic acid as a starting material, and examples include Ara-U (uracil arabinoside) and ZVA (Z-valacyclovir).
The target substance produced according to the present invention may be one type, or two or more types.

<5-1> Microorganisms that can be used for the production of the target substance Microorganisms used for the production of the target substance can assimilate the sugar obtained by the above method, for example, glucose, have the ability to produce the target substance, and are liquid The microorganism is not particularly limited as long as it is a microorganism capable of accumulating the target substance in the medium or microbial cells when cultured in the medium. The amount of the target substance to be accumulated may be any as long as it can be accumulated to the extent that it can be recovered from the medium.

  As the microorganism used in the present invention or a parent strain for breeding it, microorganisms belonging to the family Enterobacteriaceae such as Escherichia bacteria and Pantoea bacteria, coryneform bacteria, and the like can be used. Other microorganisms belonging to the family Enterobacteriaceae include the genus Enterobacter, Klebsiella, Serratia, Erwinia, Salmonella, Morganella, etc. Examples include enterobacteria belonging to γ-proteobacteria, and examples of other microorganisms include bacteria belonging to the genus Alicyclobacillus, bacteria belonging to the genus Bacillus, yeasts belonging to the genus Saccharomyces and Candida. .

  Examples of Escherichia bacteria include those listed in the book by Neidhardt et al. (Neidhardt, FCet.al., Escherichia coli and Salmonella Typhimurium, American Society for Microbiology, Washington DC, 1208, table 1), for example, Escherichia coli it can. Examples of wild strains of Escherichia coli include the K12 strain or derivatives thereof, Escherichia coli MG1655 strain (ATCC No. 47076), and W3110 strain (ATCC No. 27325). To obtain these, for example, they can be sold from the American Type Culture Collection (ATCC) (address ATCC, Address: P.O. Box 1549, Manassas, VA 20108, 1, United States of America). That is, a corresponding registration number is assigned to each strain, and distribution can be received using this registration number. The registration number corresponding to each strain is described in the catalog of American Type Culture Collection (see http://www.atcc.org/).

  Examples of Enterobacter bacteria include Enterobacter agglomerans, Enterobacter aerogenes, and the like, and Pantoea ananatis is an example of Pantoea bacteria. In recent years, Enterobacter agglomerans has been reclassified as Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii, etc. by 16S rRNA nucleotide sequence analysis. There is something. In the present invention, any substance belonging to the genus Enterobacter or Pantoea may be used as long as it is classified into the family Enterobacteriaceae. Pantoea ananatis AJ13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ13601 strain (FERM BP-7207) and their derivatives when breeding Pantoea ananatis using genetic engineering techniques Can be used. These strains were identified as Enterobacter agglomerans when they were isolated, and deposited as Enterobacter agglomerans, but as described above, they have been reclassified as Pantoea Ananatis by 16S rRNA sequence analysis, etc. .

  Coryneform bacteria are a group of microorganisms defined in the Bergey's Manual of Determinative Bacteriology, 8th edition, page 599 (1974), aerobic, Gram-positive, Non-acidic, microorganisms classified as gonococci having no sporulation ability can be used. Coryneform bacteria were previously classified as genus Brevibacterium but are now integrated as Corynebacterium (Int.J. Syst. Bacteriol., 41, 255 (1991)), and coryneform bacteria. Includes Brevibacterium and Microbatterium bacteria that are very closely related to the genus Bacteria.

Examples of such coryneform bacteria include the following.
Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium carnae Corynebacterium glutamicum Corynebacterium lylium Corynebacterium merasecola Corynebacterium thermoaminogenes ( Corynebacterium efficiens
Corynebacterium herculis Brevibacterium divaricatam Brevibacterium flavum Brevibacterium inmariophilum Brevibacterium lactofermentum Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalis Corynebacterium Umm ammoniagenes Brevibacterium album Brevibacterium cerinum Microbacterium ammonia film

Specifically, the following strains can be exemplified.
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium alkanolyticum ATCC21511
Corynebacterium carnae ATCC15991
Corynebacterium glutamicum ATCC13020, ATCC13032, ATCC13060
Corynebacterium lilium ATCC15990
Corynebacterium melasecola ATCC17965
Corynebacterium efficiens AJ12340 (FERM BP-1539)
Corynebacterium herculis ATCC13868
Brevibacterium divaricatam ATCC14020
Brevibacterium flavum ATCC13826, ATCC14067, AJ12418 (FERM BP-2205)
Brevibacterium immariophilum ATCC14068
Brevibacterium lactofermentum ATCC13869 (Corynebacterium glutamicum ATCC13869)
Brevibacterium rose ATCC13825
Brevibacterium saccharolyticum ATCC14066
Brevibacterium thiogenitalis ATCC19240
Corynebacterium ammoniagenes ATCC6871, ATCC6872
Brevibacterium album ATCC15111
Brevibacterium cerinum ATCC15112
Microbacterium ammonia film ATCC15354

To get these, for example, American Type Culture Collection (address
PO Box 1549, Manassas, VA 2010812301 Parklawn Drive, Rockville, Maryland 20852, United States of America). In addition, AJ12340 shares were issued on October 27, 1987 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center) (Ibaraki, 305-8566, Japan) It is deposited under the Budapest Treaty under the accession number of FERM BP-1539 in 1st, 1st East, 1st Street, Tsukuba City. In addition, AJ12418 stock was deposited on January 5, 1989 at the Institute of Biotechnology, Ministry of International Trade and Industry under the contract number FERM BP-2205 under the Budapest Treaty.

When Bacillus bacteria are used, examples of Bacillus bacteria include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus and the like.
Examples of Bacillus subtilis include Bacillus subtilis 168 Marburg (ATCC6051) and Bacillus subtilis PY79 (Plasmid, 1984, 12, 1-9), and examples of Bacillus amyloliquefaciens include Bacillus amyloliquefaciens T ( ATCC 23842), and Bacillus amyloliquefaciens N (ATCC 23845). Examples of Bacillus pumilus include Bacillus pumilus Gottheil No. 3218 (ATCC No. 21005) (US Pat. No. 3,616,206).

Hereinafter, a method for imparting L-amino acid or nucleic acid-producing ability to the parent strain as described above will be described.
In order to confer L-amino acid-producing ability, an auxotrophic mutant, an L-amino acid analog-resistant strain or a metabolically controlled mutant, and a recombinant strain with enhanced expression of an L-amino acid biosynthetic enzyme Can be applied to the breeding of amino acid-producing bacteria such as coryneform bacteria or Escherichia bacteria (amino acid fermentation, Academic Publishing Center, Inc., May 30, 1986, first edition) Issue, see pages 77-100). Here, in the breeding of L-amino acid-producing bacteria, properties such as auxotrophy, analog resistance, and metabolic control mutation may be used singly or in combination of two or more. In addition, L-amino acid biosynthesis enzymes whose expression is enhanced may be used alone or in combination of two or more. Furthermore, imparting properties such as auxotrophy, analog resistance, and metabolic regulation mutation may be combined with enhancement of biosynthetic enzymes.

  In order to obtain an auxotrophic mutant, an analog-resistant mutant, or a metabolically controlled mutant having L-amino acid-producing ability, the parent strain or wild strain is subjected to normal mutation treatment, that is, irradiation with X-rays or ultraviolet rays, or N-methyl. -N'-nitro-N-nitrosoguanidine and other mutants treated, etc. Among the obtained mutant strains, it shows auxotrophy, analog resistance, or metabolic control mutation, and has the ability to produce L-amino acid It can be obtained by selecting what it has.

  The L-amino acid-producing ability can also be imparted or enhanced by enhancing enzyme activity by gene recombination. Examples of the enhancement of enzyme activity include a method of modifying a bacterium so that expression of a gene encoding an enzyme involved in L-amino acid biosynthesis is enhanced. As a method for enhancing the expression of a gene, introducing an amplified plasmid in which a DNA fragment containing the gene is introduced into an appropriate plasmid, for example, a plasmid vector containing at least a gene responsible for the replication replication function of the plasmid in a microorganism, Alternatively, these genes can be achieved by making multiple copies on the chromosome by joining, transferring, etc., or by introducing mutations into the promoter regions of these genes (see International Publication No. 95/34672). .

  When the target genes are introduced onto the amplification plasmid or chromosome, the promoter for expressing these genes may be any promoter that functions in coryneform bacteria, and the promoter of the gene itself used. Or may be modified. The expression level of the gene can also be controlled by appropriately selecting a promoter that functions strongly in coryneform bacteria, or by bringing the -35 and -10 regions of the promoter closer to the consensus sequence. The method for enhancing the expression of the enzyme gene as described above is described in International Publication No. 00/18935, European Patent Application Publication No. 1010755, and the like.

  Hereinafter, a method for imparting L-amino acid-producing ability to bacteria and a bacterium imparted with L-amino acid-producing ability will be exemplified.

L-Threonine-Producing Bacteria Preferred microorganisms having L-threonine-producing ability include bacteria having enhanced activity of one or more of L-threonine biosynthetic enzymes. Examples of the L-threonine biosynthesis enzyme include aspartokinase III (lysC), aspartate semialdehyde dehydrogenase (asd), aspartokinase I (thrA) encoded by the thr operon, homoserine kinase (thrB), threonine synthase ( thrC), aspartate aminotransferase (aspartate transaminase) (aspC). The parentheses are abbreviations for the genes (the same applies to the following description). Of these enzymes, aspartate semialdehyde dehydrogenase, aspartokinase I, homoserine kinase, aspartate aminotransferase, and threonine synthase are particularly preferred. The L-threonine biosynthesis gene may be introduced into a bacterium belonging to the genus Escherichia in which threonine degradation is suppressed. Examples of the Escherichia bacterium in which threonine degradation is suppressed include, for example, the TDH6 strain lacking threonine dehydrogenase activity (Japanese Patent Laid-Open No. 2001-346578).

  The enzyme activity of the L-threonine biosynthetic enzyme is suppressed by the final product, L-threonine. Therefore, in order to construct an L-threonine-producing bacterium, it is desirable to modify the L-threonine biosynthetic gene so that it is not subject to feedback inhibition by L-threonine. The thrA, thrB, and thrC genes constitute the threonine operon, but the threonine operon forms an attenuator structure, and the expression of the threonine operon inhibits isoleucine and threonine in the culture medium. The expression is suppressed by attenuation. This modification can be achieved by removing the leader sequence or the attenuator of the attenuation region (Lynn, SP et al. 1987. J. Mol. Biol. 194: 59-69; WO 02/26993). Issue pamphlet; see International Publication No. 2005/049808 pamphlet).

  A unique promoter exists upstream of the threonine operon, but it may be replaced with a non-natural promoter (see WO98 / 04715), or the expression of a gene involved in threonine biosynthesis is expressed in lambda phage. A threonine operon as governed by a presser and promoter may be constructed. (See European Patent No. 0593792) In order to modify bacteria so that it is not subject to feedback inhibition by L-threonine, a strain resistant to α-amino-β-hydroxyvaleric acid (AHV) may be selected. Is possible.

  Thus, the threonine operon modified so as not to receive feedback inhibition by L-threonine has an increased copy number in the host or is linked to a strong promoter to improve the expression level. Is preferred. The increase in copy number can be achieved by transferring the threonine operon onto the genome by transposon, Mu-phage, etc., in addition to amplification by plasmid.

  In addition to the L-threonine biosynthetic enzyme, it is also preferable to enhance a glycolytic system, a TCA cycle, a gene related to the respiratory chain, a gene that controls gene expression, and a sugar uptake gene. Examples of these genes effective for L-threonine production include transhydronase (pntAB) gene (European Patent No. 733712), phosphoenolpyruvate carboxylase gene (pepC) (International Publication No. 95/06114 pamphlet), Phosphoenolpyruvate synthase gene (pps) (European Patent No. 877090), pyruvate carboxylase gene of Coryneform bacterium or Bacillus genus bacteria (International Publication No. 99/18228, European Patent Publication No. 1092776) Is mentioned.

  It is also preferable to enhance the expression of a gene imparting resistance to L-threonine and a gene conferring resistance to L-homoserine, or imparting L-threonine resistance and L-homoserine resistance to the host. Examples of genes that confer resistance include rhtA gene (Livshits, VA et al. 2003. Res. Microbiol. 154: 123-135), rhtB gene (European Patent Application Publication No. 0994190), rhtC gene (European patent application) Publication No. 1013765), yfiK, yeaS gene (European Patent Application Publication No. 1016710). For methods for imparting L-threonine resistance to a host, the methods described in European Patent Application Publication No. 0994190 and International Publication No. 90/04636 can be referred to.

  Examples of L-threonine producing bacteria or parent strains for deriving them include E. coli TDH-6 / pVIC40 (VKPM B-3996) (US Pat. No. 5,175,107, US Pat. No. 5,705,371), E. coli 472T23 / pYN7 (ATCC 98081) (U.S. Pat.No. 5,631,157), E. coli NRRL-21593 (U.S. Pat.No. 5,939,307), E. coli FERM BP-3756 (U.S. Pat.No. 5,474,918), E. coli FERM BP-3519 And FERM BP-3520 (U.S. Pat.No. 5,376,538), E. coli MG442 (Gusyatiner et al., 1978. Genetika (in Russian), 14: 947-956), E. coli VL643 and VL2055 (European Patent Application No. Strains belonging to the genus Escherichia, such as, but not limited to, 1149911).

  The TDH-6 strain lacks the thrC gene and is sucrose-utilizing, and the ilvA gene has a leaky mutation. This strain also has a mutation in the rhtA gene that confers resistance to high concentrations of threonine or homoserine. The B-3996 strain carries the plasmid pVIC40 in which the thrA * BC operon containing the mutated thrA gene is inserted into the RSF1010-derived vector. This mutant thrA gene encodes aspartokinase homoserine dehydrogenase I which is substantially desensitized to feedback inhibition by threonine. B-3996 shares were deposited with the accession number RIA 1867 at the All Union Scientific Center of Antibiotics (Nagatinskaya Street 3-A, 117105 Moscow, Russia) on November 19, 1987 . This strain was also deposited with the Russian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) on 7 April 1987 under the accession number B-3996. Has been.

  E. coli VKPM B-5318 (EP 0593792B) can also be used as an L-threonine producing bacterium or a parent strain for inducing it. The B-5318 strain is isoleucine non-required, and the control region of the threonine operon in the plasmid pVIC40 is replaced by a temperature sensitive lambda phage C1 repressor and a PR promoter. VKPM B-5318 was assigned to Russian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) on May 3, 1990 under the accession number VKPM B-5318. Has been deposited internationally.

