JP6221478B2 - Method for producing organic compound using microorganism having pentose utilization ability - Google Patents

Description

  The present invention relates to a pentose utilization technique, and relates to a technique for improving the pentose assimilation ability of microorganisms and an efficient organic compound production technique using the microorganisms.

Fermentative production processes of organic compounds using saccharide-based biomass as raw materials are widely used, and products obtained through the processes are used as various industrial raw materials.
Examples of carbohydrate biomass used as raw materials for these fermentation production processes include those derived from edible raw materials such as sugarcane, starch, sugar beet, corn, potato, cassava, and sugar maple.

  However, these edible material-derived sugars have concerns that the price of edible materials will rise due to the future increase in the world population, and that the supply of edible materials will be insufficient due to bad weather and climate change. There are also concerns such as ethical criticism of using edible ingredients as industrial ingredients in competition with edible uses in the event of food shortages. Therefore, the use of cellulosic biomass derived from non-edible raw materials without such concerns has attracted attention.

Cellulosic biomass is a raw material that can be obtained in a wide variety and at low cost as agricultural waste and woody waste, and does not hinder future food security.
Cellulose biomass is composed of cellulose, hemicellulose, lignin, and the like, and cellulose is mainly composed of glucose (hexose), and hemicellulose is mainly composed of pentose such as xylose and arabinose. In order to efficiently fermentatively produce organic compounds using cellulosic biomass as a raw material, highly efficient pentose utilization technology is required.

Patent Document 1 describes that by using a coryneform bacterium into which an arabinose transporter AraE is introduced, the xylose consumption rate is improved and the simultaneous use with glucose is also improved.
Non-Patent Document 1 describes that in coryneform bacteria, succinic acid production can be improved by modifying the glucose uptake pathway to a pathway via inositol transporters IolT1 and IolT2.

International Publication WO2009 / 154122 Pamphlet

Bioeng Bugs. , 2011, Sep-Oct 2 (5), p291-5

However, although the method described in Patent Document 1 improves the xylose consumption rate by introducing the arabinose transporter AraE and greatly improves the production efficiency in the case of simultaneous use with glucose, the growth of microorganisms used for production is improved. It has been clarified by examination of the present inventors that it has a defect of worsening.
In addition, although the method described in Non-Patent Document 1 describes that succinic acid production can be improved by incorporating glucose through a route via inositol transporters IolT1 and IolT2, pentose is used as a carbon source. There is no mention of what to do.

  The object of the present invention is to improve the pentose assimilation ability of microorganisms without deteriorating the growth of microorganisms, and more efficiently produce organic compounds from organic raw materials containing pentoses by using microorganisms. It is possible to do.

As a result of intensive studies to solve the above problems, the present inventors have introduced a gene encoding an inositol transporter in a microorganism used for producing an organic compound, or by enhancing the expression of the gene. The inventors have found that the ability to assimilate pentose can be improved without deteriorating the growth of microorganisms, and have completed the present invention.
That is, according to the present invention, the following inventions are provided.
[1] An organic compound-producing ability and a pentose utilization ability, and a gene encoding an inositol transporter was introduced, or expression of the gene was enhanced as compared to a non-induced strain or a non-modified strain. A method for producing an organic compound, comprising a step of producing an organic compound by allowing a microorganism or a processed product thereof to act on an organic raw material containing pentose in an aqueous medium.
[2] The expression of a gene encoding a transcription factor that suppresses the expression of a gene encoding an inositol transporter of the microorganism is reduced as compared with an unmodified strain. A method for producing the organic compound.
[3] The method for producing an organic compound according to [1] or [2], wherein the microorganism is cultured in the presence of an organic raw material containing inositol.
[4] The method for producing an organic compound according to any one of [1] to [3], wherein the aqueous medium contains inositol.
[5] The pentose is xylose, and the microorganism is further introduced with a gene encoding xylose isomerase, or the expression of the gene is enhanced [1] to [4] ] The manufacturing method of the organic compound in any one of.
[6] The method for producing an organic compound according to [5], wherein a gene encoding xylulokinase is further introduced into the microorganism, or expression of the gene is enhanced.
[7] A microorganism having an ability to produce an organic compound and a pentose utilization capacity, and a gene encoding an inositol transporter is introduced, or the expression of the gene is enhanced as compared with a non-inducible strain or a non-modified strain. A method for culturing microorganisms, comprising culturing in the presence of an organic raw material containing pentose.
[8] The method for culturing a microorganism according to [7], wherein the microorganism is cultured in the presence of an organic raw material containing inositol.
[9] A coryneform bacterium having an organic compound producing ability and a pentose assimilating ability and enhanced expression of a gene encoding an inositol transporter compared to an unmodified strain .
[10] Coryne according to [9], wherein the expression of a gene encoding a transcription factor that suppresses the expression of a gene encoding an inositol transporter is reduced as compared with an unmodified strain. Type bacteria.
[11] The coryneform bacterium according to [9] or [10], wherein the coryneform bacterium is further introduced with a xylose isomerase gene.
[12] The coryneform bacterium according to [11], wherein the coryneform bacterium is further introduced with a xylulokinase gene or enhanced expression of the gene.

According to the present invention, it is possible to provide a microorganism having an improved ability to assimilate the pentose without causing deterioration of growth.
According to the present invention, it is possible to provide a method for culturing a microorganism in which the pentose assimilation ability of the microorganism is improved without causing deterioration of growth.
By using such a microorganism of the present invention or a culture method of the present invention, an organic compound can be more efficiently produced from an organic raw material containing pentose.

The structure of the plasmid used in Examples 1-3 and Comparative Example 1 is shown. The structure of the plasmid used in Examples 1-3 and Comparative Example 1 is shown. The structure of the plasmid used in Examples 1-3 and Comparative Example 1 is shown. The structure of the plasmid used in Examples 1-3 and Comparative Example 1 is shown. The structure of the plasmid used in Examples 1-3 and Comparative Example 1 is shown. The structure of the plasmid used in Examples 1-3 and Comparative Example 1 is shown. The structure of the plasmid used in Examples 1-3 and Comparative Example 1 is shown. The structure of the plasmid used in Examples 1-3 and Comparative Example 1 is shown. The result of succinic acid production evaluation of Example 11 is shown. The result of succinic acid production evaluation of Example 12 is shown. The result of succinic acid production evaluation of Example 13 is shown. The result of the succinic acid production evaluation of Comparative Example 5 is shown. The systematic diagram of the strain produced in Examples 1-3 and Comparative Example 1 is shown.

Hereinafter, embodiments of the present invention will be described in detail.
<Microorganism used in the present invention or processed product thereof>
The microorganism used in the present invention is a microorganism having an organic compound producing ability and a pentose utilization ability, and a gene encoding an inositol transporter is introduced, or the expression of the gene is compared with a non-inducible strain or a non-modified strain. And enhanced microorganisms.

Further, in the present invention, a processed product of the microorganism can be used. Examples of the processed microorganisms include, for example, immobilized microorganisms in which microorganisms are immobilized with acrylamide, carrageenan, etc., disrupted microorganisms, centrifuged supernatants thereof, or supernatants thereof by ammonium sulfate treatment, etc. Examples include partially purified fractions.
In the present invention, “organic compound-producing ability” means that when a microorganism is cultured in a medium, the microorganism can produce and accumulate an organic compound in the medium.

The organic compound produced by the microorganism is not limited as long as the microorganism is an organic compound that can be produced and accumulated in the medium. Specifically, ethanol, propanol, butanol, 1,2-propanediol, 1,3- Alcohols such as propanediol, 1,3-butanediol, 2,3-butanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, glycerol, erythritol, xylitol, sorbitol; Amines such as 1,5-pentamethylenediamine and 1,6-hexamethylenediamine; acetic acid, butyric acid, glycolic acid, lactic acid, 3-hydroxypropionic acid, pyruvic acid, succinic acid, fumaric acid, malic acid, oxaloacetic acid, Cis-aconitic acid, citric acid, isocitric acid, 2-oxoglutaric acid, 2- Carboxylic acids such as xoisovaleric acid, glutaric acid, itaconic acid, adipic acid, levulinic acid, quinic acid, shikimic acid, acrylic acid, methacryl; alanine, valine, leucine, isoleucine, lysine, arginine, methionine, histidine, cysteine, serine Amino acids such as threonine, glutamic acid, aspartic acid, glutamine, asparagine, phenylalanine, tyrosine, proline, tryptophan; phenols such as phenol, catechol, hydroquinone; benzoic acid, 4-hydroxybenzoic acid, protocatechuic acid, phthalic acid, isophthalic acid Aromatic carboxylic acids such as acid and terephthalic acid; Nucleosides such as inosine and guanosine; Nucleosides such as inosinic acid and guanylic acid; Unsaturated hydrocarbons such as isobutylene, isoprene and butadiene Compounds, and the like.

  Among these, alcohols, amines, carboxylic acids, and phenols are preferable because known methods can be employed for fermentation production and can be used as resin raw materials, and aliphatic alcohols having 2 to 10 carbon atoms. In addition, aliphatic carboxylic acids having 2 to 10 carbon atoms are more preferable. Of these, ethanol, butanediol, and succinic acid are more preferable from the viewpoint of fermentation productivity.

Here, the microorganism having an organic compound-producing ability may be a microorganism that inherently has an organic compound-producing ability, or may be one imparted with an organic compound-producing ability by breeding.
Examples of means for imparting organic compound-producing ability by breeding include mutation treatment and gene recombination treatment. Enhancing the expression of enzyme genes in the biosynthesis pathway of organic compounds and reducing the expression of enzyme genes in the byproduct biosynthesis pathway Any known method can be employed. For example, in the case of imparting the ability to produce carboxylic acids such as succinic acid, fumaric acid, malic acid, such modifications as described below for reducing lactate dehydrogenase activity and means for enhancing pyruvate carboxylase activity are included. . In the case of imparting alcohol-producing ability such as ethanol, butanol, butanediol and the like, modifications such as those described below that reduce lactate dehydrogenase activity and means for enhancing alcohol dehydrogenase activity are included.

In the present invention, “pentose assimilation ability” means that a microorganism can use pentose as a carbon source to proliferate or produce an organic compound.
The pentose that a microorganism uses as a carbon source is not particularly limited as long as it can be used as a carbon source. Specific examples include aldopentoses such as xylose, arabinose, ribose and lyxose, and ketopentoses such as ribulose and xylulose.

Among these, aldopentoses are preferable, and xylose and arabinose contained in hemicellulose-based biomass used as a non-edible raw material are more preferable. Especially, xylose with much content in hemicellulose biomass is especially preferable.
Here, the microorganism having the pentose utilization ability may be a microorganism that originally has the pentose utilization ability, or may be one that has been imparted with a pentose utilization ability by breeding.

  Examples of means for imparting pentose assimilation ability by breeding include gene recombination treatment, and a known method such as introducing an enzyme gene of the pentose metabolic pathway can be employed. For example, when imparting xylose utilization ability, a method of introducing a xylose isomerase gene as described later, a method of introducing a xylose reductase gene and a xylitol dehydrogenase gene, and the like can be mentioned. In the case of imparting arabinose assimilation ability, a method of introducing an arabinose isomerase gene, a librokinase gene, and a ribulose 5-phosphate epimerase gene as described later can be used.

  The microorganism used in the present invention is not particularly limited as long as it has organic compound-producing ability and pentose utilization ability, but coryneform bacteria, Escherichia coli, Anaerobiospirillum bacteria, Actinobacillus genus bacteria Preferably, the microorganism is selected from the group consisting of a bacterium belonging to the genus Mannheimia, a genus Basfia, a bacterium belonging to the genus Zymomonas, a bacterium belonging to the genus Zymomobacter, a filamentous fungus, and a yeast.

  Among them, the group consisting of coryneform bacteria, Escherichia coli, Anaerobiospirillum genus bacteria, Actinobacillus genus bacteria, Mannheimia genus bacteria, Basfia genus bacteria, filamentous fungi, and yeasts At least one selected from these is preferable, more preferably coryneform bacteria, Escherichia coli, and yeast, and particularly preferably coryneform bacteria.

  The coryneform bacterium is not particularly limited as long as it is classified as such, and examples include bacteria belonging to the genus Corynebacterium, bacteria belonging to the genus Brevibacterium, bacteria belonging to the genus Arthrobacter, and the like. Preferred are those belonging to the genus Corynebacterium and Brevibacterium, and more preferred are Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium ammoniagenes ( Bacteria classified as Brevibacterium lactofermentum or Brevibacterium lactofermentum A.

  Particularly preferred specific examples of coryneform bacteria that can be used in the present invention include Brevibacterium flavum MJ-233 (FERM BP-1497), MJ-233 AB-41 (FERM BP-1498), Brevibacterium Ammonia Genes ATCC 6872, Corynebacterium glutamicum ATCC 31831, Brevibacterium lactofermentum ATCC 13869 and the like. Brevibacterium flavum is currently sometimes classified as Corynebacterium glutamicum (Lielbl W, Ehrmann M, Ludwig W, Schleiffer KH, Int J Systact Bacteriol., 1991, Vol. 41). p255-260), in the present invention, the Brevibacterium flavum MJ-233 strain and its mutant MJ-233 AB-41 are respectively Corynebacterium glutamicum MJ-233 and MJ-233 AB-41. The same stock as the stock.

  The above-mentioned Brevibacterium flavum MJ-233 was founded on April 28, 1975 at the Institute of Microbial Technology of the Ministry of International Trade and Industry (currently National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) (〒305-8856 Japan) No. 1 in Higashi 1-chome, 1-chome, Tsukuba City, Ibaraki Prefecture, Japan, with deposit number FERM P-3068, transferred to an international deposit based on the Budapest Treaty on May 1, 1981, under the deposit number of FERM BP-1497 It has been deposited.

Examples of E. coli that can be used in the present invention include Escherichia coli.
Examples of bacteria belonging to the genus Anaerobiospirillum that can be used in the present invention include Anaerobiospirillum succiniciprodusens, etc. Actinobacilli that can be used in the present invention (Actinobactus) Actinobacillus succinogenes and the like.

Examples of the bacterium belonging to the genus Mannheimia that can be used in the present invention include basin succinici producers.
Examples of the bacterium belonging to the genus Basfia that can be used in the present invention include Basfia succiniciproducens.

Examples of the genus Zymomonas that can be used in the present invention include Zymomonas mobilis.
Examples of the genus Zymobacter that can be used in the present invention include Zymobacter palmae.
Examples of filamentous fungi that can be used in the present invention include Aspergillus, Penicillium, and Rhizopus. Among these, Aspergillus niger (Aspergillus oryzae) and the like are Aspergillus niger (Aspergillus oryzae) and the like, and Penicillium chrysogenum (Pencillium chrysogenium and Penicillium crisiumum lysilis) are included in the genus Aspergillus. . In the genus Rhizopus, Rhizopus oryzae and the like can be mentioned.

  Examples of yeasts that can be used in the present invention include Saccharomyces, Shizosaccharomyces, Candida, Pichia, Kluyveromyces (Kluyveromyces), Yarrowia), Zygosaccharomyces.

Examples of the genus Saccharomyces include Saccharomyces cerevisiae, Saccharomyces uvarum, and Saccharomyces bayanmys.
Examples of the genus Shizosaccharomyces include Schizosaccharomyces pombe.

  Examples of the genus Candida include Candida albicans, Candida sonorensis, Candida glabrata, and the like. Examples of the Pichia genus include Pichia pastoris and Pichia stipitis.

  Examples of the genus Kluyveromyces include Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces thermotolerance (Kluyveromyces thermotolerance (Kluyveromyces thermotolerance). Is mentioned.

Examples of the genus Yarrowia include Yarrowia lipolytica.
Examples of the genus Zygosaccharomyces include Zygosaccharomyces bailii and Zygosaccharomyces rouxii.

The microorganism is not only a wild strain, but also a mutant strain obtained by a normal mutation treatment such as UV irradiation or NTG treatment, a recombinant strain induced by a genetic technique such as cell fusion or gene recombination. Or a stock of
As described above, the microorganism used in the present invention introduces a gene encoding an inositol transporter into a strain having an organic compound producing ability and a pentose utilization ability, as described later, or the expression of the gene is enhanced. Can be obtained by induction or modification.

In the present invention, the “inositol transporter (hereinafter sometimes referred to as“ IolT ”)” refers to a protein having an activity of taking inositol into a cell, and a gene encoding this protein is introduced, or the gene By enhancing the expression of pentose, pentose utilization ability can be improved.
The activity of incorporating inositol into cells is determined according to a known method, for example, the method of Krings et al.
2006, Vol. 188 (23), p8054-61), which can be confirmed by measuring inositol uptake activity.

  The “gene encoding IolT (hereinafter also referred to as iolT gene)” in the present invention is a gene that encodes the inositol transporter and enhances its expression, thereby improving the pentose utilization ability. Although not particularly limited, for example, iolT1 gene and iolT2 gene derived from coryneform bacteria, iolT gene derived from E. coli, iolT gene and iolF gene derived from Bacillus subtilis, ITR1 gene derived from yeast, and the like can be mentioned. Among these, the iolT1 gene or iolT2 gene derived from coryneform bacteria is preferable, and genes derived from Corynebacterium glutamicum MJ-233 strains containing the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 are more preferable (hereinafter referred to as iolT1 gene, also called the iolT2 gene).

These genes are DNAs that hybridize with DNA having the above base sequence under stringent conditions, or DNAs having 90% or more, preferably 95% or more, and more preferably 99% or more homology with the above base sequence. Such a homologue may be used. Here, the stringent conditions are 60 ° C., 1 × SSC, 0.1% SDS, preferably 60 ° C., 0.1 × SSC, 0.
Examples include conditions for hybridizing at a salt concentration corresponding to 1% SDS.

  Furthermore, as long as the iolT1 gene and the iolT2 gene have inositol uptake activity and can improve the pentose utilization ability, substitution or deletion of one or several amino acids in the amino acid sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively. It may be a gene encoding a protein having an amino acid sequence including deletion, insertion, or addition. Here, the term “several” means usually 2 or more, and usually 20 or less, preferably 10 or less, more preferably 5 or less.

  In the present invention, “introducing the iolT gene” means that this gene has been introduced using an unmodified strain such as a wild strain as a parent strain. As a means for introducing the iolT gene, for example, a wild-type strain or a parent strain can be modified by a genetic recombination method using the iolT gene. Specific examples include transformation with a vector containing a gene encoding the iolT gene, or introduction of the gene onto a chromosome by homologous recombination. In introducing the iolT gene, a plurality of types of iolT, such as iolT genes derived from different microorganisms, animals and plants, or the same but different iolT genes, for example, the iolT1 gene and the iolT2 gene derived from Corynebacterium glutamicum MJ-233 strain, etc. Genes may be used.

  The iolT gene derived from microorganisms or animals and plants is isolated from the chromosomes of microorganisms, animals and plants, etc., and the nucleotide sequence is determined based on the genes whose homology has already been determined, genes encoding proteins having inositol uptake activity, etc. Can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can also be used. These can be obtained by amplifying a region containing the promoter and the ORF portion by a hybridization method or a PCR method.

  By inserting the iolT gene isolated as described above into a known expression vector so as to allow expression, an IolT expression vector is provided. By transforming with this expression vector, a strain into which the iolT gene has been introduced can be obtained. Alternatively, a strain into which the iolT gene has been introduced can also be obtained by incorporating DNA encoding IolT into the chromosomal DNA of the host microorganism so that it can be expressed by homologous recombination. Note that transformation and homologous recombination can be performed according to ordinary methods known to those skilled in the art.

  When the iolT gene is introduced onto a chromosome or a plasmid, an appropriate promoter is incorporated upstream of the gene on the 5'-side, more preferably a terminator is incorporated downstream of the 3'-side. The promoter and terminator are not particularly limited as long as the promoter and terminator are known to function in a microorganism used as a host, and may be the promoter and terminator of the iolT gene itself or other promoters. And a terminator. Vectors, promoters and terminators that can be used in these various microorganisms are described in detail in, for example, “Basic Course of Microbiology 8 Genetic Engineering / Kyoritsu Shuppan”.

