JP6954603B2 - Manufacturing method of myo-inositol - Google Patents

Description

The present invention relates to a method for producing myo-inositol.

Inositol is a type of cyclitol having a structure (1,2,3,4,5,6-cyclohexanehexaol) in which one hydrogen atom on each carbon of cyclohexane is replaced with a hydroxy group. There are nine types of stereoisomers depending on the bonding direction of the hydroxy group: myo-inositol (MI), D-chiro-inositol (DCI) and scyllo- inositol: SI) etc. are included.

Myo-inositol is a water-soluble vitamin-like agent that is biosynthesized in the human body, widely present in animals and plants, and is usually inoculated through food. Myo-inositol is expected to have, for example, a cholesterol-lowering effect, a lipid metabolism effect, a therapeutic effect on panic syndrome, and a function of adjusting the body clock. Myo-inositol is used as an ingredient in pharmaceuticals, foods and their additives (including health foods and supplements), cosmetics and the like. In particular, it is expected to be used for the prevention or treatment of hyperlipidemia. D-kilo-inositol has a function similar to insulin that regulates blood glucose levels, and is attracting attention as a therapeutic substance for diabetes and polycystic ovary syndrome. Shiro-inositol is expected to be used as a therapeutic agent for Alzheimer's disease, a feed for preventing protein aggregation diseases (for example, prion disease) in livestock, a synthetic raw material for physiologically active substances, and a synthetic raw material for liquid crystal compounds.

For example, in Bacillus subtilis (Bacillus subtilis), the conversion reaction pathway between myo-inositol, D-kilo-inositol and white-inositol is known (Patent Document 1 and Non-Patent Documents 1 and 2). ). Therefore, myo-inositol is not only a useful substance in itself, but also a raw material for the production of D-kilo-inositol and white-inositol.

Various methods have been described for producing white-inositol from myo-inositol using Bacillus subtilis (Patent Document 1 and Non-Patent Documents 1 and 2).

On the other hand, myo-inositol is produced from glucose via glucose-6-phosphate and myo-inositol-1-phosphate by an enzyme-mediated reaction.

Many prokaryotic microorganisms such as Bacillus subtilis do not have myo-inositol-1-phosphate synthase, which is one of the enzymes involved in the production reaction system, and these microorganisms are used to obtain glucose from glucose. It is difficult to produce inositol. For this reason, attempts have been made to produce prokaryotic microorganisms that enable the production of inositol from glucose by genetic recombination. For example, production of siro-inositol from glucose by Escherichia coli transformed to carry the myo-inositol-1-phosphate synthase gene, inositol monophosphatase gene, myo-inositol dehydrogenase gene and siro-inositol dehydrogenase gene ( Patent Document 2); and it has been reported that myo-inositol was produced from glucose by Escherichia coli transformed to carry the myo-inositol-1-phosphate synthase gene and the inositol monophosphatase gene (Patent Document 3). ing.

However, there have been no reports of Bacillus subtilis producing myo-inositol from glucose, and the construction of a new production reaction system is desired.

International Publication No. 2010/050231 International Publication No. 2013/110512 Japanese Unexamined Patent Publication No. 2014-176390

Microbial Cell Factories 2013, 12: 124 Microbial Cell Factories 2017, 16:67

An object of the present invention is to provide a transformed Bacillus subtilis capable of producing myo-inositol from glucose and a method for producing myo-inositol using the transformed Bacillus subtilis.

The present invention provides a transformed Bacillus subtilis that carries a gene encoding myo-inositol-1-phosphate synthase and lacks the pbuE gene.

In one embodiment, the Bacillus subtilis further lacks the gene encoding myo-inositol-2-dehydrogenase.

In a further embodiment, the bacillus further comprises 2-keto-mio-inositol dehydratase, silinosource isomerase, myo-inositol-2-dehydrogenase, repressor protein of the inositol metabolic gene group, methylmalate semialdehyde dehydration. Elemental enzyme, 2-deoxy-5-ketogluconate isomerase, 2-deoxy-5-ketogluconate kinase, 3,5 / 4 trihydroxy-1,2-dione hydrolyzate, and 2-deoxy-5-ketogluconic acid- It lacks the gene encoding at least one protein selected from the group consisting of 6-phosphate aldolase.

The present invention is a method for producing myo-inositol, which method is:
The step of culturing the transformed Bacillus subtilis in a glucose-containing medium is included.