  The thrA gene encoding Escherichia coli aspartokinase homoserine dehydrogenase I is located at base numbers 337 to 2,799 on the genome sequence of Escherichia coli MG1655 registered under GenBank Accession No. U00096, and GenBank accession No. AAC73113 It is a gene encoding a protein registered in. The thrB gene encoding homoserine kinase of Escherichia coli is located at base numbers 2,801-3,733 on the genome sequence of Escherichia coli MG1655 strain registered in GenBank Accession No. U00096, and is registered by GenBank accession No. AAC73114 It is a gene that encodes the protein. The thrC gene encoding the threonine synthase of Escherichia coli is located at base numbers 3,734-5,020 on the genome sequence of Escherichia coli MG1655 strain registered in GenBank Accession No. U00096, and is registered under GenBank accession No. AAC73115 It is a gene that encodes the protein. These three genes are encoded as a threonine operon consisting of thrLABC downstream of the thrL gene encoding the leader peptide. In order to increase the expression of the threonine operon, it is effective to remove the attenuator region that affects transcription, preferably from the operon (WO 2005/049808, WO2003 / 097839).

  The mutant thrA gene encoding aspartokinase homoserine dehydrogenase I, which is resistant to feedback inhibition by threonine, and the thrB and thrC genes are one operon from the well-known plasmid pVIC40 present in the threonine producing strain E. coli VKPM B-3996. Can be obtained as Details of plasmid pVIC40 are described in US Pat. No. 5,705,371.

  The rhtA gene has nucleotide numbers 848,433 to 849,320 on the genome sequence of Escherichia coli MG1655 strain registered under GenBank Accession No. U00096 acquired as a gene that gives resistance to homoserine and threonine (rht: resistant to threonine / homoserine). It is a gene encoding a protein located in (complementary strand) and registered in GenBank accession No. AAC73900. It has also been found that the rhtA23 mutation that improves the expression of rthA is a G → A substitution at position −1 relative to the ATG start codon (Livshits, VA et al. 2003. Res Microbiol. 154: 123- 135, European Patent Application No. 1013765).

  The asd gene of Escherichia coli is located at base numbers 3,571,798-3,572,901 (complementary strand) on the genome sequence of Escherichia coli MG1655 strain registered in GenBank Accession No. U00096, and is registered in GenBank accession No. AAC76458 It is a gene that encodes a protein. It can be obtained by PCR using primers prepared based on the nucleotide sequence of the gene (see White, T. J. et al. 1989. Trends Genet. 5: 185-189.). The asd gene of other microorganisms can be obtained similarly.

  In addition, the aspC gene of Escherichia coli is located at base numbers 983, 742 to 984,932 (complementary strands) on the genome sequence of Escherichia coli MG1655 registered in GenBank Accession No. U00096, and is registered in GenBank accession No. AAC74014 It is a gene that encodes a protein that can be obtained by PCR. The aspC gene of other microorganisms can be obtained similarly.

L-lysine-producing bacteria Hereinafter, L-lysine-producing bacteria and their construction methods are shown as examples.
For example, L-lysine analog resistant strains or metabolic control mutants can be mentioned as strains having L-lysine producing ability. Examples of L-lysine analogs include oxalysine, lysine hydroxamate, S- (2-aminoethyl) -L-cysteine (hereinafter sometimes abbreviated as “AEC”), γ-methyllysine, α-chloro. Although caprolactam etc. are mentioned, it is not limited to these. Mutants having resistance to these lysine analogs can be obtained by subjecting bacteria belonging to the family Enterobacteriaceae or coryneform bacteria to ordinary artificial mutation treatment. Specific examples of L-lysine-producing bacteria include Escherichia coli AJ11442 (FERM BP-1543, NRRL B-12185; see JP-A-56-18596 and US Pat. No. 4,346,170), Escherichia coli VL611. Strains (JP 2000-189180 A) and the like. In addition, WC196 strain (see International Publication No. 96/17930 pamphlet) can also be used as an L-lysine producing bacterium of Escherichia coli.

  An L-lysine producing bacterium can also be constructed by increasing the enzyme activity of the L-lysine biosynthesis system. These increases in enzyme activity can be achieved by increasing the copy number of the gene encoding the enzyme in the cell or by modifying the expression regulatory sequence.

  The modification for enhancing the expression of the gene can be performed, for example, by increasing the copy number of the gene in the cell using a gene recombination technique. For example, a DNA fragment containing the gapA gene may be ligated with a vector that functions in a host bacterium, preferably a multicopy vector, to produce a recombinant DNA, which is introduced into the bacterium and transformed.

  Increasing the copy number of a gene can also be achieved by having multiple copies of the above gene on the bacterial genomic DNA. In order to introduce a gene in multiple copies on bacterial genomic DNA, homologous recombination is performed using a sequence present in multiple copies on the genomic DNA as a target. As a sequence present in multiple copies on genomic DNA, repetitive DNA and inverted repeats present at the end of a transposable element can be used. In addition, each gene may be linked in tandem beside the gapA gene present on the genome, or may be redundantly incorporated on an unnecessary gene on the genome. These gene introductions can be achieved using a temperature sensitive vector or using an integration vector.

  Alternatively, as disclosed in Japanese Patent Application Laid-Open No. 2-109985, a gene can be mounted on a transposon and transferred to introduce multiple copies onto genomic DNA. Confirmation of gene transfer on the genome can be confirmed by Southern hybridization using a part of the gene as a probe.

  Furthermore, in addition to the amplification of the gene copy number described above, the gene expression can be enhanced by the method described in the pamphlet of International Publication No. 00/18935 using expression control sequences such as each promoter of the gene on genomic DNA or plasmid. Regulators that replace powerful genes, bring the -35 and -10 regions of each gene closer to the consensus sequence, amplify regulators that increase gene expression, or reduce gene expression It can also be achieved by deleting or weakening. For example, lac promoter, trp promoter, trc promoter, tac promoter, araBA promoter, lambda phage PR promoter, PL promoter, tet promoter, T7 promoter, φ10 promoter and the like are known as strong promoters. It is also possible to introduce a base substitution or the like into the promoter region or SD region of the gapA gene and modify it to a stronger one. Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev. 1995. 1: 105-128) and the like. In addition, it is known that the substitution of several nucleotides in the spacer between the ribosome binding site (RBS) and the start codon, particularly in the sequence immediately upstream of the start codon, greatly affects the translation efficiency of mRNA, These can be modified. Expression regulatory regions such as gene promoters can also be determined using a promoter search vector or gene analysis software such as GENETYX. These promoter substitutions or modifications enhance gene expression. For example, a method using a temperature-sensitive plasmid or a Red driven integration method (WO2005 / 010175) can be used to replace the expression regulatory sequence.

  Examples of genes encoding L-lysine biosynthesis enzymes include dihydrodipicolinate synthase gene (dapA), aspartokinase gene (lysC), dihydrodipicolinate reductase gene (dapB), and diaminopimelate decarboxylase gene (lysA). , Diaminopimelate dehydrogenase gene (ddh) (international publication No. 96/40934 pamphlet), phosphoenolpyruvate carboxylase gene (ppc) (Japanese Patent Laid-Open No. 60-87788), aspartate aminotransferase gene (aspC) ( Japanese Patent Publication No.6-102028), diaminopimelate epimerase gene (dapF) (Japanese Patent Laid-Open No. 2003-135066), aspartate semialdehyde dehydrogenase gene (asd) (WO 00/61723 pamphlet), etc. Gene of enzyme of acid pathway or homoaconite hydratase gene ( It includes genes such as enzymes of aminoadipic acid pathway Open Patent Laid-open No. 2000-157276) and the like. Further, the parent strain encodes a gene involved in energy efficiency (cyo) (EP 1170376 A), a gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (US Pat. No. 5,830,716), and a protein having L-lysine excretion activity. The expression level of the ybjE gene (WO2005 / 073390), the gene encoding glutamate dehydrogenase (gdhA) (Valle F. et al. 1983. Gene 23: 199-209), or any combination of these It may be. In parentheses are abbreviations for these genes.

  Wild-type dihydrodipicolinate synthase derived from Escherichia coli is known to undergo feedback inhibition by L-lysine, and wild-type aspartokinase derived from Escherichia coli undergoes inhibition and feedback inhibition by L-lysine. It has been known. Therefore, when using the dapA gene and the lysC gene, these genes are preferably mutant genes that are not subject to feedback inhibition by L-lysine.

  Examples of DNA encoding a mutant dihydrodipicolinate synthase that is not subject to feedback inhibition by L-lysine include DNA encoding a protein having a sequence in which the histidine residue at position 118 is substituted with a tyrosine residue. As a DNA encoding a mutant aspartokinase that is not subject to feedback inhibition by L-lysine, the threonine residue at position 352 is replaced with an isoleucine residue, the glycine residue at position 323 is replaced with an asparagine residue, 318 Examples include DNA encoding AKIII having a sequence in which the methionine at the position is replaced with isoleucine (see US Pat. Nos. 5,610,010 and 6,040,160 for these variants). Mutant DNA can be obtained by site-specific mutagenesis such as PCR.

  Wide host range plasmids RSFD80, pCAB1, and pCABD2 are known as plasmids containing mutant dapA encoding mutant mutant dihydrodipicolinate synthase and mutant lysC encoding mutant aspartokinase (USA) Patent No. 6040160). The Escherichia coli JM109 strain (US Pat. No. 6,040,160) transformed with this plasmid was named AJ12396, and this strain was established on 28 October 1993 at the Institute of Biotechnology, Ministry of International Trade and Industry (METI). Deposited at the National Institute of Advanced Industrial Science and Technology (AIST) as deposit number FERM P-13936, transferred to an international deposit based on the Budapest Treaty on November 1, 1994, with the deposit number of FERM BP-4859 It is deposited with. RSFD80 can be obtained from AJ12396 strain by a known method.

  In the production of L-lysine, examples of such enzymes include homoserine dehydrogenase, lysine decarboxylase (cadA, ldcC), malic enzyme, and the like, and a strain in which the activity of the enzyme is reduced or deleted is disclosed in WO95 / 23864, It is described in WO96 / 17930 pamphlet, WO2005 / 010175 pamphlet and the like.

  In order to reduce or eliminate lysine decarboxylase activity, it is preferable to reduce the expression of both the cadA gene and ldcC gene encoding lysine decarboxylase. Decrease in the expression of both genes can be performed according to the method described in WO2006 / 078039 pamphlet.

  As a method for reducing or eliminating these enzyme activities, a mutation that reduces or eliminates the activity of the enzyme in the cell is applied to the gene of the enzyme on the genome by a usual mutation treatment method or gene recombination technique. What is necessary is just to introduce. Such mutations can be introduced, for example, by deleting a gene encoding an enzyme on the genome by genetic recombination or by modifying an expression regulatory sequence such as a promoter or Shine-Dalgarno (SD) sequence. Achieved. Also, introducing amino acid substitution (missense mutation) into the region encoding the enzyme on the genome, introducing a stop codon (nonsense mutation), or introducing a frameshift mutation that adds or deletes one or two bases. It can also be achieved by deleting a part or the entire region of the gene (Wang, JP et al. 2006. J. Agric. Food Chem. 54: 9405-9410; Winkler, WC 2005. Curr. Opin. Chem. Biol. 9: 594-602; Qiu, Z. and Goodman, MF 1997. J. Biol. Chem. 272: 8611-8617; Wente, SR and Schachman, HK 1991. J. Biol. Chem. 266: 20833-20839 ). Also, construct a gene that encodes a mutant enzyme in which all or part of the coding region has been deleted, and replace the normal gene on the genome with the gene by homologous recombination, etc. By introducing it into the gene, the enzyme activity can be reduced or eliminated.

  For example, in order to introduce a mutation that reduces or eliminates the activity of the above enzyme by genetic recombination, the following method is used. A modified gene in which a partial sequence of the target gene is modified so that it does not produce a normally functioning enzyme is prepared, and a bacterium belonging to the family Enterobacteriaceae is transformed with the DNA containing the gene. By causing recombination with the above gene, the target gene on the genome can be replaced with a mutant. Such gene replacement using homologous recombination is a method called “Red-driven integration” (Datsenko, K. A, and Wanner, BL 2000. Proc. Natl. Acad. Sci. US A 97: 6640-6645), a combination method of Red driven integration method and λ phage-derived excision system (Cho, EH, Gumport, RI, Gardner, JFJ 2002. Bacteriol. 184: 5200-5203) (WO2005 / And the like, and a method using a plasmid containing a temperature-sensitive replication origin (US Pat. No. 6,303,383; Japanese Patent Laid-Open No. 05-007491). Moreover, site-directed mutagenesis by gene replacement using homologous recombination as described above can also be performed using a plasmid that does not have replication ability on the host.

  A preferred L-lysine-producing bacterium includes Escherichia coli WC196ΔcadAΔldcC / pCABD2 (WO2006 / 078039). This strain was constructed by disrupting the cadA and ldcC genes encoding lysine decarboxylase and introducing plasmid pCABD2 (US Pat. No. 6,040,160) containing a lysine biosynthesis gene from WC196 strain. The WC196 strain was obtained from the W3110 strain derived from E. coli K-12, and encodes aspartokinase III in which feedback inhibition by L-lysine was released by replacing threonine at position 352 with isoleucine. After the wild type lysC gene on the chromosome of the W3110 strain was replaced with a mutant lysC gene (US Pat. No. 5,661,012), it was bred by conferring AEC resistance (US Pat. No. 5,827,698). The WC196 strain was named Escherichia coli AJ13069. On December 6, 1994, the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology, Patent Biodeposition Center, Ibaraki, Japan, 305-8566, Japan Deposited as FERM P-14690 in Tsukuba City Higashi 1-chome 1 1 Central 6), transferred to the international deposit under the Budapest Treaty on 29 September 1995, and assigned the accession number FERM BP-5252 (US Pat. No. 5,827,698). WC196ΔcadAΔldcC itself is also a preferred L-lysine-producing bacterium. WC196ΔcadAΔldcC was named AJ110692 and was deposited internationally on October 7, 2008 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, 1-1-1 Higashi 1-chome, Tsukuba City, Ibaraki, Japan 305-8566 Accession number FERM BP-11027 is assigned.

  pCABD2 is a mutant dapA gene encoding dihydrodipicolinate synthase (DDPS) derived from Escherichia coli having a mutation in which feedback inhibition by L-lysine is released, and a mutation in which feedback inhibition by L-lysine is released. A mutant lysC gene encoding aspartokinase III derived from Escherichia coli, dapB gene encoding dihydrodipicolinate reductase derived from Escherichia coli, and ddh encoding a diaminopimelate dehydrogenase derived from Brevibacterium lactofermentum Contains genes (International Publication Nos. WO95 / 16042 and WO01 / 53459).

