2-deoxy-scillo-inosamine-producing strain and 2-deoxy-scillo-inosamine production method

Abstract

To provide a 2-deoxy-scillo-inosamine (DOIm)-producing strain and a DOIm production method enabling efficient production of DOIm.SOLUTION: A DOIm-producing strain comprises a gene encoding a DOI synthase involved in the biosynthesis of 2-deoxy-scillo-inosose and a gene encoding a DOIm synthase involved in the biosynthesis of 2-deoxy-scillo-inosamine.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present invention relates to a 2-deoxy-siro-inosamine-producing bacterium and a method for producing 2-deoxy-siro-inosamine.

Background Art

[0002] 2-Deoxy-siro-inosamine (the following structure, hereinafter also referred to as DOIm) is produced by an aminotransferase using 2-deoxy-siro-inose (the following structure, hereinafter also referred to as DOI) as a substrate and L-glutamine as an amino group transfer source. Since DOIm serves as a raw material for aminoglycoside antibiotics (for example, Butirosin), it is a compound expected to be utilized in the production of various pharmaceuticals.

[0003]

Chemical Formula

[0004] Regarding a method for producing DOI, a method for producing DOI from glucose-6-phosphate (G-6-P) using a recombinant DOI synthase obtained using Escherichia coli has been developed (for example, see Patent Document 1). Furthermore, a method for producing DOI from sucrose as a precursor of glucose-6-phosphate using Escherichia coli in which a gene encoding DOI synthase and a gene encoding sucrose hydrolase are expressed has been developed (for example, see Patent Documents 2 and 3). Regarding a method for producing DOIm, a method for producing DOIm by an enzymatic reaction from DOI and L-glutamine using L-glutamine:2-deoxy-siro-inose aminotransferase (DOIm synthase) obtained using Escherichia coli has been developed (for example, see Patent Document 4).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

[0006] The methods described in Patent Documents 1 to 3 are intended to produce DOI from saccharides such as glucose and sucrose or their derivatives using enzymes or microorganisms, and are not intended to produce DOIm. Further, in the method described in the examples of Patent Document 4, the reaction is carried out under the condition that the DOI concentration is as low as 5 mM. In order to industrially produce DOIm, it is desirable to be able to produce DOIm at a high concentration (for example, 1% by mass or more). An object of the present disclosure is to provide a DOIm-producing bacterium capable of efficiently producing DOIm and a method for producing DOIm. [Means for Solving the Problems]

[0007] Means for solving the above problems include the following embodiments. <1> A DOIm-producing bacterium having a gene encoding a DOI synthase involved in the synthesis of 2-deoxy-sylo-inose and a gene encoding a DOIm synthase involved in the synthesis of 2-deoxy-sylo-inosamine. <2> The DOIm-producing bacterium according to <1>, wherein the DOI synthase is an enzyme that catalyzes a reaction for generating DOI from glucose-6-phosphate. <3> The DOIm-producing bacterium according to <1> or <2>, wherein the DOIm synthase is an enzyme that catalyzes a reaction in which the amino group of L-glutamine is transferred to DOI. <4>The DOI synthase is a polypeptide having the amino acid sequence shown in the following (a) or (b), and is the DOIm-producing bacterium according to any one of <1> to <3>. (a) The amino acid sequence shown in SEQ ID NO: 1 (b) An amino acid sequence having the activity as DOI synthase and having a sequence identity of 80% or more with SEQ ID NO: 1 <5>The DOIm synthase is a polypeptide having the amino acid sequence shown in the following (c) or (d), and is the DOIm-producing bacterium according to any one of <1> to <4>. (a) The amino acid sequence shown in SEQ ID NO: 2 (b) An amino acid sequence having the activity as L-glutamine:DOI aminotransferase and having a sequence identity of 80% or more with SEQ ID NO: 2 <6>The DOIm-producing bacterium according to any one of <1> to <5>, further having a gene encoding a sucrose hydrolase. <7>The DOIm-producing bacterium according to any one of <1> to <6>, further having a gene encoding a glucose transport promoting protein. <8>The DOIm-producing bacterium according to any one of <1> to <7>, wherein the activity of the enzyme involved in the metabolism of glucose-6-phosphate is inactivated or reduced. <9>The DOIm-producing bacterium according to any one of <1> to <8>, which is Escherichia coli. <10>A method for producing DOIm, comprising the step of contacting a substance selected from a combination of glucose and fructose, or sucrose, with the DOIm-producing bacterium according to any one of <1> to <9>. <11>The method for producing DOIm according to <10>, wherein the contact is carried out in the presence of ammonia. <12>The method for producing DOIm according to <11>, wherein the contact is carried out in a medium having an ammonia concentration of 0.1% by mass to 0.5% by mass.

Advantages of the Invention

[0008] According to the present disclosure, there are provided a DOIm-producing bacterium capable of efficiently producing DOIm and a method for producing DOIm.

Brief Description of the Drawings

[0009]

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0010] In the present disclosure, the numerical range indicated using "~" represents a range including the numerical values described before and after "~" as the minimum value and the maximum value, respectively.

[0011] <DOIm-producing bacterium> The DOIm-producing bacterium of the present disclosure has a gene encoding a DOI synthase involved in the synthesis of 2-deoxy-siro-inose and a gene encoding a DOIm synthase involved in the synthesis of 2-deoxy-siro-inosamine.

[0012] The DOIm-producing bacterium of the present disclosure is used for the production of DOIm. By using the DOIm-producing bacterium of the present disclosure, DOIm can be produced from saccharides used as raw materials in a single reaction step, and it has excellent DOIm production efficiency. Furthermore, by using the DOIm-producing bacterium of the present disclosure, DOIm can be produced from relatively inexpensive raw materials compared to the method of using DOI as a substrate for the DOIm synthase.

[0013] Surprisingly, when synthesizing DOIm using the DOIm-producing bacterium of the present disclosure, the production amount of components with unknown structures is suppressed compared to the case of synthesizing DOIm using DOIm synthase. The accumulation of by-products can be a factor causing a decrease in the reaction yield and purification yield of DOIm. Therefore, the DOIm-producing bacterium of the present disclosure is useful as a means for producing DOIm while suppressing the production of by-products.