According to the present invention, Bacillus subtilis can be used to produce myo-inositol from glucose. This provides a new reaction system for producing myo-inositol.

3 is a chromatogram showing the results of HPLC analysis of the medium after culturing transformed Bacillus subtilis cells of amy :: PrpsO-ino1ΔiolG (top) and amy :: PrpsO-ino1ΔioolGΔpbuE (middle) for 120 hours, respectively. It is a graph which shows the production amount of myo-inositol in the period of culturing transformed Bacillus subtilis cells of amy :: PrpsO-ino1ΔioolGΔpbuE for 120 hours.

The present invention provides a transformed Bacillus subtilis that carries a gene encoding myo-inositol-1-phosphate synthase and lacks the pbuE gene. In the present specification, "Bacillus subtilis" is also referred to as Bacillus subtilis cell.

As used herein, the term "carrying a gene" with respect to Bacillus subtilis cells means that the cell contains the gene and is in a state capable of expressing the protein encoded by the gene. For example, by introducing the gene into a wild-type cell in which the gene does not exist, a transformed cell "carrying the gene" can be produced. The “gene-carrying” transformed cell may further include modification to increase the expression level of the gene already possessed by the original cell.

As used herein, the term "gene-deficient" with respect to Bacillus subtilis cells means that the gene of interest is deleted, a mutation is introduced into the gene, or the gene is disrupted. Also includes. When a cell is "gene-deficient", the activity of the protein encoded by the target gene is reduced or eliminated as compared to, for example, a non-gene-deficient cell (eg, wild-type cell). Reduces or protein is not expressed. For example, such gene deficiencies may be naturally occurring or artificially modified. Transformed cells can be produced by artificial modification.

The reaction pathway in the production of myo-inositol from glucose is shown below. There are three stages of reaction:
(1) Glucose is converted to glucose-6-phosphate by mediation of a phosphotransferase (for example, glucose phosphotransferase) using a sugar such as glucose as a substrate;
(2) Glucose-6-phosphate is converted to myo-inositol-1-phosphate by mediation of myo-inositol-1-phosphate synthase; and (3) Myo-inositol is mediated by inositol monophosphatase. It is converted from -1-phosphate to myo-inositol.

The Bacillus subtilis of the present invention carries a gene encoding myo-inositol-1-phosphate synthase. Bacillus subtilis usually does not carry the gene encoding myo-inositol-1-phosphate synthase in wild-type cells. The gene can be introduced into Bacillus subtilis to carry the gene encoding myo-inositol-1-phosphate synthase. Bacillus subtilis originally has the reaction system of (1) above. Bacillus subtilis has an endogenous gene (for example, yktC (also known as "shuB")) (as sequence information, SWISS-PROT: Q45499, KEGG: BSU14670 or NCBI-Gene ID: 935986, NCBI-) for the inositol monophosphatase described in (3) above. ProteinID: NP_389350) may be possessed.

As the gene encoding myo-inositol-1-phosphate synthase, any gene can be used as long as the bacillus can substantially express myo-inositol-1-phosphate synthase activity. For example, the gene ino1 derived from Mycobacterium tuberculosis UT205 can be used, and information such as its sequence can be obtained from KEGG: UDA_0046c (database http://www.genome.jp/dbget-bin/www_bget?mtd:UDA_0046c). ) Or NCBI-ProteinID: Available from CCE35588. Furthermore, as gene INO1 (as sequence information, UniProtKB: P11986) derived from yeast Saccharomyces cerevisiae (strain ATCC 204508 / S288c) and gene SM11_pD1362 (sequence information) derived from alfalfa rhizobia (Sinorhizobium meliloti (strain SM11)). , UniProtKB: F7XIG1) etc. can also be used. The myo-inositol-1-phosphate synthase can also be referred to as "inositol-3-phosphate synthase". The gene encoding myo-inositol-1-phosphate synthase may be derived from an organism other than the above, or may be artificially synthesized.

A gene encoding myo-inositol-1-phosphate synthase can be a naturally occurring mutation or artificial as long as it can express substantial inositol-1-phosphate synthase activity in transformed cells. It may have a mutation introduced into or an artificial modification.