  The above-described methods for enhancing gene expression of enzymes involved in L-lysine biosynthesis and methods for reducing enzyme activity can be similarly applied to genes encoding other L-amino acid biosynthetic enzymes. .

  Examples of coryneform bacteria having L-lysine-producing ability include AEC-resistant mutant strains (Brevibacterium lactofermentum AJ11082 (NRRL B-11470), etc .: JP-B-56-1914, JP-B-56-1915 No. 57-14157, No. 57-14158, No. 57-30474, No. 58-10075, No. 59-4993, No. 61-35840, No. 62-24074, JP-B 62-36673, JP-B 5-11958, JP-B 7-112437, JP-B 7-112438); amino acids such as L-homoserine for its growth (See Japanese Patent Publication No. 48-28078, Japanese Patent Publication No. 56-6499); resistant to AEC, L-leucine, L-homoserine, L-proline, L-serine, L- Mutants that require amino acids such as arginine, L-alanine, and L-valine (see US Pat. Nos. 3,708,395 and 3,582,472) DL-α-amino-ε-caprolactam, α-amino-lauryllactam, aspartate-analog, sulfa drugs, quinoids, L-lysine-producing mutants resistant to N-lauroylleucine; oxaloacetate decarboxylase (de) L-lysine production mutants exhibiting resistance to carboxylase) or respiratory enzyme inhibitors (JP 50-53588, JP 50-31093, JP 52-102498, JP 53) -9394, JP-A-53-86089, JP-A-55-9783, JP-A-55-9759, JP-A-56-32995, JP-A-56-39778, JP-B-53-43591, JP-B-53-1833); L-lysine production mutants requiring inositol or acetic acid (JP-A-55-9784, JP-A-56-8692); Fluoro L-lysine producing mutant strains sensitive to pyruvic acid or a temperature of 34 ° C. or higher (Japanese Patent Laid-Open No. 55-9783, JP-A-53-86090); Brevibacterium spp. Or Corynebacterium spp. Production mutants (US Pat. No. 4411997) which are resistant to ethylene glycol and produce L-lysine.

Examples of L-cysteine producing bacteria L-cysteine producing bacteria or parent strains for deriving them include E. coli JM15 (US Pat. No. 6,218,168) transformed with a different cysE allele encoding a feedback inhibition resistant serine acetyltransferase. , Russian Patent Application No. 2003121601), E. coli W3110 (US Pat.No. 5,972,663) having an overexpressed gene encoding a protein suitable for excretion of a substance toxic to cells, cysteine desulfohydrase activity E. coli strains such as reduced E. coli strains (JP-A-11-155571) and E. coli W3110 (international publication No. 0127307) with increased activity of transcription regulators of the positive cysteine regulon encoded by the cysB gene. Examples include, but are not limited to, the strains to which they belong.

Examples of L-leucine-producing bacteria L-leucine-producing bacteria or parent strains for inducing them include leucine-resistant E. coil strains (for example, 57 strains (VKPM B-7386, US Pat. No. 6,124,121)) or β E. coli strains resistant to leucine analogs such as 2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, and 5,5,5-trifluoroleucine (Japanese Patent Publication No. 62-34397 and JP-A-8-70879), Although strains belonging to the genus Escherichia such as E. coli strains obtained by the genetic engineering method described in International Publication No. 96/06926, E. coli H-9068 (Japanese Patent Laid-Open No. 8-70879) can be mentioned, It is not limited to these.

  The bacterium used in the present invention may be improved by increasing the expression of one or more genes involved in L-leucine biosynthesis. As an example of such a gene, a gene of leuABCD operon represented by a mutant leuA gene (US Pat. No. 6,403,342) encoding isopropyl malate synthase preferably released from feedback inhibition by L-leucine is mentioned. . Furthermore, the bacterium used in the present invention may be improved by increasing the expression of one or more genes encoding proteins that excrete L-amino acids from bacterial cells. Examples of such genes include b2682 gene and b2683 gene (ygaZH gene) (European Patent Application Publication No. 1239041).

  L-isoleucine-producing bacteria of coryneform bacteria include coryneform bacteria (JP 2001-169788) in which a brnE gene encoding a branched-chain amino acid excretion protein is amplified, and L-isoleucine production by protoplast fusion with L-lysine-producing bacteria. Coryneform bacterium imparted with ability (JP-A 62-74293), coryneform bacterium with enhanced homoserine dehydrogenase (JP-A 62-91193), threonine hydroxamate resistant strain (JP 62-195293), α- Examples include ketomarone resistant strains (Japanese Patent Laid-Open No. 61-15695) and methyllysine resistant strains (Japanese Patent Laid-Open No. 61-15696).

Examples of L-histidine-producing bacteria L-histidine-producing bacteria or parent strains for inducing them include E. coli 24 strain (VKPM B-5945, Russian Patent No. 2003677), E. coli 80 strain (VKPM B- 7270, Russian patent 2119536), E. coli NRRL B-12116-B12121 (US Pat.No. 4,388,405), E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (US) Patent No. 6,344,347), E. coli H-9341 (FERM BP-6674) (European Patent Application Publication No. 1085087), E. coli AI80 / pFM201 (US Pat.No. 6,258,554), and other strains belonging to the genus Escherichia However, it is not limited to these.

  Examples of L-histidine-producing bacteria or parent strains for deriving the same also include strains in which expression of one or more genes encoding L-histidine biosynthetic enzymes are increased. Examples of such genes include ATP phosphoribosyltransferase gene (hisG), phosphoribosyl AMP cyclohydrolase gene (hisI), phosphoribosyl-ATP pyrophosphohydrolase gene (hisI), phosphoribosylformimino-5- Examples include aminoimidazole carboxamide ribotide isomerase gene (hisA), amide transferase gene (hisH), histidinol phosphate aminotransferase gene (hisC), histidinol phosphatase gene (hisB), and histidinol dehydrogenase gene (hisD). It is done.

  L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are known to be inhibited by L-histidine, and therefore L-histidine-producing ability is feedback-inhibited by the ATP phosphoribosyltransferase gene (hisG). Can be efficiently increased by introducing mutations that confer resistance to (Russian Patent Nos. 2003677 and 2119536).

  Specific examples of strains capable of producing L-histidine include E. coli FERM-P 5038 and 5048 into which a vector holding a DNA encoding an L-histidine biosynthetic enzyme has been introduced (Japanese Patent Laid-Open No. 56-005099). E. coli strain into which an amino acid transporting gene has been introduced (European Patent Application Publication No. 1016710), E. coli 80 to which resistance to sulfaguanidine, DL-1,2,4-triazole-3-alanine and streptomycin has been conferred Strains (VKPM B-7270, Russian Patent No. 2119536).

Examples of L-glutamic acid-producing bacteria L-glutamic acid-producing bacteria or parent strains for inducing them include, but are not limited to, strains belonging to the genus Escherichia such as E. coli VL334thrC + (EP 1172433). E. coli VL334 (VKPM B-1641) is an L-isoleucine and L-threonine auxotrophic strain having mutations in the thrC gene and the ilvA gene (US Pat. No. 4,278,765). The wild type allele of the thrC gene was introduced by a general transduction method using bacteriophage P1 grown on cells of wild type E. coli K-12 strain (VKPM B-7). As a result, L-isoleucine-requiring L-glutamic acid producing bacterium VL334thrC + (VKPM B-8961) was obtained.

  Examples of L-glutamic acid-producing bacteria or parent strains for inducing them include, but are not limited to, L-glutamic acid biosynthetic enzymes having one or two or more enhanced activities. Examples of such genes include glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), Methyl citrate synthase (prpC), phosphoenolpyruvate carbocilase (ppc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase ( eno), phosphoglyceromutase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogenase (gapA), triphosphate isomerase (tpiA), fructose bisphosphate aldolase ( fbp), phosphofructokinase (pfkA) , pfkB), glucose phosphate isomerase (pgi) and the like. Of these enzymes, glutamate dehydrogenase, citrate synthase, phosphoenolpyruvate carboxylase, and methyl citrate synthase are preferred.

  Examples of strains modified to increase expression of citrate synthetase gene, phosphoenolpyruvate carboxylase gene, and / or glutamate dehydrogenase gene include European Patent Application Publication No. 1078989, European Patent Application Publication No. 955368. And those disclosed in European Patent Application No. 952221.

  As an example of an L-glutamic acid-producing bacterium or a parent strain for deriving the same, the activity of an enzyme that catalyzes the synthesis of a compound other than L-glutamic acid by diverging from the biosynthetic pathway of L-glutamic acid is reduced or absent Stocks are also mentioned. Examples of such enzymes include isocitrate triase (aceA), α-ketoglutarate dehydrogenase (sucA), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxy acid synthase (ilvG), Examples include acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), glutamate decarboxylase (gadAB), and the like. Bacteria belonging to the genus Escherichia lacking α-ketoglutarate dehydrogenase activity or having reduced α-ketoglutarate dehydrogenase activity, and methods for obtaining them are described in US Pat. Nos. 5,378,616 and 5,573,945. .

Specific examples include the following.
E. coli W3110sucA :: Kmr
E. coli AJ12624 (FERM BP-3853)
E. coli AJ12628 (FERM BP-3854)
E. coli AJ12949 (FERM BP-4881)

  E. coli W3110sucA :: Kmr is a strain obtained by disrupting the α-ketoglutarate dehydrogenase gene (hereinafter also referred to as “sucA gene”) of E. coli W3110. This strain is completely deficient in α-ketoglutarate dehydrogenase.

In addition, examples of coryneform bacteria having reduced α-ketoglutarate dehydrogenase activity include the following strains.
Brevibacterium lactofermentum L30-2 strain (Japanese Unexamined Patent Publication No. 2006-340603)
Brevibacterium lactofermentum strain ΔS (International pamphlet No. 95/34672)
Brevibacterium lactofermentum AJ12821 (FERM BP-4172; see French patent publication 9401748)
Brevibacterium flavum AJ12822 (FERM BP-4173; French Patent Publication 9401748)
Corynebacterium glutamicum AJ12823 (FERM BP-4174; French Patent Publication 9401748)
Corynebacterium glutamicum L30-2 strain (Japanese Patent Laid-Open No. 2006-340603)

  Other examples of L-glutamic acid-producing bacteria include those belonging to the genus Escherichia and having resistance to an aspartic acid antimetabolite. These strains may be deficient in α-ketoglutarate dehydrogenase, for example, E. coli AJ13199 (FERM BP-5807) (US Pat. No. 5,908,768), and FFRM P- with reduced L-glutamate resolution. 12379 (US Pat. No. 5,393,671); AJ13138 (FERM BP-5565) (US Pat. No. 6,110,714) and the like.

  An example of L-glutamic acid-producing bacteria of Pantoea ananatis is Pantoea ananatis AJ13355 strain. This strain is a strain isolated as a strain capable of growing on a medium containing L-glutamic acid and a carbon source at low pH from the soil of Kamata City, Shizuoka Prefecture. Pantoea Ananatis AJ13355 was commissioned on February 19, 1998 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (Address: 1st, 1st, 1st East, 1-chome, Tsukuba, Ibaraki 305-8566, Japan) Deposited under the number FERM P-16644, transferred to an international deposit under the Budapest Treaty on 11 January 1999 and given the deposit number FERM BP-6614. The strain was identified as Enterobacter agglomerans at the time of its isolation and was deposited as Enterobacter agglomerans AJ13355, but in recent years, it has been identified by Pantoea ananatis (Pantoea ananatis) by 16S rRNA sequence analysis. ).

  In addition, examples of L-glutamic acid-producing bacteria of Pantoea ananatis include bacteria belonging to the genus Pantoea in which α-ketoglutarate dehydrogenase (αKGDH) activity is deficient or αKGDH activity is reduced. Such strains include AJ13356 (US Pat. No. 6,331,419) in which the αKGDH-E1 subunit gene (sucA) of AJ13355 strain is deleted, and sucA derived from SC17 strain selected from AJ13355 strain as a low mucus production mutant. There is SC17sucA (US Pat. No. 6,596,517) which is a gene-deficient strain. AJ13356 was founded on February 19, 1998, National Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, 1-chome, 1-chome, Tsukuba, Ibaraki, Japan 305-8566 No. 6) was deposited under the deposit number FERM P-16645, transferred to an international deposit under the Budapest Treaty on January 11, 1999, and given the deposit number FERM BP-6616. AJ13355 and AJ13356 are deposited as Enterobacter agglomerans in the above depository organization, but are described as Pantoea ananatis in this specification. The SC17sucA strain has been assigned a private number AJ417, and was deposited on February 26, 2004 at the above-mentioned National Institute of Advanced Industrial Science and Technology as the accession number FERM BP-08646.

  Furthermore, examples of L-glutamic acid-producing bacteria of Pantoea ananatis include SC17sucA / RSFCPG + pSTVCB strain, AJ13601 strain, NP106 strain, and NA1 strain. The SC17sucA / RSFCPG + pSTVCB strain is different from the SC17sucA strain in that the plasmid RSFCPG containing the citrate synthase gene (gltA), the phosphoenolpyruvate carboxylase gene (ppsA), and the glutamate dehydrogenase gene (gdhA) derived from Escherichia coli, This is a strain obtained by introducing a plasmid pSTVCB containing a citrate synthase gene (gltA) derived from bacteria lactofermentum. The AJ13601 strain is a strain selected from this SC17sucA / RSFCPG + pSTVCB strain as a strain resistant to a high concentration of L-glutamic acid at low pH. The NP106 strain is a strain obtained by removing the plasmid RSFCPG + pSTVCB from the AJ13601 strain. On August 18, 1999, AJ13601 shares were registered with the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st, 1st East, Tsukuba City, Ibaraki 305-8566, Japan). Deposited as 17516, transferred to an international deposit under the Budapest Treaty on July 6, 2000, and assigned the deposit number FERM BP-7207.