[0014] The DOIm-producing bacterium of the present disclosure can produce DOIm in the presence of glucose and fructose. Glucose becomes glucose-6-phosphate in the bacterial cell and is converted to DOI by DOIm synthase. Fructose becomes fructose-1,6-bisphosphate in the bacterial cell and is utilized as an energy source for the DOIm-producing bacterium.

[0015] Glucose and fructose to be contacted with the DOIm-producing bacterium may be added as glucose and fructose to the medium containing the DOIm-producing bacterium, or may be added in other states. For example, it may be added to the medium containing the DOIm-producing bacterium as a substance (such as sucrose) that can be changed to glucose and fructose by the action of the DOIm-producing bacterium or other microorganisms.

[0016] The DOIm-producing bacterium of the present disclosure has a gene (btrC) encoding DOI synthase (BtrC). DOI synthase is an enzyme involved in the synthesis of DOI, and more specifically, it is an enzyme that catalyzes the reaction of generating DOI from glucose-6-phosphate. As the gene of DOI synthase, any gene can be used as long as it is derived from an organism having this gene. For example, those derived from Bacillus bacteria can be mentioned. From the viewpoint of DOIm production efficiency, a gene encoding a 42 kDa subunit derived from Bacillus circulans (GenBank accession number AB066276) can be preferably used.

[0017] The DOI synthase may be a polypeptide having the amino acid sequence shown in (a) or (b) below. (a) The amino acid sequence shown in SEQ ID NO: 1 (b) An amino acid sequence having the activity as a DOI synthase and having a sequence identity of 80% or more with SEQ ID NO: 1

[0018] (a) The amino acid sequence of SEQ ID NO: 1 shown is the amino acid sequence of the DOI synthase possessed by Bacillus circulans (ATCC 4513) described in GenBank accession number AB066276. (b) The amino acid sequence shown may have a sequence identity of 85% or more, 90% or more, 95% or more, or 99% or more with SEQ ID NO: 1.

[0019] (SEQ ID NO: 1) MTTKQICFADRCFNFAFGEHVLESVESYIPRDEFDQYIMISDSGVPDSIVHYAAEYFGKLAPVHILRFQGGEEYKTLSTVTNLQERAIALGANRRTAIVAVGGGLTGNVAGVAAGMMFRGIALIHVPTTFLAASDSVLSIKQAVNLTSGKNLVGFYYPPRFVFADTRILSESPPRQVKAGMCELVKNMLILENDNKEFTEDDLNSANVYSPKQLETFINFCISAKMSVLSEDIYEKKKGLIFEYGHTIGHAIELAEQGGITHGEAIAVGMIYAAKIANRMNLMPEHDVSAHYWLLNKIGALQDIPLKSDPDSIFHYLIHDNKRGYIKLDEDNLGMILLSGVGKPAMYNQTLLTPVRKTLIKEVIREGL

[0020] In the present disclosure, the value (%) of sequence identity of the amino acid sequence of a polypeptide refers to the value (Identity) shown as a percentage of the number of matching amino acid residues divided by the number of aligned amino acid residues when the amino acid sequence of the reference polypeptide and the amino acid sequence of the polypeptide to be measured are aligned so as to maximize the match. When aligning the amino acid sequence of the reference polypeptide and the amino acid sequence of the polypeptide to be measured, deletions (gaps) may be included. The sequence identity of the amino acid sequence may be a value calculated using a method well known to those skilled in the art, for example, Blastp (protein - protein BLAST), which is a search program of BLAST (Basic Local Alignment Search Tool).

[0021] The DOIm - producing bacterium of the present disclosure has a gene (btrR) encoding DOIm synthase (BtrR). DOIm synthase is an enzyme involved in the synthesis of DOIm. More specifically, it is an enzyme (L - glutamine:DOI amino - group transferase) that catalyzes the reaction in which the amino group of L - glutamine is transferred to DOI.

[0022] DOIm synthase may be a polypeptide having the amino acid sequence shown in the following (c) or (d). (c) The amino acid sequence shown in SEQ ID NO: 2 (d) An amino acid sequence having activity as L - glutamine:DOIm synthase and having a sequence identity of 80% or more with SEQ ID NO: 2

[0023] (c) The amino acid sequence of SEQ ID NO: 2 shown is the amino acid sequence of an enzyme derived from Bacillus circulans SANK72073. (d) The amino acid sequence shown may have a sequence identity of 85% or more, 90% or more, 95% or more, or 99% or more with SEQ ID NO: 2.

[0024] (SEQ ID NO: 2) MTIPFDHWPEWPQHSDRTRRKIEEVFQSNRWAISGYWTGEESMERKFAKAFADFNGVPYCVPTTSGSTALMLALEALGIGEGDEVIVPSLTWIATATAVLNVNALPVFVDVEADTYCIDPQLIKSAITDKTKAIIPVHLFGSMANMDEINEIAQEHNLFVIEDCAQSHGSVWNNQRAGTIGDIGAFSCQQGKVLTAGEGGIIVTKNPRLFELIQQLRADSRVYCDDSSELMHGDMQLVKKGDIQGSNYCLSEFQSAILLDQLQELDDKNAIREKNAMFLNDALSKIDGIKVMKRPPQVSRQTYYGYVFRFDPVKFGGLNADQFCEILREKLNMGTFYLHPPYLPVHKNPLFCPWTKNRYLKSVRKTEAYWRGLHYPVSERASGQSIVIHHAILLAEPSHLSLLVDAVAELARKFCVTH

[0025] The DOIm-producing bacterium of the present disclosure may further have a gene (cscA) encoding sucrose hydrolase (CscA). The sucrose hydrolase in the present disclosure refers to a general term for enzymes that catalyze the reaction of generating glucose and fructose from sucrose. Note that this enzyme is not originally possessed by Escherichia coli such as K-12 strain and B strain.

[0026] The DOIm-producing bacterium having a gene encoding sucrose hydrolase has the activity of hydrolyzing sucrose. When sucrose is decomposed by sucrose hydrolase, glucose and fructose are generated. That is, the DOIm-producing bacterium having a gene encoding sucrose hydrolase can produce DOIm not only in the presence of glucose and fructose but also in the presence of sucrose. Sucrose is, for example, available at a lower cost than glucose and fructose as the main component of molasses. Therefore, the DOIm-producing bacterium that can use sucrose as a raw material has great industrial utility value.