In one embodiment, the protein encoded by the gene encoding inositol-1-phosphate synthase has a natural amino acid sequence (eg, the published amino acid described above) as long as it has inositol-1-phosphate synthase activity. In the sequence or the amino acid sequence of SEQ ID NO: 1), it may be a mutant protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and this gene encodes such a mutant protein. It may be a thing. Such amino acid mutations (eg, deletions, substitutions or additions) may be any one or a combination of two or more. The total number of these mutations is one or several, but is not particularly limited as long as it has substantially the desired function or effect, and may depend on the size of the protein. The total number of mutations is, for example, 1 or more and 10 or less, 1 or more and 5 or less, 1 or more and 4 or less, 1 or more and 3 or less, or 1 or more and 2 or less, but is not limited thereto. .. In addition, the total number of mutations can be, for example, within the range satisfying the sequence identity described below.

In another embodiment, the gene encoding inositol-1-phosphate synthase has the above-mentioned natural amino acid sequence and, for example, BLAST (J. Mol. Biol., 215, 403 (1990)) or FASTA (Methods). When calculated using analysis software such as in Enzymology, 183, 63 (1990)), for example, 74% or more, 78% or more, 80% or more, 85% or more, 90% or more, 92% or more, 95%. As described above, it may be a gene encoding a protein having 98% or more or 99% or more sequence identity and having inositol-1-phosphate synthase.

In yet another embodiment, the gene encoding inositol-1-phosphate synthase is a DNA stringent condition consisting of a base sequence complementary to the base sequence of the gene encoding the protein described herein. It may contain DNA that hybridizes with. The DNA that hybridizes under stringent conditions is, for example, a colony hybridization method, a plaque hybridization method, or a plaque hybridization method using a DNA having a base sequence described in a published database or a DNA fragment thereof as a probe. It means DNA obtained by using Southern blot hybridization or the like. More specifically, the DNA that hybridizes under stringent conditions is, for example, 65 in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from a colony or plaque is immobilized. After hybridization at ° C., the filter is washed at 65 ° C. in a 0.1 to 2-fold concentration SSC solution (the composition of the 1-fold concentration SSC solution is 150 mM NaCl, 15 mM sodium citrate). DNA that can be obtained by this can be mentioned. Hybridization can be performed by well-known methods such as those described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Ed ,, Cold Spring Harbor Laboratory (2001). The higher the temperature or the lower the salt concentration, the higher the stringency, and a polynucleotide with higher homology (sequence identity) can be isolated.

In still another embodiment, the gene encoding inositol-1-phosphate synthase is obtained, for example, when calculated using the base sequence of the gene encoding the protein described in the present specification and the above analysis software. , 70% or more, 75% or more, 78% or more, 80% or more, 85% or more, 90% or more, 92% or more, 95% or more, 98% or more, or 99% or more base sequences having sequence identity. It may encode a protein having and substantially having inositol-1-phosphate synthase activity.

In yet another embodiment, the gene encoding inositol-1-phosphate synthase also includes a gene having a base sequence modified by a degenerate-based substitution of the genetic code. The base sequence of the gene encoding the predetermined amino acid sequence (for example, the above-mentioned published amino acid sequence or the amino acid sequence of SEQ ID NO: 1) is predetermined without changing the amino acid sequence of the protein by substitution based on the degeneracy of the genetic code. It is also possible to replace at least one base of the base sequence encoding the amino acid sequence of the above with another base. Genes can also be obtained by artificial synthesis based on sequences optimized for the codon frequency of the host. For example, the nucleotide sequence of SEQ ID NO: 2 used in the following examples (positions 39 to 1145; the encoded amino acid sequence is shown in SEQ ID NO: 3) is a tuberculosis-derived inositol-1-phosphate synthase gene ino1. This sequence is optimized in consideration of the codon frequency of Bacillus subtilis.

Such genes are modeled on, for example, nucleic acids derived from DNA extracted from various organisms, various cDNA libraries, genomic DNA libraries, etc. using primers designed based on disclosed or known base sequences. It can be obtained as a nucleic acid fragment by performing PCR amplification. Further, it can be obtained as a nucleic acid fragment by hybridization using a nucleic acid derived from the above library or the like as a template and a DNA fragment which is a part of a gene encoding the enzyme to be expressed or expressed in the present invention as a probe. Can be done. Alternatively, the gene may be synthesized as a nucleic acid fragment by various nucleic acid sequence synthesis methods known in the art such as a chemical synthesis method.