  Furthermore, as a method for conferring L-glutamic acid producing ability to coryneform bacteria, a method of amplifying the yggB gene encoding mechanosensitive channel (International Publication WO2006 / 070944), a mutation in which a mutation is introduced into the coding region It is also possible to use a method for introducing a type yggB gene. The yggB gene is located at base numbers 1,337,692 to 1,336,091 (complementary strand) on the genome sequence of Corynebacterium glutamicum ATCC 13032 strain registered under GenBank Accession No. NC_003450, and GenBank accession No. NP_600492, also called NCgl1221 It is a gene that encodes a registered membrane protein.

As another method for imparting or enhancing L-glutamic acid-producing ability, a method for imparting resistance to an organic acid analog or a respiratory inhibitor and a method for imparting sensitivity to a cell wall synthesis inhibitor may be mentioned. For example, a method of imparting monofluoroacetic acid resistance (Japanese Patent Laid-Open No. 50-113209), a method of imparting adenine resistance or thymine resistance (Japanese Patent Laid-Open No. 57-065198), and a method of weakening urease (Japanese Patent Laid-Open No. 52-038088) A method for imparting resistance to malonic acid (Japanese Patent Laid-Open No. 52-038088), a method for imparting resistance to benzopyrone or naphthoquinones (Japanese Patent Laid-Open No. 56-1889), a method of imparting HOQNO resistance (Japanese Patent Laid-Open No. 56-140895) ), A method for imparting resistance to α-ketomalonic acid (Japanese Patent Laid-Open No. 57-2689), a method for imparting resistance to guanidine (Japanese Patent Laid-Open No. 56-35981), a method for imparting sensitivity to penicillin (Japanese Patent Laid-Open No. 4-88994), etc. Is mentioned.
Specific examples of such resistant bacteria include the following strains.
Brevibacterium flavum AJ3949 (FERM BP-2632: see JP-A-50-113209)
Corynebacterium glutamicum AJ11628 (FERM P-5736; see JP 57-065198)
Brevibacterium flavum AJ11355 (FERM P-5007; see JP-A-56-1889)
Corynebacterium glutamicum AJ11368 (FERM P-5020; see JP 56-1889)
Brevibacterium flavum AJ11217 (FERM P-4318; see JP-A-57-2689)
Corynebacterium glutamicum AJ11218 (FERM P-4319; see JP-A-57-2689)
Brevibacterium flavum AJ11564 (FERM P-5472; see JP 56-140895 A)
Brevibacterium flavum AJ11439 (FERM P-5136; see JP-A-56-35981)
Corynebacterium glutamicum H7684 (FERM BP-3004; see JP 04-88994 A)
Brevibacterium lactofermentum AJ11426 (FERM P-5123; see JP-A-56-048890)
Corynebacterium glutamicum AJ11440 (FERM P-5137; see JP-A-56-048890)
Brevibacterium lactofermentum AJ11796 (FERM P-6402; see JP-A-58-158192)

Examples of L-phenylalanine producing bacteria L-phenylalanine producing bacteria or parent strains for deriving them include E. coli AJ12739 (tyrA :: Tn10, tyrR) lacking chorismate mutase-prefenate dehydrogenase and tyrosine repressor ( VKPM B-8197) (WO 03/044191), E. coli HW1089 (ATCC 55371) carrying a mutant pheA34 gene encoding chorismate mutase-prefenate dehydratase with desensitized feedback inhibition (US Pat.No. 5,354,672) Strains belonging to the genus Escherichia such as E. coli MWEC101-b (KR8903681), E. coli NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (US Pat.No. 4,407,952). For example, but not limited to. In addition, E. coli K-12 [W3110 (tyrA) / pPHAB] (FERM BP-3566), E. coli K that retains the gene encoding chorismate mutase-prefenate dehydratase whose feedback inhibition has been released. -12 [W3110 (tyrA) / pPHAD] (FERM BP-12659), E. coli K-12 [W3110 (tyrA) / pPHATerm] (FERM BP-12662) and E. coli K-12 named AJ 12604 [W3110 (tyrA) / pBR-aroG4, pACMAB] (FERM BP-3579) can also be used (EP 488424 B1). Furthermore, L-phenylalanine producing bacteria belonging to the genus Escherichia having an increased activity of the protein encoded by the yedA gene or the yddG gene can also be used (US Patent Application Publication Nos. 2003/0148473 and 2003/0157667, International Publication No. 03/044192). .

  Coryneform bacteria that produce phenylalanine include Corynebacterium glutamica BPS-13 strains (FERM BP-1777, K77 (FERM BP-2062) and K78 (FERM BP) with reduced phosphoenolpyruvate carboxylase or pyruvate kinase activity. -2063) (European Patent Publication No. 331145, JP 02-303495 A), tyrosine-requiring strain (JP 05-049489) and the like.

  In addition, as a phenylalanine-producing bacterium, by-modifying by-products into cells, for example, improving the expression level of L-tryptophan uptake genes tnaB, mtr and L-tyrosine uptake gene tyrP Also, a strain that efficiently produces L-phenylalanine can be obtained (EP1484410).

Examples of L-tryptophan-producing bacteria L-tryptophan-producing bacteria or parent strains for inducing them include E. coli JP4735 / pMU3028 (DSM10122) and JP6015 / pMU91 lacking tryptophanyl-tRNA synthetase encoded by the mutant trpS gene. (DSM10123) (U.S. Pat.No. 5,756,345), E. coli having a serA allele encoding phosphoglycerate dehydrogenase not subject to feedback inhibition by serine and a trpE allele encoding an anthranilate synthase not subject to feedback inhibition by tryptophan. SV164 (pGH5) (US Pat.No. 6,180,373), E. coli AGX17 (pGX44) (NRRL B-12263) and AGX6 (pGX50) aroP (NRRL B-12264) lacking tryptophanase (US Pat.No. 4,371,614) E. coli AGX17 / pGX50, pACKG4-pps (WO9708333, U.S. Pat.No. 6,319,696) with increased ability to produce phosphoenolpyruvate Strains include belonging to Erihia genus, but is not limited thereto. L-tryptophan-producing bacteria belonging to the genus Escherichia with increased activity of the protein encoded by the yedA gene or the yddG gene can also be used (US Patent Application Publications 2003/0148473 and 2003/0157667).

  Examples of L-tryptophan-producing bacteria or parent strains for inducing them include anthranilate synthase (trpE), phosphoglycerate dehydrogenase (serA), 3-deoxy-D-arabinohepturonic acid-7-phosphorus Acid synthase (aroG), 3-dehydroquinate synthase (aroB), shikimate dehydrogenase (aroE), shikimate kinase (aroL), 5-enolic acid pyruvylshikimate 3-phosphate synthase (aroA), chorismate synthase (aroC ), Prephenate dehydratase, chorismate mutase and tryptophan synthase (trpAB). One or more strains having enhanced activity are also included. Prefenate dehydratase and chorismate mutase are encoded by the pheA gene as a bifunctional enzyme (chorismate mutase / prephenate dehydrogenase (CM / PDH)). Among these enzymes, phosphoglycerate dehydrogenase, 3-deoxy-D-arabinohepturonic acid-7-phosphate synthase, 3-dehydroquinate synthase, shikimate dehydratase, shikimate kinase, 5-enolic acid Pyruvylshikimate 3-phosphate synthase, chorismate synthase, prefenate dehydratase, chorismate mutase-prefenate dehydrogenase are particularly preferred. Since both anthranilate synthase and phosphoglycerate dehydrogenase are subject to feedback inhibition by L-tryptophan and L-serine, mutations that cancel the feedback inhibition may be introduced into these enzymes. Specific examples of strains having such mutations include E. coli SV164 carrying desensitized anthranilate synthase and a mutant serA gene encoding phosphoglycerate dehydrogenase desensitized to feedback inhibition Examples include a transformant obtained by introducing plasmid pGH5 (International Publication No. 94/08031) into E. coli SV164.

  Examples of L-tryptophan-producing bacteria or parent strains for deriving them include strains into which a tryptophan operon containing a gene encoding an inhibitory anthranilate synthase has been introduced (JP-A 57-71397, JP-A 62-244382, US Pat. No. 4,371,614). Furthermore, L-tryptophan-producing ability may be imparted by increasing the expression of a gene encoding tryptophan synthase in the tryptophan operon (trpBA). Tryptophan synthase consists of α and β subunits encoded by trpA and trpB genes, respectively. Furthermore, L-tryptophan production ability may be improved by increasing the expression of the isocitrate triase-malate synthase operon (WO 2005/103275).

  Coryneform bacteria include Corynebacterium glutamicum AJ12118 (FERM BP-478 Patent 016881002), a coryneform bacterium introduced with a tryptophan operon (Japanese Patent Laid-Open No. 63240794), coryneform bacteria, which are resistant to sulfaguanidine. Coryneform bacteria (Japanese Patent Laid-Open No. 01994749) into which a gene encoding shikimate kinase derived therefrom has been introduced can be used.

Examples of L-proline-producing bacteria L-proline-producing bacteria or parent strains for deriving the same are E. coli 702ilvA (VKPM B-8012) that lacks the ilvA gene and can produce L-proline (European Patent Publication) Examples include, but are not limited to, strains belonging to the genus Escherichia such as No. 1,172,433).

  The bacterium used in the present invention may be improved by increasing the expression of one or more genes involved in L-proline biosynthesis. An example of a gene preferable for L-proline-producing bacteria includes a proB gene (German Patent No. 3127361) encoding glutamate kinase that is desensitized to feedback inhibition by L-proline. Furthermore, the bacterium used in the present invention may be improved by increasing the expression of one or more genes encoding proteins that excrete L-amino acids from bacterial cells. Examples of such genes include b2682 gene and b2683 gene (ygaZH gene) (European Patent Publication No. 1,239,041).

  Examples of bacteria belonging to the genus Escherichia having L-proline producing ability include NRRL B-12403 and NRRL B-12404 (British Patent No. 2075056), VKPM B-8012 (Russian Patent Application 2000124295), German Patent No. 3127361 And E. coli strains such as those described in Bloom FR et al (The 15th Miami winter symposium, 1983, p. 34).

Examples of L-arginine producing bacteria L-arginine producing bacteria or parent strains for deriving them include E. coli strain 237 (VKPM B-7925) (US Patent Application Publication No. 2002/058315) and mutant N- Derived strains carrying acetylglutamate synthase (Russian Patent Application No. 2,001,112,869), E. coli 382 strain (VKPM B-7926) (European Patent Publication No.1,170,358), argA gene encoding N-acetylglutamate synthetase introduced Strains belonging to the genus Escherichia such as, but not limited to, arginine producing strains (European Patent Publication No. 1,170,361).

  Examples of L-arginine-producing bacteria or parent strains for deriving the same also include strains in which expression of one or more genes encoding L-arginine biosynthetic enzymes are increased. Examples of such genes include N-acetylglutamylphosphate reductase gene (argC), ornithine acetyltransferase gene (argJ), N-acetylglutamate kinase gene (argB), acetylornithine transaminase gene (argD), ornithine carbamoyltransferase gene ( argF), arginosuccinate synthetase gene (argG), arginosuccinate lyase gene (argH), carbamoylphosphate synthetase gene (carAB).

Examples of L-valine producing bacteria L-valine producing bacteria or parent strains for inducing the same include, but are not limited to, strains modified to overexpress the ilvGMEDA operon (US Pat. No. 5,998,178). Not. It is preferable to remove the ilvGMEDA operon region required for attenuation so that the operon expression is not attenuated by the produced L-valine. Furthermore, it is preferred that the ilvA gene of the operon is disrupted and the threonine deaminase activity is reduced.
Examples of L-valine-producing bacteria or parent strains for deriving the same also include mutant strains having aminoacyl t-RNA synthetase mutations (US Pat. No. 5,658,766). For example, E. coli VL1970 having a mutation in the ileS gene encoding isoleucine tRNA synthetase can be used. E. coli VL1970 was registered with Russian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) on June 24, 1988 under the accession number VKPM B-4411. It has been deposited.
Further, a mutant strain (International Publication No. 96/06926) that requires lipoic acid for growth and / or lacks H + -ATPase can be used as a parent strain.

  Examples of L-valine-producing bacteria of coryneform bacteria include strains modified so that expression of a gene encoding an enzyme involved in L-valic acid biosynthesis is enhanced. Examples of the enzyme involved in L-valinate biosynthesis include, for example, an enzyme encoded by the ilvBNC operon, that is, an acetohydroxyacid synthase encoded by ilvBN and an isomeroreductase encoded by ivlC (International Publication No. 00/50624) Is mentioned. Since the ilvBNC operon is regulated by operon expression by L-valine and / or L-isoleucine and / or L-leucine, the attenuation is released to release the suppression of the expression by L-valine produced. Is desirable.

  Coryneform bacteria having L-valine-producing ability may be performed by reducing or eliminating the activity of at least one enzyme involved in a substance metabolic pathway that reduces L-valine production. For example, it is conceivable to reduce the activity of threonine dehydratase involved in L-leucine synthesis and the enzyme involved in D-pantosenate synthesis (WO 00/50624).

Another method for imparting L-valine-producing ability includes a method for imparting resistance to amino acid analogs and the like.
For example, L-isoleucine and L-methionine auxotrophs, and mutant strains (FERM P-1841, FERM P having resistance to D-ribose, purine ribonucleoside or pyrimidine ribonucleoside and capable of producing L-valine) -29, JP-B 53-025034), mutants resistant to polyketoids (FERM P-1763, FERM P-1764, JP-B 06-065314), and in a medium containing acetic acid as the only carbon source. -Mutants (FERM BP-3006, FERM BP-3007, Patent 3006929) that are resistant to valine and that are sensitive to pyruvic acid analogs (fluoropyruvic acid, etc.) in a medium that uses glucose as the only carbon source. .

Examples of L-isoleucine-producing bacteria and L-isoleucine-producing bacteria or parent strains for inducing the same include mutants having resistance to 6-dimethylaminopurine (Japanese Patent Laid-Open No. 5-304969), thiisoleucine, isoleucine hydroxamate Mutants having resistance to isoleucine analogs such as the above, and mutants having resistance to DL-ethionine and / or arginine hydroxamate (Japanese Patent Laid-Open No. 5-130882), but are not limited thereto. Furthermore, a recombinant strain transformed with a gene encoding a protein involved in L-isoleucine biosynthesis such as threonine deaminase and acetohydroxy acid synthase can also be used as a parent strain (JP-A-2-458, FR 0356739, and US Pat. No. 5,998,178).