[0027] The gene for sucrose hydrolase belongs to the sucrose non-PTS gene group. In the present disclosure, the "sucrose non-PTS gene group" refers to a group of genes involved in the non-PTS system among the sucrose assimilation pathways of microorganisms. Specifically, it is a gene group composed of the gene (cscR) encoding the repressor protein (CscR), the gene (cscA) encoding sucrose hydrolase (CscA), the gene (cscK) encoding fructokinase (CscK), and the gene (cscB) encoding sucrose permease (CscB). In the present disclosure, the gene for sucrose hydrolase preferably has a combination containing at least cscA among the above genes. For example, only cscA, a combination of cscA and cscK, a combination of cscA and cscB, a combination of cscA and cscR, a combination of cscA, cscB, and cscR, a combination of cscA, cscK, and cscR, a combination of cscA, cscK, and cscB, and a combination of cscA, cscK, cscB, and cscR can be mentioned.

[0028] From the perspective of efficiently producing DOIm, the DOIm-producing bacterium of the present disclosure preferably has only cscA among the genes included in the sucrose non-PTS gene group and does not contain other genes.

[0029] The DOIm-producing bacterium has the activity of sucrose hydrolase imparted by introducing at least cscA (preferably only cscA) among the sucrose non-PTS gene group, and thus decomposes sucrose existing outside the cell into glucose and fructose on the cell membrane and releases them extracellularly. The released glucose and fructose are taken into the cytoplasm while being phosphorylated via the glucose PTS and fructose PTS originally possessed by Escherichia coli. As a result, fructose existing outside the cell appears in the cell as fructose-1-phosphate, and then is converted into fructose-1,6-bisphosphate by fructose-1-phosphate kinase (FruK) existing in the cell and enters the glycolysis pathway.

[0030] As the gene of the sucrose hydrolase to be introduced into the DOIm-producing bacterium, DNA having the nucleotide sequence of the gene encoding the sucrose hydrolase obtained from an organism possessing this enzyme, or a synthetic DNA sequence synthesized based on its known nucleotide sequence can be used. Preferred examples include those derived from bacteria of the genus Erwinia, Proteus, Vibrio, Agrobacterium, Rhizobium, Staphylococcus, Bifidobacterium, and Escherichia. Among these, DNA having the nucleotide sequence of the gene derived from Escherichia coli O-157 strain is preferred. It is preferable that the gene of the sucrose hydrolase has a signal sequence added thereto for translocating the sucrose hydrolase into the periplasm of the bacterial cell.

[0031] As the gene of the repressor protein that can be introduced into the DOIm-producing bacterium, DNA having the nucleotide sequence of the gene encoding the repressor protein obtained from an organism possessing this enzyme, or a synthetic DNA sequence synthesized based on its known nucleotide sequence can be used. Preferred examples include those derived from bacteria of the genus Erwinia, Proteus, Vibrio, Agrobacterium, Rhizobium, Staphylococcus, Bifidobacterium, and Escherichia. Among these, DNA having the nucleotide sequence of the gene derived from Escherichia coli O-157 strain is preferred.

[0032] As the gene of the fructokinase that can be introduced into the DOIm-producing bacterium, DNA having the nucleotide sequence of the gene encoding the fructokinase obtained from an organism possessing this enzyme, or a synthetic DNA sequence synthesized based on its known nucleotide sequence can be used. Preferred examples include those derived from bacteria of the genus Erwinia, Proteus, Vibrio, Agrobacterium, Rhizobium, Staphylococcus, Bifidobacterium, and Escherichia. Among these, DNA having the nucleotide sequence of the gene derived from Escherichia coli O-157 strain is preferred.

[0033] As the gene of sucrose permease that can be introduced into a DOI-producing bacterium, DNA having the nucleotide sequence of a gene encoding sucrose permease obtained from an organism possessing this enzyme, or a synthetic DNA sequence synthesized based on its known nucleotide sequence can be used. Preferred examples include those derived from bacteria of the genus Erwinia, Proteus, Vibrio, Agrobacterium, Rhizobium, Staphylococcus, Bifidobacterium, and Escherichia. Among these, it is DNA having the nucleotide sequence of a gene derived from Escherichia coli O-157 strain.

[0034] The sucrose hydrolase may be a polypeptide having the amino acid sequence shown in the following (e) or (f). (e) The amino acid sequence shown in SEQ ID NO: 3 (f) An amino acid sequence having activity as a sucrose hydrolase and having a sequence identity of 80% or more with SEQ ID NO: 3

[0035] The amino acid sequence of SEQ ID NO: 3 shown in (e) is the amino acid sequence corresponding to the nucleotide sequence described in positions 3274383 - 3275816 of the Escherichia coli O-157 strain genomic sequence described in GenBank accession number AE005174. The amino acid sequence shown in (f) may have a sequence identity of 85% or more, 90% or more, 95% or more, or 99% or more with SEQ ID NO: 3.

[0036] (SEQ ID NO: 3) MTQSRLHAAQNALAKLHERRGNTFYPHFHLAPPAGWMNDPNGLIWFNDRYHAFYQHHPMSEHWGPMHWGHATSDDMIHWQHEPIALAPGDENDKDGCFSGSAVDDNGVLSLIYTGHVWLDGAGNDDAIREVQCLATSRDGIHFEKQGVILTPPEGIMHFRDPKVWREADTWWMVVGAKDPGNTGQILLYRGSSLREWTFDRVLAHADAGESYMWECPDFFSLGDQHYLMFSPQGMNAEGYSYRNRFQSGVIPGMWSPGRLFAQSGHFTELDNGHDFYAPQSFVAKDGRRIVIGWMDMWESPMPSKREGWAGCMTLARELSESNGKLLQRPVHEAESLRQQHQSISPRTISNKYVLQENAQAVEIQLQWALKNSDAEHYGLQLGTGMRLYIDNQSERLVLWRYYPHENLDGYRSIPLPQRDTLALRIFIDTSSVEVFINDGEAVMSSRIYPQPEERELSLYASHGVAVVQHGALWQLG

[0037] The DOIm-producing bacterium of the present disclosure may have a sugar uptake ability enhancement system. The sugar uptake ability in the present disclosure means the sugar transport ability through the biological membrane, and this ability can include the action on either the sugar transport from the outside to the inside of the biological membrane or the sugar transport from the inside to the outside of the biological membrane. The sugars in sugar transport include pentoses or hexoses. Specifically, glucose, mannose, arabinose, galactose, fructose, etc. can be mentioned, and preferably glucose can be mentioned.