Hereinafter, a method for introducing the iolT gene in coryneform bacteria will be described. When a coryneform bacterium is used, the iolT gene can be expressed in the coryneform bacterium by introducing a DNA fragment containing the iolT gene into a plasmid vector containing a gene that controls the replication and replication function of the plasmid in the coryneform bacterium. Recombinant plasmids can be obtained. By transforming a coryneform bacterium, for example, Corynebacterium glutamicum MJ-233 strain, with this recombinant vector, a coryneform bacterium into which the iolT gene has been introduced can be obtained. Transformation can be performed, for example, by the electric pulse method (Vertes AA, Inui M, Kobay
ashi M, Kurusu Y, Yukawa H, Res. Microbiol. , 1993, Vol. 144 (3), p181-185).

  A plasmid vector capable of introducing a gene into a coryneform bacterium is not particularly limited as long as it contains at least a gene that controls the replication and proliferation function in the coryneform bacterium. Specific examples thereof include, for example, plasmid pCRY30 described in JP-A-3-210184; plasmids pCRY21, pCRY2KE, pCRY2KX described in JP-A-2-72876 and US Pat. No. 5,185,262. pCRY31, pCRY3KE, and pCRY3KX; plasmids pCRY2 and pCRY3 described in JP-A-1-191686; pAM330 described in JP-A-58-67679; pHM1519 described in JP-A-58-77895; PAJ655, pAJ611, and pAJ1844 described in JP-A-58-192900; pCG1 described in JP-A-57-134500; pCG2 described in JP-A-58-35197; JP-A-57-183799 Recorded in pCG4 and pCG11, etc. can be mentioned. Among them, plasmid vectors used in the host-vector system of coryneform bacteria have a gene responsible for the replication and replication function of the plasmid in coryneform bacteria and a gene responsible for the plasmid stabilization function in coryneform bacteria. For example, plasmids pCRY30, pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX are preferably used.

  In the integration into the above recombinant plasmid or chromosome, the promoter for expressing the iolT gene may be any promoter as long as it functions in coryneform bacteria, and may be the promoter of the iolT gene itself used. The expression level of the iolT gene can also be adjusted by appropriately selecting a promoter. As mentioned above, although the example using a coryneform bacterium was described, the introduction of the iolT gene can be achieved by the same method also when using other bacteria.

In the present invention, “enhance the expression of ioI T gene” means that the expression of this gene is enhanced as compared to a non-inducible strain or a non-modified strain such as a wild strain or a parent strain. The expression of the iolT gene is preferably enhanced by 1.5 times or more, more preferably enhanced by 3 times or more compared to the non-inducible strain or the non-modified strain.
The enhanced expression of the iolT gene can be confirmed by measuring the amount of mRNA by Northern hybridization or quantitative PCR.

  Methods for enhancing the expression of the iolT gene include, for example, a method of treating the parent strain with a mutagen, a method of increasing the copy number of the iolT gene, a method of modifying the promoter of the iolT gene, and a transcription factor that suppresses the expression of the iolT gene. Examples thereof include a method for reducing the expression of a gene to be encoded and a method for inducing the expression of the iolT gene by culturing using an organic raw material containing inositol. In addition, in order to enhance the expression of the iolT gene, a plurality of types of iolT genes derived from different microorganisms, animals and plants, or the same but different iolT genes, for example, the iolT1 gene and iolT2 gene derived from Corynebacterium glutamicum MJ-233 The iolT gene may be used. Furthermore, a plurality of methods for enhancing the expression of the iolT gene may be combined.

Hereinafter, a method for enhancing the expression of the iolT gene will be described. For the strain with enhanced expression of the iolT gene, the parent strain is treated with a mutagen such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrite, which is usually used for mutagenesis. It can be obtained by selecting a strain with increased expression.
A strain with enhanced expression of the iolT gene can also be obtained by modifying it using the iolT gene. Specifically, it can be achieved by increasing the copy number of the iolT gene, which can be achieved by transforming with a vector containing the iolT gene or introducing the gene onto the chromosome by homologous recombination. This can be achieved by making multiple copies on the chromosome.

Furthermore, a strain in which the expression of the iolT gene is enhanced can also be achieved by introducing a mutation into the promoter of the iolT gene on a chromosome or on a plasmid, or by making it highly expressed by replacing it with a stronger promoter.
Any strain transformed with a vector containing the iolT gene, a strain introduced with the gene on the chromosome by homologous recombination, a strain introduced with a mutation in the promoter of the gene, or a strain substituted with a stronger promoter Can also be produced in the same manner as the above-described method for introducing the iolT gene.

  A strain with enhanced expression of the iolT gene can also be obtained by modifying so as to reduce the expression of a gene encoding a transcription factor that suppresses the expression of the iolT gene. Here, “a transcription factor that suppresses the expression of the iolT gene (hereinafter also referred to as IolR)” refers to a protein having an activity of suppressing the transcription of the iolT gene, and the expression of the gene encoding this protein is reduced. Thus, the expression of the iolT gene can be enhanced.

  The “gene encoding IolR (hereinafter also referred to as iolR gene)” is not particularly limited as long as it is a gene that encodes IolR and can enhance the expression of the iolT gene by reducing its expression. For example, a gene derived from Corynebacterium glutamicum MJ-233 strain containing the base sequence shown in SEQ ID NO: 5 can be mentioned.

  This gene may be a DNA that hybridizes with a DNA having the above base sequence under stringent conditions, or a DNA having 90% or more, preferably 95% or more, more preferably 99% or more homology with the above base sequence. It may be a homologue. Here, the stringent conditions are 60 ° C., 1 × SSC, 0.1% SDS, preferably 60 ° C., 0.1 × SSC, 0. Examples include conditions for hybridizing at a salt concentration corresponding to 1% SDS.

  Furthermore, as long as the iolR gene has an activity of suppressing transcription of the iolT gene, the protein having an amino acid sequence including substitution, deletion, insertion, or addition of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 6 It may be a gene encoding. Here, the term “several” means usually 2 or more, and usually 20 or less, preferably 10 or less, more preferably 5 or less.

  In the present invention, “reducing the expression of the iolR gene” means that the expression of this gene is reduced as compared to an unmodified strain such as a wild strain or a parent strain. The expression of the iolR gene is preferably reduced to 30% or less of the unmodified strain, more preferably 10% or less. The expression of the iolR gene may be reduced below the detection limit.

The reduction in the expression of the iolR gene can be confirmed by measuring the amount of mRNA by Northern hybridization or quantitative PCR.
Methods for reducing the expression of the iolR gene include, for example, a method of treating the parent strain with a mutagen, a method of destroying the iolR gene on the chromosome, a method of introducing a mutation into the iolR gene on the chromosome, and modifying the promoter of the iolR gene The method of doing is mentioned. A plurality of methods for reducing the expression of these iolR genes may be combined.

Hereinafter, a method for reducing the expression of the iolR gene will be described. For the strain with reduced iolR gene expression, the parent strain is treated with a normal mutagen such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid. It can be obtained by selecting strains with reduced expression.
A strain with reduced iolR gene expression can also be obtained by disrupting the iolR gene on the chromosome. The iolR gene is disrupted by homologous recombination into the chromosome or by using the sacB gene (Schaffer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Puhler).
A, Gene 1994 Vol. 145 (1), p69-73).

Furthermore, a strain in which the expression of the iolR gene is reduced can also be obtained by introducing a mutation into the iolR gene on the chromosome to reduce the activity of suppressing the transcription of the iolT gene possessed by IolR. Mutation introduction into the iolR gene
A strain in which the expression of the iolR gene is reduced can also be obtained by modifying the promoter of the iolR gene on the chromosome. Specifically, it can also be achieved by introducing a mutation into the promoter of the iolT gene on the chromosome or substituting it with a weaker promoter to suppress the expression.

  Hereinafter, a method for disrupting the iolR gene in coryneform bacteria will be described. The iolR gene can be obtained by, for example, cloning a synthetic oligonucleotide based on the above sequence and performing a PCR reaction using the chromosomal DNA of Corynebacterium glutamicum as a template. Chromosomal DNA is obtained from bacteria that are DNA donors, for example, by the method of Saito and Miura (Saito H, Miura K, Biochim Biophys Acta., 1963, Vol. 72, p619-629, Biotechnology Experiments, Japan Society for Biotechnology, Ed., Pp. 97-98, Bafukan, 1992).

  The iolR gene prepared as described above or a part thereof can be used for gene disruption. However, since the gene used for gene disruption only needs to have homology (identity) to the extent that homologous recombination occurs with the iolR gene on the chromosomal DNA of the bacterium to be disrupted, it has homology with SEQ ID NO: 5. Homologous genes can also be used. Here, the homology that causes homologous recombination is preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more. In addition, homologous recombination can occur as long as it is a DNA that can hybridize with the above gene under stringent conditions (complementary strand of SEQ ID NO: 5). Here, the stringent conditions are 60 ° C., 1 × SSC, 0.1% SDS, preferably 60 ° C., 0.1 × SSC, 0.1%, which are the usual washing conditions for Southern hybridization. A condition for hybridization at a salt concentration corresponding to SDS is mentioned.

  Using a gene as described above, for example, deleting a partial sequence of the iolR gene, producing a deleted iolR gene modified so as not to produce a normal IolR protein, and preparing a coryneform bacterium with the DNA containing the gene The iolR gene on the chromosome can be destroyed by transforming and causing recombination between the deleted gene and the gene on the chromosome. Gene disruption by gene replacement using such homologous recombination has already been established, and there are a method using linear DNA and a method using a plasmid containing a temperature-sensitive replication origin (US Pat. No. 6,303,383). Or Japanese Patent Laid-Open No. 05-007491). In addition, gene disruption by gene replacement using homologous recombination as described above can be performed using a plasmid that does not have replication ability on the host. As a plasmid that does not have replication ability in coryneform bacteria, A plasmid having replication ability in Escherichia coli is preferable, and examples thereof include pHSG299 (manufactured by Takara Bio Inc.), pHSG399 (manufactured by Takara Bio Inc.), and the like.

  A strain with enhanced expression of the iolT gene can also be obtained by inducing the expression of the iolT gene by culturing in the presence of an organic raw material containing inositol. In enhancing the expression of the iolT gene by this means, a strain that has been genetically modified as described above may be used, but a strain that has not been genetically modified can also be used.

  The inositol that can be used in the present invention is not particularly limited as long as the expression of the iolT gene can be induced. Specific examples include cis-inositol, epi-inositol, allo-inositol, myo-inositol, muco-inositol, neo-inositol, D-chiro-inositol, L-chiro-inositol, scyllo-inositol. Among these, the most common myo-inositol is preferable since it exists in nature.

Inositol is known to be abundant in natural foods such as cereals and fruits, and organic raw materials such as extracts and processed products may be used. Specific examples include corn steep liquor and yeast extract.
When culturing in the presence of an organic raw material containing inositol, other organic raw materials may be added as necessary. The organic raw material other than inositol is not particularly limited as long as it is a carbon source that can be assimilated by the microorganism used in the present invention. Usually, carbohydrates such as galactose, lactose, glucose, fructose, sucrose, starch or cellulose; Fermentable sugars such as polyalcohols such as xylitol or ribitol are used, and among these, glucose, sucrose or fructose is preferable, and glucose or sucrose is particularly preferable.

These organic raw materials may be added alone, or two or more kinds may be added in any combination.
When culturing in the presence of an organic raw material containing inositol, it may be cultured in a solid medium such as an agar medium containing inositol or in a liquid medium containing inositol. About other culture conditions, it can carry out similarly to the culture method using the organic raw material containing the pentose mentioned later.

  In addition, the microbial cell obtained in this way can be used for the culture | cultivation using the organic raw material containing the pentose mentioned later, and an organic compound production reaction. Here, the cells may be cultured in the presence of an organic raw material containing inositol and pentose, and grown while inducing the expression of the iolT gene. Alternatively, an organic compound may be produced while inducing the expression of the iolT gene by conducting an organic compound production reaction using an organic raw material containing inositol and pentose.

The microorganism used in the present invention may be a microorganism that inherently has pentose utilization ability, or may be one that has been imparted with pentose utilization ability by breeding.
Hereinafter, as specific examples of imparting pentose utilization ability by breeding, a modification example relating to provision of xylose utilization ability and a modification example relating to impartation of arabinose utilization ability will be described.
A microorganism to which xylose assimilation ability is imparted is obtained by using a microorganism having an organic compound-producing ability as described above as a parent strain, and introducing a gene encoding a protein having xylose isomerase (hereinafter also referred to as XylA) activity into the parent strain. Can be obtained.

  Here, “XylA activity” refers to an activity (EC: 5.3.1.5) that catalyzes a reaction of isomerizing xylose to produce xylulose. XylA activity was imparted or enhanced by known methods such as the method of Gao et al. (Gao Q, Zhang M, McMillan JD, Kompala DS, Appl. Biochem. Biotechnol., 2002, Vol. 98 (100), p341. -55) can be confirmed by measuring XylA activity.

A specific method for producing a strain having XylA activity imparted or enhanced can be achieved by introducing the xylA gene by a plasmid or by introducing it into a chromosome by a known homologous recombination method.
The xylA gene used for imparting or enhancing XylA activity is not particularly limited as long as it encodes a protein having XylA activity, and examples thereof include a gene derived from Escherichia coli.

  In addition, xylA gene derived from bacteria other than E. coli, other microorganisms, and animals and plants can also be used. The xylA gene derived from microorganisms or animals and plants has been isolated from the chromosomes of microorganisms, animals and plants, etc. based on genes whose homology has already been determined, genes encoding proteins having XylA activity, and the nucleotide sequences have been determined. Things can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can also be used. These can be obtained by amplifying a region containing the promoter and the ORF portion by a hybridization method or a PCR method.

  An XylA expression vector is provided by inserting the gene encoding XylA isolated as described above into a known expression vector so that the gene can be expressed. By transforming with this expression vector, a strain to which XylA activity is imparted or enhanced can be obtained. Alternatively, a strain in which XylA activity is imparted or enhanced can be obtained by incorporating DNA encoding XylA into a chromosomal DNA of a host microorganism so that it can be expressed by homologous recombination. Note that transformation and homologous recombination can be performed according to ordinary methods known to those skilled in the art.

  When the XylA gene is introduced onto a chromosome or a plasmid, an appropriate promoter is incorporated upstream of the gene on the 5'-side, more preferably a terminator is incorporated downstream of the 3'-side. The promoter and terminator are not particularly limited as long as the promoter and terminator are known to function in microorganisms used as hosts, and may be the promoter and terminator of the xylA gene itself, or other promoters. And a terminator. Vectors, promoters and terminators that can be used in these various microorganisms are described in detail in, for example, “Basic Course of Microbiology 8 Genetic Engineering / Kyoritsu Shuppan”.

In addition, in imparting xylose availability, a microorganism modified to impart or enhance xylulokinase (hereinafter also referred to as XylB) activity in addition to imparting or enhancing XylA activity may be used.
Here, “XylB activity” refers to an activity (EC: 2.7.1.17) that catalyzes a reaction in which xylulose is phosphorylated to produce xylulose 5-phosphate. XylB activity was imparted or enhanced by known methods, for example, the method of Eliasson et al. Microbiol.Biotechnol., 2000, Vol.53, p376-82) can be confirmed by measuring XylB activity.

A strain to which XylB activity is imparted or enhanced can be produced in the same manner as described above for imparting or enhancing XylA activity.
The xylB gene used for imparting or enhancing XylB activity is not particularly limited as long as it encodes a protein having XylB activity, and examples thereof include genes derived from Escherichia coli and Corynebacterium glutamicum. be able to.

  Furthermore, bacteria other than the above, other microorganisms, and xylB genes derived from animals and plants can also be used. The xylB gene derived from microorganisms or animals and plants was isolated from the chromosomes of microorganisms, animals and plants, etc., and the nucleotide sequence was determined based on genes whose base sequences had already been determined, genes encoding proteins having XylB activity based on homology, etc. Things can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can also be used. These can be obtained by amplifying a region containing the promoter and the ORF portion by a hybridization method or a PCR method.

When XylA activity and XylB activity are imparted or enhanced, the introduced xylA gene and xylB gene may be present on the same locus, or may be present on different loci. Examples of two genes existing on the same locus include, for example, an operon formed by linking each gene.
A microorganism imparted with xylose utilization ability uses the above-mentioned microorganism as a parent strain, and the parent strain has a gene encoding a protein having xylose reductase (hereinafter also referred to as XR) activity and xylitol dehydrogenase (hereinafter also referred to as XDH) activity. It can also be obtained by introducing a gene encoding a protein having the same.

  Here, “XR activity” refers to an activity (EC: 1.1.1.21) that catalyzes a reaction of reducing xylose to produce xylitol. XR activity was imparted or enhanced by a known method, for example, the method of Sasaki et al. (Sasaki M, Jojima T, Inui M, Yukawa H, Appl Microbiol Biotechnol., 2010, Vol. 86 (4), p1057-66. ) Can be confirmed by measuring the XR activity.

A strain to which XR activity is imparted or enhanced can be prepared in the same manner as described above for imparting or enhancing XylA activity.
The xr gene used for imparting or enhancing XR activity is not particularly limited as long as it encodes a protein having XR activity.
XYL1 gene derived from Stipitis).

  Furthermore, xr genes derived from microorganisms or animals and plants other than those described above can also be used. The xr gene derived from microorganisms or animals and plants was isolated from the chromosomes of microorganisms, animals and plants, etc., and the nucleotide sequences were determined based on genes whose base sequences had already been determined, genes encoding proteins having XR activity based on homology, etc. Things can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can also be used. These can be obtained by amplifying a region containing the promoter and the ORF portion by a hybridization method or a PCR method.

Next, “XDH activity” refers to an activity (EC: 1.1.1.9) that catalyzes a reaction of dehydrogenating xylitol to produce xylulose. XDH activity was imparted or enhanced by known methods such as the method of Rizzi et al. (Rizzi M, Harwart K, Erlemann P, Bui-Thahn NA, Dellweg H, J
Ferment Bioeng. , 1989, Vol. 67, p20-24) and can be confirmed by measuring the XDH activity.

A strain imparted or enhanced with XDH activity can be produced in the same manner as the method for imparting or enhancing XylA activity described above.
The xdh gene used for imparting or enhancing XDH activity is not particularly limited as long as it encodes a protein having XDH activity. For example, XYL2 gene derived from Pichia stipitis can be mentioned.

  Furthermore, xdh genes derived from microorganisms or animals and plants other than those described above can also be used. The xdh gene derived from microorganisms or animals and plants was isolated from the chromosomes of microorganisms, animals and plants, etc., and the nucleotide sequences were determined based on the genes whose homology was already determined, the genes encoding the proteins having XDH activity. Things can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can also be used. These can be obtained by amplifying a region containing the promoter and the ORF portion by a hybridization method or a PCR method.

In addition, in imparting xylose utilization ability, in addition to imparting or enhancing XDH activity and XR activity, a microorganism modified to impart or enhance XylB activity may be used. The provision or enhancement of XylB activity is as described above.
A microorganism to which arabinose utilization ability is imparted uses the above-mentioned microorganism as a parent strain, and the parent strain has a gene encoding a protein having arabinose isomerase (hereinafter also referred to as AraA) activity and ribokinase (hereinafter also referred to as AraB) activity. And a gene encoding a protein having ribulose 5-phosphate epimerase (hereinafter also referred to as AraD) activity.

Here, “AraA activity” refers to an activity (EC: 5.3.1.4) that catalyzes a reaction of isomerizing arabinose to produce ribulose. AraA activity was imparted or enhanced by known methods such as Patrick et al. (Patrick JW, Lee).
N, J .; Biol. Chem. , 1968, Vol. 243, p4312- 19) and can be confirmed by measuring AraA activity.

A strain to which AraA activity is imparted or enhanced can be produced in the same manner as the method for imparting or enhancing XylA activity described above.
The araA gene used for imparting or enhancing AraA activity is not particularly limited as long as it encodes a protein having AraA activity. For example, a gene derived from Escherichia coli or Corynebacterium glutamicum is used. Can be mentioned.

  Furthermore, araA genes derived from bacteria other than those described above, other microorganisms, and animals and plants can also be used. The araA gene derived from microorganisms or animals and plants was isolated from the chromosomes of microorganisms, animals and plants, etc., and the nucleotide sequence was determined based on the genes, homology, etc. of which the nucleotide sequences had already been determined. Things can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can also be used. These can be obtained by amplifying a region containing the promoter and the ORF portion by a hybridization method or a PCR method.