  L-isoleucine-producing bacteria of coryneform bacteria include coryneform bacteria (JP 2001-169788) in which a brnE gene encoding a branched-chain amino acid excretion protein is amplified, and L-isoleucine production by protoplast fusion with L-lysine-producing bacteria. Coryneform bacterium imparted with ability (JP-A 62-74293), coryneform bacterium with enhanced homoserine dehydrogenase (JP-A 62-91193), threonine hydroxamate resistant strain (JP 62-195293), α- Examples include ketomarone resistant strains (Japanese Patent Laid-Open No. 61-15695) and methyllysine resistant strains (Japanese Patent Laid-Open No. 61-15696).

Examples of L-methionine-producing bacteria L-methionine-producing bacteria or parent strains for inducing them include, but are not limited to, L-threonine-requiring strains and mutants having resistance to norleucine (Japanese Patent Laid-Open No. 2000-2000). 139471). Furthermore, a strain lacking a methionine repressor, a recombinant strain transformed with a gene encoding a protein involved in L-methionine biosynthesis, such as homoserine transsuccinylase, cystathionine γ-synthase, can also be used as a parent strain. (Japanese Patent Laid-Open No. 2000-139471).

Next, a method for imparting nucleic acid-producing ability to a microorganism and a nucleic acid-producing bacterium will be exemplified.
Bacterial microorganisms having nucleic acid-producing ability can be obtained by, for example, imparting purine nucleoside requirements or resistance to drugs such as purine analogs to the above-described bacterial microorganisms (Japanese Patent Publication No. 38-23099). JP-B-54-17033, JP-B-55-45199, JP-B-57-14160, JP-B-57-41915, JP-B-59-42895). For example, Bacillus bacteria having auxotrophy and drug resistance are used for normal mutation treatments such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or EMS (ethane methanesulfonate). It can be obtained by treatment with a mutagen.

Examples of Bacillus bacteria that produce purine nucleosides include the following.
As a specific example of the inosine-producing strain belonging to the genus Bacillus, the Bacillus subtilis KMBS16 strain can be used. The strain contains a deletion of purR gene encoding purine operon repressor (purR :: spc), deletion of purA gene encoding succinyl-AMP synthase (purA :: erm), and deoD gene encoding purine nucleoside phosphorylase. It is a known derivative of Bacillus subtilis trpC2 strain (168 Marburg) into which a deficiency (deoD :: kan) has been introduced (JP 2004-242610, US2004166575A1). Also, Bacillus subtilis strain AJ3772 (FERM P-2555) (Japanese Patent Laid-Open No. 62-014794) can be used.

  Examples of Bacillus bacteria having the ability to produce guanosine include Bacillus bacteria with increased IMP dehydrogenase activity (Japanese Patent Laid-Open No. 3-58787), vectors incorporating a purine analog resistance or decoinin resistance gene, and adenine-requiring mutants And Bacillus bacteria introduced in (4).

Examples of Bacillus bacteria that produce purine nucleotides include the following.
As inosinic acid-producing bacteria, inosine-producing strains with reduced phosphatase activity of Bacillus subtilis have been reported (Uchida, K. et al., Agr. Biol. Chem., 1961, 25, 804-805, Fujimoto, M. Uchida, K., Agr. Biol. Chem., 1965, 29, 249-259). As a guanylate-producing bacterium, it has adenine requirement, is resistant to decoinin or methionine sulfoxide, and can produce 5′-guanylic acid (guanosine-5′-monophosphate, hereinafter also referred to as “GMP”). And a mutant strain of the genus Bacillus (Japanese Patent Publication No. 56-12438).

  In addition, xanthylic acid-producing bacteria can be constructed using a method used for breeding coryneform bacteria centering on Corynebacterium ammmoniagenes. For example, by obtaining a PRPP amidotransferase-enhanced strain (Japanese Patent Laid-Open No. 8-168383), an aliphatic amino acid resistant strain (Japanese Patent Laid-Open No. 4-262790), a dehydroproline resistant strain (Korea Patent Publication No. 2003-56490), xanthylic acid production Fungi can be constructed.

  Moreover, the following method is mentioned as a method of breeding the Bacillus genus bacteria which have purine-type substance production ability. Examples include a method for increasing the intracellular activity of an enzyme involved in purine biosynthesis common to purine nucleosides and purine nucleotides, that is, purine biosynthesis enzyme. The intracellular activity of the enzyme is preferably increased as compared with an unmodified strain of Bacillus bacterium, for example, a wild-type Bacillus bacterium. “The activity increases” corresponds to, for example, a case where the number of enzyme molecules per cell increases or a case where the specific activity per enzyme molecule increases. For example, the activity can be increased by increasing the expression level of the enzyme gene. Examples of the enzyme involved in the purine biosynthesis include phosphoribosyl pyrophosphate amide transferase, phosphoribosyl pyrophosphate synthetase (PRPP synthetase [EC: 2.7.6.1]) and the like.

  A part of catabolite produced by metabolism by a sugar source such as glucose incorporated into the pentose phosphate system becomes ribose-5-phosphate via ribulose-5-phosphate. Biosynthesized ribose-5-phosphate produces purine nucleoside, histidine, and phosphoribosyl pyrophosphate (PRPP), an essential precursor for biosynthesis of tryptophan. Specifically, ribose-5-phosphate is converted to PRPP by phosphoribosyl pyrophosphate synthetase. Therefore, the ability to produce purine substances can be imparted to or enhanced in bacteria belonging to the genus Bacillus by modifying the activity of phosphoribosyl pyrophosphate synthetase to increase.

  “The activity of the phosphoribosyl pyrophosphate synthetase is increased” means that the activity of the phosphoribosyl pyrophosphate synthetase is increased relative to a non-modified strain such as a wild strain or a parent strain. The activity of phosphoribosyl pyrophosphate synthetase should be measured by, for example, the method of Switzer et al. (Methods Enzymol., 1978, 51, 3-11), the method of Roth et al. (Methods Enzymol., 1978, 51, 12-17) Can do. Bacteria belonging to the genus Bacillus having an increased activity of phosphoribosyl pyrophosphate synthase can be obtained by, for example, phosphoribosyl pyrophosphate by a method using a plasmid or a method of integrating on a chromosome in the same manner as described in JP-A-2004-242610. It can be produced by highly expressing a gene encoding a synthetase in a Bacillus bacterium.

  On the other hand, when PRPP, a precursor essential to purine nucleoside, histidine, and tryptophan biosynthesis, is produced, a part of it is converted into a purine nucleotide and purine nucleoside by a group of enzymes involved in purine biosynthesis. The genes encoding such enzyme groups include the Bacillus subtilis purine operon, specifically the purEKB-purC (orf) QLF-purMNH (J) -purD operon gene (Ebbole DJ and Zalkin H, J. Biol Chem., 1987, 262, 17, 8274-87 (now also called purEKBCSQLFMNHD: Bacillus subtilis and Its Closest Relatives, Editor in Chief: AL Sonenshein, ASM Press, Washington DC, 2002. GenBank Accession No. NC_000964) And the Escherichia coli pur regulon gene (Escherichia and Salmonella, Second Edition, Editor in Chief: FC Neidhardt, ASM Press, Washington DC, 1996).

  Therefore, the ability to produce purine substances can be imparted or enhanced by enhancing the expression of these genes. The purine operon genes that can be used in the present invention are not limited to these genes, and genes derived from other microorganisms and animals and plants can also be used.

  Examples of a method for increasing the expression level of the purine operon include a method in which the purine operon gene is highly expressed in a Bacillus bacterium by a method using a plasmid or a method of integrating on a chromosome.

  As a second method for increasing the expression level of the purine operon, replacement of the promoter unique to the purine operon with a stronger promoter, or substitution of the -35 and -10 regions of the unique promoter with a consensus sequence can be mentioned. .

  For example, in Bacillus subtilis (Strain B. subtilis 168 Marburg; ATCC6051), the -35 sequence of the purine operon is a consensus sequence (TTGACA), but the -10 sequence is TAAGAT, which is different from the consensus sequence TATAAT ( Ebbole, DJ and H. Zalikn, J. Biol. Chem., 1987, 262, 8274-8287). Therefore, the transcriptional activity of the purine operon can be increased by making the -10 sequence (TAAGAT) into a consensus sequence, or by modifying it so that it becomes TATAAT, TATGAT, or TAAAAT close to the consensus sequence. The replacement of the promoter sequence can be performed by the same method as the gene replacement described below.

  As a third method of increasing the expression level of the purine operon, there is a method of decreasing the expression level of the purine operon repressor (USP 6,284,495). “Expression of the purine operon repressor” includes both transcription of the purine operon gene and translation of the transcript. Further, “reducing the expression level” includes that the expression level is lower than that in an unmodified strain, for example, a wild-type Bacillus bacterium, and that the expression is substantially lost.

  In order to reduce the expression level of the purine operon repressor (purine repressor), for example, the Bacillus bacterium is treated with an ultraviolet ray irradiation or a mutagen that is usually used for mutagenesis such as NTG or EMS. A method of selecting a mutant strain in which the expression level of the repressor is reduced can be employed.

  Further, Bacillus bacteria having a reduced expression level of the purine repressor may include, for example, a homologous recombination method using genetic recombination (Experiments in Molecular Genetics, Cold Spring Harbor Laboratory press (1972); According to Matsuyama, S. and Mizushima, S., J. Bacteriol., 1985, 162, 1196-1202), a gene encoding a purine repressor on the chromosome (purR; GenBank Accession NC_000964 (the coding region has nucleotide numbers 54439 to 55293). ) With a gene that does not function normally (hereinafter sometimes referred to as a “disrupted gene”).

  Furthermore, the ability to produce purine substances can also be enhanced by weakening the incorporation of purine substances into cells. For example, purine nucleoside uptake into cells can be attenuated by blocking reactions involved in purine nucleoside uptake into cells. The reaction involved in the incorporation of the purine nucleoside into the cell is, for example, a reaction catalyzed by a nucleoside permease.

  Furthermore, when producing a purine nucleoside, the activity of an enzyme that degrades purine substances may be reduced in order to enhance the purine nucleoside production ability. Examples of such an enzyme include purine nucleoside phosphorylase.

  Purine nucleotides biosynthesized from PRPP by enzymes involved in purine biosynthesis are dephosphorylated and converted to purine nucleosides. In order to accumulate the purine nucleoside efficiently, it is preferable to further degrade the purine nucleoside to reduce the activity of purine nucleoside phosphorylase such as hypoxanthine. That is, it is desirable to modify so that the purine nucleoside phosphorylase using purine nucleosides such as inosine as a substrate is weakened or deleted.

  Specifically, reduction of purine nucleoside phosphorylase activity can be achieved by disrupting the deoD gene and pupG gene encoding purine nucleoside phosphorylase in Bacillus bacteria. The Bacillus bacterium of the present invention may be modified so as to destroy the deoD gene and the pupG gene as described above alone or simultaneously. As the deoD gene and pupG gene, for example, genes derived from the genus Bacillus (deoD; GenBank Accession No. NC_000964 (coding region is 2134672-2135370), pupG; GenBank Accession No. NC_000964 (coding region is 2445610-2446422) can be used.

  In addition, the activity of succinyl-AMP synthase may be decreased in order to enhance the purine substance production ability. An example of a gene encoding succinyl-AMP synthase is the purA gene. Examples of the purA gene include those having a base sequence registered under GenBank Accession No. NC — 000964 (the coding region is the complementary strand base number 4153460-4155749).

  Furthermore, the activity of inosine monophosphate (IMP) dehydrogenase may be reduced in order to enhance the ability to produce purine substances. Examples of the gene encoding IMP dehydrogenase include the guaB gene. Examples of the guaB gene include those having a base sequence registered in GenBank Accession No. NC — 000964 (coding region is 15913-17376).

 Further, as a method for enhancing the ability to produce purine substances, it is conceivable to amplify a gene that encodes a protein having an activity of discharging purine substances. Examples of the bacterium in which such a gene is amplified include a Bacillus bacterium in which the rhtA gene is amplified (Japanese Patent Laid-Open No. 2003-219876).

  When breeding the above-mentioned L-amino acid-producing bacterium by genetic recombination, the gene used is not limited to the gene having the genetic information described above or a gene having a known sequence, but a conservative variant, that is, an encoded protein As long as the function of the gene is not impaired, a gene having a conservative mutation such as a homologue or artificially modified gene of the gene can also be used. That is, it may be a gene encoding a protein having a sequence including substitution, deletion, insertion or addition of one or several amino acids at one or several positions in the amino acid sequence of a known protein. Conservative variants and the like are the same as described for the xylanase and the gene encoding it.

<5-2> Cultivation of microorganisms in the production of the target substance The sugar solution produced by the method of the present invention or a fraction thereof (hereinafter sometimes referred to as “sugar”) as a carbon source is used for the purpose. A microorganism having a substance-producing ability is cultured, and a target substance is collected from a medium or a microorganism cell.
A method similar to a normal fermentation method can be used except that the sugar is used as a carbon source.

  The method of the present invention can use any of batch culture, fed-batch culture, and continuous culture, and the sugar obtained by the present invention is contained in the initial medium. It may be contained in the feed medium or in both of them.

  Fed-batch culture refers to a culture method in which a medium is fed continuously or intermittently into a culture container and the medium is not removed from the container until the end of the culture. Continuous culture refers to a method in which a medium is fed continuously or intermittently into a culture container and the medium (usually equivalent to the medium to be fed) is extracted from the container. The initial medium means a medium (medium at the start of the culture) used for batch culture (batch culture) before feeding the fed-batch medium in fed-batch culture or continuous culture. It means a medium supplied to a fermenter when fed-batch culture or continuous culture is performed. In addition, batch culture (batch culture) means a method in which a new medium is prepared every time, a strain is planted there, and no medium is added until harvest.

  The sugar may be used at any concentration that is suitable for producing the target substance. The sugar concentration in the medium is preferably about 0.05 w / v% to 50 w / v%, more preferably about 0.1 w / v% to 40 w / v%, particularly preferably 0.2 w / v% to 20 w / v%. To be included in the medium. The sugar produced by the method of the present invention can be used alone or in combination with other carbon sources such as glucose obtained by other methods or fructose, sucrose, molasses, starch hydrolysate, etc. It can also be used. In this case, the sugar obtained by the method of the present invention and the other carbon source can be mixed at an arbitrary ratio. Preferred as other carbon sources are fructose, sucrose, lactose, galactose, molasses, starch hydrolysates, sugars such as sugar liquid obtained by hydrolysis of other biomass, alcohols such as ethanol and glycerol, Organic acids such as fumaric acid, citric acid and succinic acid.