[0038] "Having a sugar uptake ability enhancement system" in the present disclosure refers to a state in which the amount of sugar taken up from outside the cell into the cell has increased. In the present invention, the "sugar uptake ability enhancement system" preferably means a mechanism for improving the glucose uptake ability. The glucose uptake ability enhancing system may be a system that takes up extracellular glucose in its original form into the cell, different from the PTS system or non-PTS system originally possessed by the cells. The glucose taken up into the cell becomes glucose-6-phosphate by a phosphorylation enzyme such as glucokinase (Glk) possessed by the cells and can be utilized as a substrate for the DOI synthase.

[0039] From the viewpoint of the production efficiency of DOIm, the glucose uptake ability enhancing system is preferably a system for enhancing the activity of glucose transport promoting protein. The glucose transport promoting protein in the present invention refers to a general term for proteins having the function of transporting sugars such as glucose and fructose from the outside to the inside of the biological membrane.

[0040] As the gene (glf) of the glucose transport promoting protein (Glf), a DNA having the nucleotide sequence of a gene encoding a glucose transport promoting protein derived from an organism having this protein or a synthetic DNA sequence synthesized based on its known nucleotide sequence can be used. Examples of organisms having the glucose transport promoting protein include yeast and Zymomonas bacteria, preferably Zymomonas bacteria, and more preferably Zymomonas mobilis.

[0041] The glucose transport promoting protein may be a polypeptide having the amino acid sequence shown in the following (g) or (h). (g) The amino acid sequence shown in SEQ ID NO: 4 (h) An amino acid sequence having the activity as a glucose transport promoting protein and having a sequence identity of 80% or more with SEQ ID NO: 4

[0042] The amino acid sequence of SEQ ID NO: 4 shown in (g) is the amino acid sequence of the glucose transport promoting protein possessed by Zymomonas mobilis (ATCC 29191) described in GenBank accession number M60615. The amino acid sequence shown in (h) may have a sequence identity of 85% or more, 90% or more, 95% or more, or 99% or more with SEQ ID NO: 4.

[0043] (SEQ ID NO: 4) MSSESSQGLVTRLALIAAIGGLLFGYDSAVIAAIGTPVDIHFIAPRHLSATAAASLSGMVVVAVLVGCVTGSLLSGWIGIRFGRRGGLLMSSICFVAAGFGAALTEKLFGTGGSALQIFCFFRFLAGLGIGVVSTLTPTYIAEIAPPDKRGQMVSGQQMAIVTGALTGYIFTWLLAHFGSIDWVNASGWCWSPASEGLIGIAFLLLLLTAPDTPHWLVMKGRHSEASKILARLEPQADPNLTIQKIKAGFDKAMDKSSAGLFAFGITVVFAGVSVAAFQQLVGINAVLYYAPQMFQNLGFGADTALLQTISIGVVNFIFTMIASRVVDRFGRKPLLIWGALGMAAMMAVLGCCFWFKVGGVLPLASVLLYIAVFGMSWGPVCWVVLSEMFPSSIKGAAMPIAVTGQWLANILVNFLFKVADGSPALNQTFNHGFSYLVFAALSILGGLIVARFVPETKGRSLDEIEEMWRSQK

[0044] In a preferred embodiment of the DOIm-producing bacterium of the present disclosure, from the perspective of the ability to decompose sucrose, the activity of the sucrose-hydrolyzing enzyme is derived from Escherichia bacteria, and from the perspective of improving the productivity of DOI, the activity of the glucose transport-promoting protein is derived from Zymomonas bacteria. In a more preferred embodiment of the DOIm-producing bacterium of the present disclosure, the activity of the sucrose-hydrolyzing enzyme is derived from Escherichia coli O-157 bacteria, and the activity of the glucose transport-promoting protein is derived from Zymomonas mobilis bacteria.

[0045] In the DOIm-producing bacterium of the present disclosure, from the perspective of the production efficiency of DOIm, it is preferable that the activity of the enzyme involved in the metabolism of glucose-6-phosphate possessed by the host bacterium is in an inactivated or reduced state. By inactivating or reducing the activity of enzymes involved in the metabolism of glucose-6-phosphate, the consumption of glucose-6-phosphate as an energy source by the bacterial cells is suppressed, and the utilization efficiency as a substrate for DOI synthase is improved.

[0046] Enzymes involved in the metabolism of glucose-6-phosphate are selected from the group consisting of, for example, phosphoglucose isomerase (Pgi), glucose-6-phosphate-1-dehydrogenase (Zwf), and phosphoglucomutase (Pgm), and ribosome modification factor (Rmf) involved in the control of protein synthesis in the stationary phase. Examples of the mode in which the activity of the above enzymes is inactivated or reduced include a mode in which any one, two, or three of the genes encoding the above enzymes are simultaneously gene-disrupted; a mode in which the gene encoding the RMF protein involved in the control of protein synthesis in the stationary phase is singly gene-disrupted; and a mode in which any one, two, or three of the genes encoding the above enzymes are disrupted and the gene encoding the RMF protein is disrupted. Such modes are described, for example, in WO 2006 / 109479 pamphlet.

[0047] A preferred mode of the DOI-producing bacterium of the present disclosure is a state in which the activities of phosphoglucose isomerase and glucose-6-phosphate-1-dehydrogenase are simultaneously inactivated or reduced. A more preferred mode of the DOI-producing bacterium of the present disclosure is a state in which the activities of phosphoglucose isomerase, glucose-6-phosphate-1-dehydrogenase, and phosphoglucomutase are simultaneously inactivated or reduced.

[0048] In the present disclosure, "inactivation" refers to a state in which the activity of the target enzyme measured by an existing measurement system is below the detection limit. In the present disclosure, "reduction" refers to a state in which the activity of the target enzyme is significantly decreased compared to the state before those treatments by genetic recombination of the gene encoding the target enzyme. The activity of the enzyme in the present disclosure may be the activity measured by any of the existing measurement systems.

[0049] The means for inactivating the enzyme involved in the metabolism of glucose-6-phosphate can be used without particular limitation as long as it is a means commonly used for this purpose. For example, it may be gene disruption by homologous recombination of the gene encoding the enzyme. The method of gene disruption is not particularly limited, and known techniques can be adopted without particular limitation.