  Next, “AraB activity” refers to an activity (EC: 2.7.1.16) that catalyzes a reaction in which ribulose is phosphorylated to produce ribulose 5-phosphate. AraB activity was imparted or enhanced by known methods such as the method of Lee et al. (Lee N, Englesberg E, Proc. Natl. Acad. Sci., 1962, Vol. 48, p335-48). It can be confirmed by measuring.

A strain to which AraB activity is imparted or enhanced can be produced in the same manner as the method for imparting or enhancing XylA activity described above.
The araB gene used for imparting or enhancing AraB activity is not particularly limited as long as it encodes a protein having AraB activity. For example, a gene derived from Escherichia coli or Corynebacterium glutamicum is used. Can be mentioned.

  Furthermore, araB genes derived from bacteria other than those mentioned above, other microorganisms, and animals and plants can also be used. The araB gene derived from microorganisms or animals and plants was isolated from the chromosomes of microorganisms, animals and plants, etc., and the nucleotide sequence was determined based on the genes, homology, etc., of which the nucleotide sequences had already been determined. Things can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can also be used. These can be obtained by amplifying a region containing the promoter and the ORF portion by a hybridization method or a PCR method.

Furthermore, “AraD activity” refers to an activity (EC: 5.1.3.4) that catalyzes a reaction of isomerizing ribulose 5-phosphate to produce xylulose 5-phosphate. AraD activity is imparted or enhanced by known methods such as the method of Deanda et al.
, Zhang M, Eddy C, Picataggio S, Appl Environ Microbiol. , 1996, Vol. 62 (12), p4465-70) and can be confirmed by measuring AraD activity.

A strain to which AraD activity is imparted or enhanced can be produced in the same manner as the method for imparting or enhancing XylA activity described above.
The araD gene used for imparting or enhancing AraD activity is not particularly limited as long as it encodes a protein having AraD activity. For example, a gene derived from Escherichia coli, Corynebacterium glutamicum is used. Can be mentioned.

  Furthermore, araD genes derived from bacteria other than those described above, other microorganisms, and animals and plants can also be used. The araD gene derived from microorganisms or animals and plants was isolated from the chromosomes of microorganisms, animals and plants, etc., and the nucleotide sequence was determined based on genes whose homology was already determined, genes encoding AraD activity proteins based on homology, etc. Things can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can also be used. These can be obtained by amplifying a region containing the promoter and the ORF portion by a hybridization method or a PCR method.

  When AraA activity, AraB activity, and AraD activity are imparted or enhanced, the araA gene, araB gene, and araD gene to be introduced may be present on the same locus or on different loci. Also good. An example in which two or three genes are present on the same locus is, for example, an operon formed by linking each gene.

The microorganism used in the present invention may be a bacterium obtained by combining two or more types of modifications among the modifications for imparting pentose utilization. When making a plurality of modifications, the order does not matter.
The microorganism used in the present invention may be a microorganism inherently capable of producing an organic compound or a microorganism imparted with an organic compound producing ability by breeding.

Hereinafter, as specific examples of imparting the ability to produce organic compounds by breeding, modified examples relating to the production of carboxylic acids such as succinic acid, fumaric acid and malic acid and modified examples relating to the production of alcohols such as ethanol, butanol and butanediol will be described.
In the carboxylic acid production method of the present invention, it is preferable to use a microorganism modified so as to reduce lactate dehydrogenase (hereinafter also referred to as LDH) activity. Here, “LDH activity” refers to an activity (EC: 1.1.1.17) that catalyzes a reaction of reducing pyruvic acid to produce lactic acid. “LDH activity is reduced” means that LDH activity is reduced as compared to an unmodified strain. The LDH activity is preferably reduced to 30% or less per unit cell weight, more preferably 10% or less, compared to the unmodified strain. LDH activity may be completely lost. The decrease in LDH activity was confirmed by known methods such as the method of Kanarek et al. (Kanarek L, Hill RL, J
Biol Chem. , 1964, Vol. 239, p4202-4206) and can be confirmed by measuring LDH activity.

The strain with reduced LDH activity is treated with the above-mentioned microorganism as a parent strain and treated with a mutagen usually used for mutagenesis such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid, Each can be obtained by selecting strains with reduced LDH activity. Moreover, you may modify | change using the gene which codes LDH. Specifically, this can be achieved by disrupting the ldh gene on the chromosome or altering an expression regulatory sequence such as a promoter or Shine-Dalgarno (SD) sequence.

Specific methods for producing strains with reduced LDH activity include methods using homologous recombination to chromosomes (see JP-A-11-206385, etc.), and methods using the sacB gene (Schaffer A, Tach A, Jager W). , Kalinowski J, Thierbach G, Puhler A, Gene 1994 Vol. 145 (
1), p69-73) and the like.

  In the carboxylic acid production method of the present invention, a microorganism modified so that pyruvate carboxylase (hereinafter also referred to as PC) activity is enhanced may be used. Here, “PC activity” refers to an activity (EC: 6.4.1.1) that catalyzes a reaction in which pyruvic acid is carboxylated to produce oxaloacetic acid. “PC activity is enhanced” means that PC activity is increased as compared to an unmodified strain. The PC activity is preferably increased 1.5 times or more per unit cell weight, more preferably 3 times or more, compared to the unmodified strain. The PC activity was increased by measuring the PC activity by a known method, for example, the method of Fisher et al. (Fisher SH, Masasanik B, J Bacteriol., 1984, Vol. 158 (1), p55-62). Can be confirmed.

  The strain with enhanced PC activity is treated with the above-mentioned microorganism as a parent strain, and is treated with a mutagen that is usually used for mutagenesis such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid, Each can be obtained by selecting a strain having enhanced PC activity. Moreover, you may modify | change using the gene which codes PC. Specifically, it can be achieved by increasing the copy number of the pc gene. Increasing the copy number can be achieved by using a plasmid or making multiple copies on the chromosome by a known homologous recombination method. . The enhancement of PC activity can also be achieved by high expression by introducing mutations into the promoter of the pc gene on the chromosome or on the plasmid, replacement with a stronger promoter, and the like.

The pc gene used for enhancing PC activity is not particularly limited as long as it encodes a protein having PC activity, and examples thereof include a gene derived from Corynebacterium glutamicum.
In addition, pc genes derived from bacteria other than coryneform bacteria, other microorganisms, and animals and plants can also be used. A pc gene derived from a microorganism or animal or plant was isolated from a chromosome of a microorganism, animal or plant, etc., and the nucleotide sequence was determined based on a gene having a base sequence already determined, a gene encoding a protein having PC activity based on homology, etc. Things can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can also be used. These can be obtained by amplifying a region containing the promoter and the ORF portion by a hybridization method or a PCR method.

  A PC expression vector is provided by inserting the gene encoding PC isolated as described above into a known expression vector so as to allow expression. By transforming with this expression vector, a strain with enhanced PC activity can be obtained. Alternatively, a PC activity-enhanced strain can also be obtained by integrating a DNA encoding PC into a chromosomal DNA of a host microorganism so as to allow expression, such as by homologous recombination. Note that transformation and homologous recombination can be performed according to ordinary methods known to those skilled in the art.

When a PC gene is introduced onto a chromosome or a plasmid, an appropriate promoter is incorporated 5′-upstream of the gene, more preferably a terminator is incorporated 3′-downstream of the gene. The promoter and terminator are not particularly limited as long as the promoter and terminator are known to function in microorganisms used as hosts, and may be the promoter and terminator of the pc gene itself, or other promoters. And a terminator. Vectors, promoters and terminators that can be used in these various microorganisms are described in detail in, for example, “Basic Course of Microbiology 8 Genetic Engineering / Kyoritsu Shuppan”.

In the alcohol production method of the present invention, as in the carboxylic acid production method, it is preferable to use a microorganism modified so that LDH activity is reduced. Strains with reduced LDH activity can be prepared in the same manner as described above.
In the method for producing alcohol according to the present invention, a microorganism modified so as to enhance alcohol dehydrogenase (hereinafter also referred to as ADH) activity may be used. Here, “ADH activity” refers to an activity (EC: 1.1.1.1, 1.1.1.2) that catalyzes a reaction in which an aldehyde is reduced to produce an alcohol. “ADH activity is enhanced” means that ADH activity is increased as compared to an unmodified strain. The ADH activity is preferably increased 1.5 times or more per unit cell weight, more preferably 3 times or more, compared with the unmodified strain. The enhancement of ADH activity was confirmed by known methods such as the method of Kotrbova-Kozak et al. ), P1347-56) can be confirmed by measuring ADH activity.

A strain with enhanced ADH activity can be prepared in the same manner as the method for enhancing PC activity described above.
The adh gene used for enhancing ADH activity is not particularly limited as long as it encodes a protein having ADH activity. For example, adhB gene derived from Zymomonas mobilis, adhE2 gene derived from Clostridium acetobutylicum Can be mentioned.

  Furthermore, adh genes derived from bacteria other than the above, other microorganisms, and animals and plants can also be used. The adh gene derived from microorganisms or animals and plants was isolated from the chromosomes of microorganisms, animals and plants, etc., and the nucleotide sequences were determined based on the genes whose nucleotide sequences had already been determined, genes encoding ADH activity based on homology, etc. Things can be used. In addition, after the base sequence is determined, a gene synthesized according to the sequence can also be used. These can be obtained by amplifying a region containing the promoter and the ORF portion by a hybridization method or a PCR method.

The microorganism used in the present invention may be a microorganism obtained by combining two or more kinds of modifications for imparting organic compound-producing ability. When making a plurality of modifications, the order does not matter.
The microorganism used in the present invention may be a microorganism obtained by combining a modification for imparting organic compound-producing ability and a modification for imparting pentose utilization ability. When making a plurality of modifications, the order does not matter.

<Microbial culture method>
The culture method of the present invention is a microorganism culture method characterized by using an organic raw material containing pentose as a carbon source. The microorganism obtained by the culturing method of the present invention can then recover an organic compound produced by acting on an organic raw material. As an organic raw material at this time, an organic raw material containing pentose may be used, or another organic raw material may be used. Examples of organic compounds that can be produced and examples of preferred organic compounds are as described above.

In carrying out the culture method of the present invention using the microorganism, it may be cultured in a solid medium such as an agar medium containing pentose, or may be cultured in a liquid medium containing pentose. By performing seed culture and main culture described later, the microorganisms used for the organic compound production reaction can be grown.
The seed culture is performed in order to prepare the cells of the microorganism to be used for the main culture. As a medium used for seed culture, a normal medium used for culturing microorganisms can be used, but a medium containing a nitrogen source, an inorganic salt, or the like is preferable. Here, the nitrogen source is not particularly limited as long as it is a nitrogen source that can be assimilated and propagated by the present microorganism. Various organic and inorganic nitrogen compounds such as extract, meat extract and corn steep liquor can be mentioned. As the inorganic salt, various phosphates, sulfates, magnesium, potassium, manganese, iron, zinc, and other metal salts are used. In addition, vitamins such as biotin, thiamine, pantothenic acid, inositol, and nicotinic acid, factors that promote growth such as nucleotides and amino acids are added as necessary. Furthermore, if necessary, an organic raw material containing pentose as a carbon source may be added to the medium, or an organic raw material such as glucose may be added.

  The seed culture is preferably performed at a general optimum growth temperature. The general optimum growth temperature means a temperature at which the growth rate is the fastest under the conditions used for the production of the organic compound. The specific culture temperature is usually 25 ° C to 40 ° C, preferably 30 ° C to 37 ° C. In the case of coryneform bacteria, the temperature is usually 25 ° C to 35 ° C, more preferably 28 ° C to 33 ° C, and particularly preferably about 30 ° C.

The seed culture is preferably performed at a general optimum pH for growth. The general optimum growth pH refers to a pH having the fastest growth rate under the conditions used for the production of organic compounds. The specific culture pH is usually pH 4 to 10, preferably pH 6 to 8. In the case of coryneform bacteria, the pH is usually 6-9, with pH 6.5-8.5 being preferred.
The culture time for seed culture is not particularly limited as long as a certain amount of cells can be obtained, but is usually 6 hours or more and 96 hours or less. In the seed culture, oxygen is preferably supplied by aeration or stirring.

The bacterial cells after the seed culture can be used for the main culture described later. However, the seed culture may be omitted, and the cells cultured on the slope with a solid medium such as an agar medium may be directly used for the main culture. Moreover, you may repeat seed culture several times as needed.
The main culture is performed in order to prepare the microbial cells to be subjected to the organic compound production reaction described later, and is mainly intended to increase the amount of the microbial cells. When performing the seed culture described above, the main culture is performed using the cells obtained by seed culture.

  The medium used for the main culture can be a normal medium used for culturing microorganisms, but is preferably a medium containing a nitrogen source, an inorganic salt, or the like. Here, the nitrogen source is not particularly limited as long as it is a nitrogen source that can be assimilated and propagated by the present microorganism. Specifically, ammonium salt, nitrate, urea, soybean hydrolyzate, casein degradation product, peptone, yeast Various organic and inorganic nitrogen compounds such as extract, meat extract and corn steep liquor can be mentioned. As the inorganic salt, various phosphates, sulfates, magnesium, potassium, manganese, iron, zinc, and other metal salts are used. In addition, vitamins such as biotin, thiamine, pantothenic acid, inositol, and nicotinic acid, factors that promote growth such as nucleotides and amino acids are added as necessary. In order to suppress foaming during culture, it is preferable to add an appropriate amount of a commercially available antifoaming agent to the medium.

  In the main culture, an organic raw material containing pentose is used as a carbon source. If necessary, other organic raw materials may be added. The organic raw material other than pentose used in the main culture is not particularly limited as long as the microorganism can be assimilated and proliferated. Usually, galactose, lactose, glucose, fructose, sucrose, saccharose, starch, cellulose, etc. Fermentable carbohydrates such as carbohydrates; polyalcohols such as glycerol, mannitol, xylitol, ribitol and the like are used, among which glucose, sucrose, or fructose is preferable, and glucose or sucrose is particularly preferable.

Moreover, the starch saccharified liquid containing the said fermentable saccharide | sugar, molasses etc. are also used, and what is the saccharide liquid which the said fermentable saccharide extracted from plants, such as sugarcane, sugar beet, and a sugar maple, is preferable.
These organic raw materials may be added alone or in combination.
The concentration of the organic raw material used is not particularly limited, but it is advantageous to add it within a range that does not inhibit growth, and is usually 0.1 to 10% (W / V), preferably 0.5 It can be used within a range of ˜5% (W / V). Further, an additional addition of the organic raw material may be performed in accordance with the decrease of the organic raw material accompanying the growth.

The main culture is preferably performed at a general optimum growth temperature. The specific culture temperature is usually 25 ° C to 40 ° C, preferably 30 ° C to 37 ° C. In the case of coryneform bacteria, the temperature is usually 25 ° C to 35 ° C, more preferably 28 ° C to 33 ° C, and particularly preferably about 30 ° C.
Further, the main culture is preferably performed at a general optimum growth pH. The specific culture pH is usually pH 4 to 10, preferably pH 6 to 8. In the case of coryneform bacteria, the pH is usually 6-9, with pH 6.5-8.5 being preferred.

The culture time for the main culture is not particularly limited as long as a certain amount of cells can be obtained, but is usually 6 hours or more and 96 hours or less. In the main culture, it is preferable to supply oxygen by aeration or stirring.
In addition, in the main culture, as a method for preparing bacterial cells more suitable for the production of organic compounds, the culture is performed so that the carbon source depletion and sufficiency described in JP-A-2008-259451 are alternately repeated in a short time. Methods can also be used.
The cells after the main culture can be used for the organic compound production reaction described later, but the culture solution may be used directly, or may be used after the cells are collected by centrifugation, membrane separation or the like.

<Method for producing organic compound>
The method for producing an organic compound of the present invention comprises the step of producing an organic compound by allowing the microorganism or a treated product thereof to act on an organic raw material containing pentose in an aqueous medium. Is the method. Among them, it is preferable to produce an organic compound by allowing the microorganism or a processed product thereof to act on an organic raw material containing pentose, and recover this. Examples of organic compounds that can be produced and examples of preferred organic compounds are as described above.

  Here, the aqueous medium is an aqueous solution that performs an organic compound production reaction, and is preferably an aqueous solution containing a nitrogen source, an inorganic salt, and the like as described later. In the aqueous medium, an organic compound production reaction can be performed by reacting the microorganism or a processed product thereof with an organic raw material. In this specification, the aqueous medium means all liquids contained in the reaction vessel.

When using the microorganism in the method for producing an organic compound of the present invention, it is possible to directly use a slant-cultured solid medium such as an agar medium, but if necessary, use a microorganism previously cultured in a liquid medium May be. That is, the microorganism may be proliferated in advance and the microorganism may produce an organic compound. By performing seed culture and main culture as described above, the microorganism can be proliferated in advance. As the organic raw material used at this time, an organic raw material containing pentose may be used. Organic raw materials may be used.

An organic compound may be produced by reacting an organic raw material containing pentose while growing a seed culture or main culture microorganism in a reaction solution, or proliferating by performing seed culture or main culture in advance. You may manufacture an organic compound by making the microbial cell made to react with an organic raw material in the aqueous medium containing the organic raw material containing a pentose.
Moreover, in the organic compound manufacturing method of this invention, the processed material of microorganisms can also be used. Examples of the processed microorganisms include, for example, immobilized microorganisms in which microorganisms are immobilized with acrylamide, carrageenan, etc., disrupted microorganisms, centrifuged supernatants thereof, or supernatants thereof by ammonium sulfate treatment, etc. Examples include partially purified fractions.

  In the organic compound production method of the present invention, an organic raw material containing pentose is used. If necessary, other organic raw materials may be added. The organic raw material other than the pentose used in the organic compound production method is not particularly limited as long as it is a carbon source that can be used by the microorganism to produce an organic compound, but is usually galactose, lactose, glucose, fructose, sucrose, starch Alternatively, carbohydrates such as cellulose; fermentable carbohydrates such as polyalcohols such as glycerol, mannitol, xylitol, and ribitol are used, and among these, glucose, sucrose, or fructose is preferable, and glucose or sucrose is particularly preferable.

In addition, a starch saccharified solution or molasses containing the fermentable carbohydrate is also used, and specifically, a sugar solution extracted from a plant such as sugar cane, sugar beet or sugar maple is preferable.
These organic raw materials may be added alone or in combination.
The use concentration of the organic raw material is not particularly limited, but it is advantageous to make it as high as possible within the range not inhibiting the formation of the organic compound, and is usually 5% (W / V) or more, preferably On the other hand, it is usually 30% (W / V) or less, preferably 20% (W / V) or less. Further, an additional organic raw material may be added in accordance with a decrease in the organic raw material as the reaction proceeds.

  The aqueous medium is not particularly limited, and may be, for example, a medium for culturing microorganisms or a buffer solution such as a phosphate buffer, but the reaction solution may be a nitrogen source or an inorganic salt. An aqueous solution containing is preferable. Here, the nitrogen source is not particularly limited as long as it is a nitrogen source that can be assimilated by the microorganism to produce an organic compound. Specifically, ammonium salt, nitrate, urea, soybean hydrolysate, casein degradation product And various organic and inorganic nitrogen compounds such as peptone, yeast extract, meat extract and corn steep liquor. As the inorganic salt, various phosphates, sulfates, magnesium, potassium, manganese, iron, zinc, and other metal salts are used. In addition, vitamins such as biotin, thiamine, pantothenic acid, inositol, and nicotinic acid, factors that promote growth such as nucleotides and amino acids are added as necessary. In order to suppress foaming during the reaction, it is preferable to add an appropriate amount of a commercially available antifoaming agent to the reaction solution.

The aqueous medium preferably contains carbonate ion, bicarbonate ion, or carbon dioxide gas (carbon dioxide gas) in addition to the organic raw material, nitrogen source, inorganic salt, and the like described above. Carbonate ions or bicarbonate ions are supplied from magnesium carbonate, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, etc., which can also be used as a neutralizing agent. It can also be supplied from these salts or carbon dioxide gas. Specific examples of the carbonate or bicarbonate salt include, for example, magnesium carbonate,
Examples include ammonium carbonate, sodium carbonate, potassium carbonate, ammonium bicarbonate, sodium bicarbonate, potassium bicarbonate and the like.

