RU2823674C1 - Method for producing heparosan and bacterium of genus escherichia having ability to produce heparosan - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a bacterium from the genus Escherichia, which produces heparosan and has a genetic modification that increases expression of the kpsS gene compared to expression for an unmodified bacterium and does not have a genetic modification increasing expression of the kpsC gene compared to expression for an unmodified bacterium. Also disclosed is a method of producing heparosan using said bacteria.

EFFECT: invention provides effective production of heparosan.

11 cl, 3 dwg, 3 tbl, 6 ex

Description

Field of technology

[0001] The present invention relates to a method for producing N-acetylheparosan (hereinafter referred to as heparosan in the present description), which is a capsular polysaccharide of a bacterium of the genus Escherichia , by enzymatic production using this bacterium.

State of the art

[0002] Heparin, which is a sulfated polysaccharide, is an anticoagulant agent and is used to treat thromboembolism and disseminated intravascular coagulation syndrome, and to prevent clotting during artificial dialysis and extracorporeal circulation, etc. In industry, most of the heparin used is isolated and purified from the intestinal mucosa of pigs.

[0003] Since a fatal case occurred in 2008 due to contamination of porcine-derived heparin with impurities, research and development of non-animal-derived heparin with production control and quality control were required. As a specific example, a method in which enzymatically produced and purified N-acetylheparosan, which is a capsular polysaccharide from Gram-negative microorganisms, is chemically N-deacetylated and N-sulfated, followed by enzymatic epimerization and sulfation to obtain heparin having the same structure and anticoagulant activity as those derived from porcine (NPL 1 and 2).

[0004] In the above method, heparosan, as a basic structure, is a sugar chain consisting of a repeating disaccharide structure of glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc). For producing heparosan, a method of using Escherichia coli strain K5 originally having the ability to produce heparosan (PTL 1 and NPL 1), a method of using Escherichia coli strain Nissle 1917 also having the ability to produce heparosan (NPL 2), and a method of using Escherichia coli originally not having the ability to produce heparosan (PTL 4 and NPL 3 and 4) have been published.

[0005] Heparosan is classified as a group 2 capsular polysaccharide, and gene groups from region I, region II, and region III, which form a cluster in the genome, are known to be involved in the synthesis and transport of heparosan in Escherichia coli strain K5 (NPL 4).

[0006] Among the proteins encoded by these gene groups, KpsS and KpsC are said to mediate the transfer of multiple 3-deoxy-D-mannooctulosic acid (Kdo) residues to phosphatidylglycerol on the inner membrane, and the glycosyltransferases KfiA and KfiC add the nucleotide sugar precursor, thus continuing the synthesis of heparosan (NPL 5 and 6).

[0007] In addition, KfiD is involved in the synthesis of the UDP-GlcA precursor; KpsF and KpsU are involved in the synthesis of CMP-Kdo, which is a substrate for the synthesis of the Kdo linker; and KpsM, KpsT, KpsE, and KpsD are involved in the transport of heparosan synthesized on the inner membrane to the outer environment of bacterial cells (NPL 6).

[0008] As a method for carrying out heparosan fermentation using the BL21 strain and the K-12 strain of Escherichia coli , which do not have the ability to produce heparosan, as hosts, a method of introducing a KfiABCD gene cluster from region II obtained from the K5 strain of Escherichia coli into the host is known. In addition, genes improving heparosan production, such as rfaH, nusG, and rpoE, (PTL 4, and NPL 3 and 4), have been published.

References

Patent documents

[0009] [PTL 1] Japanese Patent No. 5830464

[PTL 2] US Patent No. 8771995

[PTL 3] WO2018/048973

[PTL 4] US Patent No. 9975928

Non-patent documents

[0010] [NPL 1] Biotechnology and Bioengineering 107 (2010) 964-973

[NPL 2] Applied Microbiology and Biotechnology 103 (2019) 6771-6782

[NPL 3] Metabolic Engineering 14 (2012) 521-527

[NPL 4] Carbohydrate Research 360 (2012) 19-24

[NPL 5] Proceedings of the National Academy of Sciences of USA 110 (2013) 20753-20758

[NPL 6] Carbohydrate Research 378 (2013) 35-44

The essence of the invention

Technical problem

[0011] As described above, research and development of heparin products not derived from animal sources have been conducted with production control and quality control, but the conventional production methods have been insufficient. However, how each of the genes encoded by regions I, II, and III affects the production of heparosan by bacteria of the genus Escherichia having the ability to produce heparosan is still unknown.