[0050] The bacterium used for producing the DOIm-producing bacterium is not particularly limited as long as the introduction and modification of each of the above genes are possible. The bacterium is preferably a type that does not originally have the ability to assimilate sucrose. Examples of such bacteria include bacteria of the genus Escherichia, and Escherichia coli is preferably used. That is, the DOIm-producing bacterium may be Escherichia coli.

[0051] In the present disclosure, the "ability to assimilate sucrose" refers to the ability to take in sucrose as it is, either by reducing its molecular weight or increasing its molecular weight (preferably reducing its molecular weight), into the living body, or the ability to metabolically convert it into another substance. In the present disclosure, the assimilation of sucrose includes decomposing sucrose into a more low-molecular compound. Specifically, it includes decomposing sucrose into glucose and fructose.

[0052] Examples of Escherichia coli used for producing the DOIm-producing bacterium include the K-12 strain, B strain, C strain, and strains derived therefrom. As Escherichia coli, among the types of Escherichia coli that do not originally have the ability to assimilate sucrose, the K-12 strain, B strain, or strains derived therefrom are preferably used.

[0053] <Method for producing DOIm> The method for producing DOIm of the present disclosure is a step of contacting a substance selected from a combination of glucose and fructose, or sucrose, with the DOIm-producing bacterium of the present disclosure described above (hereinafter, also referred to as the contacting step).

[0054] According to the method of the present disclosure, DOIm can be obtained from a combination of glucose and fructose, or sucrose, in a single step. Since the method of the present disclosure can use inexpensive substances as raw materials compared to the method of producing DOIm using DOI as a raw material, it is excellent in economy.

[0055] Surprisingly, when DOIm is synthesized by the method of the present disclosure, the amount of components with unknown structures produced is suppressed compared to the case of synthesizing DOIm using DOIm synthase. The accumulation of by-products can be a factor causing a decrease in the reaction yield and purification yield of DOIm. Therefore, the method of the present disclosure is useful as a means for producing DOIm while suppressing the production of by-products.

[0056] The substance to be contacted with the DOIm-producing bacterium in the contacting step may be only a combination of glucose and fructose, only sucrose, or a combination of sucrose, glucose, and fructose. From the viewpoint of economy, the substance to be contacted with the DOIm-producing bacterium is preferably sucrose. When the DOIm-producing bacterium is contacted with sucrose, the DOIm-producing bacterium preferably has a gene encoding sucrose hydrolase.

[0057] The glucose, fructose, or sucrose used in the method of the present disclosure may be in a state contained in a plant-derived raw material. The plant-derived raw material is a carbon source obtained from a plant and is not particularly limited as long as it can be converted into DOIm by a DOIm-producing bacterium. In the present disclosure, it refers to organs such as roots, stems, trunks, branches, leaves, flowers, seeds, etc., plants containing them, decomposition products of those plant organs, and further, among the carbon sources obtained from plants, plant organs, or their decomposition products, those that microorganisms can use as a carbon source in culture are also included in the plant-derived raw materials.

[0058] As carbon sources included in plant-derived raw materials, in addition to glucose, fructose, and sucrose, saccharides such as starch, xylose, arabinose, etc., lignocellulose decomposition products, cellulose hydrolysates, components derived from vegetable oils (glycerol, fatty acids, etc.) can be mentioned.

[0059] As plant-derived raw materials, agricultural crops such as grains, corn, rice, wheat, soybeans, sugarcane, beets, cotton, etc., or combinations thereof can be preferably mentioned, and the form of use as the raw material is not particularly limited, such as unprocessed products, squeezed juices, pulverized products, etc. Also, it may be in the form of only the above carbon source. As plant-derived raw materials, corn steep liquor (CSL) generated as a by-product when manufacturing corn starch may be used.

[0060] The contact between the DOIm-producing bacteria and the plant-derived raw material in the contact step is generally carried out by culturing the DOIm-producing bacteria in a medium containing the plant-derived raw material.

[0061] The contact density between the plant-derived raw material and the DOIm-producing bacteria varies depending on the activity of the DOIm-producing bacteria, but generally, as the concentration of the plant-derived raw material in the medium, the initial sugar concentration in terms of glucose can be 20% by mass or less based on the total mass of the mixture. From the perspective of the sugar tolerance of the DOIm-producing bacteria, it is preferable that the initial sugar concentration is 15% by mass or less. The other components may be added in the amounts usually added to a microbial medium and are not particularly limited.

[0062] The content of the DOIm-producing bacteria in the medium varies depending on the type and activity of the DOIm-producing bacteria, but generally, the initial bacterial concentration can be 0.1% by mass to 30% by mass based on the culture solution, and preferably 1% by mass to 10% by mass from the perspective of controlling the culture conditions.

[0063] From the perspective of enhancing the production efficiency of DOIm, the method of the present disclosure may be carried out in the presence of ammonia. Specifically, it may be carried out in a state where the medium used for culturing the DOIm-producing bacteria contains ammonia. When the medium contains ammonia, the DOIm-producing bacterium synthesizes L-glutamine from ammonia, and the synthesized L-glutamine is used as a source for amino group transfer to DOI. As a result, the production efficiency of DOIm is improved.

[0064] From the viewpoint of optimizing the production efficiency of DOIm, the concentration of ammonia in the medium is preferably in the range of 0.1% by mass to 0.5% by mass. The ammonia supplied to the medium may be in the form of an organic ammonium salt, an inorganic ammonium salt, ammonia gas, aqueous ammonia, or the like.

[0065] The method of adding ammonia to the medium to increase the production efficiency of DOIm is advantageous from the viewpoint of economy, for example, as compared with the method of adding L-glutamine to the medium. The method of using L-glutamine synthesized from ammonia by the action of the DOIm-producing bacterium as a source for amino group transfer to DOI is also advantageous from the viewpoint of optimizing the concentration of L-glutamine in the medium as compared with the method of adding L-glutamine to the medium from the outside. When the concentration of L-glutamine in the medium is optimized, for example, the generation of impurities derived from L-glutamine that is not consumed in the production of DOIm by the DOIm-producing bacterium is suppressed.