  The concentration of carbonate ion or bicarbonate ion in the aqueous medium is usually 1 mM or more, preferably 2 mM or more, more preferably 3 mM or more, and usually 500 mM or less, preferably 300 mM or less, more preferably 200 mM or less. When carbon dioxide gas is contained, it is usually preferred to contain 50 mg or more, preferably 100 mg or more, more preferably 150 mg or more of carbon dioxide gas per liter of aqueous medium, while usually 25 g or less, preferably It is preferable to contain 15 g or less, more preferably 10 g or less of carbon dioxide gas.

  The pH of the aqueous medium during the organic compound production reaction is preferably adjusted to a range where the activity is most effectively exhibited according to the type of the microorganism used. Specifically, when coryneform bacteria are used, the pH of the reaction solution is usually 5.5 or higher, preferably 6 or higher, more preferably 6.6 or higher, and even more preferably 7.1 or higher. Usually, it is preferably 10 or less, preferably 9.5 or less, more preferably 9.0 or less.

  The pH of the aqueous medium is determined when the organic compound produced is an acidic substance, such as sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, ammonium carbonate, ammonium bicarbonate, sodium hydroxide, hydroxide It can be adjusted by adding calcium, magnesium hydroxide, ammonia (ammonium hydroxide), or a mixture thereof. When the organic compound to be produced is a basic substance, by adding an inorganic acid such as hydrochloric acid, sulfuric acid or phosphoric acid, an organic acid such as acetic acid, a mixture thereof, or by supplying carbon dioxide gas Can be adjusted.

  Further, inositol may be contained in the aqueous medium. Thereby, the expression of iolT gene can be induced and the expression can be enhanced. In enhancing the expression of the iolT gene by this means, a strain that has been genetically modified as described above may be used, but a strain that has not been genetically modified can also be used. Examples of inositol that can be used here are the same as those exemplified in <Microorganisms or processed product used in the present invention> such as myo-inositol.

The amount of microbial cells used in the organic compound production reaction is not particularly limited, but the weight of wet cells is usually 1 g / L or more, preferably 10 g / L or more, more preferably 20 g / L or more. 700 g / L or less, preferably 500 g / L or less, more preferably 400 g / L or less.
The time for the organic compound production reaction is not particularly limited, but is usually 1 hour or longer, preferably 3 hours or longer, and is usually 168 hours or shorter, preferably 72 hours or shorter.

  The organic compound production reaction may be performed at the same temperature as the optimum growth temperature of the microorganism to be used, but it is advantageous to carry out the reaction at a temperature higher than the optimum growth temperature, usually 2 ° C to 20 ° C, preferably 7 to 15 ° C higher temperature Specifically, in the case of coryneform bacteria, it is usually 35 ° C. or higher, preferably 37 ° C. or higher, more preferably 39 ° C. or higher, and usually 45 ° C. or lower, preferably 43 ° C. or lower, more preferably 41 It is below ℃. During the organic compound production reaction, it is not always necessary to set the temperature within the range of 35 ° C to 45 ° C.

The organic compound generation reaction may be performed with aeration and stirring, but is preferably performed in an anaerobic atmosphere without aeration and without supplying oxygen. Under the anaerobic atmosphere here, for example, the container is sealed and reacted without aeration, the reaction is performed by supplying an inert gas such as nitrogen gas, or the inert gas containing carbon dioxide gas is vented. Can be obtained.
The organic compound production method of the present invention is not particularly limited, but can be applied to any of batch reaction, semi-batch reaction, and continuous reaction.

  By the microbial reaction as described above, an organic compound can be generated and accumulated in the reaction solution. The accumulated organic compound can be recovered from the reaction solution according to a conventional method (recovery step). Specifically, for example, when the accumulated organic compound is a carboxylic acid such as succinic acid, fumaric acid or malic acid, solid substances such as cells are removed by centrifugation, filtration, etc., and then ion exchange is performed. The carboxylic acid can be recovered by desalting with a resin or the like, and purification from the solution by crystallization (crystallization) or column chromatography. If the accumulated organic compound is an alcohol such as ethanol, butanol, or butanediol, solids such as cells are removed by centrifugation, filtration, etc., and then concentrated by distillation or the like, and the solution is dehydrated into a membrane. For example, alcohol can be recovered.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

[Comparative Example 1]
For Brevibacterium flavum MJ233 strain, disruption of lactate dehydrogenase gene, enhancement of pyruvate carboxylase gene, introduction of xylose isomerase gene and xylulokinase gene were performed as follows, and Brevibacterium flavum MJ233 / XylAB / A PC-4 / ΔLDH strain was prepared.

<Disruption of lactate dehydrogenase gene>
(A) Extraction of Brevibacterium flavum MJ233 strain genomic DNA Brevibacterium flavum MJ233 strain A medium containing 2% glucose [urea: 4 g, ammonium sulfate: 14 g, 1 potassium phosphate: 0.5 g, phosphate 2 potassium 0.5 g, magnesium sulfate heptahydrate: 0.5 g, ferrous sulfate heptahydrate: 20 mg, manganese sulfate pentahydrate: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 200 μg Yeast extract: 1 g, casamino acid: 1 g, dissolved in 1,000 mL of distilled water] The cells were cultured with 10 mL until the late logarithmic growth phase and collected. The obtained cells were suspended in 0.15 mL of a buffer solution [20 mM Tris-HCl pH 8.0, 10 mM NaCl, 1 mM EDTA · 2Na] containing lysozyme at a concentration of 10 mg / mL. Next, proteinase K was added to the suspension so that the final concentration was 100 μg / mL, and the mixture was incubated at 37 ° C. for 1 hour. Further, sodium dodecyl sulfate was added to a final concentration of 0.5%, and the mixture was incubated at 50 ° C. for 6 hours for lysis. After adding an equal amount of phenol / chloroform solution to this lysate and shaking gently at room temperature for 10 minutes, the whole amount was centrifuged (5,000 × g, 20 minutes, 10 to 12 ° C.) to obtain a supernatant fraction. The fraction was collected. After adding sodium acetate to 0.3M, 2 volumes of ethanol was added and mixed, and the precipitate collected by centrifugation (15,000 × g, 2 minutes) was washed with 70% ethanol, Air dried. To the obtained DNA, 5 mL of TE buffer [10 mM Tris-HCl pH 7.5, 1 mM EDTA · 2Na] was added and allowed to stand overnight at 4 ° C., and used as template DNA for subsequent PCR.

(B) Cloning of the lactate dehydrogenase gene The ldh gene of Brevibacterium flavum MJ233 strain was obtained from the Corynebacterium glutamicum ATCC13032 strain whose entire genome sequence was reported using the DNA prepared in (A) as a template. Sequence around the gene (GenBank Accession
No. This was carried out by PCR using synthetic DNA (SEQ ID NO: 7 and SEQ ID NO: 8) designed based on (BA000036).

The composition of the reaction solution was as follows: template DNA 1 μl, Taq DNA polymerase (Takara Bio) 0
. 2 μl, 1 × concentration buffer, 0.2 μM each primer, 0.25 μM dN
TPs were mixed to make a total volume of 20 μl. As the reaction temperature condition, a DNA thermal cycler PTC-200 (MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 55 ° C. for 20 seconds, and 72 ° C. for 1 minute was repeated 30 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 5 minutes.

  The amplification product was confirmed by separation by 0.75% agarose gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.95 kb was detected. Recovery of the target DNA fragment from the gel was performed using a QIAquick Gel Extraction Kit (QIAGEN). The recovered DNA fragment was mixed with a PCR product cloning vector pGEM-TEasy (Promega), and DNA Ligation Kit ver. 2 (Takara Bio), and the resulting plasmid DNA was used to transform E. coli DH5α strain. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin and 50 μg / mL X-Gal. A clone that formed white colonies on this medium was subjected to liquid culture in TB medium [Terrific Broth 47 g / L, Glycerol 5 g / L] (containing 50 μg / mL ampicillin), and then using QIAprep Spin Miniprep Kit (QIAGEN) Plasmid DNA was prepared. The plasmid DNA thus obtained was cleaved with restriction enzymes SacI and SphI. As a result, an inserted fragment of about 1.0 kb was confirmed, and this was named pGEMT / CgLDH.

(C) Construction of plasmid for disrupting lactate dehydrogenase gene The internal sequence of the coding region of LDH consisting of about 0.25 kb was cut out by cutting pGEMT / CgLDH prepared in (B) above with restriction enzymes EcoRV and XbaI. The remaining DNA fragment of about 3.7 kb was blunted with Klenow Fragment (Takara Bio) and DNA Ligation Kit ver. 2 (Takara Bio) was used to circulate and transform Escherichia coli DH5α strain. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin. The obtained clone was subjected to liquid culture in TB medium [Terrific Broth 47 g / L, Glycerol 5 g / L] (containing 50 μg / mL ampicillin), and then QIAprep.
Plasmid DNA was prepared using Spin Miniprep Kit (QIAGEN). The plasmid DNA thus obtained was cleaved with restriction enzymes SacI and SphI. As a result, an inserted fragment of about 0.75 kb was confirmed, and this was named pGEMT / ΔLDH.

Next, a DNA fragment of about 0.75 kb generated by cleaving the above pGEMT / ΔLDH with restriction enzymes SacI and SphI is separated and recovered by 0.75% agarose gel electrophoresis, and an ldh gene fragment containing a defective region is obtained. Prepared. This DNA fragment was mixed with pKMB1 (Japanese Patent Laid-Open No. 2005-95169) cleaved with restriction enzymes SacI and SphI, and DNA
Ligation Kit ver. After ligation using 2 (Takara Bio), Escherichia coli DH5α strain was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin and 50 μg / mL X-Gal. A clone that formed white colonies on this medium was subjected to liquid culture in TB medium (containing 50 μg / mL kanamycin), and then plasmid DNA was prepared using QIAprep Spin Miniprep Kit (QIAGEN). The plasmid DNA thus obtained was cleaved with restriction enzymes SacI and SphI. As a result, an inserted fragment of about 0.75 kb was confirmed, and this was named pKMB1 / ΔLDH. The construction process of pKMB1 / ΔLDH is shown in FIG.

(D) Disruption of lactate dehydrogenase gene Plasmid DNA used for transformation of Brevibacterium flavum MJ233 strain was transformed into Escherichia coli transformed by the calcium chloride method (Journal of Molecular Biology, 1970, 53, 159) using pKMB1 / ΔLDH. Prepared from JM110 strain.

Brevibacterium flavum MJ233 strain is prepared by conventional methods (Wolf Hetal, J. Bacteriol., 1983, 156 (3), p1165-70, Kurusu Yetal., Agric Biol Chem., 1990, 54 (2), p443-7. ), The endogenous plasmid was removed (curing) and used for the subsequent transformation.
Transformation of Brevibacterium flavum MJ233 strain was carried out by the electric pulse method (Res. Microbiol., 1993, 144, p181-5), and the obtained transformant was treated with an LBG agar medium containing 50 μg / mL kanamycin [tryptone. 10 g, 5 g yeast extract, 5 g NaCl, 20 g glucose, and 15 g agar were dissolved in 1 L distilled water].

  Since the strain grown on this medium is a plasmid in which pKMB1 / ΔLDH is not replicable in Brevibacterium flavum MJ233 strain, the ldh gene of the plasmid and the same gene on the genome of Brevibacterium flavum MJ233 strain As a result of homologous recombination with the kanamycin, the kanamycin resistance gene and SacB gene derived from the plasmid should be inserted on the same genome.

Next, the homologous recombination strain was subjected to liquid culture in an LBG medium containing 50 μg / mL kanamycin. A portion equivalent to about 1 million cells of this culture was smeared on a 10% sucrose-containing LBG medium. As a result, about 10 strains that were considered to be sucrose-insensitive due to elimination of the SacB gene by the second homologous recombination were obtained.
Among the strains obtained in this manner, those in which the ldh gene is replaced with a mutant derived from pKMB1 / ΔLDH and those in which the wild type is restored are included. Confirmation of whether the ldh gene is a mutant type or a wild type can be easily confirmed by subjecting the cells obtained by culturing in the LBG medium to a direct PCR reaction and detecting the ldh gene. When analyzed using primers (SEQ ID NO: 9 and SEQ ID NO: 10) for PCR amplification of the ldh gene, a wild-type DNA fragment of 720 bp and a mutant type having a deletion region should have a DNA fragment of 471 bp. As a result of analyzing the strain that became insensitive to sucrose by the above method, a strain having only the mutant gene was selected, and the strain was named Brevibacterium flavum MJ233 / ΔLDH.

(E) Measurement of lactate dehydrogenase activity Brevibacterium flavum MJ233 / ΔLDH strain prepared in (D) above was added to A medium containing 2% glucose [urea: 4 g, ammonium sulfate: 14 g, 1 potassium phosphate: 0.5 g. , Dipotassium phosphate 0.5 g, magnesium sulfate heptahydrate: 0.5 g, ferrous sulfate heptahydrate: 20 mg, manganese sulfate pentahydrate: 20 mg, D-biotin: 200 μg, hydrochloric acid Thiamine: 200 μg, yeast extract: 1 g, casamino acid: 1 g, dissolved in 1,000 mL of distilled water] and inoculated aerobically at 30 ° C. for 15 hours. The obtained culture broth was centrifuged (3,000 × g, 4 ° C., 20 minutes) to recover the cells, and then washed with 50 mM sodium phosphate buffer (pH 7.3).

Next, 0.5 g (wet weight) of washed cells was suspended in 2 mL of the sodium phosphate buffer, and subjected to an ultrasonic crusher (Branson) under ice cooling to obtain a crushed cell. The crushed material is centrifuged (
(10,000 × g, 4 ° C., 30 minutes), and the supernatant was obtained as a crude enzyme solution. As a control, a crude enzyme solution of Brevibacterium flavum MJ233 strain was similarly prepared and subjected to the following activity measurement.

The LDH enzyme activity was measured by detecting the coenzyme NADH being oxidized to NAD ⁺ by the change in absorbance at 340 nm with the production of lactic acid using pyruvate as a substrate for both crude enzyme solutions. The reaction was performed at 37 ° C. in the presence of 50 mM potassium phosphate buffer (pH 7.2), 10 mM pyruvic acid and 0.4 mM NADH. As a result, the LDH activity in the crude enzyme solution prepared from Brevibacterium flavum MJ233 / ΔLDH was 1/10 or less compared to the LDH activity in the crude enzyme solution prepared from Brevibacterium flavum MJ233 strain. there were.

<Enhancement of pyruvate carboxylase gene>
(A) Preparation of Promoter Fragment for Coryneform Bacteria Acquisition of TZ4 promoter (DNA fragment described in SEQ ID NO: 4 of JP-A-7-95891), which is a DNA fragment having strong promoter activity in coryneform bacteria, was obtained in the above Comparative Example. 1. Synthetic DNA designed based on the sequence described in JP-A-7-95891 using the genomic DNA of Brevibacterium flavum MJ233 prepared in (A) of <1) <Disruption of lactate dehydrogenase gene> It was performed by PCR using (SEQ ID NO: 11 and SEQ ID NO: 12).

The composition of the reaction solution was 1 μl of template DNA, 0.2 μl of Pfx DNA polymerase (Invitrogen), 1 × concentration buffer, 0.3 μM each primer, 1 mM MgSO ₄ , 0.25 μM dNTPs, and the total volume was 20 μl. As the reaction temperature conditions, a DNA thermal cycler PTC-200 (MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds, and 72 ° C. for 30 seconds was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 2 minutes.

The amplification product was confirmed by separation by 2.0% agarose gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 250 bp was detected. Recovery of the target DNA fragment from the gel was performed using a QIAquick Gel Extraction Kit (QIAGEN). The recovered DNA fragment is T4 Polynucleotide.
After phosphorylating the 5 ′ end with Kinase (Takara Bio), DNA Ligation Kit ver. 2 (Takara Bio) was used to bind to the SmaI site of E. coli vector pUC19 (Takara Bio), and the resulting plasmid DNA was used to transform E. coli DH5α strain. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin and 50 μg / mL X-Gal.

  Six clones that formed white colonies on this medium were subjected to liquid culture in TB medium [Terrific Broth 47 g / L, Glycerol 5 g / L] (containing 50 μg / mL ampicillin), and then QIAprep Spin Miniprep Kit (QIAGEN) was used. The plasmid DNA was purified and the nucleotide sequence was determined. Among them, a clone in which the TZ4 promoter was inserted so as to have a transcriptional activity in the reverse direction to the lac promoter of pUC19 was selected and named pUC / TZ4.

Next, a DNA fragment prepared by cleaving pUC / TZ4 with restriction enzymes BamHI and PstI consists of synthetic DNA (SEQ ID NO: 13 and SEQ ID NO: 14) phosphorylated at the 5 ′ end, and BamHI and A DNA linker having a sticky end to PstI was mixed, and DNA Ligation Kit ver. After ligation using 2 (Takara Bio), Escherichia coli DH5α strain was transformed with the obtained plasmid DNA. This DNA linker includes a ribosome binding sequence (AGGAGG) and a cloning site (PacI, NotI, ApaI in this order from the upstream).

  A clone that formed white colonies on this medium was subjected to liquid culture in TB medium [Terrific Broth 47 g / L, Glycerol 5 g / L] (containing 50 μg / mL ampicillin), and then using QIAprep Spin Miniprep Kit (QIAGEN) The plasmid DNA was purified. From the obtained plasmid DNA, one that was cleaved by the restriction enzyme NotI was selected and named pUC / TZ4-SD.

  The pUC / TZ4-SD constructed in this way was cleaved with the restriction enzyme PstI, the ends were blunted with Klenow Fragment (Takara Bio), and then cleaved with the restriction enzyme KpnI to obtain a promoter fragment of about 300 bp. It isolate | separated by 2.0% agarose gel electrophoresis and collect | recovered using QIAquick Gel Extraction Kit (QIAGEN).

(B) Construction of Coryneform Bacterial Expression Vector pHSG298par-rep (Japanese Patent Laid-Open No. 12-93183) was used as a plasmid that can stably replicate independently in coryneform bacteria. This plasmid comprises a replication region and a region having a stabilizing function of the natural plasmid pBY503 possessed by Brevibacterium stachynis IFO12144, a kanamycin resistance gene derived from the Escherichia coli vector pHSG298 (Takara Bio), and a replication region of Escherichia coli. The DNA prepared by cleaving pHSG298par-rep with the restriction enzyme SseI, blunting the ends with Klenow Fragment (Takara Bio), and then cleaving with the restriction enzyme KpnI > TZ4 promoter fragment prepared in (A), and DNA Ligation Kit ver. After ligation using 2 (Takara Bio), Escherichia coli DH5α strain was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin.

  The strain grown on this medium is subjected to liquid culture with TB medium [Terrific Broth 47 g / L, Glycerol 5 g / L] (including 50 μg / mL kanamycin), and then the plasmid DNA is purified using QIAprep Spin Miniprep Kit (QIAGEN) did. From the obtained plasmid DNA, one that was cleaved by the restriction enzyme NotI was selected, and the plasmid was named pTZ4.

(C) Cloning of pyruvate carboxylase gene The pc gene of Brevibacterium flavum MJ233 strain was obtained by using the DNA prepared in (A) of <Disruption of lactate dehydrogenase gene> in Comparative Example 1 as a template and the entire genome sequence. Was performed by PCR using synthetic DNA (SEQ ID NO: 15 and SEQ ID NO: 16) designed based on the sequence around the gene of Corynebacterium glutamicum ATCC13032 (GenBank Accession No. BA00000036).

The composition of the reaction solution was 1 μl of template DNA, 0.2 μl of Pfx DNA polymerase (Invitrogen), 1 × concentration buffer, 0.3 μM each primer, 1 mM MgSO ₄ , 0.25 μM dNTPs, and the total volume was 20 μl. As a reaction temperature condition, a DNA thermal cycler PTC-200 (MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds and 68 ° C. for 4 minutes was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute and 20 seconds, and the heat retention at 68 ° C. in the final cycle was 10 minutes.
After completion of the PCR reaction, 0.1 μl of Ex Taq DNA polymerase (Takara Bio) was added and incubated at 72 ° C. for 30 minutes. The amplification product was confirmed by separation by 0.75% agarose gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 3.7 kb was detected.