[0012] Accordingly, an object of the present invention is to provide a method for efficiently producing heparosan by improving the efficiency of heparosan production by genetically modifying a bacterium of the genus Escherichia having the ability to produce heparosan.

Solution to the problem

[0013] The present inventors found that the efficiency of producing heparosan is improved by using a bacterium of the genus Escherichia which has a specific genetic modification and has the ability to produce heparosan, and thus completed the present invention.

[0014] Thus, the present invention is as follows.

1. A method for producing heparosan, comprising: culturing a bacterium of the genus Escherichia , which has the following genetic modification (1) and has the ability to produce heparosan, in a medium for producing heparosan:

(1) genetic modification that increases the expression of the kpsS gene.

2. The method for producing heparosan according to 1, wherein the bacterium from the genus Escherichia additionally has at least one of the following genetic modifications (2) and (3):

(2) a genetic modification that increases the expression of at least one gene selected from the kfiA gene, the kfiB gene, the kfiC gene and the kfiD gene, and

(3) genetic modification causing loss of function of the yhbJ gene.

3. The method for producing heparosan according to 1 or 2, wherein the genetic modification (1) is at least one of modifying the expression control region of the kpsS gene and increasing the copy number of the kpsS gene.

4. The method for producing heparosan according to 2 or 3, wherein the genetic modification (2) is at least one of modifying the expression control region of at least one gene selected from the kfiA gene, the kfiB gene, the kfiC gene and the kfiD gene, and increasing the copy number of at least one gene selected from the kfiA gene, the kfiB gene, the kfiC gene and the kfiD gene.

5. The method for producing heparosan according to any of 2-4, wherein the genetic modification (3) is a deletion of the yhbJ gene.

6. The method for producing heparosan according to any of 1-5, wherein the bacterium of the genus Escherichia is Escherichia coli .

7. The method for producing heparosan according to any one of 1 to 6, wherein the kpsS gene is a DNA comprising a nucleotide sequence shown in SEQ ID NO: 33, or a DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 33, and having the property of increasing the heparosan-producing ability of a bacterium of the genus Escherichia having the heparosan-producing ability when the expression level is increased in the bacterium.

8. The method for producing heparosan according to any one of 2 to 7, wherein the kfiA gene is a DNA comprising a nucleotide sequence shown in SEQ ID NO: 34 or a DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 34 and having the property of increasing the heparosan-producing ability of a bacterium of the genus Escherichia having the heparosan-producing ability when the expression level is increased in the bacterium, the kfiB gene is a DNA comprising a nucleotide sequence shown in SEQ ID NO: 35 or a DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 35 and having the property of increasing the heparosan-producing ability of a bacterium of the genus Escherichia having the heparosan-producing ability when the expression level is increased in the bacterium, the kfiC gene is a DNA, comprising a nucleotide sequence shown in SEQ ID NO: 36, or a DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 36 and having the property of increasing the heparosan producing ability of a bacterium of the genus Escherichia having the heparosan producing ability when the expression level is increased in the bacterium, and the kfiD gene is a DNA comprising a nucleotide sequence shown in SEQ ID NO: 37, or a DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 37 and having the property of increasing the heparosan producing ability of a bacterium of the genus Escherichia having the heparosan producing ability when the expression level is increased in the bacterium.

9. The method for producing heparosan according to any one of 2 to 8, wherein the yhbJ gene is a DNA comprising the nucleotide sequence shown in SEQ ID NO: 38, or a DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 38, and having the property of increasing the heparosan-producing ability of a bacterium of the genus Escherichia having the heparosan-producing ability when the expression level is reduced in the bacterium.

10. The method for producing heparosan according to any of 1-9, wherein the bacterium of the genus Escherichia does not have the following genetic modification (4):

(4) genetic modification that increases the expression of the kpsC gene.

11. A bacterium of the genus Escherichia that has the ability to produce heparosan and has the following genetic modification (1):

(1) genetic modification that increases the expression of the kpsS gene.