[0066] In the method of the present disclosure, L-glutamine synthesized by the DOIm-producing bacterium in the presence of ammonia can be used for the synthesis of DOIm. Therefore, the medium may not contain L-glutamine, but the medium may contain L-glutamine to improve the production efficiency of DOIm. If necessary, the medium may contain known additives. The medium used in the method of the present disclosure is preferably a liquid medium in consideration of industrial production.

[0067] The culture conditions in the contact step vary depending on the state of the DOIm-producing bacterium and the culture apparatus, but generally, the culture temperature can be in the range of 20°C to 40°C, preferably 25°C to 35°C. The pH of the medium can be in the range of 4.0 to 9.0, preferably 5.0 to 7.5, more preferably 6.0 to 7.0. The method for producing DOIm using a DOIm-producing bacterium can be preferably carried out near neutrality, which is more suitable for culturing Escherichia coli, as compared with the method for generating DOI using a DOI-producing bacterium or the like. This is because DOIm has better stability than DOI.

[0068] During culturing, it is generally common to use a culture tank capable of controlling temperature, pH, aeration conditions, and stirring speed, but the present disclosure is not limited to using a culture tank. When culturing using a culture tank, if necessary, a seed culture may be performed as a pre-culture in advance, and this may be inoculated into a medium in a culture tank prepared in advance in a required amount.

[0069] When culturing a DOIm-producing bacterium to produce DOIm, it is not necessary to perform aeration at all, but it is better to perform aeration in order to obtain more favorable results. The aeration conditions mentioned here do not necessarily require air to pass through the culture solution. Depending on the shape of the culture tank, surface aeration in which the air layer above the culture solution is ventilated while moderately stirring the culture solution is also included, which means flowing a gas containing oxygen into the inside of the culture tank.

[0070] The method for recovering DOIm obtained in the contacting step is not particularly limited and can be carried out by known methods. The recovered DOIm may be, for example, in a liquid (such as an aqueous solution) state or in a solid (such as powder, tablet, etc.) state.

Examples

[0071] Hereinafter, embodiments according to the present disclosure will be described based on examples, but the present disclosure is not limited thereto. Also, unless otherwise specified, “%” in the examples is based on mass.

[0072] <Preparation of DOIm-producing bacterium> (1) Preparation of DOI-producing bacterium An Escherichia coli strain as a DOI-producing bacterium was prepared according to the method described in International Publication No. 2010 / 053052. The prepared Escherichia coli strain is an MG1655 strain (MG1655ΔpgiΔzwf / pGAP-btsC-cscA-glf) in which the phosphoglucose isomerase gene (pgi) and glucose-6-phosphate-1-dehydrogenase (zwf) are deleted, and the genes of sucrose hydrolase (cscA), glucose transport promoting protein (glf), and DOI synthase (btrC) are introduced respectively.

[0073] In the above Escherichia coli strain, the DOI synthase is a polypeptide having the amino acid sequence of SEQ ID NO: 1, the sucrose hydrolase is a polypeptide having the amino acid sequence of SEQ ID NO: 3, and the glucose transport promoting protein is a polypeptide having the amino acid sequence of SEQ ID NO: 4.

[0074] (2) Introduction of the DOIm synthase gene into an ampicillin-resistant vector The gene of the artificially synthesized DOIm synthase (SEQ ID NO: 2) (Thermo Fisher Scientific Inc.) and the plasmid pGAP described in International Publication No. 2010 / 053052 were each treated with restriction enzymes BamHI and XbaI (New England Biolabs Japan). After mixing both fragments and ligating them using ligase (Toyobo), they were transformed into Escherichia coli DH5α competent cells (Toyobo), and transformants growing on an LB agar plate containing 50 μg / mL of ampicillin were obtained. The obtained colonies were cultured overnight at 30 °C in an LB liquid medium containing 50 μg / mL of ampicillin, and the plasmid pGAP-btrR was recovered from the obtained cells to construct an ampicillin-resistant DOIm synthase gene expression vector.

[0075] (3) Construction of a cloning vector for chloramphenicol-resistant Escherichia coli pMW119 (Nippon Gene) was treated with restriction enzymes AaII and NdeI (New England Biolabs Japan), and a fragment containing each region of par, ori, and repA was excised. The excised 5'-end and 3'-ends were blunted using DNA polymerase (Toyobo). The region from the promoter to the terminator of plasmid pGAP described in International Publication No. 2010 / 053052 was amplified by PCR. Subsequently, the par-ori-repA region and the PCR product fragment were ligated, and this plasmid was designated as pMWG48. To obtain the chloramphenicol resistance gene, oligonucleotide primers having the nucleotide sequences of TCGGCACGTAAGAGGTTCC (SEQ ID NO: 5) and CGGGTCGAATTTGCTTTCG (SEQ ID NO: 6) were synthesized respectively (Thermo Fisher Scientific Inc.), and PCR was performed using synthetic pTH18cs1 (GenBank: AB019610.2, Thermo Fisher Scientific Inc.) and the above two primers to amplify the fragment. The obtained fragment was used as the chloramphenicol resistance gene fragment. Oligonucleotide primers having the nucleotide sequences of CTAGATCTGACAGTAAGACGGGTAAGCC (SEQ ID NO: 7) and CTAGATCTCAGGGTTATTGTCTCATGAGC (SEQ ID NO: 8) were synthesized respectively (Thermo Fisher Scientific Inc.), and PCR was performed using pMWG48 and the above two primers to amplify a sequence of approximately 2.5 kbp containing each region of par, repA, gapA promoter, and rrn terminator of pMWG48. The chloramphenicol resistance gene fragment and the region of approximately 2.5 kbp were ligated, and this plasmid was designated as the vector pMWG48kc for chloramphenicol-resistant Escherichia coli.

[0076] (4) Construction of a vector expressing the DOIm synthase gene The artificially synthesized DOIm synthase gene (Thermo Fisher Scientific Inc.) and that of pMWG48kc were each treated with restriction enzymes EcoRI and HindIII (New England Biolabs Japan). After mixing both fragments and ligating them using ligase (Toyobo), they were transformed into Escherichia coli DH5α competent cells (Toyobo), and transformants that grew on an LB agar plate containing 34 μg / mL of chloramphenicol were obtained. The obtained colonies were cultured overnight at 30 °C in an LB liquid medium containing 34 μg / mL of chloramphenicol. The plasmid pMWG48kc-btrR was recovered from the obtained bacterial cells to construct a chloramphenicol-resistant DOIm synthase gene expression vector.