Recovery of the target DNA fragment from the gel was performed using a QIAquick Gel Extraction Kit (QIAGEN). The recovered DNA fragment was mixed with a PCR product cloning vector pGEM-TEasy (Promega), and DNA Ligation Kit ver. 2 (Takara Bio), and the resulting plasmid DNA was used to transform E. coli DH5α strain. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin and 50 μg / mL X-Gal.
A clone that formed white colonies on this medium was subjected to liquid culture in TB medium [Terrific Broth 47 g / L, Glycerol 5 g / L] (containing 50 μg / mL ampicillin), and then using QIAprep Spin Miniprep Kit (QIAGEN) Plasmid DNA was prepared. The plasmid DNA thus obtained was cleaved with restriction enzymes PacI and ApaI. As a result, an inserted fragment of about 3.7 kb was confirmed, and this was named pGEM / MJPC.

  The base sequence of the insert fragment of pGEM / MJPC was determined using BigDye Terminator v3 Cycle Sequencing Kit and base sequence decoder 377XL (Applied Biosystems). The amino acid sequence deduced from the nucleotide sequence obtained as a result showed extremely high homology (99.4%) with the PC derived from Corynebacterium glutamicum ATCC13032. Therefore, the insert fragment of pGEM / MJPC was found to be a Brevibacterium. It was determined to be a pc gene derived from Um flavum MJ233 strain.

(D) Construction of plasmid for enhancing pyruvate carboxylase activity A pc gene fragment consisting of about 3.7 kb produced by cleaving the pGEM / MJPC prepared above with restriction enzymes PacI and ApaI was subjected to 0.75% agarose gel electrophoresis. Separated and recovered. This DNA fragment was mixed with pTZ4 cleaved with the same restriction enzyme, and DNA Ligation Kit ver. 2 (Takara Bio), and the resulting plasmid DNA was used to transform E. coli DH5α strain. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin.

  The obtained clone was subjected to liquid culture in TB medium [Terrific Broth 47 g / L, Glycerol 5 g / L] (containing 50 μg / mL kanamycin), and then a plasmid DNA was prepared using QIAprep Spin Miniprep Kit (QIAGEN). The plasmid DNA thus obtained was cleaved with restriction enzymes PacI and ApaI. As a result, an inserted fragment of about 3.7 kb was confirmed, and this was named pMJPC1. The construction process of pMJPC1 is shown in FIG.

(E) Construction of Pyruvate Carboxylase Gene Promoter Replacement Plasmid A DNA fragment in the N-terminal region of the pc gene derived from Brevibacterium flavum MJ233 strain was obtained by (A) in <Disruption of lactate dehydrogenase gene> in Comparative Example 1 above. Synthetic DNA (SEQ ID NO: 17 and SEQ ID NO: 17) that was designed based on the sequence around the gene of Corynebacterium glutamicum ATCC 13032 (GenBank Accession No. BA000036), which was prepared by using the DNA prepared in step 1 as a template. 18). The DNA of SEQ ID NO: 18 used was phosphorylated at the 5 ′ end.

The composition of the reaction solution was 1 μl of template DNA, 0.2 μl of Pfx DNA polymerase (Invitrogen), 1 × concentration buffer, 0.3 μM each primer, 1 mM MgSO ₄ , 0.25 μM dNTPs, and the total volume was 20 μl. As a reaction temperature condition, a DNA thermal cycler PTC-200 (MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds, and 72 ° C. for 1 minute was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 4 minutes.

The amplification product was confirmed by separation by 0.75% agarose gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.9 kb was detected. The DNA fragment of interest was recovered from the gel using a QIAquick Gel Extraction Kit (QIAGEN), which was used as the pc gene N-terminal fragment.
On the other hand, TZ4 promoter fragment constitutively highly expressed from Brevibacterium flavum strain MJ233 was obtained by PCR using plasmid pMJPC1 as a template and the synthetic DNAs described in SEQ ID NO: 19 and SEQ ID NO: 20. The DNA of SEQ ID NO: 20 used was phosphorylated at the 5 ′ end.

The composition of the reaction solution was 1 μl of template DNA, 0.2 μl of Pfx DNA polymerase (Invitrogen), 1 × concentration buffer, 0.3 μM each primer, 1 mM MgSO ₄ , 0.25 μM dNTPs, and the total volume was 20 μl. As the reaction temperature conditions, a DNA thermal cycler PTC-200 (MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds, and 72 ° C. for 30 seconds was repeated 25 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 3 minutes.

The amplification product was confirmed by separation by 1.0% agarose gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.5 kb was detected. The DNA fragment of interest was recovered from the gel using a QIAquick Gel Extraction Kit (QIAGEN), which was used as a TZ4 promoter fragment.
The pc gene N-terminal fragment prepared above and the TZ4 promoter fragment were mixed, and DNA Ligation Kit ver. After ligation using 2 (Takara Bio), it was cleaved with restriction enzyme PstI, separated by 1.0% agarose gel electrophoresis, and a DNA fragment of about 1.0 kb was recovered using QIAquick Gel Extraction Kit (QIAGEN). This DNA fragment and E. coli plasmid pHSG299 (Takara Bio) were mixed with DNA prepared by digestion with PstI, and DNA Ligation Kit ver. 2 (Takara Bio), and the resulting plasmid DNA was used to transform E. coli DH5α strain. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin and 50 μg / mL X-Gal.

  A clone that formed white colonies on this medium was subjected to liquid culture in TB medium [Terrific Broth 47 g / L, Glycerol 5 g / L] (containing 50 μg / mL kanamycin), and then using QIAprep Spin Miniprep Kit (QIAGEN) Plasmid DNA was prepared. The plasmid DNA thus obtained was cleaved with the restriction enzyme PstI. As a result, an inserted fragment of about 1.0 kb was confirmed, and this was named pMJPC17.1.

The DNA fragment in the 5 ′ upstream region of the pc gene derived from Brevibacterium flavum MJ233 strain was obtained by using the DNA prepared in (A) of <Lactate dehydrogenase gene disruption> in Comparative Example 1 as a template, and the entire genome sequence was Sequences around the gene of the reported Corynebacterium glutamicum ATCC13032 strain (GenBank Accession)
No. This was performed by PCR using synthetic DNAs (SEQ ID NO: 21 and SEQ ID NO: 22) designed based on (BA000036).

The composition of the reaction solution was 1 μl of template DNA, 0.2 μl of Pfx DNA polymerase (Invitrogen), 1 × concentration buffer, 0.3 μM each primer, 1 mM MgSO ₄ , 0.25 μM dNTPs, and the total volume was 20 μl. As the reaction temperature conditions, a DNA thermal cycler PTC-200 (MJ Research) was used, and a cycle consisting of 94 ° C. for 20 seconds, 60 ° C. for 20 seconds, and 72 ° C. for 30 seconds was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 1 minute 20 seconds, and the heat retention at 72 ° C. in the final cycle was 5 minutes.

The amplification product was confirmed by separation by 1.0% agarose gel electrophoresis and visualization by ethidium bromide staining to detect a fragment of about 0.7 kb. Recovery of the target DNA fragment from the gel was performed using a QIAquick Gel Extraction Kit (QIAGEN). The recovered DNA fragment is T4 Polynucleotide.
After phosphorylating the 5 ′ end with Kinase (Takara Bio), DNA Ligation Kit ver. 2 (Takara Bio) was used to bind to the SmaI site of E. coli vector pUC119 (Takara Bio), and the resulting plasmid DNA was used to transform E. coli DH5α strain. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL ampicillin and 50 μg / mL X-Gal.

  A clone that formed white colonies on this medium was subjected to liquid culture in TB medium [Terrific Broth 47 g / L, Glycerol 5 g / L] (containing 50 μg / mL ampicillin), and then using QIAprep Spin Miniprep Kit (QIAGEN) Plasmid DNA was prepared. The obtained plasmid DNA was subjected to PCR reaction using the primers of SEQ ID NO: 22 and SEQ ID NO: 23. As a result of confirming the presence or absence of the inserted DNA fragment in this way, a plasmid that recognized an amplification product of about 0.7 kb was selected and named pMJPC5.1.

  Next, the above-mentioned pMJPC17.1 and pMJPC5.1 were digested with restriction enzyme XbaI and mixed, and DNA Ligation Kit ver. 2 (Takara Bio). The DNA fragment cleaved with restriction enzymes SacI and SphI was separated by 0.75% agarose gel electrophoresis, and a DNA fragment of about 1.75 kb was recovered using QIAquick Gel Extraction Kit (QIAGEN). A DNA fragment in which the TZ4 promoter was inserted between the 5 ′ upstream region and the N-terminal region of this pc gene was mixed with DNA prepared by cleaving pKMB1 (Japanese Patent Laid-Open No. 2005-95169) with SacI and SphI, and DNA Ligation Kit ver. 2 (Takara Bio), and the resulting plasmid DNA was used to transform E. coli DH5α strain. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 50 μg / mL kanamycin and 50 μg / mL X-Gal.

  A clone that formed white colonies on this medium was subjected to liquid culture in TB medium (containing 50 μg / mL kanamycin), and then plasmid DNA was prepared using QIAprep Spin Miniprep Kit (QIAGEN). The plasmid DNA thus obtained was cleaved with restriction enzymes SacI and SphI. As a result, an insert fragment of about 1.7 kb was confirmed, and this was named pMJPC17.3. The construction process of pMJPC17.3 is shown in FIG.

(F) Enhancement of pyruvate carboxylase gene Plasmid DNA used for transformation of the Brevibacterium flavum MJ233 / ΔLDH strain prepared in (D) of <Disruption of lactate dehydrogenase gene> in Comparative Example 1 above is pMJPC17.3. And re-prepared from Escherichia coli JM110 strain transformed by the calcium chloride method (Journal of Molecular Biology, 1970, 53, 159).

Transformation of the Brevibacterium flavum MJ233 / ΔLDH strain was performed by the electric pulse method (Res. Microbiol., 1993, 144, p181-5), and the obtained transformant was added to an LBG agar medium containing 25 μg / mL kanamycin [ Tryptone 10 g, yeast extract 5 g, NaCl 5 g, glucose 20 g, and agar 15 g
Was dissolved in 1 L of distilled water].

  Since the strain grown on this medium is a plasmid in which pMJPC17.3 is not replicable in Brevibacterium flavum MJ233, the same gene on the genome of Brevibacterium flavum MJ233 As a result of homologous recombination between the kanamycin and the kanamycin resistance gene and sacB gene derived from the plasmid should be inserted on the genome.

  Next, the homologous recombination strain was subjected to liquid culture in an LBG medium containing 25 μg / mL kanamycin. A portion equivalent to about 1 million cells of this culture was smeared on a 10% sucrose-containing LBG medium. As a result, sacB gene was lost by the second homologous recombination, and several tens of strains considered to be insensitive to sucrose were obtained.

  Among the strains thus obtained, those in which the TZ4 promoter derived from pMJPC17.3 is inserted upstream of the pc gene and those that have returned to the wild type are included. Whether the pc gene is a promoter-substituted type or a wild type can be easily confirmed by subjecting the cells obtained by culturing in the LBG medium directly to the PCR reaction and detecting the pc gene. When analyzed using primers (SEQ ID NO: 24 and SEQ ID NO: 25) for PCR amplification of the TZ4 promoter and pc gene, a 678 bp DNA fragment should be observed in the promoter-substituted form. As a result of analyzing the sucrose-insensitive strain by the above method, a strain into which the TZ4 promoter was inserted was selected, and the strain was named Brevibacterium flavum MJ233 / PC-4 / ΔLDH.

(G) Measurement of pyruvate carboxylase activity The Brevibacterium flavum MJ233 / PC-4 / ΔLDH strain prepared in (F) of <Enhancement of pyruvate carboxylase gene> in Comparative Example 1 above is an A containing 2% glucose. Medium [urea: 4 g, ammonium sulfate: 14 g, monopotassium phosphate: 0.5 g, dipotassium phosphate 0.5 g, magnesium sulfate heptahydrate: 0.5 g, ferrous sulfate heptahydrate: 20 mg , Manganese sulfate pentahydrate: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 200 μg, yeast extract: 1 g, casamino acid: 1 g, dissolved in 1,000 mL of distilled water] and inoculated at 30 ° C. for 15 hours Cultured with aerobic shaking. The obtained culture broth was centrifuged (3,000 × g, 4 ° C., 20 minutes), and the cells were collected and washed with 50 mM potassium phosphate buffer (pH 7.5).

Next, 0.5 g (wet weight) of the washed cells was suspended in 2 mL of the above potassium phosphate buffer, and subjected to an ultrasonic crusher (Branson) under ice cooling to obtain a crushed cell. The crushed material was ultracentrifuged (10
(10,000 × g, 4 ° C., 90 minutes), and the supernatant was obtained as a crude enzyme solution. As a control, a crude enzyme solution of Brevibacterium flavum MJ233 / ΔLDH strain was similarly prepared and subjected to the following activity measurement.

PC enzyme activity was measured using 100 mM Tris-HCl buffer (pH 7.5).
1 mg / 10 mL biotin, 5 mM magnesium chloride, 50 mM sodium bicarbonate, 5 mM sodium pyruvate, 5 mM adenosine triphosphate, 0.32 mM NADH, 20 units / 1.5 mL malate dehydrogenase (Wako Pure Chemicals, yeast) and crude The reaction was carried out at 25 ° C. in a reaction solution containing an enzyme solution. 1 U was defined as the amount of enzyme that decreased 1 μmol of NADH per minute. The specific activity of the crude enzyme solution prepared from Brevibacterium flavum MJ233 / PC-4 / ΔLDH strain was 0.1 U / mg protein. On the other hand, the specific activity of the crude enzyme solution prepared from the parent strain Brevibacterium flavum MJ233 / ΔLDH was below the detection limit of this activity measurement method.

<Introduction of xylose isomerase gene and xylulokinase gene>
(A) Extraction of Escherichia coli genomic DNA Escherichia coli JM109 strain was cultured in a TB medium [Terrific Broth 47 g / L, Glycerol 5 g / L] until the late logarithmic growth phase and collected. The obtained cells were suspended in 0.15 mL of a buffer solution [20 mM Tris-HCl pH 8.0, 10 mM NaCl, 1 mM EDTA · 2Na] containing lysozyme at a concentration of 10 mg / mL. Next, proteinase K was added to the suspension so that the final concentration was 100 μg / mL, and the mixture was incubated at 37 ° C. for 1 hour. Further, sodium dodecyl sulfate was added to a final concentration of 0.5%, and the mixture was incubated at 50 ° C. for 6 hours for lysis. After adding an equal amount of phenol / chloroform solution to this lysate and shaking gently at room temperature for 10 minutes, the whole amount was centrifuged (5,000 × g, 20 minutes, 10 to 12 ° C.) to obtain a supernatant fraction. The fraction was collected. After adding sodium acetate to 0.3M, 2 volumes of ethanol was added and mixed, and the precipitate collected by centrifugation (15,000 × g, 2 minutes) was washed with 70% ethanol, Air dried. To the obtained DNA, 5 mL of TE buffer [10 mM Tris-HCl pH 7.5, 1 mM EDTA · 2Na] was added and allowed to stand overnight at 4 ° C., and used as template DNA for subsequent PCR.

(B) Cloning of the xylose isomerase-xylulokinase gene operon The xylAB operon of Escherichia coli JM109 strain was obtained by using the DNA prepared in (A) of Comparative Example 1 as a template and Escherichia coli K12- This was performed by PCR using synthetic DNA (SEQ ID NO: 26 and SEQ ID NO: 27) designed based on the sequence around the operon of MG1655 strain (GenBank Accession No. U00096).

The composition of the reaction solution was 1 μl of template DNA, 0.5 μl of Pfx DNA polymerase (Invitrogen), 1 × concentration buffer, 0.4 μM each primer, 1 mM MgSO ₄ , and 0.25 μM dNTPs were mixed to make a total volume of 50 μl. As a reaction temperature condition, a DNA thermal cycler PTC-200 (MJ Research) was used, and a cycle consisting of 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 3 minutes was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 2 minutes, and the heat retention at 68 ° C. in the final cycle was 5 minutes.

Confirmation of the amplification product was performed by separation by 0.9% agarose gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 3.0 kb was detected. The obtained DNA fragment of the xylAB operon was purified using ChargeSwitch PCR Clean-Up Kit (Invitrogen) and then cleaved with restriction enzymes BamHI and ApaI. The resulting DNA fragment of about 2.9 kb was detected by 0.9% agarose gel electrophoresis and then visualized by ethidium bromide staining.
The gel DNA was recovered from the gel using a Recovery Kit (Zymo Research). This DNA fragment was mixed with DNA prepared by cleaving pTZ4 with restriction enzymes BamHI and ApaI, and DNA Ligation Kit ver. 2 (Takara Bio). Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA and smeared on an LB agar medium containing 50 μg / mL kana machine. After clones grown on this medium are liquid-cultured with TB medium [Terrific Broth 47 g / L, Glycerol 5 g / L] (including 50 μg / mL kanamycin), plasmid DNA is prepared using QIAprep Spin Miniprep Kit (QIAGEN) did. The plasmid DNA thus obtained was cleaved with restriction enzymes BamHI and ApaI. As a result, an inserted fragment of about 2.9 kb was confirmed, and this was named pZylAB1.

(C) Construction of plasmid for introducing xylose isomerase-xylulokinase gene operon Cloning of ldh gene in order to introduce E. coli-derived XylAB operon into the chromosomal ldh gene deletion site of Brevibacterium flavum MJ233 / PC-4 / ΔLDH strain Went. The Ldh gene of Brevibacterium flavum MJ233 strain was obtained using the DNA prepared in (A) of <Disruption of lactate dehydrogenase gene> in Comparative Example 1 as a template, and the entire genome sequence of which has been reported. This was performed by PCR using synthetic DNA (SEQ ID NO: 28 and SEQ ID NO: 29) designed based on the sequence around the gene of Glutamicum ATCC 13032 (GenBank Accession No. BA00000036).

The composition of the reaction solution was 1 μl of template DNA, 0.5 μl of Pfx DNA polymerase (Invitrogen), 1 × concentration buffer, 0.4 μM each primer, 1 mM MgSO ₄ , and 0.25 μM dNTPs were mixed to make a total volume of 50 μl. As a reaction temperature condition, a DNA thermal cycler PTC-200 (MJ Research) was used, and a cycle consisting of 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 2 minutes was repeated 35 times. However, the heat retention at 94 ° C. in the first cycle was 2 minutes, and the heat retention at 68 ° C. in the final cycle was 5 minutes.

  Confirmation of the amplified product was carried out by separation by 0.9% agarose gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 2.1 kb was detected. The obtained DNA fragment of the ldh gene was purified using ChargeSwitch PCR Clean-Up Kit (Invitrogen) and bound to the XbaI site of pKMB1 (Japanese Patent Laid-Open No. 2005-95169) using In-Fusion Cloning Kit (Takara Bio). Escherichia coli DH5α strain was transformed with the obtained plasmid DNA. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 25 μg / mL kanamycin and 25 μg / mL X-Gal.

  Clone that formed white colonies on this medium was liquid-cultured in TB medium [Terrific Broth 47 g / L, Glycerol 5 g / L] (containing 25 μg / mL kanamycin), and then QIAprep Spin Miniprep Kit (QIAGEN) was used. Plasmid DNA was prepared. The plasmid DNA thus obtained was cleaved with restriction enzymes XhoI and BglI. As a result, an inserted fragment of about 2.2 kb was confirmed, and this was named pKB-LDH2.

The XylAB operon linked to the constitutively highly expressed TZ4 promoter derived from Brevibacterium flavum MJ233 strain was obtained by PCR using the plasmid pZylAB1 as a template and the synthetic DNA described in SEQ ID NO: 30 and SEQ ID NO: 31. went.
The composition of the reaction solution was such that template DNA 1 μl, PrimeSTAR Max DNA polymerase (Takara Bio) 0.5 μl, 1 × concentration buffer, 0.4 μM each primer were mixed to make the total volume 50 μl. As the reaction temperature conditions, a DNA thermal cycler PTC-200 (MJ Research) was used, and a cycle consisting of 94 ° C. for 15 seconds, 55 ° C. for 20 seconds, and 72 ° C. for 45 seconds was repeated 30 times. However, the heat retention at 94 ° C. in the first cycle was 2 minutes.