  As the medium to be used, a medium conventionally used in the fermentation production of L-amino acids using microorganisms can be used except that the sugar obtained by the method of the present invention is used as a carbon source. That is, in addition to a carbon source, a normal medium containing a nitrogen source, inorganic ions, and other organic components as required can be used. Here, as the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium acetate, and urea, or organic nitrogen such as nitrate and soybean hydrolysate, ammonia gas, aqueous ammonia, and the like can be used. Further, peptone, yeast extract, meat extract, malt extract, corn steep liquor, soybean hydrolyzate and the like can also be used. Only 1 type of these nitrogen sources may be contained in the culture medium, and it may contain 2 or more types. These nitrogen sources can be used for both the initial medium and the fed-batch medium. In addition, the same nitrogen source may be used for both the initial culture medium and the feed medium, or a different nitrogen source from the initial culture medium may be used.

  The medium preferably contains a phosphate source and a sulfur source in addition to a carbon source and a nitrogen source. As the phosphoric acid source, phosphoric acid polymers such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate and pyrophosphoric acid can be used. The sulfur source may be any one containing sulfur atoms, but sulfates such as sulfates, thiosulfates and sulfites, and sulfur-containing amino acids such as cysteine, cystine and glutathione are desirable. However, ammonium sulfate is desirable.

  In addition to the above components, the medium may contain a growth promoting factor (a nutrient having a growth promoting effect). As the growth-promoting factor, trace metals, amino acids, vitamins, nucleic acids, peptone, casamino acid, yeast extract, soybean protein degradation products and the like containing these can be used. Examples of trace metals include iron, manganese, magnesium, calcium and the like, and examples of vitamins include vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12. These growth promoting factors may be contained in the initial culture medium or in the fed-batch medium.

  In addition, when using an auxotrophic mutant that requires an amino acid or the like for growth, the medium is preferably supplemented with the required nutrients. In particular, L-lysine-producing bacteria that can be used in the present invention have many L-lysine biosynthetic pathways as described later, and many have weakened L-lysine resolution. -It is desirable to add 1 type (s) or 2 or more types chosen from homoserine, L-isoleucine, and L-methionine. The initial medium and fed-batch medium may have the same or different medium composition. The initial culture medium and the fed-batch medium may have the same or different sulfur concentration. Furthermore, when the feeding of the feeding medium is performed in multiple stages, the composition of each feeding medium may be the same or different.

  In addition, as long as the culture medium used by this invention is a culture medium containing a carbon source, a nitrogen source, and another component as needed, any of a natural culture medium and a synthetic culture medium may be sufficient.

  Cultivation is preferably carried out for 1 to 7 days under aerobic conditions, and the culture temperature is preferably 20 ° C to 45 ° C, preferably 24 ° C to 45 ° C, particularly preferably 33 to 42 ° C. The culture is preferably aeration culture, and the oxygen concentration is preferably adjusted to 5 to 50%, desirably about 10% with respect to the saturation concentration. Further, the pH during the culture is preferably 5-9. For adjusting the pH, inorganic or organic acidic or alkaline substances such as calcium carbonate, ammonia gas, aqueous ammonia and the like can be used.

  By culturing preferably for about 10 to 120 hours under the above conditions, a significant amount of the target substance is accumulated in the culture solution.

  Moreover, when producing basic amino acids such as L-lysine, the pH during the cultivation is controlled to 6.5 to 9.0, and the pH of the medium at the end of the cultivation is set to 7.2 to 9.0. Control the pressure to be positive, or supply carbon dioxide or a mixed gas containing carbon dioxide to the medium, and there is a culture period in which bicarbonate ions and / or carbonate ions in the medium are present at least 2 g / L or more. In addition, fermentation may be performed by a method in which the bicarbonate ion and / or carbonate ion is used as a counter ion of a cation mainly composed of a basic amino acid, and production may be performed by a method of recovering the target basic amino acid (special feature). Open 2002-65287, US2002-0025564A, EP1813677A).

  Moreover, in L-glutamic acid fermentation, it can also culture | cultivate, making L-glutamic acid precipitate in a culture medium using the liquid medium adjusted to the conditions which L-glutamic acid precipitates. Examples of conditions under which L-glutamic acid precipitates include pH 5.0 to 4.0, preferably pH 4.5 to 4.0, more preferably pH 4.3 to 4.0, and particularly preferably pH 4.0. (European Patent Application Publication No. 1078989)

  Recovery of the target substance from the culture solution can usually be carried out by combining ion exchange resin method, precipitation method and other known methods. When the target substance accumulates in the microbial cells, the target substance is recovered from the supernatant obtained by crushing the microbial bodies with, for example, ultrasonic waves and removing the microbial cells by centrifugation, for example, by an ion exchange resin method. can do. When the target substance is an amino acid or a nucleic acid, the target substance to be recovered may be a free form or a salt containing a sulfate, hydrochloride, carbonate, ammonium salt, sodium salt, or potassium salt.

In addition, the target substance collected in the present invention may contain microbial cells, medium components, moisture, and microbial metabolic byproducts in addition to the target substance. The purity of the collected target substance is 50% or more, preferably 85% or more, particularly preferably 95% or more (US5,431,933, JP1214636B,
US4,956,471, US4,777,051, US4946654, US5,840358, US6,238,714, US2005 / 0025878).

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, the technical scope of this invention is not limited by this Example.

[Example 1] Isolation of xylanase gene by metagenomic cloning (1) DNA isolation Compost mainly composed of sugarcane bagasse produced by a livestock farmer in Tanegashima was collected. This bagasse compost is a mixture of sugarcane bagasse and cow dung and fermented for several months. Compost was made as appropriate on an outdoor concrete floor, and there were several bagasse composts with different fermentation periods. In bagasse compost having a fermentation period of about 6 months, the decomposition of bagasse progressed so much that no bagasse pieces could be visually confirmed. This bagasse compost was collected as a gene source for constructing a metagenomic library.

E. coli W3110 as Gram-negative bacteria, Lactobacillus murinus rat cecum-derived isolate as Gram-positive bacteria, Saccharomyces carlsbergensis CBS1513 strain as yeast, Aspergillus niger as filamentous fungus niger) JCM2261 strain cells and bagasse compost were used to study DNA extraction methods. As a DNA extraction method, a method using Blood & Cell culture DNA Midi kit (QIAGEN) or UltraClean Mega Soil DNA Isolation Kit (MO BIO Laboratories, UltraClean is a trademark of the company) and a freeze-thaw method were examined.
DNA extraction using the kits was performed according to the protocol attached to each kit. However, when using the UltraClean Mega Soil DNA Isolation Kit, 500 μl of 100 mg / ml lysozyme chloride solution (Nacalai Tesque) and 220 μl of 3 mg / ml before bead crushing treatment
DNA extraction was performed according to the protocol except that N-acetylmuramidase (Mutanolysin from Streptomyces globisporus ATCC 21553 (Sigma-Aldrich Co.)) solution was added and lysis pretreatment was performed at 37 ° C. for 30 minutes. In addition, the protocol described a method of stirring vigorously during the lysis operation and a method of shaking slowly, so both methods were examined.

  The freeze-thaw method was performed by the following method. Add lysozyme chloride (Nacalai Tesque) and sodium dodecyl sulfate to suspension of bacterial cells or sample in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to final concentrations of 5 mg / ml and 50 mg / ml, respectively. Then, after reacting at 37 ° C for 1 hour, the plastic tube containing the reaction solution is immersed in ethanol that has been cooled in a -80 ° C freezer and placed in the -80 ° C freezer for 30 minutes, and then set to 65 ° C. Freezing and thawing by immersing in a water bath for 30 minutes was repeated three times. After freeze-thawing, protease K (QIAGEN) was added to a final concentration of 1 mg / ml and reacted at 60 ° C. for 1 hour. Then add the same amount of TE-saturated phenol: chloroform: isoamyl alcohol (25: 24: 1) as the reaction solution, mix gently for 30 minutes, and then centrifuge (6000 × g, 20 minutes) to collect the supernatant. This operation was repeated three times. Subsequently, an equal volume of chloroform / isoamyl alcohol (24: 1) to the recovered supernatant was added, and after gently mixing for 20 minutes, the supernatant was recovered by centrifugation (6000 × g, 20 minutes). Add sodium acetate (Nacalai Tesque) solution to the collected supernatant to a final concentration of 0.3M, add 2.5 times the amount of pre-cooled ethanol and mix gently, then centrifuge (9400 × g, 30 minutes) The supernatant was removed and the DNA precipitate was collected. 70% ethanol that had been cooled in advance was added to the collected DNA and mixed well, then centrifuged (9400 × g, 30 minutes) to remove the supernatant, and the DNA precipitate was washed. The precipitate was air-dried at room temperature and then dissolved in TE solution. The extracted total DNA solution was subjected to pulse field gel electrophoresis, and after completion of the electrophoresis, a gel containing a DNA fragment in the vicinity of 20 kbp was cut out. For pulse field gel electrophoresis, CHEF-DR III System (Bio-Rad Laboratories, Inc.) is used, 1% agarose gel (Lonza Rockland, Inc., Seakem GTG agarose) is used in 0.5 × TBE buffer. Electrophoresis was performed under the conditions of a buffer temperature of 14 ° C., an angle of 120 °, a switch time of 1-30 seconds, and a voltage of 6 V / cm.

  DNA extraction using the Blood & Cell culture DNA Midi kit had lower DNA extraction efficiency than other methods. In addition, it was determined that the Gram-negative bacterium had significantly higher DNA extraction efficiency than the other microorganisms in the freeze-thaw method, and it was difficult to extract the microbial DNA present in the sample without bias. When using the UltraClean Mega Soil DNA Isolation Kit, the variability of DNA extraction effects by microbial species is relatively small, and the efficiency of DNA extraction from other microbial species excluding Gram-negative bacteria and bagasse compost is almost the same as the freeze-thaw method Met. In addition, in DNA extraction from bagasse compost, when the mixture was vigorously stirred during the lysis operation, the extracted DNA was fragmented and the preferred size DNA fragment (20-40 kbp) was not obtained. Since the extracted DNA was not fragmented and a DNA fragment of a preferred size was obtained, this method was used as the DNA extraction method. To construct a metagenomic library, 8 ml of DNA extract was obtained by extracting DNA from 10 g of bagasse compost using the UltraClean Mega Soil DNA Isolation Kit as described above. Add 0.32ml 5M NaCl solution and 16.6ml ethanol to the DNA extract, perform ethanol precipitation, air-dry and then re-dissolve in 0.2ml 10mM TE buffer (pH 8.0) to concentrate the DNA at -20 ℃ Saved with.

(2) Purification of DNA and construction of a DNA library In order to investigate the effects of enzyme reaction inhibitors that are presumed to be mixed in DNA solution extracted from bagasse compost, a consensus sequence primer set (JCM 27f [SEQ ID NO: 28] and JCM 1492R [SEQ ID NO: 29]) PCR reaction (96 ° C. 10 seconds, [96 ° C. 30 seconds, 50 ° C. 30 seconds, 72 ° C. 2 minutes] 30 cycles, 72 ° C. 5 Minutes 30 seconds). ExTaq DNA polymerase (TAKARA) was used for PCR reaction, H ₂ O 22.9 μl, 10 × buffer 3 μl, dNTPs 2.4 μl, ExTaq DNA polymerase 0.2 μl, 20 pmol / μl primer 0.5 μl, template DNA solution 0.5 μl, total amount PCR reaction was performed with a reaction solution composition of 30 μl. As a result of the PCR reaction, no PCR product of 16S rRNA gene was obtained, so there was concern that the enzyme reaction inhibitor contained in the PCR would affect the library construction. Therefore, for the purpose of removing various enzyme reaction inhibitors in the operation of DNA library construction, DNA was electrophoresed and DNA was recovered from the gel after electrophoresis. The total DNA solution isolated from bagasse compost was subjected to pulse field gel electrophoresis, and a gel containing a DNA fragment in the vicinity of 20 kbp was cut out after completion of the electrophoresis. For pulse field gel electrophoresis, CHEF-DR III System (Bio-Rad Laboratories, Inc.) is used, 1% agarose gel (Lonza Rockland, Inc., Seakem GTG agarose) is used in 0.5 × TBE buffer. Electrotransfer was performed under the conditions of a buffer temperature of 14 ° C., an angle of 120 °, a switch time of 1-30 seconds, and a voltage of 6 V / cm.

  Put the excised gel piece into a dialysis tube (Funakoshi Co., Ltd., Spectra / Pore CE dialysis tube) filled with 20 ml of 0.25 × TBE buffer, and apply voltage in a minigel electrophoresis tank filled with 0.25 × TBE buffer. The DNA fragment was recovered from the gel in 0.25 × TBE buffer in the dialysis membrane. Collect 0.25 × TBE buffer containing DNA fragments, add 20 ml phenol / chloroform solution [TE saturated phenol: chloroform: isoamyl alcohol = 25: 24: 1] and infiltrate slowly for 30 minutes, then centrifuge (9400 × g, 20 And the supernatant was collected. A 20 ml chloroform solution [chloroform: isoamyl alcohol = 24: 1] was added to the collected supernatant, slowly permeated for 30 minutes, and then centrifuged (9400 × g, 20 minutes), and the supernatant was collected again. To the collected supernatant, 1.9 ml of 3M sodium acetate and 15.2 ml of isopropanol were added to perform alcohol precipitation, and the DNA fragment was collected. PCR was performed on the 16s rRNA gene using the recovered DNA as a template by the same method as described above. As a result, an amplification product was obtained, and removal of the enzyme reaction inhibitor was confirmed. Using the recovered DNA as a template, the CopyControl Fosmid Library Production Kit (EPICENTRE Biotechnologies. CopyControl is a trademark of the company) is used to construct a bagasse compost DNA library of the recovered DNA fragments according to the protocol attached to the kit. Stored as a glycerol solution at -80 ° C.