[0077] (5) Construction of DOIm-producing bacteria Using the chloramphenicol-resistant DOIm synthase gene expression vector (pMWG48kc-btrR), transformation of an Escherichia coli strain prepared according to the method described in International Publication No. 2010 / 053052 was performed. By culturing overnight at 37 °C on an LB agar plate containing 50 μg / mL of ampicillin and 34 μg / mL of chloramphenicol, a DOIm-producing bacterium was obtained.

[0078] <Method for analyzing DOIm> In the tests described below, quantitative analysis of DOIm was carried out by the following method. The enzyme reaction solution or fermentation broth was diluted with ion-exchanged water and centrifuged at 15,000 rpm for 5 minutes. The obtained supernatant was subjected to an HPLC system equipped with a column RSpack NN-814 (Shodex) connected to a guard column RSpack NN-G (Shodex). The mobile phase (13 mM sodium dihydrogen phosphate, pH 3.0) was separated at a flow rate of 0.8 mL / min. After separation by the column, derivatization was performed with an orthophthalaldehyde (OPA) solution (0.6 mM OPA, 1.3 mM N-acetyl-L-cysteine, 40 mM boric acid, 0.06% aqueous Briji solution, 1% EtOH, 2% KOH) by the post-column method, and DOIm was quantified using a fluorescence detector (excitation: 340 nm, emission: 455 nm). Analysis was performed using an HPLC system equipped with LC-NetII / ADC, autosampler AS-2055 Plus, pump PU-2080 Plus, column oven CO-2060 Plus, and fluorescence detector FP2025 Plus. According to the above analysis method, compounds having an amino group such as DOIm can be quantified.

[0079] <Comparative Example 1: Production of DOIm Using Enzyme> Under the conditions using DOI and L-glutamine at concentrations assumed for industrial production, the synthesis of DOIm using DOIm synthase was carried out. To obtain the enzyme solution of DOIm, an Escherichia coli clone transformed with the ampicillin-resistant DOIm synthase gene pGAP-btrR was inoculated into 100 mL of LB liquid medium containing 50 μg / mL of ampicillin and cultured with shaking overnight at 30 °C. The cultured bacterial solution was centrifuged at 8,000 rpm for 20 minutes in 50 mL portions, and the cells were collected. The collected cells from 50 mL of the culture solution were suspended in 5 mL of ion-exchanged water. This suspension was used as the DOIm enzyme solution. After adjusting the pH of the substrate solution containing DOI (3.3%) and L-glutamine (3.3%) to 7.0 with 2N NaOH, the DOIm enzyme solution (6 mL) was added to prepare a mixed solution, and an enzyme reaction was carried out at 30 °C to obtain an enzyme reaction solution. 48 hours after the start of the reaction, the amount of DOIm produced in the enzyme reaction mixture was quantified using the above analytical method. The results are shown in Figure 1. Additionally, the results of quantifying an L-glutamine standard using the above analytical method are shown in Figure 2. As shown in Figure 1, a peak derived from DOIm was observed in the enzyme reaction solution, confirming that the DOIm synthetic enzyme produces DOIm in the presence of DOI and L-glutamine. Furthermore, a peak derived from a compound of unknown structure was observed at a position different from the peak derived from L-glutamine (see Figure 2), which was of similar intensity to the DOIm peak. It is presumed that the compound of unknown structure corresponding to this peak was produced by a chemical reaction between components present in the enzyme reaction solution. The concentration of DOIm in the enzyme reaction solution was 42.8 mM, and the yield of DOIm calculated from the concentration of DOI contained in the enzyme solution before the start of the reaction (204.5 mM) was 20.9%. These results suggest that increasing the concentrations of DOI and L-glutamine in the enzymatic synthesis of DOIm results in the production of compounds with unknown structures, limiting the yield of DOIm.

[0080] <Example 1: Production of DOIm using DOIm-producing bacteria> DOIm was produced using DOIm-producing bacteria under conditions that would not result in high concentrations of L-glutamine through fermentation. As a preculture, the DOIm-producing bacteria obtained by the above method was inoculated into 20 mL of LB liquid medium containing 50 μg / mL ampicillin and 34 μg / mL chloramphenicol in an Erlenmeyer flask, and cultured overnight with shaking at 30°C and 120 rpm to obtain a culture solution. CSL (0.05%), fructose (1.7%) and glucose (2.6%) were added to M9 medium (1x) to prepare a culture medium. The culture solution (1 mL) of the DOIm-producing bacteria was added to an Erlenmeyer flask containing this medium (21 mL), and the culture was carried out under conditions of 30°C and 250 rpm. The components in the fermentation broth obtained 48 hours after the start of cultivation were quantified by the above analysis method. The results are shown in Fig. 3. As shown in Fig. 3, it was confirmed that DOIm was produced without the peak derived from the compound with unknown structure detected in Comparative Example 1 being confirmed. Also, the production amount of DOIm was 9 g / L.

[0081] Cultivation was carried out under the same conditions except that the DOI-producing bacterium used for the preparation of the DOIm-producing bacterium was used instead of the DOIm-producing bacterium, and the components in the fermentation broth obtained 48 hours after the start of cultivation were quantified by the above analysis method. The results are shown in Fig. 4. As shown in Fig. 4, production of DOIm was not confirmed when the DOI-producing bacterium was cultured. From the above results, it was confirmed that the gene of the DOIm synthase introduced into the DOIm-producing bacterium is involved in the production of DOIm.

[0082] In the test using the DOIm-producing bacterium, it is considered that L-glutamine and other nitrogen sources contained in CSL were utilized for the production of DOIm along with the metabolism of the DOIm-producing bacterium. In the test using the DOI-producing bacterium, it is considered that L-glutamine and other nitrogen sources contained in CSL were consumed in the metabolism of the DOI-producing bacterium without being utilized for the synthesis of DOIm.