Further, for a gene, for example, DNA encoding a disclosed or known amino acid sequence is modified by a conventional mutagenesis method, a site-specific mutagenesis method, a molecular evolution method using error-prone PCR, or the like. Can be obtained by. Examples of such a method include a known method such as the Kunkel method or the Gapped duplex method, or a method similar thereto. For example, a mutation introduction kit using a site-specific mutagenesis method (for example, Mutant-K (Takara Bio Inc.)). Mutations are introduced using (manufactured by Takara Bio Inc.), Mutant-G (manufactured by Takara Bio Inc.), or using the LA PCR in vitro Mutagenesis series kit of Takara Bio Inc.

An expression cassette containing a gene encoding inositol-1-phosphate synthase can be constructed. In this expression cassette, the promoter can be operably linked upstream of the gene encoding inositol-1-phosphate synthase (5'region of the gene). As the promoter, any promoter that functions in Bacillus subtilis can be used. For example, the promoter of rpsO (positions 1 to 38 of the base sequence of SEQ ID NO: 2), the promoter of ybfK, and the like can be mentioned, but are not limited thereto. In the expression cassette, a terminator can be further operably linked downstream of the gene encoding inositol-1-phosphate synthase (3'region of the gene). As the terminator, any terminator that functions in Bacillus subtilis (for example, tufA terminator (positions 1146 to 1219 of the base sequence of SEQ ID NO: 2)) can be used. Further, the expression cassette can contain a marker gene (for example, a drug resistance gene as described below) that enables selection of transformed cells in an appropriate arrangement. The expression cassette may be inserted at any position on the chromosome for introduction into Bacillus subtilis, or may be incorporated into a vector that replicates and maintains independently of the chromosome (eg, a plasmid vector).

Synthesis and ligation (binding) of genes or DNA can be performed by techniques commonly used by those skilled in the art. Regarding the binding of each component, for example, the DNA of each component to which an appropriate restriction enzyme recognition sequence has been assigned by the PCR method can be cleaved with the restriction enzyme and ligated using ligase or the like.

For the introduction of the expression cassette into cells of Bacillus subtilis, for example, a homologous recombination method, a phage transduction method, or the like is used. In the homologous recombination method, for example, drug resistance genes (for example, chloramphenicol resistance gene, canamycin resistance gene, tetracycline resistance gene, erythromycin resistance gene, spectinomycin resistance gene, ampicillin resistance gene, hyglomycin resistance gene, etc.) are used. This is a method in which a viable cell is obtained by introducing an expression cassette containing the gene and then selecting the gene with a corresponding drug. After introduction into Bacillus subtilis cells, the expression cassette may be inserted at any location on the chromosome or in a vector that replicates and maintains independently of the chromosome (eg, a plasmid vector), eg, desired. The target cells can be obtained by confirming that the expression cassette has been inserted into the power portion using a device such as PCR.

As the Bacillus subtilis cell as a host into which the expression cassette is inserted, a wild-type cell (originally, Bacillus subtilis cell that does not carry a gene encoding inositol-1-phosphate synthase) can be used. Alternatively, it may be an auxotrophic mutant strain, a drug resistance mutant strain, or may have already been transformed to have various marker genes related to the mutation. In addition, "gene-deficient" cells as described below may be used as the host.

The Bacillus subtilis of the present invention lacks the pbuE gene. The pbuE gene encodes the protein PbuB (Purine Efflux Pump), which was named as having the function of responsible for the extracellular excretion of purine nucleosides in Bacillus subtilis (as sequence information, KEGG: BSU05800 or NCBI-GeneID: 937996, NCBI- ProteinID: NP_388461). In reaction path of (2), inositol 1-phosphate synthase activity is dependent on ^{NAD + / NADH, NAD + /} NADH is a derivative of purine nucleoside.

Means of gene deletion include, for example, artificial deletion (deletion) of a gene, introduction of a mutation, or destruction or inactivation of a gene. As the genetic manipulation for that purpose, for example, a phage transduction method, a homologous recombination method, a plasmid vector insertion method and the like can be used. Inactivation of a target gene in Bacillus subtilis using the plasmid vector insertion method (a circular plasmid DNA is inserted into a specific site of chromosomal DNA by homologous recombination at one site) is described, for example, in Microbiology 1998, 144: 3097-3104. It can be done according to the method described in. Deletion of the gene of interest by homologous recombination can be performed, for example, according to the method described in Genes Genet. Syst. 2009, 84: 315-318. As a method using homologous recombination, gene knockout for gene disruption, gene knockin for introduction of mutation, and the like can also be used. By including a drug resistance gene in advance in a DNA fragment to be taken up by homologous recombination, and by selecting surviving cells with a corresponding drug, transformed cells in which homologous recombination has occurred can be obtained. Furthermore, for example, by confirming the sequence of the target region of homologous recombination using an instrument such as PCR, the target transformed cell can be obtained.