(3) Expression screening Screening of target genes was performed using xylan degradation activity as an index. E. coli having the above bagasse compost DNA library was added to LB agar medium (tryptone 10.0 g / L, Bacto-yeast extract 5.0 g / L, NaCl 10.0 g / L, supplemented with 12.5 μg / ml chloramphenicol. , Agar 20 g / L) (hereinafter referred to as “LBcm agar medium”), and after overnight culture at 37 ° C., the emerged colonies were 0.6 μm using a nylon membrane (Amersham, Hybribond-N + membrane). The LBcm agar medium containing% xylan (Sigma-Aldrich Co.) was copied and cultured overnight at 37 ° C. After confirming the appearance of colonies, add 300 μl of 10-fold diluted Induction solution (included in CopyControl ^™ Fosmid Library Production Kit) to the agar medium, and again incubate at 37 ° C for 1 week. The presence or absence of halo formation by xylan decomposition was observed, and the fungus was sequentially used from the colonies where halo formation was confirmed. The purified E. coli clones were confirmed to form halos on LBcm agar medium containing 0.6% xylan after purification.

(4) Identification of xylanase gene A fosmid vector was extracted from an E. coli strain having a bagasse compost fosmid that exhibits xylan-degrading activity using the QIAGEN Plasmid Kit (QIAGEN GmbH). The extracted fosmid vector was digested with BamHI, HindIII, KpnI, SalI, or Sau3AI (Takara Bio Inc.) according to the protocol attached to each enzyme, and the digested fragment was then converted into a pUC18 vector (Takara Bio Inc.). Subcloned and screened for E. coli clones showing xylan degradation on LB agar medium containing 0.6% xylan and 100 μg / ml ampicillin. Plasmids were prepared from clones showing xylan degradation, and total nucleotide sequencing was entrusted to Takara Bio Inc. Based on the determined base sequence information, Genetyx version 6.1.2 (GENETYX CORPORATION) found an open reading frame (ORF), translated it into an amino acid sequence, and then performed homology search using a protein database. 8 kinds of base sequences (including SEQ ID NOs: 19, 21, and 23) were determined. There was no known amino acid sequence that matched these translated amino acid sequences, and it was considered to be a novel xylanase gene sequence. Table 1 shows the homology between these translated amino acid sequences and the homology (%) between these amino acid sequences and known sequences.

  Eight kinds of base sequences presumed to encode xylanase are Xyn10-1, Xyn10-2, Xyn11-1 (SEQ ID NO: 19), Xyn11-2 (SEQ ID NO: 21), Xyn11-3, Xyn11-4, and Xyn11-5. (SEQ ID NO: 23), named Xyn11-6. Clones E. coli JM109 / Xyn11-1, E. coli JM109 / Xyn11-2, and E. coli JM109 / Xyn11-5 expressing xylanases of Xyn11-1, Xyn11-2, and Xyn11-5 are AJ110764, They were named AJ110765 and AJ110769. AJ110764 and AJ110765 on January 29, 2010, AJ110769 on March 11, 2010, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (Higashi 1-chome, Tsukuba City, Ibaraki Prefecture, 305-8566, Japan) No. 1 1 Central No. 6), each with the deposit numbers FERM P-21897, FERM P-21898, and FERM P-21939, transferred to the international deposit under the Budapest Treaty on 25 January 2011, The accession numbers of FERM BP-11325, FERM BP-11326, and FERM BP-11327 are assigned respectively.

(5) Escherichia coli TA cloning of the xylanase gene Based on the xylanase gene sequence determined as described above, a DNA primer for amplifying the DNA region of the ORF is prepared, and the diluted solution of the subclone DNA is used as a template for KOD-plus-ver. PCR was performed according to the standard protocol of 2 (TOYOBO CO., LTD.). DNA primers that amplify the DNA regions of Xyn11-1 (SEQ ID NO: 19), Xyn11-2 (SEQ ID NO: 21), and Xyn11-5 (SEQ ID NO: 23) are described in SEQ ID NOs: 1-6. The PCR product was subjected to agarose gel electrophoresis, and a band of the expected molecular weight was cut out and purified according to the standard protocol of QIAEX II Gel Extraction Kit (QIAGEN GmbH). Using TaKaRa Ex Taq (Takara Bio Inc.), dA was added to the purified PCR product, and ligation with T4 ligase was performed according to the standard protocol of pGEM-T Easy vector (Promega Corporation). The ligation reaction was 16 hours at 4 ° C. Competent high JM109 (TOYOBO CO., LTD.) Was transformed according to a conventional method using 5 μL of the obtained ligation mixture. The resulting transformants were purified and examined for halo formation on LB agar medium containing 0.6% xylan and 50 μg / ml ampicillin. Six ORFs except Xyn11-3 and Xyn11-6 were cloned. Halo formation was confirmed in the transformant. The subclone DNA fragments that found Xyn11-3 and Xyn11-6 also contain ORFs that are expected to encode other xylanases, so the halo formation of the subclones is not Xyn11-3 and Xyn11-6. It was thought to be due to these different ORFs. Therefore, Xyn11-3 and Xyn11-6 were considered to be genes that cannot be expressed or functioned in E. coli.

(6) Expression system construction by Corynebacterium glutamicum C. glutamicum was selected as a host for expressing each of the above xylanase genes. A characteristic of the protein secretion system of C. glutamicum is that there is almost no secreted protein and the expressed heterologous protein accumulates in the medium with high purity. The protein secretion pathway includes the general secretion pathway (Sec system) in which proteins are folded outside the cell (see WO01 / 23591) and the twin-arginine translocation pathway (Tat system) in which proteins are folded and secreted in the cell (WO2005). / 103278) is known, and since which system works well depending on the target gene, both secretion systems were constructed.

  The amino acid sequence of each xylanase was input to SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/), and signal peptide prediction was performed. As a result, it was predicted that the 55th and 56th amino acid residues were cleaved from the N-terminal amino acid residue of Xyn11-5 (SEQ ID NO: 24). Each DNA fragment was ligated so as to be ligated to the Sec and Tat signal peptides in the expression vector. Since Xyn11-1 (SEQ ID NO: 20) and Xyn11-2 (SEQ ID NO: 22) were not expected to have a signal sequence, the first amino acid residue and after were linked to Sec and Tat signal peptides. In other xylanases, signal sequences were predicted in Xyn10-1, Xyn10-2, Xyn11-4 and Xyn11-6 in the same manner as Xyn11-5, so the sequence after the cleavage site was linked to the Sec and Tat signal peptides. It was.

C. glutamicum YDK010 strain was used as an expression host for the Sec system. The strain is a disrupted strain of the gene encoding a cell surface protein of YS ^r strain is C. glutamicum mutant (PS2) (International Publication Pamphlet WO01 / twenty-three thousand five hundred ninety-one described). On the other hand, as an expression host of the Tat system, C. glutamicum
WDK010 strain was used. This strain is a cell surface protein (PS2) gene disruption strain derived from C. glutamicum 2256 strain (ATCC 13869).

WDK010 was built by the following process.
Genomic DNA was extracted from C. glutamicum (Brevibacterium lactofermentum) AJ12036 strain described in WO02 / 081694. The AJ12036 strain is a strain obtained by removing the originally present plasmid pAM330 from the C. glutamicum 2256 strain. Using the genomic DNA of this AJ12036 strain as a template, PCR amplification was performed using primer YSrpsL5 (5'-AGGTCAGTGGCGAGTTTCTT-3 ') (SEQ ID NO: 26) and primer YSrpsL3 (5'-GGTAGGTAGCGCCACCAACA-3') (SEQ ID NO: 27) A 1 kbp fragment containing a part of the gene encoding PS2 was obtained. PCR conditions are as follows.

Pyrobest DNA polymerase (TAKARA) is used.
H ₂ O 37.8 μl, PCR buffer 5 μl, dNTPs 4 μl, Pyrobest DNA polymerase 0.2 μl, 10 μM each primer 1 μl, 100 ng / μl DNA (template) 1 μl, total volume 50 μl
<PCR reaction conditions>
Cycle 1 (x1) 98 ° C; 5 minutes
Cycle 2 (x30) 98 ℃; 10 seconds, 57 ℃; 0.5 minutes, 72 ℃; 1 minute

  Using this DNA fragment and the C. glutamicum ATCC13869 strain, a gene replacement strain was prepared in the same manner as described in Example 9 of WO02 / 081694. It was confirmed that the obtained gene-substituted strain grew on a CM2G agar medium containing 100 mg / L streptomycin. Further, using this gene replacement strain, a cell surface protein (PS2) gene complete disruption strain was prepared in the same manner as described in Example 9 of WO02 / 081694, and this strain was named WDK010 strain.

  The expression plasmid was constructed according to the method described in App. Env. Micro., 2003, 69, 358-366. For the Sec system, the plasmid pPSPTG1 described in App. Env. Micro., 2003, 69, 358-366 is used as a template for PCR amplification of the region encoding the CspB promoter and signal peptide (SlpA signal, derived from Corynebacterium ammoniagenes). DNA primers (SEQ ID NOs: 7, 8) were designed. For the Tat system, using the plasmid pPTPPG described in Appl Microbiol Biotechnol., 2008, 78: 67-74 as a template, a primer for PCR amplification of the region encoding the CspB promoter and signal peptide (TorA signal, derived from Escherichia coli) (SEQ ID NO: 7, 9) designed. In both the Sec expression system and the Tat expression system, a BamHI site was added upstream of the promoter sequence in the amplified DNA fragment.

  Regarding the DNA fragment of each xylanase gene, Xyn11-1 (SEQ ID NO: 19) and Xyn11-2 (SEQ ID NO: 21) are the first amino acid residue, Xyn11-5 (SEQ ID NO: 19), using the plasmid extracted from the above TA clone as a template. SEQ ID NO: 23) was obtained by PCR using a primer (SEQ ID NOs: 10 to 18) that amplifies from the 56th amino acid residue to the amino acid residue immediately before the stop codon. At that time, primers were designed so that 20 bases having homology with Sec and Tat signal sequences were added to the 5 ′ end of the primer, and an XbaI site was added downstream of each stop codon. As for other xylanase genes, the first amino acid is related to Xyn11-3 for which no signal sequence was predicted, and the amino acids below the cleavage site for predicted Xyn10-1, Xyn10-2, Xyn11-4 and Xyn11-6. Primers that amplify up to the amino acid before the stop codon were designed.

  Regarding the DNA fragment of each xylanase gene, Xyn11-1 (SEQ ID NO: 19) and Xyn11-2 (SEQ ID NO: 21) are the first amino acid residue, Xyn11-5 (SEQ ID NO: 19), using the plasmid extracted from the above TA clone as a template. SEQ ID NO: 23) was obtained by PCR using a primer (SEQ ID NOs: 10 to 18) that amplifies from the 56th amino acid residue to the amino acid residue immediately before the stop codon. At that time, primers were designed so that 20 bases having homology with Sec and Tat signal sequences were added to the 5 ′ end of the primer, and an XbaI site was added downstream of each stop codon.

PCR was performed according to the standard protocol of KOD-plus-ver.2 (TOYOBO CO., LTD.). The PCR product of the promoter and signal peptide was purified according to the standard protocol of AMpure (Beckman Coulter, Inc.). The PCR product was subjected to agarose gel electrophoresis, and a band of the expected molecular weight was cut out and purified according to the standard protocol of QIAEX II Gel Extraction Kit (QIAGEN GmbH).
Next, the PCR product of the promoter and signal peptide and the PCR product of the obtained xylanase gene mixed at 1: 1 (volume ratio) as a template, the primer corresponding to the promoter sequence (SEQ ID NO: 7) and the stop codon Primer (SEQ ID NO: 16 [corresponds to Xyn11-1 (SEQ ID NO: 19)], SEQ ID NO: 17 [corresponds to Xyn11-2 (SEQ ID NO: 21)], SEQ ID NO: 18 [corresponds to Xyn11-5 (SEQ ID NO: 23)], etc. ) Was used for crossover PCR. Thereby, the DNA fragment encoding the promoter, the signal sequence, and the xylanase was amplified.

This crossover PCR product is digested with restriction enzymes BamHI and XbaI, then agarose gel electrophoresis is performed, a DNA band of the desired molecular weight is excised, and QIAEX II Gel Extraction
Purified according to the standard protocol of Kit (QIAGEN GmbH). These DNA fragments were inserted into the BamHI-XbaI site of pPK4, a Corynebacterium-E. Coli shuttle vector described in JP-A-9-322774 (EP0841395), to obtain Sec and Tat obtained xylanase secretion expression plasmids. These plasmids were introduced into the YDK010 strain by the Sec system and the WDK010 strain by the electroporation method, and the CM2G agar medium containing 25 mg / l kanamycin (yeast extract 10 g, tryptone 10 g, glucose 5 g, A strain was selected that grew on 5 g NaCl, 15 g agar, adjusted to pH 7.2 with 1 L water.

The selected strain was inoculated into CM2G liquid medium (yeast extract 10 g, tryptone 10 g, glucose 5 g, NaCl 5 g, adjusted to pH 7.2 with 1 L with water and adjusted to pH 7.2), and pre-cultured overnight at 30 ° C. MMTG liquid medium (glucose 120g, magnesium sulfate heptahydrate 3g, ammonium sulfate 30g, potassium dihydrogen phosphate 1.5g, iron sulfate heptahydrate 0.03g, manganese sulfate tetrahydrate 0.03g, thiamine hydrochloride 0.45mg, Biotin 0.45mg, DL-methionine 0.15g, calcium carbonate 50g, 1L with water and adjusted to pH 7.0) 25mg / l kanamycin is added, 4ml is put into a large test tube, and 5% of the pre-culture solution is added. Inoculated at a rate of (v / v) and cultured at 30 ° C. for 72 hours.
The culture supernatant after completion of the culture was dropped onto a xylan-containing agarose plate and halo was formed to confirm xylan decomposition activity. As a result, Xyn11-3 and Xyn11 whose activity could not be confirmed with the E. coli clone described in (5) In the case of -6, the activity could not be confirmed even in the coryneform bacterium expression system. Even in the case of Xyn10-2, whose activity was confirmed in the E. coli clone, the activity in the coryneform bacterial expression system could not be confirmed. Xyn11-1 (SEQ ID NO: 19), Xyn11-2 (SEQ ID NO: 21), Xyn11-5 (SEQ ID NO: 23), Xyn10-1, and Xyn11-4 whose activity was confirmed were analyzed on SDS-PAGE. It was confirmed that there was protein expression at the expected molecular weight. From the enzyme activity and protein expression level, Xyn11-1 selected the Tat system, and Xyn11-2, Xyn11-5, Xyn10-1, and Xyn11-4 selected the Sec system.