  Confirmation of the amplified product was performed by separation by 0.7% agarose gel electrophoresis and visualization by ethidium bromide staining to detect a fragment of about 3.2 kb. The obtained DNA fragment of the XylAB operon linked to the TZ4 promoter was phosphorylated at the 5 'end with T4 Polynucleotide Kinase (Takara Bio), and then DNA Ligation Kit ver. 2 (Takara Bio) was used to bind to the EcoRV site of pKB-LDH2, and the resulting plasmid DNA was used to transform E. coli DH5α strain. The recombinant Escherichia coli thus obtained was smeared on an LB agar medium containing 25 μg / mL kanamycin.

The clones grown on this medium are liquid-cultured with TB medium [Terrific Broth 47 g / L, Glycerol 5 g / L] (containing 25 μg / mL kanamycin), and then plasmid DNA is prepared using QIAprep Spin Miniprep Kit (QIAGEN) did. The base sequence of the plasmid DNA insert fragment thus obtained is BigDye Terminator v3 Cycle Sequencing.
Determination was performed using Kit and a base sequence decoding device 377XL (Applied Biosystems). The resulting base sequence (XylAB operon) was completely identical to the genome sequence of Escherichia coli K12-MG1655 strain, and it was confirmed that the XylAB operon was not mutated, and this was named pZylAB3. The construction process of pZylAB3 is shown in FIG.

(D) Introduction of xylose isomerase-xylulokinase gene operon For transformation of Brevibacterium flavum MJ233 / PC-4 / ΔLDH strain prepared in (F) of <Enhancement of pyruvate carboxylase gene> in Comparative Example 1 above The plasmid DNA used was re-prepared from E. coli JM110 strain transformed with pZylAB3 by the calcium chloride method (Journal of Molecular Biology, 1970, 53, 159).

The Brevibacterium flavum MJ233 / PC-4 / ΔLDH strain was transformed by the electric pulse method (Res. Microbiol., 1993, 144, p181-5), and the obtained transformant contained 25 μg / mL kanamycin. LBG agar medium [tryptone 10 g, yeast extract 5 g, NaCl 5 g, glucose 20 g, and agar 15 g dissolved in 1 L of distilled water] was smeared.

  Since the strain grown on this medium is a plasmid in which pZylAB3 cannot replicate in the Brevibacterium flavum MJ233 strain, the plasmid ldh gene and the same gene on the Brevibacterium flavum MJ233 strain genome As a result of homologous recombination between them, the kanamycin resistance gene and sacB gene derived from the plasmid should be inserted on the genome.

  Next, the homologous recombination strain was subjected to liquid culture in an LBG medium containing 25 μg / mL kanamycin. A portion equivalent to about 1 million cells of this culture was smeared on a 10% sucrose-containing LBG medium. As a result, sacB gene was lost by the second homologous recombination, and several tens of strains considered to be insensitive to sucrose were obtained.

Among the strains thus obtained, those in which the ZylAB operon linked to the TZ4 promoter derived from pZylAB3 has been inserted into the ldh gene-deficient site and those that have returned to the same sequence as the parent strain are included. Whether or not the ZylAB operon linked to the TZ4 promoter has been inserted is determined by subjecting the cells obtained by culturing in the LBG medium to a direct PCR reaction and detecting the ZylAB operon linked to the TZ4 promoter. Can be easily confirmed. When analyzed using primers (SEQ ID NO: 32 and SEQ ID NO: 33) for PCR amplification of the TZ4 promoter and the ZylAB operon, a clone containing the ZylAB operon linked to the TZ4 promoter should have a 4,196 bp DNA fragment. It is. As a result of analyzing the strain that became insensitive to sucrose by the above method, a strain into which the ZylAB operon linked to the TZ4 promoter was inserted was selected, and the strain was selected as Brevibacterium flavum MJ233 / XylAB / PC-4 / It was named ΔLDH.

(E) Xylose utilization test MM agar medium containing 20 g / L xylose of Brevibacterium flavum MJ233 / XylAB / PC-4 / ΔLDH strain prepared in (D) of Comparative Example 1 above [urea 2 g, (NH ₄ ) ₂ SO ₄ 7 g, KH ₂ PO ₄ 0.5 g, K ₂ HPO ₄ 0.5 g, MgSO ₄ .7H ₂ O 0.5 g, FeSO ₄ .7H ₂ O ₆ mg, MnSO ₄ .4-5H ₂ O 6 mg, biotin 200 μg , Thiamine 200 μg, agar 15 g, dissolved in 1 L of distilled water] and statically cultured at 30 ° C. for 3 days. As a control, Brevibacterium flavum MJ233 / PC-4 / ΔLDH strain was cultured in the same manner. As a result, it was confirmed that the Brevibacterium flavum MJ233 / XylAB / PC-4 / ΔLDH strain can grow on a medium containing xylose as a single carbon source. On the other hand, the parent strain Brevibacterium flavum MJ233 / PC-4 / ΔLDH strain could not grow.

[Example 1]
<Enhancement of gene encoding inositol transporter IolT1>
(A) Construction of iolT1 Gene Promoter Replacement Plasmid A DNA fragment in the 5 ′ upstream region of the iolT1 gene derived from Brevibacterium flavum MJ233 strain was obtained in (A) of <Disruption of lactate dehydrogenase gene> in Comparative Example 1 above. Synthetic DNAs (SEQ ID NO: 34 and SEQ ID NO: 35) designed on the basis of the sequence around the gene of Corynebacterium glutamicum ATCC13032 strain (GenBank Accession No. BA000036) using the prepared DNA as a template and the entire genome sequence being reported ).

The composition of the reaction solution was 1 μl of template DNA, 25 μl of PrimeSTAR Max DNA polymerase (Takara Bio), and 0.4 μM. Each primer was mixed to make a total volume of 50 μl. The reaction temperature was determined by using a DNA thermal cycler PTC-200 (MJ Research). First, the sample was kept at 94 ° C. for 1 minute, followed by a cycle consisting of 98 ° C. for 10 seconds, 55 ° C. for 10 seconds and 72 ° C. for 10 seconds. Repeated 30 times.

The amplification product was confirmed by separation by 0.75% agarose gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.5 kb was detected. Recovery of the target DNA fragment from the gel was performed using QIAquick Gel Extraction Kit (QIAGEN), and this was used as the upstream region fragment of the iolT1 gene.
Meanwhile, the constitutively highly expressed TZ4 promoter fragment derived from Brevibacterium flavum MJ233 strain was obtained by using the plasmid pZylAB1 prepared in (B) of Comparative Example 1 as a template, as described in SEQ ID NO: 36 and SEQ ID NO: 37. This was performed by PCR using synthetic DNA.

The composition of the reaction solution was 1 μl of template DNA, 25 μl of PrimeSTAR Max DNA polymerase (Takara Bio), and 0.4 μM. Each primer was mixed to make a total volume of 50 μl. The reaction temperature was determined by using a DNA thermal cycler PTC-200 (MJ Research). First, the sample was kept at 94 ° C. for 1 minute, followed by a cycle consisting of 98 ° C. for 10 seconds, 55 ° C. for 10 seconds and 72 ° C. for 10 seconds. Repeated 30 times.

The amplification product was confirmed by separation by 0.75% agarose gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.3 kb was detected. The DNA fragment of interest was recovered from the gel using a QIAquick Gel Extraction Kit (QIAGEN), which was used as a TZ4 promoter fragment.
Furthermore, the DNA fragment of the N-terminal region of the iolT1 gene derived from Brevibacterium flavum MJ233 strain was obtained by using the DNA prepared in (A) of <Disruption of lactate dehydrogenase gene> in Comparative Example 1 as a template and the entire genome sequence. Was performed by PCR using synthetic DNA (SEQ ID NO: 38 and SEQ ID NO: 39) designed based on the sequence around the gene of Corynebacterium glutamicum ATCC13032 (GenBank Accession No. BA00000036).

The composition of the reaction solution was 1 μl of template DNA, 25 μl of PrimeSTAR Max DNA polymerase (Takara Bio), and 0.4 μM. Each primer was mixed to make a total volume of 50 μl. The reaction temperature was determined by using a DNA thermal cycler PTC-200 (MJ Research). First, the sample was kept at 94 ° C. for 1 minute, followed by a cycle consisting of 98 ° C. for 10 seconds, 55 ° C. for 10 seconds and 72 ° C. for 10 seconds. Repeated 30 times.

The amplification product was confirmed by separation by 0.75% agarose gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.5 kb was detected. Recovery of the target DNA fragment from the gel was performed using QIAquick Gel Extraction Kit (QIAGEN), which was used as the N-terminal fragment of the iolT1 gene.
The DNA fragment in which the TZ4 promoter was inserted between the 5 ′ upstream region and the N-terminal region of the iolT1 gene derived from Brevibacterium flavum strain MJ233 was obtained using the three amplification products obtained above as a template and SEQ ID NO: 34 And crossover PCR using the synthetic DNA of SEQ ID NO: 39.

The composition of the reaction solution was 1 μl of template DNA, 25 μl of PrimeSTAR Max DNA polymerase (Takara Bio), and 0.4 μM. Each primer was mixed to make a total volume of 50 μl. The reaction temperature was determined by using a DNA thermal cycler PTC-200 (MJ Research). First, the sample was kept at 94 ° C. for 1 minute, followed by a cycle consisting of 98 ° C. for 10 seconds, 55 ° C. for 15 seconds, and 72 ° C. for 10 seconds. Repeated 30 times.

The amplification product was confirmed by separation by 0.75% agarose gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 1.3 kb was detected.
The obtained TZ4 promoter insertion fragment upstream of the iolT1 gene was purified using QIAquick PCR Purification Kit (QIAGEN) and then cleaved with restriction enzymes SalI and SphI. The resulting DNA fragment of about 1.3 kb was separated by 0.7% agarose gel electrophoresis and then visualized by ethidium bromide staining to detect the Zymoclean Gel DNA Recovery Kit (Zymo).
Recovered from the gel using Research. This DNA fragment was mixed with DNA prepared by cleaving pKMB1 (Japanese Patent Laid-Open No. 2005-95169) with restriction enzymes SalI and SphI, and DNA Ligation Kit ver. 2 (Takara Bio). Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA and smeared on an LB agar medium containing 25 μg / mL kana machine.

Clone that formed white colonies on this medium was liquid-cultured with TB medium [Terrific Broth 47 g / L, Glycerol 5 g / L] (containing 25 μg / mL kanamycin), and then QIAprep Spin Miniprep Kit (QIAGEN) was used. Plasmid DNA was prepared. Plasmid DNA thus obtained
Was digested with restriction enzymes SalI and SphI. As a result, an insert fragment of about 1.3 kb was confirmed, which was named pIolT1-2. The construction process of pIolT1-2 is shown in FIG.

(B) Preparation of Inositol Transporter IolT1 Expression-Enhanced Strain Plasmid DNA used for transformation of Brevibacterium flavum MJ233 / XylAB / PC-4 / ΔLDH strain prepared in (D) of Comparative Example 1 described above is pIolT1-2 Was re-prepared from E. coli strain JM110 transformed by the calcium chloride method (Journal of Molecular Biology, 1970, 53, 159).
The Brevibacterium flavum MJ233 / XylAB / PC-4 / ΔLDH strain was transformed by the electric pulse method (Res. Microbiol., 1993, 144, p181-5), and the obtained transformant was treated with 25 μg / mL kanamycin. LBG agar medium [tryptone 10 g, yeast extract 5 g, NaCl 5 g, glucose 20
g and 15 g of agar were dissolved in 1 L of distilled water].

  Since the strain grown on this medium is a plasmid in which pIolT1-2 cannot replicate in the Brevibacterium flavum MJ233 strain, the iolT1 gene of the plasmid and the same gene on the genome of Brevibacterium flavum MJ233 strain As a result of homologous recombination between the kanamycin and the kanamycin resistance gene and sacB gene derived from the plasmid should be inserted on the genome.

  Next, the homologous recombination strain was subjected to liquid culture in an LBG medium containing 25 μg / mL kanamycin. A portion equivalent to about 1 million cells of this culture was smeared on a 10% sucrose-containing LBG medium. As a result, sacB gene was lost by the second homologous recombination, and several tens of strains considered to be insensitive to sucrose were obtained.

  The strains thus obtained include those in which the TZ4 promoter derived from pIolT1-2 is inserted upstream of the iolT1 gene and those that have returned to the wild type. Confirmation of whether the iolT1 gene is a promoter-substituted type or a wild type can be easily confirmed by subjecting the cells obtained by culturing in the LBG medium to a direct PCR reaction and detecting the iolT1 gene. When analyzed using primers (SEQ ID NO: 40 and SEQ ID NO: 41) for PCR amplification of the TZ4 promoter and the iolT1 gene, a 1,562 bp DNA fragment should be observed in the promoter substitution type. As a result of analyzing the strain that became insensitive to sucrose by the above method, a strain in which the TZ4 promoter was inserted was selected, and the strain was named Brevibacterium flavum MJ233 / IolT1 / XylAB / PC-4 / ΔLDH. .

[Example 2]
<Enhancement of gene encoding inositol transporter IolT2>
(A) Construction of iolT2 Gene Promoter Replacement Plasmid A DNA fragment in the 5 ′ upstream region of the iolT2 gene derived from Brevibacterium flavum MJ233 strain was obtained in (A) of <Disruption of lactate dehydrogenase gene> in Comparative Example 1 above. Synthetic DNA (SEQ ID NO: 42 and SEQ ID NO: 43) designed based on the sequence around the gene of Corynebacterium glutamicum ATCC13032 strain (GenBank Accession No. BA000036) using the prepared DNA as a template ).

The composition of the reaction solution was 1 μl of template DNA, 25 μl of PrimeSTAR Max DNA polymerase (Takara Bio), and 0.4 μM. Each primer was mixed to make a total volume of 50 μl. The reaction temperature conditions were as follows: DNA thermal cycler PTC-200 (MJ Research)
ch) was first kept at 94 ° C. for 1 minute, and then a cycle consisting of 98 ° C. for 10 seconds, 55 ° C. for 10 seconds and 72 ° C. for 10 seconds was repeated 30 times.

The amplification product was confirmed by separation by 0.75% agarose gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.5 kb was detected. Recovery of the target DNA fragment from the gel was performed using QIAquick Gel Extraction Kit (QIAGEN), and this was used as the ioI T2 gene upstream region fragment.
Meanwhile, the constitutively highly expressed TZ4 promoter fragment derived from Brevibacterium flavum MJ233 strain was obtained by using the plasmid pZylAB1 prepared in (B) of Comparative Example 1 as a template, as described in SEQ ID NO: 44 and SEQ ID NO: 45 This was performed by PCR using synthetic DNA.

The composition of the reaction solution was 1 μl of template DNA, 25 μl of PrimeSTAR Max DNA polymerase (Takara Bio), and 0.4 μM. Each primer was mixed to make a total volume of 50 μl. The reaction temperature was determined by using a DNA thermal cycler PTC-200 (MJ Research). First, the sample was kept at 94 ° C. for 1 minute, followed by a cycle consisting of 98 ° C. for 10 seconds, 55 ° C. for 10 seconds and 72 ° C. for 10 seconds. Repeated 30 times.

The amplification product was confirmed by separation by 0.75% agarose gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.3 kb was detected. The DNA fragment of interest was recovered from the gel using a QIAquick Gel Extraction Kit (QIAGEN), which was used as a TZ4 promoter fragment.
Furthermore, the DNA fragment of the N-terminal region of the iolT2 gene derived from Brevibacterium flavum MJ233 strain was obtained by using the DNA prepared in (A) of <Disruption of lactate dehydrogenase gene> in Comparative Example 1 as a template and the entire genome sequence. Was performed by PCR using synthetic DNA (SEQ ID NO: 46 and SEQ ID NO: 47) designed based on the sequence around the gene of Corynebacterium glutamicum ATCC13032 (GenBank Accession No. BA00000036).

The composition of the reaction solution was 1 μl of template DNA, 25 μl of PrimeSTAR Max DNA polymerase (Takara Bio), and 0.4 μM. Each primer was mixed to make a total volume of 50 μl. The reaction temperature was determined by using a DNA thermal cycler PTC-200 (MJ Research). First, the sample was kept at 94 ° C. for 1 minute, followed by a cycle consisting of 98 ° C. for 10 seconds, 55 ° C. for 10 seconds and 72 ° C. for 10 seconds. Repeated 30 times.

The amplification product was confirmed by separation by 0.75% agarose gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.7 kb was detected. The DNA fragment of interest was recovered from the gel using a QIAquick Gel Extraction Kit (QIAGEN), which was used as the N-terminal fragment of the iolT2 gene.
The DNA fragment in which the TZ4 promoter is inserted between the 5 ′ upstream region and the N-terminal region of the iolT2 gene derived from Brevibacterium flavum MJ233 strain is obtained using the three amplification products obtained above as a template and SEQ ID NO: 42. And crossover PCR using the synthetic DNA of SEQ ID NO: 47.

The composition of the reaction solution was 1 μl of template DNA, 25 μl of PrimeSTAR Max DNA polymerase (Takara Bio), and 0.4 μM. Each primer was mixed to make a total volume of 50 μl. The reaction temperature was determined by using a DNA thermal cycler PTC-200 (MJ Research). First, the sample was kept at 94 ° C. for 1 minute, followed by a cycle consisting of 98 ° C. for 10 seconds, 55 ° C. for 10 seconds and 72 ° C. for 20 seconds. Repeated 30 times.

The amplification product was confirmed by separation by 0.75% agarose gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 1.5 kb was detected.
The obtained TZ4 promoter insertion fragment upstream of the iolT2 gene was purified using QIAquick PCR Purification Kit (QIAGEN) and then cleaved with restriction enzymes BamHI and XbaI. The resulting DNA fragment of about 1.5 kb was detected by separation by 0.75% agarose gel electrophoresis and then visualized by ethidium bromide staining, and was removed from the gel using Zymoclean Gel DNA Recovery Kit (Zymo Research). It was collected. This DNA fragment was mixed with DNA prepared by cleaving pKMB1 (Japanese Patent Application Laid-Open No. 2005-95169) with restriction enzymes BamHI and XbaI, and DNA Ligation Kit ver. 2 (Takara Bio). Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA and smeared on an LB agar medium containing 25 μg / mL kana machine.

  Clone that formed white colonies on this medium was liquid-cultured in TB medium [Terrific Broth 47 g / L, Glycerol 5 g / L] (containing 25 μg / mL kanamycin), and then QIAprep Spin Miniprep Kit (QIAGEN) was used. Plasmid DNA was prepared. The plasmid DNA thus obtained was cleaved with restriction enzymes BamHI and XbaI. As a result, an inserted fragment of about 1.5 kb was confirmed, and this was named pIolT2-2. The construction process of pIolT2-2 is shown in FIG.

(B) Enhancement of gene encoding inositol transporter IolT2 Plasmid DNA used for transformation of Brevibacterium flavum MJ233 / XylAB / PC-4 / ΔLDH strain prepared in (D) of Comparative Example 1 above is pIolT2- 2 was re-prepared from E. coli strain JM110 transformed by the calcium chloride method (Journal of Molecular Biology, 1970, 53, 159).
The Brevibacterium flavum MJ233 / XylAB / PC-4 / ΔLDH strain was transformed by the electric pulse method (Res. Microbiol., 1993, 144, p181-5), and the obtained transformant was treated with 25 μg / mL kanamycin. LBG agar medium [tryptone 10 g, yeast extract 5 g, NaCl 5 g, glucose 20
g and 15 g of agar were dissolved in 1 L of distilled water].

  Since the strain grown on this medium is a plasmid in which pIolT2-2 is not replicable in Brevibacterium flavum MJ233, the same gene on the genome of Brevibacterium flavum MJ233 As a result of homologous recombination between the kanamycin and the kanamycin resistance gene and sacB gene derived from the plasmid should be inserted on the genome.

  Next, the homologous recombination strain was subjected to liquid culture in an LBG medium containing 25 μg / mL kanamycin. A portion equivalent to about 1 million cells of this culture was smeared on a 10% sucrose-containing LBG medium. As a result, sacB gene was lost by the second homologous recombination, and several tens of strains considered to be insensitive to sucrose were obtained.