In one embodiment, the Bacillus subtilis of the present invention further lacks the gene encoding myo-inositol-2-dehydrogenase. Myo-inositol-2-dehydrogenase is the first enzyme of myo-inositol metabolism encoded by the iolG gene, which is one of the inositol metabolism system genes (iol group). Myo-inositol-2-dehydrogenase is a NAD + -dependent reaction of myo-inositol (MI), 2-keto-myo-inositol (2KMI) (also referred to as "shiro-inositol"). Convert to. Myo-inositol-2-dehydrogenase also converts D-chiro-inositol (DCI) to 1-keto-D-chiro-inositol (1KDCI). It also catalyzes the reaction.

In another embodiment, Bacillus subtilis of the present invention, further, 2-keto - myo - inositol dehydratase (Iole), white Ino isomerase (Ioli), inositol metabolism genes repressor protein (iolR), methyl Semialdehyde malonate dehydrogenase (iolA), 2-deoxy-5-ketogluconate isomerase (iolB), 2-deoxy-5-ketogluconate kinase (iolC), 3,5 / 4 trihydroxy-1,2-dione It lacks the gene encoding at least one protein selected from the group consisting of hydrolytic enzyme (iolD) and 2-deoxy-5-ketogluconic acid-6-phosphate aldolase (iolJ). The above-mentioned enzyme or protein is encoded by a gene of the inositol metabolic system gene group (iol group) and is involved in the metabolic system, like the iolG gene. The Bacillus subtilis of the present invention lacks genes encoding at least one, preferably two or more, more preferably all of the above proteins. The Bacillus subtilis of the present invention may also be deficient in genes encoding inositol transporter (iolF), inositol metabolism-related enzyme (iolH) whose function is unidentified, and the like. The Bacillus subtilis deficient in the genes encoding all the above proteins is, for example, Bacillus subtilis MYI04 described in Non-Patent Document 1. MYI04 lacks the iolABCDEFHIJ operons, iolX and iolR (Non-Patent Document 1).

The Bacillus subtilis of the present invention preferably lacks at least iolG with respect to the above-mentioned ioll group genes.

The present invention further provides a method for producing myo-inositol. The method for producing myo-inositol includes a step of culturing the transformed Bacillus subtilis of the present invention in a medium containing glucose. This production method may also include a step of removing Bacillus subtilis cells from the culture filtrate obtained in the culture step and a step of isolating myo-inositol from the culture filtrate from which the cells have been removed.

The "medium containing glucose" may be a medium containing a sugar other than glucose. For example, it may contain glucose produced by decomposing a polysaccharide having a larger molecular weight, which comprises glucose as a constituent sugar, by an enzyme or the like during the culture of the transformed Bacillus subtilis of the present invention.

The composition of the medium is not particularly limited except that it contains glucose. Any medium may be used as long as it contains a carbon source, a nitrogen source, an organic nutrient source, inorganic salts and the like in addition to glucose as a raw material, and either a synthetic medium or a natural medium can be used. A culture medium (liquid medium) is preferable.

As the medium, for example, a medium formulated as follows can be used (the "%" of the blended amount is "% (w / v)" in each case): glucose is 0.1% to 40%, preferably. 1% to 30%, more preferably 1% to 10%; 0.1% to 20%, preferably 0.3% to 5%, and the total amount of nitrogen source. 0.01% to 5%, preferably 0.5% to 2%. As the carbon source, for example, at least one of malic acid, glycerol, sucrose, maltose, starch and the like can be used. As the nitrogen source, at least one such as soyton, casamino acid, peptone, yeast extract, ammonium sulfate, ammonium chloride, ammonium nitrate, urea and the like can be used.

In addition, if necessary, generate ions such as sodium ion, potassium ion, calcium ion, magnesium ion, cobalt ion, manganese ion, zinc ion, iron ion, copper ion, molybdenum ion, phosphate ion, and sulfate ion. Inorganic salts can be added to the medium. The amount of the inorganic salt added can be arbitrarily determined by those skilled in the art.

The hydrogen ion concentration in the culture solution does not need to be particularly adjusted, but is preferably pH 5 to 10, more preferably pH 6 to 9.