12. A bacterium from the genus Escherichia according to 11, additionally having at least one of the following genetic modifications (2) and (3):

(2) a genetic modification that increases the expression of at least one gene selected from the kfiA gene, the kfiB gene, the kfiC gene and the kfiD gene, and

(3) genetic modification causing loss of function of the yhbJ gene.

13. A bacterium of the genus Escherichia 11 or 12, which does not have the following genetic modification (4):

(4) genetic modification that increases the expression of the kpsC gene.

Advantageous effects of invention

[0015] According to the method for producing heparosan of the present invention, heparosan derived from non-animal sources can be produced with excellent production efficiency by using a bacterium of the genus Escherichia that has a specific genetic modification and has the ability to produce heparosan.

Brief description of the drawings

[0016] [FIG. 1] FIG. 1 shows a schematic diagram of the heparosan synthesis gene cluster on the chromosome of Escherichia coli strain K5.

[Fig. 2] FIG. 2 shows a schematic diagram of the enzymes involved in the production of heparosan.

[Fig. 3] FIG. 3 shows a schematic diagram of the heparosan biosynthetic pathway.

Description of embodiments

[0017] Further in this description, the terms used in this description have the meanings generally used in this field, unless otherwise specified.

[0018] The bacterium of the present invention

In the method for producing heparosan according to the present invention, a bacterium of the genus Escherichia is used, which has the following genetic modification (1) and has the ability to produce heparosan (hereinafter referred to as the bacterium of the present invention):

(1) genetic modification that increases the expression of the kpsS gene.

[0019] The bacterium of the present invention preferably further has at least one of the following genetic modifications (2) and (3):

(2) a genetic modification that increases the expression of at least one gene selected from the kfiA gene, the kfiB gene, the kfiC gene and the kfiD gene, and

(3) a genetic modification causing loss of function of the yhbJ gene. Accordingly, examples of genetic modifications in bacteria of the genus Escherichia include (1) and (2) described above, (1) and (3) described above, and (1) - (3) described above.

[0020] The bacterium of the present invention preferably does not have the following genetic modification (4):

(4) genetic modification that increases the expression of the kpsC gene.

[0021] As an example of a bacterium of the genus Escherichia , FIG. 1 shows a schematic diagram of the heparosan synthesis gene cluster on the chromosome of Escherichia coli strain K5 [J. Nzakizwanayo et al., PLOS ONE, (2015)]. In addition, FIG. 2 shows a schematic diagram of the enzymes involved in heparosan production.

[0022] As shown in FIG. 1, kpsS and kpsC are genes encoded by region I, among a group of genes from region I, region II, and region III. As shown in FIG. 2, kpsS and kpsC are involved in the initiation of heparosan synthesis. In heparosan production, kpsS, together with kpsC, plays a role in adding multiple Kdo linkers to phosphatidylglycerol on the inner membrane.

[0023] As the kpsS gene, the kpsS gene originating from the genus Escherichia is preferred. Specific examples thereof include the kpsS gene of Escherichia coli strain K5. The nucleotide sequence of the kpsS gene of Escherichia coli strain K5 and the amino acid sequence of the protein encoded by the gene can be obtained from public databases. The kpsS gene of Escherichia coli strain K5 is registered as GenBank accession number CAA52659.1.

[0024] Examples of kpsS genes include DNA comprising a nucleotide sequence shown in SEQ ID NO: 33, or DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 33, and having the property of increasing the heparosan-producing ability of a bacterium of the genus Escherichia having the heparosan-producing ability when the expression level is increased in the bacterium.

[0025] As shown in FIG. 1, kfiA, kfiB, kfiC, and kfiD are genes encoded by region II among a group of genes from region I, region II, and region III. As shown in FIG. 2, kfiA, kfiB, kfiC, and kfiD are involved in the synthesis of heparosan, and play a role in adding saccharide and thus synthesizing heparosan.

[0026] As the kfiA, kfiB, kfiC, or kfiD gene, the kfiA, kfiB, kfiC, or kfiD gene originating from the genus Escherichia is preferable. Specific examples thereof include the kfiA, kfiB, kfiC, or kfiD gene from Escherichia coli strain K5. The nucleotide sequence of the kfiA, kfiB, kfiC, or kfiD gene from Escherichia coli strain K5 and the amino acid sequence of the protein encoded by the gene can be obtained from public databases. kfiA is registered as GenBank accession number CAA54711.1; kfiB is registered as GenBank accession number CAE55824.1; kfiC is registered as GenBank accession number CAA54709.1; and kfiD is registered as GenBank accession number CAA54708.1.