Among the strains obtained in this manner, a strain in which the TZ4 promoter derived from pIolT2-2 is inserted upstream of the iolT2 gene and a strain that has returned to the wild type are included. Confirmation of whether the iolT1 gene is a promoter-substituted type or a wild type can be easily confirmed by subjecting the cells obtained by culturing in the LBG medium to a direct PCR reaction and detecting the iolT2 gene. When analyzed using primers (SEQ ID NO: 48 and SEQ ID NO: 49) for PCR amplification of the TZ4 promoter and the iolT2 gene, a 1,570 bp DNA fragment should be observed in the promoter substitution type. As a result of analyzing the strain that became insensitive to sucrose by the above method, a strain into which the TZ4 promoter was inserted was selected, and the strain was named Brevibacterium flavum MJ233 / IolT2 / XylAB / PC-4 / ΔLDH. .

[Example 3]
<Disruption of gene encoding transcription factor IolR that suppresses the expression of iolT1 gene>
(A) Construction of plasmid for disrupting iolR gene The iolR gene fragment of Brevibacterium flavum MJ233 strain was obtained by using the DNA prepared in (A) of <Lactate dehydrogenase gene disruption> in Comparative Example 1 as a template. It was carried out by PCR using synthetic DNA (SEQ ID NO: 50 and SEQ ID NO: 51) designed based on the sequence of the gene of Corynebacterium glutamicum ATCC 13032 (GenBank Accession No. BA00000036) for which the genome sequence was reported.

  The composition of the reaction solution was such that template DNA 1 μl, PrimeSTAR DNA polymerase (Takara Bio) 0.5 μl, 1-fold concentration buffer, 0.4 μM each primer were mixed to make the total volume 50 μl. The reaction temperature was determined by using a DNA thermal cycler PTC-200 (MJ Research). First, the sample was kept at 96 ° C. for 1 minute, followed by a cycle consisting of 98 ° C. for 10 seconds, 55 ° C. for 10 seconds, and 72 ° C. for 40 seconds. Repeated 30 times.

Confirmation of the amplification product was performed by separation by 0.7% agarose gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.5 kb was detected. The DNA fragment of the internal sequence of the obtained iolR gene was QIAquick PCR Purification
After purification using Kit (QIAGEN), it was cleaved with restriction enzymes SacI and EcoRI. The resulting DNA fragment of about 0.5 kb was separated by 0.7% agarose gel electrophoresis and then visualized by ethidium bromide staining and detected from the gel using the Zymoclean Gel DNA Recovery Kit (Zymo Research). It was collected. This DNA fragment was mixed with DNA prepared by cutting E. coli vector pHSG298 (Takara Bio) with restriction enzymes SacI and EcoRI, and DNA Ligation Kit ver. 2 (Takara Bio). Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA and smeared on an LB agar medium containing 25 μg / mL kana machine.

  Clone that formed white colonies on this medium was liquid-cultured in TB medium [Terrific Broth 47 g / L, Glycerol 5 g / L] (containing 25 μg / mL kanamycin), and then QIAprep Spin Miniprep Kit (QIAGEN) was used. Plasmid DNA was prepared. The plasmid DNA thus obtained was digested with restriction enzymes SacI and EcoRI. As a result, an inserted fragment of about 0.5 kb was confirmed, and this was named pD-IolR-1. The construction process of pD-IolR-1 is shown in FIG.

(B) Disruption of gene encoding transcription factor IolR that suppresses expression of iolT1 gene Used for transformation of Brevibacterium flavum MJ233 / XylAB / PC-4 / ΔLDH strain prepared in (D) of Comparative Example 1 above Plasmid DNA was re-prepared from E. coli JM110 strain transformed with pD-IolR-1 by the calcium chloride method (Journal of Molecular Biology, 1970, 53, 159).
The Brevibacterium flavum MJ233 / XylAB / PC-4 / ΔLDH strain was transformed by the electric pulse method (Res. Microbiol., 1993, 144, p181-5), and the obtained transformant was treated with 25 μg / mL kanamycin. LBG agar medium [tryptone 10 g, yeast extract 5 g, NaCl 5 g, glucose 20
g and 15 g of agar were dissolved in 1 L of distilled water].

  Since the strain grown on this medium is a plasmid in which pD-IolR-1 cannot replicate in the Brevibacterium flavum MJ233 strain, the iolR gene of the plasmid and the Brevibacterium flavum MJ233 genome As a result of homologous recombination with the above gene, a kanamycin resistance gene derived from the plasmid should be inserted into the genome. Whether or not the kanamycin resistant strain thus obtained has undergone homologous recombination between the iolR gene present on its genome and the gene present on plasmid pD-IolR-1 is as follows: Colony PCR was performed using the synthetic DNAs of SEQ ID NO: 23, SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54 as primers.

As the template DNA, a solution in which colonies were suspended in 50 μL of sterile water was used. The composition of the reaction solution was 1 μl of template DNA, MightyAmp DNA polymerase Ver. 2 (Takara Bio) 0.2 μl, 1 × concentration attached buffer, 0.4 μM Each primer was mixed to make the total volume 10 μl. As the reaction temperature conditions, a DNA thermal cycler PTC-200 (MJ Research) was used, and a cycle consisting of 98 ° C. for 10 seconds, 60 ° C. for 15 seconds and 68 ° C. for 50 seconds was repeated 30 times. However, the heat retention at 98 ° C. in the first cycle was 2 minutes.
As a result of analyzing the kanamycin-resistant strain by the above method, a strain capable of obtaining a PCR amplification product of 760 bp for the combination of SEQ ID NO: 23 and SEQ ID NO: 52 and 500 bp for the combination of SEQ ID NO: 53 and SEQ ID NO: 54 was selected. It was named as Bacteria flavum MJ233 / ΔIolR / XylAB / PC-4 / ΔLDH.

[Comparative Example 2]
<Introduction of gene encoding pentose transporter AraE>
(A) Extraction of Corynebacterium glutamicum ATCC 31831 genomic DNA Corynebacterium glutamicum ATCC 31831 was extracted from A medium containing 2% glucose [urea: 4 g, ammonium sulfate: 14 g, monopotassium phosphate: 0.5 g, phosphoric acid. 2 potassium 0.5 g, magnesium sulfate heptahydrate: 0.5 g, ferrous sulfate heptahydrate: 20 mg, manganese sulfate pentahydrate: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 200 μg Yeast extract: 1 g, casamino acid: 1 g, dissolved in 1,000 mL of distilled water] The cells were cultured with 10 mL until the late logarithmic growth phase and collected. The obtained cells were suspended in 0.15 mL of a buffer solution [20 mM Tris-HCl pH 8.0, 10 mM NaCl, 1 mM EDTA · 2Na] containing lysozyme at a concentration of 10 mg / mL. Next, proteinase K was added to the suspension so that the final concentration was 100 μg / mL, and the mixture was incubated at 37 ° C. for 1 hour. Further, sodium dodecyl sulfate was added to a final concentration of 0.5%, and the mixture was incubated at 50 ° C. for 6 hours for lysis. After adding an equal amount of phenol / chloroform solution to this lysate and shaking gently at room temperature for 10 minutes, the whole amount was centrifuged (5,000 × g, 20 minutes, 10 to 12 ° C.) to obtain a supernatant fraction. The fraction was collected. After adding sodium acetate to 0.3M, 2 volumes of ethanol was added and mixed, and the precipitate collected by centrifugation (15,000 × g, 2 minutes) was washed with 70% ethanol, Air dried. To the obtained DNA, 5 mL of TE buffer [10 mM Tris-HCl pH 7.5, 1 mM EDTA · 2Na] was added and allowed to stand overnight at 4 ° C., and used as template DNA for subsequent PCR.

(B) Construction of plasmid for introducing pentose transporter araE gene The araE gene derived from Corynebacterium glutamicum ATCC31831 was linked to the TZ4 promoter, and Brevibacterium flavum MJ233 / XylAB produced in (D) of Comparative Example 1 above. / PC-4 / ΔLDH strain for introduction into the ldh gene deletion site on the chromosome (downstream of the already introduced E. coli-derived XylAB operon), homologous recombination between the internal sequence of the ldh gene and the 3 ′ downstream region of the ldh gene Used as an area.
The internal sequence of the ldh gene derived from Brevibacterium flavum MJ233 strain was obtained by using the DNA prepared in (A) of <Lactate dehydrogenase gene disruption> in Comparative Example 1 as a template, and the entire genome sequence has been reported. This was performed by PCR using synthetic DNA (SEQ ID NO: 55 and SEQ ID NO: 56) designed based on the sequence (GenBank Accession No. BA00000036) around the gene of the bacterial glutamicum ATCC13032 strain.

The composition of the reaction solution was 1 μl of template DNA, 25 μl of PrimeSTAR Max DNA polymerase (Takara Bio), and 0.4 μM. Each primer was mixed to make a total volume of 50 μl. The reaction temperature was determined by using a DNA thermal cycler PTC-200 (MJ Research). First, the sample was kept at 94 ° C. for 1 minute, followed by a cycle consisting of 98 ° C. for 10 seconds, 55 ° C. for 10 seconds and 72 ° C. for 10 seconds. Repeated 30 times.

Confirmation of the amplification product was performed by separation by 0.7% agarose gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.4 kb was detected. Recovery of the target DNA fragment from the gel was performed using QIAquick Gel Extraction Kit (QIAGEN), which was used as an internal sequence fragment of the ldh gene.
Next, the constitutively highly expressed TZ4 promoter fragment derived from Brevibacterium flavum MJ233 strain was obtained by using the plasmid pZylAB1 prepared in (B) of Comparative Example 1 as a template, and described in SEQ ID NO: 57 and SEQ ID NO: 58 This was performed by PCR using the synthetic DNA.

The composition of the reaction solution was 1 μl of template DNA, 25 μl of PrimeSTAR Max DNA polymerase (Takara Bio), and 0.4 μM. Each primer was mixed to make a total volume of 50 μl. The reaction temperature was determined by using a DNA thermal cycler PTC-200 (MJ Research). First, the sample was kept at 94 ° C. for 1 minute, followed by a cycle consisting of 98 ° C. for 10 seconds, 55 ° C. for 10 seconds and 72 ° C. for 10 seconds. Repeated 30 times.

Confirmation of the amplification product was performed by separation by 0.7% agarose gel electrophoresis and visualization by ethidium bromide staining to detect a fragment of about 0.3 kb. The DNA fragment of interest was recovered from the gel using a QIAquick Gel Extraction Kit (QIAGEN), which was used as a TZ4 promoter fragment.
On the other hand, the araE gene derived from Corynebacterium glutamicum ATCC 31831 strain was obtained by using the DNA prepared in (A) of Comparative Example 2 as a template and the sequence around the gene of Corynebacterium glutamicum ATCC 31831 strain (GenBank Accession No. This was performed by PCR using synthetic DNAs (SEQ ID NO: 59 and SEQ ID NO: 60) designed based on AB447371).

  The composition of the reaction solution was 1 μl of template DNA, 25 μl of PrimeSTAR Max DNA polymerase (Takara Bio), and 0.4 μM. Each primer was mixed to make a total volume of 50 μl. The reaction temperature was determined by using a DNA thermal cycler PTC-200 (MJ Research), first keeping the temperature at 94 ° C. for 1 minute, followed by 30 cycles of 98 ° C. for 10 seconds, 55 ° C. for 15 seconds, and 72 ° C. for 2 minutes. Repeated times. Confirmation of the amplification product was carried out by separation by 0.7% agarose gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 1.6 kb was detected.

Furthermore, the 3 ′ downstream region of the ldh gene derived from Brevibacterium flavum MJ233 strain was obtained using the DNA prepared in (A) of <Lactate dehydrogenase gene disruption> in Comparative Example 1 as a template, and the entire genome sequence was reported. Sequence around the gene of the strain Corynebacterium glutamicum ATCC13032 (GenBank Accession)
No. This was performed by PCR using synthetic DNA (SEQ ID NO: 61 and SEQ ID NO: 62) designed based on (BA000036).

The composition of the reaction solution was 1 μl of template DNA, 25 μl of PrimeSTAR Max DNA polymerase (Takara Bio), and 0.4 μM. Each primer was mixed to make a total volume of 50 μl. The reaction temperature was determined by using a DNA thermal cycler PTC-200 (MJ Research). First, the sample was kept at 94 ° C. for 1 minute, followed by a cycle consisting of 98 ° C. for 10 seconds, 55 ° C. for 10 seconds and 72 ° C. for 10 seconds. Repeated 30 times.

Confirmation of the amplification product was performed by separation by 0.7% agarose gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 0.5 kb was detected. Recovery of the target DNA fragment from the gel was performed using QIAquick Gel Extraction Kit (QIAGEN), and this was used as the ldh gene downstream region fragment.
The fragment obtained by binding the ldh gene downstream region to the araE gene linked to the TZ4 promoter was obtained by crossover using the three amplification products obtained above as templates and the synthetic DNAs of SEQ ID NO: 57 and SEQ ID NO: 62. Performed by PCR.

The composition of the reaction solution was 1 μl of template DNA, 25 μl of PrimeSTAR Max DNA polymerase (Takara Bio), and 0.4 μM. Each primer was mixed to make a total volume of 50 μl. The reaction temperature was determined by using a DNA thermal cycler PTC-200 (MJ Research). First, the sample was kept at 94 ° C. for 1 minute, followed by a cycle consisting of 98 ° C. for 10 seconds, 55 ° C. for 10 seconds and 72 ° C. for 40 seconds. Repeated 30 times.

The amplification product was confirmed by separation by 0.75% agarose gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 2.4 kb was detected. Recovery of the target DNA fragment from the gel was performed using a QIAquick Gel Extraction Kit (QIAGEN).
Obtaining a fragment comprising the araE gene linked to the TZ4 promoter and the region used for homologous recombination at both ends thereof using the internal sequence fragment of the ldh gene obtained above and the amplification product of crossover PCR as a template, SEQ ID NO: 55 And crossover PCR using the synthetic DNA of SEQ ID NO: 62.

The composition of the reaction solution was 1 μl of template DNA, 25 μl of PrimeSTAR Max DNA polymerase (Takara Bio), and 0.4 μM. Each primer was mixed to make a total volume of 50 μl. The reaction temperature was determined by using a DNA thermal cycler PTC-200 (MJ Research). First, the sample was kept at 94 ° C. for 1 minute, followed by a cycle consisting of 98 ° C. for 10 seconds, 55 ° C. for 10 seconds and 72 ° C. for 40 seconds. Repeated 30 times. The amplification product was confirmed by separation by 0.75% agarose gel electrophoresis and visualization by ethidium bromide staining, and a fragment of about 2.8 kb was detected.

  The obtained DNA fragment (fragment containing the araE gene linked to the TZ4 promoter and the region used for homologous recombination at both ends thereof) was purified using QIAquick PCR Purification Kit (QIAGEN) and then cleaved with restriction enzymes BamHI and SalI. did. The resulting DNA fragment of about 3.0 kb was separated by 0.7% agarose gel electrophoresis and then visualized by ethidium bromide staining, and was detected from the gel using the Zymoclean Gel DNA Recovery Kit (Zymo Research). It was collected. This DNA fragment was mixed with pKMB1 (Japanese Patent Laid-Open No. 2005-95169) cleaved with the same restriction enzyme, and DNA Ligation Kit ver. 2 (Takara Bio). Escherichia coli (DH5α strain) was transformed with the obtained plasmid DNA and smeared on an LB agar medium containing 25 μg / mL kana machine.

  Clone that formed white colonies on this medium was liquid-cultured in TB medium [Terrific Broth 47 g / L, Glycerol 5 g / L] (containing 25 μg / mL kanamycin), and then QIAprep Spin Miniprep Kit (QIAGEN) was used. Plasmid DNA was prepared. The plasmid DNA thus obtained was digested with restriction enzymes BamHI and SalI. As a result, an insertion fragment of about 3.0 kb was confirmed, and this was named pAraE-2. The construction process of pAraE-2 is shown in FIG.

(C) Introduction of pentose transporter araE gene pAraE-2 is used as a plasmid DNA for transformation of Brevibacterium flavum MJ233 / XylAB / PC-4 / ΔLDH strain prepared in (D) of Comparative Example 1 above. From Escherichia coli JM110 strain transformed by the calcium chloride method (Journal of Molecular Biology, 1970, 53, 159).

The Brevibacterium flavum MJ233 / XylAB / PC-4 / ΔLDH strain was transformed by the electric pulse method (Res. Microbiol., 1993, 144, p181-5), and the obtained transformant was treated with 25 μg / mL kanamycin. LBG agar medium [tryptone 10 g, yeast extract 5 g, NaCl 5 g, glucose 2
0 g and 15 g of agar were dissolved in 1 L of distilled water].

  Since the strain grown on this medium is a plasmid in which pIolT2-2 is not replicable in Brevibacterium flavum MJ233, the same gene on the genome of Brevibacterium flavum MJ233 As a result of homologous recombination between the kanamycin and the kanamycin resistance gene and sacB gene derived from the plasmid should be inserted on the genome.

  Next, the homologous recombination strain was subjected to liquid culture in an LBG medium containing 25 μg / mL kanamycin. A portion equivalent to about 1 million cells of this culture was smeared on a 10% sucrose-containing LBG medium. As a result, sacB gene was lost by the second homologous recombination, and several tens of strains considered to be insensitive to sucrose were obtained.

Among the strains obtained in this way, the araE gene (TZ4-araE) linked to the TZ4 promoter derived from pAraE-2 was inserted into the ldh gene deletion site on the chromosome and returned to the wild type. Is included. Confirmation of the TZ4-araE introduction type or the wild type can be easily confirmed by subjecting the cells obtained by culturing in the LBG medium directly to the PCR reaction and detecting TZ4-araE. When analyzed using primers (SEQ ID NO: 57 and SEQ ID NO: 63) for PCR amplification of TZ4-araE, a DNA fragment of 3,229 bp should be observed in the TZ4-araE-introduced type. As a result of analyzing the strain that became insensitive to sucrose by the above method, a strain into which the TZ4 promoter was inserted was selected, and the strain was named Brevibacterium flavum MJ233 / AraE / XylAB / PC-4 / ΔLDH. .
A systematic diagram of the strains of Examples 1 to 3 and Comparative Example 1 prepared as described above is shown in FIG.

[Example 4]
<Transcriptional analysis of the iolT1 gene and iolT2 gene of the iolT1 gene-enhanced strain>
In order to analyze the transcription amounts of iolT1 gene and iolT2 gene of Brevibacterium flavum MJ233 / IolT1 / XylAB / PC-4 / ΔLDH strain prepared in Example 1 (B), flask culture was performed as follows to analyze the bacteria. Body was prepared, followed by total RNA preparation, reverse transcription reaction, and quantitative PCR.

(A) Seed culture A medium [urea: 4 g, ammonium sulfate: 14 g, monopotassium phosphate: 0.5 g, dipotassium phosphate 0.5 g, magnesium sulfate heptahydrate: 0.5 g, ferrous sulfate 7 hydrate: 20 mg, manganese sulfate pentahydrate: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 200 μg, yeast extract: 1 g, casamino acid: 1 g, dissolved in 1,000 mL of distilled water] 1,000 mL After sterilization by heating at 121 ° C. for 20 minutes and cooling to room temperature, 15 mL was put into a 200 mL Erlenmeyer flask, and 600 μl of a 50% glucose aqueous solution previously sterilized was added. Brevibacterium flavum MJ233 / IolT1 / XylAB / PC-4 / ΔLDH strain was inoculated and cultured with shaking at 30 ° C. for 8 hours.

(B) Main culture After adding 100 mL of A medium to a 500 mL Erlenmeyer flask and adding 4 mL of a 50% aqueous glucose solution sterilized in advance, the culture solution obtained by the above culture is adjusted so that OD ₆₆₀ becomes 0.02. And inoculated at 30 ° C. for 18 hours with shaking.

(C) Preparation of total RNA Total RNA was prepared from the cells prepared in (B) above using RNeasy plus Mini Kit (QIAGEN) and RNase-Free DNase Set (QIAGEN).