As the culture conditions, the culture temperature is, for example, 12 ° C. to 45 ° C., preferably 15 ° C. to 37 ° C., and the culture can be aerobic culture, for example, shaking the culture solution or airing the culture solution. Alternatively, oxygen gas can be blown into the culture. The culturing period may be continued until myo-inositol shows the maximum or required amount of accumulation, for example, 1 to 7 days, preferably 1 to 5 days.

The culture medium after culturing contains Bacillus subtilis cells and myo-inositol. For example, as a method for collecting myo-inositol from a culture solution, a general method for isolating and purifying an ordinary water-soluble neutral substance can be applied. That is, by removing the bacterial cells from the culture solution and then treating the culture supernatant with activated carbon, an ion exchange resin, or the like, impurities other than myo-inositol can be almost removed. Then, the target substance can be isolated by using a method such as recrystallization. The Bacillus subtilis cells collected after culturing can be subjected to a new culture again.

Specifically, for example, the culture supernatant in which myo-inositol has accumulated is filled with a strongly acidic cation exchange resin, for example, Duolite (registered trademark) C-20 (H + type) for the purpose of removing undesired components. The passing liquid is collected by passing it through the column, and then the deionized water is passed through this column to wash and collect the washing liquid, and the obtained passing liquid and the washing liquid are combined. The solution thus obtained is passed through a column packed with a strong basic anion exchange resin, for example, Duolite (registered trademark) A116 (OH-type) to collect the passing liquid, and then deionized water is passed through this column for washing. Then, the cleaning solution is collected, and the obtained passing solution and the cleaning solution are combined to obtain an aqueous solution containing myo-inositol and containing almost no other impurities. Pure myo-inositol crystals can be crystallized by adding an appropriate amount of ethanol to a concentrated solution of myo-inositol obtained by concentrating this aqueous solution and leaving it at room temperature or low temperature overnight. Further, when performing the column operation, a column filled with activated carbon can be used for the purpose of decolorization.

In the present invention, white-inositol can be produced from myo-inositol against the transformed Bacillus subtilis by using any of the methods described in Patent Document 1 and Non-Patent Documents 1 and 2. May be modified to.

The transformed Bacillus subtilis of the present invention is used to produce myo-inositol from glucose. This provides a means for stably supplying myo-inositol itself, which is useful as a material for pharmaceuticals, foods, cosmetics and the like. Myo-inositol can also be used as a raw material for the production of siro-inositol or D-kilo-inositol.

Hereinafter, the present invention will be described in detail with reference to Examples. However, the present invention is not limited thereto.

(Example 1: Preparation of transformed Bacillus subtilis cells)
The iolG gene of Bacillus subtilis MYI04 described in Non-Patent Document 1 was deleted by the following method.

This method conformed to the method described in Genes Genet. Syst. 2009, 84: 315-318.

First, from the MYI04 chromosomal DNA, the yxdJ gene region existing downstream of the iolG gene was obtained by PCR amplification using a pair of oligo DNA primers AF (SEQ ID NO: 4) and AR (SEQ ID NO: 5) (referred to as DNA fragment A). ). Similarly, from the MYI04 chromosomal DNA, the ioluS DiolR gene region existing upstream of the iolG gene was obtained by PCR amplification using a pair of oligo DNA primers BF (SEQ ID NO: 6) and BR (SEQ ID NO: 7) (with DNA fragment B). ). Further, the iolG gene region was obtained from the MYI04 chromosomal DNA by PCR amplification using a pair of oligo DNA primers CF (SEQ ID NO: 8) and CR (SEQ ID NO: 9) (referred to as DNA fragment C). Furthermore, from the Bacillus subtilis TMO311 chromosomal DNA described in Genes Genet. Syst. 2009, 84: 315-318, a pair of oligo DNA primers mazFF (SEQ ID NO: 10) and mazFR (SEQ ID NO: 11) are used for the mazF cassette region. Obtained by PCR amplification (referred to as DNA fragment D).

The four DNA fragments prepared in this way, A, B, D and C, were ligated in this order by recombinant PCR. A kanamycin resistance gene is contained in the mazF cassette, and when this recombinant PCR DNA is incorporated by the natural transformation ability of MYI04, homologous recombination occurs in the regions corresponding to DNA fragments A and C. As a result, recombinants in which the entire recombinant PCR DNA was incorporated into the chromosome were selected by acquiring kanamycin resistance.