[0083] <Reference experiment: L-glutamine accumulation culture with wild-type Escherichia coli> Since DOIm is produced from DOI and L-glutamine, it is considered that increasing the amount of L-glutamine in the fermentation broth to such an extent that components with unknown structures are not produced leads to an improvement in DOIm productivity. Escherichia coli is known to accumulate nitrogen as L-glutamine under culture conditions with an excess of nitrogen source. Therefore, it is considered that adding ammonia as a nitrogen source to the medium leads to an increase in the L-glutamine accumulation amount and thus an improvement in DOIm productivity. Therefore, a comparison of the L-glutamine accumulation amounts of wild-type Escherichia coli was made between the case where the medium contains ammonia and the case where it does not. As the medium, a mixture obtained by adding an aqueous solution of 1 M magnesium chloride (0.1 g) and an aqueous solution of 45% glucose (1 g) to 20 g of M9 medium (1x) was used. As the ammonia source, an aqueous solution (pH 7.0) containing 10% ammonium chloride and 0.1 M ammonium phosphate was used. Using the medium (23.1 g) to which the ammonia source (2 g) was added and the medium (21.1 g) to which no ammonia source was added, the wild-type Escherichia coli was cultured under the conditions of 30 °C and 250 rpm. The amount of L-glutamine accumulated when the ammonia source was added to the medium 48 hours after the start of the culture was 0.64 g / L, and the amount of L-glutamine accumulated when no ammonia source was added to the medium was 0.07 g / L. From the above results, it was confirmed that the L-glutamine accumulation amount of wild-type Escherichia coli increased when the medium contained ammonia.

[0084] <Example 2: Influence of Ammonia Addition on DOIm Productivity> Based on the results of the reference experiment, it was examined by the following test whether the productivity of DOIm would be improved by including ammonia in the medium used for culturing the DOIm-producing bacterium. The DOIm-producing bacterium was inoculated into 2 mL of LB liquid medium containing 50 μg / mL of ampicillin and 34 μg / mL of chloramphenicol in a 15 mL tube, and cultured with shaking overnight at 30 °C and 120 rpm to obtain a preculture solution. 1 mL of the preculture solution was added to an Erlenmeyer flask containing the medium (20 mL) to which the ammonia source was added, and the culture was carried out under the conditions of 30 °C and 250 rpm. As the medium, a medium prepared by adding 1 mL of 50% sucrose aqueous solution, 4 mL of 1 M potassium phosphate buffer, and sterilized water (in an amount such that the total amount of the medium was 20 mL) to 10 mL of YT medium (2x) was used. The ammonia concentration of the medium was adjusted by adding a 10% aqueous solution of ammonium chloride to the medium.

[0085] The amount of DOIm produced in the fermentation broth obtained 48 hours after the start of cultivation was quantified by the above analysis method. The results are shown in Table 1. As shown in Table 1, when the medium contains ammonia, the amount of DOIm produced increases compared to the case where the medium does not contain ammonia, and it is suggested that the ammonia concentration in the medium is preferably in the range of 0.1% to 0.5% by mass from the viewpoint of the production efficiency of DOIm. The reason why the amount of DOIm produced shown in Table 1 is smaller than that in Example 1 is that the medium used in Example 1 contains more nutrients. In this example, ammonium chloride was used as the ammonia source, but other ammonia sources may be used.

[0086]

Table 1

[0087] <Example 3: Extraction and Purification of DOIm> The extraction and purification of DOIm were carried out using the fermentation broth containing DOIm obtained in Example 1 and Example 2. 10% sulfuric acid was added to the fermentation broth, the pH was adjusted to around 3.0, and it was allowed to stand overnight to perform a dead bacteria treatment by acidification. The fermented broth after the dead bacteria treatment was centrifuged at 8,000 rpm for 20 minutes. The supernatant obtained by centrifugation was subjected to an ion exchange resin (DIAION TM UBK530K, Mitsubishi Chemical) and eluted with ion-exchanged water. The elution fraction was analyzed by the above analysis method, and the DOIm elution fraction was recovered. The DOIm elution fraction was concentrated under reduced pressure at 30 hPa to 40 hPa and 40 °C using a rotary evaporator (NE-100RG, Tokyo Rika Kikai). DOIm was crystallized by adding methanol to the concentrated solution, and a DOIm powder was obtained by filtration. Crystallization by adding methanol to the DOIm powder was further performed twice to increase the purity of DOIm. As shown in the above results, the DOIm produced using the DOIm-producing bacterium of the present disclosure could be recovered without technical problems. In this example, crystallization was carried out using methanol, but other organic solvents such as ethanol and isopropanol may also be used.

Claims

1. A DOI-producing bacterium having a gene encoding a DOI synthase involved in the synthesis of 2-deoxy-siro-inose and a gene encoding a DOI synthase involved in the synthesis of 2-deoxy-siro-inosamine.

2. The DOI-producing bacterium according to claim 1, wherein the DOI synthase is an enzyme that catalyzes a reaction to produce DOI from glucose-6-phosphate.

3. The DOI-producing bacterium according to claim 1, wherein the DOI synthase is an enzyme that catalyzes a reaction in which the amino group of L-glutamine is transferred to DOI.

4. The DOI-producing bacterium according to claim 1, wherein the DOI synthase is a polypeptide having an amino acid sequence shown in the following (a) or (b). (a) The amino acid sequence shown in SEQ ID NO: 1 (b) An amino acid sequence having an activity as a DOI synthase and having a sequence identity of 80% or more with SEQ ID NO: 1

5. The DOI-producing bacterium according to claim 1, wherein the DOI synthase is a polypeptide having an amino acid sequence shown in the following (c) or (d). (c) The amino acid sequence shown in SEQ ID NO: 2 (d) An amino acid sequence having an activity as a DOI synthase and having a sequence identity of 80% or more with SEQ ID NO: 2

6. The DOI-producing bacterium according to claim 1, further having a gene encoding a sucrose hydrolase.

7. The DOI-producing bacterium according to claim 1, further having a gene encoding a glucose transport promoting protein.

8. The DOI-producing bacterium according to claim 1, wherein the activity of an enzyme involved in the metabolism of glucose-6-phosphate is inactivated or reduced.

9. The DOI-producing bacterium according to claim 1, which is Escherichia coli.

10. A method for producing DOI m, comprising a step of contacting a substance selected from a combination of glucose and fructose, or sucrose, with the DOI m-producing bacterium according to any one of claims 1 to 9.

11. The method for producing DOI m according to claim 10, wherein the contact is carried out in the presence of ammonia.

12. The method for producing DOI m according to claim 11, wherein the contact is carried out in a medium having an ammonia concentration of 0.1% by mass to 0.5% by mass.

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