[Example 2] Saccharification of bagasse with xylanase In order to evaluate the action of each xylanase to assist saccharification of sugarcane bagasse, the following effect test for improving the saccharification ability of bagasse was conducted.
Bagasse was obtained from Tanegashima, and bagasse pulverization was performed under the following conditions.

Drying: Sun drying Grinding contractor: Nara Machinery Co., Ltd. Grinding machine: Nara type free crusher M-4 type Grinding condition: Rotational speed = 5500 / min, Screen: Dia screen 0.9mm
Sieveing: 0.15 mm and 0.355 mm mesh sieves were used, and 0.15-0.355 mm bagasse was collected and used for the test.

  C. glutamicum culture supernatant prepared by the method described in Example 1 was concentrated 10-fold using a 10 kDa cut-out ultrafiltration membrane Vivaspin 20 (Sartorium Stedim Biotech GmbH), and 50 mM sodium acetate buffer (pH 5.0). ). As positive controls, xylanase preparations NS50030 (Xyn30) and NS50014 (Xyn14) included in Novozymes biomass kit (Novozymes A / S) were introduced, while 50 mM sodium acetate buffer (pH 5.0) and pPK4 were introduced as negative controls. The YDK010 culture supernatant was used for evaluation. The sample was evaluated at N = 3.

  The amount of protein in each sample was quantified according to the standard protocol of Protein Assay CBB Solution (Nacalai Tesque). In addition, using ImageJ (http://rsb.info.nih.gov/ij/), the ratio of the target band to the total protein in the SDS-PAGE image was calculated and used to calculate the enzyme concentration. 0.143g / ml Celluclast (Novozymes A / S) and 0.033g / ml Novozyme188 (Novozymes A / S) as cellulase mix and 5mg / ml Geneticine as antibiotics dissolved in 50mM sodium acetate buffer (pH5.0) Prepared.

  To a glass vial with a butyl rubber stopper, 0.1 g dry bagasse and 4 ml of 50 mM sodium acetate buffer (pH 5.0) or 50 mM phosphate buffer (pH 8.0) were added, and autoclaved at 121 ° C for 20 minutes. 0.5 ml of cellulase mix solution sterilized using 22 PES filters MILLEX GP (Millipore) and 0.5 ml of the test sample were added, and saccharification reaction was carried out for 4 days while gently shaking at 50 ° C. The enzyme amount was adjusted to 65 mg to 150 mg per 5 mL reaction system. After completion of the reaction, the supernatant of the reaction solution was collected by centrifugation (14000 rpm, 10 min), and the glucose concentration was measured by Biotech Analyzer AS310 (Sakura Seye Co., Ltd.). A reaction system in which bagasse was not added was placed in all test sections, and the value obtained by subtracting the amount of glucose as a blank was used as the amount of free glucose derived from bagasse, and the ratio of the amount of free glucose to the amount of glucose containing bagasse was determined as the saccharification rate. In addition, when 50 mM phosphate buffer (pH 8.0) was added, the final pH of the sample was around 7, so the bagasse saccharification test was performed under pH 7.0 conditions.

The concentration of glucose produced as a result of the saccharification reaction in the xylanase-free group (50 mM sodium acetate buffer (denoted as “NC” in the figure)) was used as a reference. And the relative ratio of the saccharification rate of each sample when the saccharification rate at this time is set to 1, and those averaged are shown in FIG. 1 (pH 5) and FIG. 2 (pH 7). When NS50014 (Xyn14) (denoted as “X14” in the figure) and NS50030 (Xyn30) (denoted as “X30” in the figure) are added, the average of the test data for three times, N = 9 , Xyn11-1, Xyn11-2, Xyn11-5 (each indicated as "X11-1", "X11-2", "X11-5" in the figure) is the average of the test data for two times, N = 6. The standard deviation is indicated by an error bar.
As a result, under the condition of pH 7, the addition of the xylanase of the present invention showed a higher saccharification rate than the addition of the commercially available xylanase, and the effect of assisting the saccharification reaction of cellulase was clear. Further, it was found that the pH 5 condition tends to promote the saccharification although it is smaller than the pH 7. Similar tests were performed on other xylanases (Xyn10-1 and Xyn11-4), but none of them showed a significant difference from commercially available xylanases.

[Example 3] Evaluation of enzymatic properties of xylanase In order to evaluate the enzymatic properties of each xylanase, the following tests were conducted.
(1) Optimum reaction pH
A 1% (w / v) substrate solution was prepared from xylan from birch (Xylan from Birchwood, Sigma) and citrate-phosphate buffer (pH 3.5-8.0, pH 0.5 increments), and xylan was autoclaved. Was well dispersed in the solution.

  As an enzyme solution, C. glutamicum culture supernatant prepared by the method described in Example 1 was concentrated 10 times using a 10 kDa cut-out ultrafiltration membrane Vivaspin 20 (Sartorium Stedim Biotech GmbH), and 50 mM sodium acetate. The one substituted with buffer (pH 5.0) was used.

  Enzymatic reaction was carried out according to the method of Berrin et al. (Protein Expression and Purification, 19, 179-187, 2000). Mixing 180μL of 1% substrate solution and 20μL of enzyme solution in a microplate and using a thermal cycler at 50 ℃ for 5 minutes Reacted. The reaction was performed at N = 3 per enzyme for each pH. 50 μL of the solution after the reaction was taken, mixed with 100 μL of 1% DNS solution, and reacted at 95 ° C. for 5 minutes. The 1% DNS solution refers to a solution composed of 1% dinitrosalicylic acid, 0.05% sodium sulfite, 1% sodium hydroxide, 20% potassium sodium tartrate, and this DNS solution is generally used for reducing sugar content. Used.

  The absorbance (540 nm) of the reaction solution was measured, and the amount of produced xylooligosaccharide was calculated from a calibration curve prepared with a xylose solution. As a result of prior verification, no saccharide contained in the enzyme solution was detected, but a reducing sugar that was considered to be an acid hydrolysis product was detected particularly in the acidic region in the substrate solution. Therefore, blanks were prepared by adding distilled water instead of the enzyme solution to all pH substrates, and the value obtained by subtracting the background sugar derived from the substrate solution was used as the amount of produced sugar. The amount of sugar produced at each pH when the sample showing the maximum amount of sugar produced was taken as 1 was determined as specific activity.

  A graph of the optimum reaction pH is shown in FIG. Each new xylanase showed a similar optimum pH pattern, and the optimum pH was around 7.0. On the other hand, the optimum pH of commercially available xylanase Xyn14 (Novozymes, NS50014) was around 6.5.

(2) Optimal reaction temperature
A 1% (w / v) substrate solution was prepared using Xylan from Birchwood (Sigma) and citrate-phosphate buffer (pH 7.0), and xylan was well dispersed in the solution by autoclaving. The enzyme solution was prepared by the method described in the previous section. Enzymatic reaction is performed according to the method described in the previous section. 90 μL of 1% substrate solution and 10 μL of enzyme solution diluted appropriately with distilled water are mixed in a microplate, and 35 to 85 ° C (in 5 ° C increments) using a thermal cycler. For 5 minutes. The reaction was performed at N = 3 per enzyme for each temperature (N = 2 only for the commercially available enzyme Xyn14 as a control). 50 μL of the solution after the reaction was taken and mixed with 100 μL of the DNS solution, color reaction was carried out at 95 ° C. for 5 minutes, and the absorbance (540 nm) of the reaction solution was measured. Blanks were prepared by adding distilled water instead of the enzyme solution for all temperature samples, and the value obtained by subtracting the background value derived from the substrate solution was used as the amount of sugar produced. The amount of sugar produced at each temperature when the sample showing the maximum amount of sugar produced was taken as 1 was shown as the specific activity.
A graph of the optimum reaction temperature is shown in FIG. Xyn11-1 showed the highest activity at 65 ° C, Xyn11-2 and the commercially available enzyme Xyn14 at 70 ° C, and Xyn11-5 at 75 ° C.

(3) Temperature stability in a short time The enzyme solution prepared by the method described in the previous section was diluted with citrate-phosphate buffer (pH 7.0), and it was confirmed that the pH was 7. The enzyme solution was reacted at 50 to 75 ° C. (in increments of 5 ° C.) for 10 minutes to obtain a heat-treated enzyme. 90 μL of 1% substrate solution (pH 7.0) and 10 μL of heat-treated enzyme solution or non-heat-treated enzyme solution were mixed in a microplate and reacted at 50 ° C. for 5 minutes using a thermal cycler. The reaction was performed at N = 3 per enzyme for each heat treatment temperature. 50 μL of the solution after the reaction was taken and mixed with 100 μL of the DNS solution, color reaction was carried out at 95 ° C. for 5 minutes, and the absorbance (540 nm) of the reaction solution was measured. For all temperature samples, a blank was prepared by adding the buffer used for dilution instead of the enzyme solution, and the value obtained by subtracting the background value derived from the substrate solution was defined as the amount of produced sugar. The amount of sugar produced at each heat treatment temperature when the average value of the sample to which the non-heat treatment enzyme solution was added was assumed to be 1 as the specific activity. The commercial enzyme Xyn14 was examined at 35, 50, 70, and 80 ° C.
A graph of temperature stability in a short time is shown in FIG. The new enzyme Xyn11-2 and the commercially available enzyme Xyn14 showed almost the same activity profile. On the other hand, Xyn11-5 retained a relatively high activity even when treated at high temperature, and retained 78% activity even when treated at 75 ° C. for 10 minutes.

(4) Long-term temperature stability Since the saccharification reaction of bagasse is a long-time reaction performed at 50 ° C for 4 days, the temperature stability against heat treatment for a longer time was examined. In the method described in the previous section, the heat treatment time of the enzyme solution was extended from 10 minutes to 16 hours, and the other conditions were tested in the same manner as in the previous section. The amount of sugar produced at each heat treatment temperature when the average value of the sample to which the specific heat treatment enzyme solution was added was taken as 1 was shown as the specific activity.

  The temperature stability over a long time is shown in FIG. Even after treatment at 50 ° C. for 16 hours, the novel enzyme retained 86-97% activity, whereas the commercially available enzyme decreased to 69%. In particular, Xyn11-5 retained a relatively high activity even when treated at a high temperature, and retained 61% activity even when treated at 70 ° C. for 16 hours. The fact that the novel xylanase retained higher activity than the commercially available enzyme Xyn14 in the treatment at 50 ° C. for 16 hours supports the result of the bagasse saccharification ability improving effect shown in Example 2. Since the process of saccharifying bagasse is a long-time reaction, it is an industrially effective property that the temperature stability of the new enzyme is maintained high for a long time.

Claims (19)

DNA encoding the following protein (A) or (B):
(A) a protein having an amino acid sequence of SEQ ID NO: 20, 22, or 25 or an amino acid sequence consisting of at least positions 56 to 455 of SEQ ID NO: 24 and having xylanase activity;
(B) an amino acid sequence of SEQ ID NO: 20, 22, or 25, or an amino acid sequence consisting of at least positions 56 to 455 of SEQ ID NO: 24, wherein one or several amino acids are substituted, deleted, inserted or added A protein having a sequence and having xylanase activity.   The DNA according to claim 1, wherein the amino acid sequence consisting of at least positions 56 to 455 of SEQ ID NO: 24 is the amino acid sequence of SEQ ID NO: 24. The DNA according to claim 1, which is the following DNA (a) or (b):
(A) a DNA having a base sequence of SEQ ID NO: 19 or 21, or a base sequence consisting of at least positions 166 to 1368 of SEQ ID NO: 23;
(B) DNA encoding a protein having 90% or more homology with the nucleotide sequence of SEQ ID NO: 19 or 21, or the nucleotide sequence consisting of at least positions 166 to 1368 of SEQ ID NO: 23 and having xylanase activity.   The DNA according to claim 3, wherein the base sequence consisting of at least positions 166 to 1368 of SEQ ID NO: 23 is the base sequence of SEQ ID NO: 23. The following protein (A) or (B):
(A) a protein having an amino acid sequence of SEQ ID NO: 20, 22, or 25, or an amino acid sequence consisting of at least positions 56 to 455 of SEQ ID NO: 24 and having xylanase activity;
(B) an amino acid sequence of SEQ ID NO: 20, 22, or 25, or an amino acid sequence consisting of at least positions 56 to 455 of SEQ ID NO: 24, wherein one or several amino acids are substituted, deleted, inserted or added A protein having a sequence and having xylanase activity.   The protein according to claim 5, wherein the amino acid sequence consisting of at least positions 56 to 455 of SEQ ID NO: 24 is the amino acid sequence of SEQ ID NO: 24.   A fusion protein in which the protein according to claim 5 or 6 is bound to a peptide selected from a marker peptide, a peptide tag, a secretion signal peptide, and a part thereof.   DNA encoding the protein according to claim 7.   A recombinant vector comprising the DNA according to any one of claims 1 to 4 and claim 8.   A transformed cell comprising the recombinant vector of claim 9.   A method for producing xylanase, comprising culturing the transformed cell according to claim 10 in a medium and collecting a protein having xylanase activity from the obtained culture.   The recombinant vector according to claim 7, wherein the recombinant vector is capable of expressing a fusion protein containing a secretion signal, and the transformed cell has an ability to secrete the fusion protein, and a xylanase The method for producing a recombinant protein according to claim 11, wherein the recombinant protein having activity is collected from the culture supernatant.   A method for producing a sugar solution, wherein the protein according to claim 5 or 6, or the fusion protein according to claim 7 and cellulase are reacted with a biomass resource containing xylan to produce sugar.   The method according to claim 13, wherein the reaction is performed at 50 to 75 ° C.   15. A target substance obtained by culturing a microorganism producing a target substance in a medium containing the sugar solution produced by the method according to claim 13 or 14 or a fraction thereof as a carbon source, and collecting the target substance from the culture. Manufacturing method.   The method according to claim 15, wherein the microorganism is a bacterium selected from microorganisms belonging to Corynebacterium glutamicum and Escherichia coli.   The method according to claim 15 or 16, wherein the target substance is an L-amino acid.   A method for decomposing biomass resources, wherein the protein according to claim 5 or 6 or the fusion protein according to claim 7 is reacted with a biomass resource containing xylan.   The method according to claim 18, wherein the reaction is performed at 50 to 75 ° C.

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