The chromosome of this recombinant has regions corresponding to DNA fragment B overlapping in two places, and homologous recombination within the same chromosome occurs frequently between these regions. Then, as a result of such homologous recombination within the same chromosome, the chromosomal DNA containing the DNA fragment C (iolG gene region) and D (mazF cassette region) sandwiched between the overlapping DNA fragment B regions is cut out and disappears. The mazF cassette contains mazF, which is an IPTG-induced toxin gene, and cells in which the above-mentioned chromosome excision did not occur die in the presence of IPTG. In addition, the mazF cassette is also lost by excision of this chromosome, so that the cell also loses resistance to kanamycin.

Therefore, after culturing the above recombinant for a certain period of time, those that grow by transferring to a medium containing IPTG are selected, and in addition to the susceptibility of such cells to kanamycin, the base sequence of the chromosomal region is confirmed, and MYI04 ΔiolG in which the iolG gene of the above was deleted was obtained.

The myo-inositol-1-phosphate synthase gene of Mycobacterium tuberculosis was introduced into the above ΔiolG by the following method.

From the information in the database (http://www.genome.jp/dbget-bin/www_bget?mtd:UDA_0046c), the myo-inositol-1-phosphate synthase gene (ino1) of Mycobacterium tuberculosis can be found in SEQ ID NO: 1. It was prepared as a synthetic DNA that gives an amino acid sequence. The synthetic DNA was codon-adjusted for translation in Bacillus subtilis, and the rpsO Shine-Dalgarno sequence and tufaA terminator were artificially added to the 5'and 3'regions, respectively. The base sequence of this synthetic DNA is shown in SEQ ID NO: 2, and the encoded amino acid sequence is shown in SEQ ID NO: 3.

From the KU106 chromosome of Bacillus subtilis described in Non-Patent Document 1, a chromosomal DNA region containing the C-terminal region of the amyE locus, the chloramphenicol resistance gene and the rpsO gene promoter is combined with a pair of oligo DNA primers a011 (SEQ ID NO: 12) and Obtained by PCR amplification using a650 (SEQ ID NO: 13) (referred to as DNA fragment E). In addition, the synthetic DNA giving the myo-inositol-1-phosphate synthase gene of the above-mentioned tuberculosis bacterium was amplified by PCR using oligo DNA primers a651 (SEQ ID NO: 14) and a652 (SEQ ID NO: 15) (DNA fragment). Called F). Further, from the KU106 chromosome, the region containing the N-terminal region of the amyE locus was amplified by PCR using primers a014 (SEQ ID NO: 16) and a015 (SEQ ID NO: 17) (referred to as DNA fragment G).

Then, using the DNA in which these three DNA fragments E, F and G were ligated in this order by recombinant PCR, they were incorporated into ΔiolG by their natural transformation ability and transformed into chloramphenicol resistance. As a result, the myo-inositol-1-phosphate synthase gene of tuberculosis bacterium is incorporated into the amyE locus of ΔiolG so as to be transcribed and expressed by the rpsO promoter together with the chloramphenicol resistance gene, and the accurate incorporation is performed in the base sequence. After confirmation, amy :: PrpsO-ino1ΔioolG was obtained.

Furthermore, the pbuE gene of the above-mentioned amy :: PrpsO-ino1ΔioolG was disrupted by the following method. This method conformed to the method described in Microbiology 1998, 144: 3097-3104.

Bacillus subtilis mutant strain YDHLd (pbuE gene disruption strain: https://shigen.nig.ac.jp/) obtained from the Bacillus subtilis mutant strain bank managed by the National Institute of Genetics Nuclear Biogenes Laboratory by the National BioResource Project. Using the chromosomal DNA of bsub / resource / strainGeneDisrupted / detail / 2185; jsessionid = BCB7B4AF23FB68E7667ABF98AA9AA57A), amy :: PrpsO-ino1ΔiolG was transformed to become erythromycin resistant. As a result, the pbuE gene of this cell is disrupted and inactivated by the insertion of the erythromycin resistance gene cassette. The cells obtained as a result were designated as amy :: PrpsO-ino1ΔioolGΔpbuE.

(Example 2: Detection of inositol in the medium after culturing the transformed Bacillus subtilis)
Bacillus subtilis was precultured overnight at 37 ° C. in Lysogeny Broth (LB) medium with shaking at 180 rpm (antibiotics added as needed).