After culturing in the above (B), a bacterial cell solution is prepared using the collected culture solution so as to be 10 ⁸ to 10 ⁹ , and obtained by centrifuging at 8,000 rpm for 2 minutes at 4 ° C. The bacterial cells were suspended in distilled sterilized water and centrifuged again at 8,000 rpm for 2 minutes at 4 ° C. The obtained cells are suspended in a solution obtained by adding 6 μl of 2-mercaptoethanol to 600 μl of RLT Buffer attached to the kit, and this suspension is transferred to a tube of MagNA Lyser Green Beads (Roche), and MagNA Lyser (Roche) is placed on the tube. The mixture was crushed at 6,000 rpm for 1 minute at room temperature. This cell disruption solution was placed in a genomic DNA-removed mini spin column, and total RNA was prepared according to the instruction manual attached to the RNeasy plus Mini Kit. As an option, 80 μl of DNase mix solution was added after column adsorption of total RNA, and allowed to stand at room temperature for 15 minutes to decompose genomic DNA. Finally, RNase-free water kept at 50 ° C. was added, allowed to stand at room temperature for 1 minute, and total RNA was eluted by centrifugation.

(D) Reverse transcription reaction Using the total RNA prepared in (C) above as a template, using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen), a reverse transcription reaction was performed according to the attached instruction manual, Single stranded cDNA was synthesized. For reverse transcription, 300 ng of total RNA, 50 ng as primer
/ Μl Random hexamers were used.

(D) Quantitative PCR
Quantitative PCR reaction was performed using SYBR premix Ex Taq (Perfect Real Time) (Takara Bio) according to the attached instruction manual, and first using Light-Cycler (Roche Diagnostics) at 95 ° C. After keeping the temperature for 10 seconds, a cycle consisting of 95 ° C. for 5 seconds, 55 ° C. for 5 seconds, and 68 ° C. for 20 seconds was repeated 40 times. The temperature change rate was 20 ° C./sec. Melting curve analysis was performed at (1) 95 ° C., 0 seconds, (2) 65 ° C., 10 seconds, (3) 95 ° C., 0 seconds, and the temperature change rate was 20 ° C./(1) and (2). And (3) was 0.1 ° C./second.
As PCR primers, synthetic DNAs of SEQ ID NO: 64 and SEQ ID NO: 65 were used for quantitative PCR of the iolT1 gene, and synthetic DNAs of SEQ ID NO: 66 and SEQ ID NO: 67 were used for quantitative PCR of the iolT2 gene. Further, 16S rRNA was used as an internal standard, and synthetic DNAs of SEQ ID NO: 68 and SEQ ID NO: 69 were used.

  For quantitative analysis, Light-Cycler Software Ver. The analysis was performed by the Second Derivative Maximum method using 3.5 (Roche Diagnostics).

[Example 5]
<Transcriptional analysis of iolT1 gene and iolT2 gene of iolT2 gene-enhanced strain>
The same procedure as in Example 4 was performed except that the Brevibacterium flavum MJ233 / IolT2 / XylAB / PC-4 / ΔLDH strain prepared in (B) of Example 2 was used.

[Example 6]
<Transcriptional analysis of iolT1 gene and iolT2 gene of iolR gene-disrupted strain>
The same procedure as in Example 4 was performed except that the Brevibacterium flavum MJ233 / ΔIolR / XylAB / PC-4 / ΔLDH strain prepared in (B) of Example 3 was used.

[Example 7]
<Transcriptional analysis of iolT1 gene and iolT2 gene of inositol culture strain>
In order to adjust the cells by culturing the Brevibacterium flavum MJ233 / XylAB / PC-4 / ΔLDH strain prepared in Comparative Example 1 in the presence of inositol and analyze the transcription amounts of the iolT1 gene and the iolT2 gene, Flask culture was performed as described above.
The seed culture was performed in the same manner as in Example 4. In the main culture, 16.7 mL of 12% myo-inositol aqueous solution was added instead of adding 4 mL of 50% glucose aqueous solution as a carbon source, and the culture time was 22.5. The same operation as in Example 4 was performed except that time was used.

  Preparation of total RNA, reverse transcription reaction, and quantitative PCR were performed in the same manner as in Example 4.

[Comparative Example 3]
<Transcriptional analysis of iolT1 gene and iolT2 gene of iolT gene non-modified strain>
The same procedure as in Example 4 was performed except that the Brevibacterium flavum MJ233 / XylAB / PC-4 / ΔLDH strain prepared in Comparative Example 1 was used.

  The results of Examples 4 to 7 and Comparative Example 3 are shown in Table 1. The numerical values of iolT1 gene and iolT2 gene expression represent the ratio to Comparative Example 3.

From Table 1, in Example 4, where transcription analysis of Brevibacterium flavum MJ233 / IolT1 / XylAB / PC-4 / ΔLDH strain prepared in Example 1 was performed, transcription of the iolT1 gene relative to Comparative Example 3 of the parent strain It was confirmed that the amount increased about 62 times.
In Example 5 in which transcription analysis of Brevibacterium flavum MJ233 / IolT2 / XylAB / PC-4 / ΔLDH strain prepared in Example 2 was performed, the transcription amount of the iolT2 gene was about 103 compared to Comparative Example 3 of the parent strain. It was confirmed that the number increased twice.

In Example 6 in which transcription analysis of Brevibacterium flavum MJ233 / ΔIolR / XylAB / PC-4 / ΔLDH strain prepared in Example 3 was performed, the transcription amount of the iolT1 gene was about 12 compared to the comparative example 3 of the parent strain. It was confirmed that the number increased twice.
In Example 7, in which the transcription analysis of the same Brevibacterium flavum MJ233 / XylAB / PC-4 / ΔLDH strain cultured in the presence of inositol was performed as in Comparative Example 3, iolT1 was compared with Comparative Example 3 cultured in glucose. It was confirmed that the gene transcription amount increased about 69 times and the iolT2 gene transcription amount increased about 10 times.

[Example 8]
<Evaluation of succinic acid production by iolT1 gene-enhanced strain>
In order to evaluate the succinic acid production of Brevibacterium flavum MJ233 / IolT1 / XylAB / PC-4 / ΔLDH strain prepared in (B) of Example 1, a flask was cultured as follows to prepare the cells. Thereafter, a succinic acid production reaction was carried out.

(A) Seed culture A medium [urea: 4 g, ammonium sulfate: 14 g, monopotassium phosphate: 0.5 g, dipotassium phosphate 0.5 g, magnesium sulfate heptahydrate: 0.5 g, ferrous sulfate 7 hydrate: 20 mg, manganese sulfate pentahydrate: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 200 μg, yeast extract: 1 g, casamino acid: 1 g, dissolved in 1,000 mL of distilled water] 1,000 mL After sterilization by heating at 121 ° C. for 20 minutes and cooling to room temperature, 15 mL was put into a 200 mL Erlenmeyer flask, and 600 μl of a 50% glucose aqueous solution previously sterilized was added. Brevibacterium flavum MJ233 / IolT1 / XylAB / PC-4 / ΔLDH strain was inoculated and cultured with shaking at 30 ° C. for 6 hours.

(B) Main culture After adding 100 mL of A medium to a 500 mL Erlenmeyer flask and adding 4 mL of a 50% aqueous glucose solution sterilized in advance, the culture solution obtained by the above culture is adjusted so that OD ₆₆₀ is 0.2. And inoculated at 30 ° C. for 16.5 hours with shaking.

(C) Succinic acid production reaction The culture solution obtained in the main culture of (B) above was collected by centrifugation at 5,000 × g for 7 minutes, and the cell suspension [magnesium sulfate heptahydrate 1 g, ferrous sulfate heptahydrate: 40 mg, manganese sulfate pentahydrate: 40 mg, D-biotin: 400 μg, thiamine hydrochloride: 400 μg, monoammonium phosphate: 0.8 g, diammonium phosphate: 0.8 g, 1 g of potassium chloride, dissolved in 1000 mL of distilled water] was suspended so that OD ₆₆₀ was 40. Subsequently, 2 mL of 50% xylose aqueous solution, 1.58 g of ammonium bicarbonate, and 23 mL of distilled water were mixed to prepare a substrate solution. The 5 mL reactor was mixed with 0.5 mL of the bacterial cell suspension and 0.5 mL of the substrate solution, and reacted at 40 ° C. under anaerobic conditions. As a result, the succinic acid accumulation concentration after 6 hours was 6.7 g / L, and the xylose concentration was 7.8 g / L. The succinic acid and xylose were quantified by HPLC analysis. The analysis conditions are as follows.

Column: ULTRON PS-80H 8.0 mm ID x 30 cm manufactured by Shinwa Kogyo Co., Ltd.
Eluent: 60% aqueous solution of perchloric acid (1.8 mL-perchloric acid / 1 L-H ₂ O)
Temperature: 60 ° C
Detection: RI, UV (210 nm)

[Example 9]
<Succinic acid production evaluation of iolT2 gene-enhanced strain>
The same procedure as in Example 8 was performed except that the Brevibacterium flavum MJ233 / IolT2 / XylAB / PC-4 / ΔLDH strain prepared in (B) of Example 2 was used. As a result, the succinic acid accumulation concentration after 6 hours was 3.5 g / L, and the xylose concentration was 12.6 g / L.

[Example 10]
<Succinic acid production evaluation of inositol culture strain>
In order to adjust microbial cells by culturing Brevibacterium flavum MJ233 / XylAB / PC-4 / ΔLDH strain prepared in Comparative Example 1 in the presence of inositol and evaluate succinic acid production, flask culture was performed as follows. went.

Seed culture was carried out in the same manner as in Example 8. In this culture, 16.7 mL of 12% myo-inositol aqueous solution was added instead of adding 4 mL of 50% glucose aqueous solution as a carbon source. As well as.
The succinic acid production reaction was performed in the same manner as in Example 8. As a result, the succinic acid accumulation concentration after 6 hours was 14.0 g / L, and the xylose concentration was 0.3 g / L.

[Comparative Example 4]
<Evaluation of succinic acid production of iolT gene non-modified strain>
The same procedure as in Example 8 was performed except that the Brevibacterium flavum MJ233 / XylAB / PC-4 / ΔLDH strain prepared in Comparative Example 1 was used. As a result, the succinic acid accumulation concentration after 6 hours was 2.7 g / L, and the xylose concentration was 11.8 g / L.
As a result of Examples 8 to 10 and Comparative Example 4, succinic acid accumulation concentration and xylose concentration 6 hours after the start of the reaction are shown in Table 2.

From Table 2, in Example 8 in which succinic acid production evaluation of Brevibacterium flavum MJ233 / IolT1 / XylAB / PC-4 / ΔLDH strain prepared in Example 1 was performed, succinic acid was compared to Comparative Example 4 of the parent strain. Production speed improved by about 148%.
In Example 9, in which succinic acid production evaluation of Brevibacterium flavum MJ233 / IolT2 / XylAB / PC-4 / ΔLDH strain prepared in Example 2 was performed, the succinic acid production rate was about Improved by 30%.

In addition, in Example 10 in which the succinic acid production evaluation of a strain obtained by culturing the same Brevibacterium flavum MJ233 / XylAB / PC-4 / ΔLDH strain as in Comparative Example 3 in the presence of inositol was performed, Comparative Example 4 cultured in glucose was used. In contrast, the succinic acid production rate was improved by about 419%.
From the above results, it was revealed that the succinic acid production rate when xylose was used as a raw material was improved by enhancing the expression of the iolT1 gene or iolT2 gene encoding the inositol transporter.

[Example 11]
<Evaluation of succinic acid production in one-pot reaction of iolT1 gene-enhanced strain>
In order to integrally evaluate the cell growth and succinic acid production of the Brevibacterium flavum MJ233 / IolT1 / XylAB / PC-4 / ΔLDH strain prepared in Example 1 (B), the succination in a one-pot reaction was performed as follows. An acid production evaluation was performed.

(A) Seed culture A medium [urea: 4 g, ammonium sulfate: 14 g, monopotassium phosphate: 0.5 g, dipotassium phosphate 0.5 g, magnesium sulfate heptahydrate: 0.5 g, ferrous sulfate 7 hydrate: 20 mg, manganese sulfate pentahydrate: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 200 μg, yeast extract: 1 g, casamino acid: 1 g, dissolved in 1,000 mL of distilled water] 1,000 mL After sterilization by heating at 121 ° C. for 20 minutes and cooling to room temperature, 15 mL was put into a 200 mL Erlenmeyer flask, and 600 μl of a 50% glucose aqueous solution previously sterilized was added. Brevibacterium flavum MJ233 / IolT1 / XylAB / PC-4 / ΔLDH strain was inoculated and cultured with shaking at 30 ° C. for 6 hours.

(B) One-pot reaction (main culture / succinic acid production reaction)
100 mL of A medium is put into a 500 mL Erlenmeyer flask, 8 mL of 50% xylose aqueous solution sterilized in advance is added, and the culture solution obtained by the above culture is inoculated so that OD ₆₆₀ becomes 0.1. Cultured with shaking at ℃. After 26.5 hours from the start of the culture, 3.16 g of ammonium hydrogen carbonate was added, the mouth of the Erlenmeyer flask was sealed with parafilm, and the temperature was changed to 40 ° C. and reacted for 20 hours. As a result, the OD ₆₆₀ after 26.5 hours from the start of the culture was 14.9, the xylose concentration was 20.9 g / L, the succinic acid accumulation concentration after 46.5 hours was 18.3 g / L, and the xylose concentration. Was 0.0 g / L. A graph showing changes in OD ₆₆₀ , succinic acid accumulation concentration, and xylose concentration is shown in FIG. The succinic acid and xylose were quantified by HPLC analysis according to the following method.

Column: ULTRON PS-80H 8.0 mm ID x 30 cm manufactured by Shinwa Kogyo Co., Ltd.
Eluent: 60% aqueous solution of perchloric acid (1.8 mL-perchloric acid / 1 L-H ₂ O)
Temperature: 60 ° C
Detection: RI, UV (210 nm)

[Example 12]
<Evaluation of succinic acid production in one-pot reaction of iolT2 gene-enhanced strain>
The same procedure as in Example 11 was performed except that the Brevibacterium flavum MJ233 / IolT2 / XylAB / PC-4 / ΔLDH strain prepared in (B) of Example 2 was used. As a result, the OD ₆₆₀ after 26.5 hours from the start of the culture was 15.7, the xylose concentration was 20.5 g / L, the succinic acid accumulation concentration after 46.5 hours was 17.2 g / L, and the xylose concentration. Was 0.0 g / L. A graph showing the transition of OD ₆₆₀ , succinic acid accumulation concentration, and xylose concentration is shown in FIG.

[Example 13]
<Evaluation of succinic acid production in one-pot reaction of inositol culture strain>
In order to adjust microbial cells by culturing the Brevibacterium flavum MJ233 / XylAB / PC-4 / ΔLDH strain prepared in Comparative Example 1 in the presence of inositol and evaluate succinic acid production in a one-pot reaction, it is as follows. Flask culture was performed.

The seed culture was performed in the same manner as in Example 8 except that 600 μl of 50% glucose aqueous solution and 3 mL of 10% myo-inositol aqueous solution were added as a carbon source. The one-pot reaction was performed in the same manner as in Example 11. As a result, OD ₆₆₀ after 26.5 hours from the start of culture was 16.1, xylose concentration was 20.6 g / L, succinic acid accumulation concentration after 46.5 hours was 16.8 g / L, xylose concentration Was 0.9 g / L. A graph showing changes in OD ₆₆₀ , succinic acid accumulation concentration, and xylose concentration is shown in FIG.

[Comparative Example 5]
<Evaluation of succinic acid production in one-pot reaction of araE transgenic strain>
The same procedure as in Example 11 was performed except that the Brevibacterium flavum MJ233 / XylAB / PC-4 / ΔLDH strain prepared in Comparative Example 2 was used. As a result, the OD ₆₆₀ after 26.5 hours from the start of culture was 6.2, the xylose concentration was 26.5 g / L, the succinic acid accumulation concentration after 46.5 hours was 3.8 g / L, and the xylose concentration. Was 17.0 g / L. A graph showing changes in OD ₆₆₀ , succinic acid accumulation concentration, and xylose concentration is shown in FIG.
As a result of Examples 11 to 13 and Comparative Example 5, Table 3 shows OD ₆₆₀ and xylose concentrations at 26.5 hours after the start of culture, and succinic acid accumulation concentrations and xylose concentrations at 46.5 hours after the start of culture.

From Table 3, in Example 11 in which the succinic acid production evaluation in the one-pot reaction of Brevibacterium flavum MJ233 / IolT1 / XylAB / PC-4 / ΔLDH strain prepared in Example 1 was performed, Brevibacterium prepared in Comparative Example 2 was used. Compared to Comparative Example 5 in which the bacterial flavum MJ233 / AraE / XylAB / PC-4 / ΔLDH strain was evaluated, the reached OD ₆₆₀ was improved by 140% and the succinic acid accumulation concentration was improved by about 382%.

In Example 12, where succinic acid production was evaluated in the one-pot reaction of the Brevibacterium flavum MJ233 / IolT2 / XylAB / PC-4 / ΔLDH strain prepared in Example 2, the Brevibacterium flavum prepared in Comparative Example 2 was used. As compared with Comparative Example 5 in which the MJ233 / AraE / XylAB / PC-4 / ΔLDH strain was evaluated, the reached OD ₆₆₀ was improved by 153%, and the succinic acid accumulation concentration was improved by about 353%.

Further, in Example 13 in which succinic acid production evaluation was performed in a one-pot reaction of a strain obtained by culturing Brevibacterium flavum MJ233 / XylAB / PC-4 / ΔLDH strain prepared in Comparative Example 1 in the presence of inositol, Comparative Example 2 As compared with Comparative Example 5 in which the Brevibacterium flavum MJ233 / AraE / XylAB / PC-4 / ΔLDH strain prepared in Example 1 was evaluated, the ultimate OD ₆₆₀ was improved by 160% and the succinic acid accumulation concentration was improved by about 342%.

From the above results, in Patent Document 1, by introducing the pentose transporter AraE, the xylose consumption rate is improved. However, the reached OD ₆₆₀ is reduced in the growth using xylose as a carbon source, and thus the succinic acid production amount is reduced. It turned out to be worse. On the other hand, when the expression of the iolT1 gene or the iolT2 gene encoding the inositol transporter was enhanced, it was revealed that the amount of succinic acid produced was improved without such growth deterioration.

Claims (9)

Has the ability to produce organic compounds and xylose utilization, and has the nucleotide sequence of SEQ ID NO: 1 or 2
Is introduced gene encoding inositol transporter comprising a DNA having a sequence homology of 90% or more, or, microorganisms modified as the expression of the gene is enhanced as compared with an unmodified strain
A method of producing an organic compound by causing an organic compound to act on an organic raw material containing xylose in an aqueous medium. The organic compound according to claim 1, wherein the expression of a gene encoding a transcription factor that suppresses the expression of a gene encoding an inositol transporter of the microorganism is reduced as compared to an unmodified strain. Manufacturing method. Before SL microorganisms, further production of the gene encoding a xylose isomerase is introduced, or wherein the expression of the gene has been modified to enhance an organic compound according to claim 1 or claim 2 Method. The method for producing an organic compound according to claim 3 , wherein the microorganism is further modified so that a gene encoding xylulokinase is introduced or the expression of the gene is enhanced. Has the ability to produce organic compounds and xylose utilization, and has the nucleotide sequence of SEQ ID NO: 1 or 2
Gene introduction encoding inositol transporter comprising a DNA having a sequence homology of 90% or more, or, a modified microbe that expression of the gene is enhanced as compared with an unmodified strain,
A method for culturing microorganisms, comprising culturing in the presence of an organic raw material containing xylose . A coryneform bacterium having an ability to produce an organic compound and an ability to assimilate xylose , and is modified so that expression of a gene encoding an inositol transporter is enhanced as compared with an unmodified strain . The coryneform bacterium according to claim 6 , wherein the expression of a gene encoding a transcription factor that suppresses the expression of a gene encoding an inositol transporter is reduced compared to an unmodified strain. The coryneform bacterium according to claim 6 or 7 , wherein the coryneform bacterium is further modified so that a xylose isomerase gene is introduced. The coryneform bacterium further introduces xylulokinase gene, or characterized in that the expression of said gene is modified to enhance coryneform bacterium according to claim 8.