For inositol bioconversion, 0.5% (w / v) bactoeast extract (Becton, Dickinson and Co., Sparks, MD, USA), 2% (w / v) glucose, and 2% bactosoyton ( A biotransformation medium consisting of Becton, Dickinson and Co.) was used. A 20 mL bioconversion medium placed in a 100 mL Erlenmeyer flask was inoculated with a preculture solution of Bacillus subtilis at 600 nm so as to give a turbidity of 0.1, and cultured at 37 ° C. with shaking at 180 rpm.

As described above, the amy :: PrpsO-ino1ΔiolG strain and the amy :: PrpsO-ino1ΔioolGΔpbuE strain were cultured in a biotransformation medium (glucose-added medium) for 120 hours in the presence of glucose.

After culturing, cells were first removed from the medium by centrifugation and then filtered through an Amicon Ultra-0.5 mL 3K Centrifugal Filter (Millipore, Billerica, MA, USA) to completely eliminate solids. The liquid thus obtained was transferred to 2 mL of acetonitrile / water (80/20) kept at 28 ° C. using a COSMOSIL Sugar-D column (4.6 × 250 mm) (Nacalai Tesque, Kyoto). It was analyzed by a differential refractometer detector through high performance liquid chromatography (HPLC) (LaChrom Elite: HITACHI High Technologies, Tokyo, Japan) as a phase. The stereoisomers were identified using the outflow retention time when the standard substances myo-inositol and siro-inositol were subjected to high performance liquid chromatography under the same conditions as an index, and the concentration was calculated from the peak area of the differential refractometer.

FIG. 1 is a chromatogram showing the results of HPLC analysis of the culture medium after culturing the transformed Bacillus subtilis cells of amy :: PrpsO-ino1ΔiolG (top) and amy :: PrpsO-ino1ΔioolGΔpbuE (middle) in a glucose-added medium for 120 hours, respectively. Is. FIG. 1 also shows a chromatogram (bottom) showing the peaks of the standard substances myo-inositol and siro-inositol. After 120 hours of culturing in the biotransformation medium, the production of myo-inositol was not confirmed in the amy :: PrpsO-ino1ΔioolG strain, but the production of myo-inositol was confirmed in the amy :: PrpsO-ino1ΔioolGΔpbuE strain.

(Example 3: Production of myo-inositol by culturing transformed Bacillus subtilis)
As described in Example 2, the amy :: PrpsO-ino1ΔioolGΔpbuE strain was cultured in a biological conversion medium (glucose-added medium), and the amount of myo-inositol 24 hours, 48 hours, and 120 hours after the start of the culture was determined by HPLC. Analyzed and measured.

FIG. 2 is a graph showing the amount of myo-inositol produced during the period in which transformed Bacillus subtilis cells of amy :: PrpsO-ino1ΔioolGΔpbuE were cultured in a glucose-added medium for 120 hours. The horizontal axis shows the culture time (unit: time), and the vertical axis shows the production amount of myo-inositol (unit:% (w / v)). The production of myo-inositol after 120-hour culture of the amy :: PrpsO-ino1ΔioolGΔpbuE strain was 0.01% (w / v), and the conversion efficiency from 2% glucose was 0.5%.

The present invention can be used for industrial production of myo-inositol. In addition, it is useful for the supply of raw materials for the manufacture of pharmaceuticals, foods and cosmetics.

Claims (4)

A transformed Bacillus subtilis that carries a gene encoding myo-inositol-1-phosphate synthase and lacks the pbuE gene. The transformed Bacillus subtilis according to claim 1, which further lacks the gene (iolG) encoding myo-inositol-2-dehydrogenase. Furthermore, 2-keto - myo - inositol dehydratase (Iole), white Ino isomerase (Ioli), inositol metabolism genes repressor protein (iolR), methylmalonic acid semialdehyde dehydrogenase (Iola), 2- Deoxy-5-ketogluconate isomerase (iolB) , 2-deoxy-5-ketogluconate kinase (iolC) , 3,5 / 4 trihydroxy-1,2-dione hydrolyzate (iolD) , and 2-deoxy-5 The transformed bacillus according to claim 1 or 2, which lacks a gene encoding at least one protein selected from the group consisting of -ketogluconic acid-6-phosphate aldolase (iolJ). A method for producing myo-inositol,
A method comprising the step of culturing the transformed Bacillus subtilis according to any one of claims 1 to 3 in a medium containing glucose.

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