[0027] Examples of kfiA genes include DNA comprising a nucleotide sequence shown in SEQ ID NO: 34, or DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 34, and having the property of increasing the heparosan-producing ability of a bacterium of the genus Escherichia having the heparosan-producing ability when the expression level is increased in the bacterium.

[0028] Examples of kfiB genes include DNA comprising a nucleotide sequence shown in SEQ ID NO: 35, or DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 35, and having the property of increasing the heparosan-producing ability of a bacterium of the genus Escherichia having the heparosan-producing ability when the expression level is increased in the bacterium.

[0029] Examples of kfiC genes include DNA comprising a nucleotide sequence shown in SEQ ID NO: 36, or DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 36, and having the property of increasing the heparosan-producing ability of a bacterium of the genus Escherichia having the heparosan-producing ability when the expression level is increased in the bacterium.

[0030] Examples of kfiD genes include DNA comprising a nucleotide sequence shown in SEQ ID NO: 37, or DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 37, and having the property of increasing the heparosan-producing ability of a bacterium of the genus Escherichia having the heparosan-producing ability when the expression level is increased in the bacterium.

[0031] FIG. 3 shows a schematic diagram of the biosynthetic pathway of heparosan. As shown in FIG. 3, GlmS is the first enzyme in the pathway for supplying UDP-N-acetylglucosamine, which is a precursor of heparosan, and is an enzyme that catalyzes the reaction of fructose-6-phosphate to glucosamine-6-phosphate. YhbJ is an enzyme that negatively controls GlmS.

[0032] As the yhbJ gene, the yhbJ gene originating from the genus Escherichia is preferred. Specific examples thereof include the yhbJ gene from Escherichia coli strain K-12. The nucleotide sequence of the yhbJ gene from Escherichia coli strain K-12 and the amino acid sequence of the protein encoded by the gene can be obtained from public databases. The yhbJ gene from Escherichia coli strain K-12 is registered as GenBank accession number BAE77249.1.

[0033] Examples of yhbJ genes include DNA comprising a nucleotide sequence shown in SEQ ID NO: 38, or DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 38, and having the property of increasing the heparosan-producing ability of a bacterium of the genus Escherichia having the heparosan-producing ability when the expression level is reduced in the bacterium.

[0034] Each of the genes in (1) to (3) above can be easily obtained from public databases by, for example, a BLAST search or a FASTA search using the nucleotide sequence of each gene described above. In addition, a homologue of each gene can be obtained by, for example, PCR using a chromosome of a microorganism such as a bacterium as a template and using an oligonucleotide prepared based on these known gene sequences as a primer.

[0035] Each of the genes in (1) to (3) above may be gene variants, as long as the original functions (e.g., activity or property) of the protein encoded by the genes are retained. It is possible to test whether or not the protein encoded by the gene variants retains its original function; specifically, for example, when the original function is to improve the ability to produce heparosan, by introducing the gene variant into a microorganism belonging to prokaryotes having the ability to produce heparosan.

[0036] Variants of each of the genes in (1) to (3) above can be obtained according to a site-directed mutagenesis method by modifying the coding region of the gene such that amino acid residues at specific positions of the encoded protein are replaced, deleted, inserted, or added. In addition, variants of each of the genes in (1) to (3) above can also be obtained, for example, by mutagenesis treatment.

[0037] Provided that their original functions are retained, each of the genes in (1) to (3) above may be genes encoding a protein having an amino acid sequence in which one or more amino acids at one or more positions are replaced, deleted, inserted or added. For example, in the encoded protein, its N-terminus and/or C-terminus may be extended or shortened. The phrase "one or more" differs depending on the position and type of the amino acid residue in the three-dimensional structure of the protein. Specific examples thereof include 1-50, 1-40, 1-30, and it is preferably 1-20, more preferably 1-10, even more preferably 1-5, and particularly preferably 1-3.

[0038] A substitution, deletion, insertion or addition of one or more amino acids as described above is a conservative mutation that normally preserves the function of the protein. Representative conservative mutations are conservative substitutions. A conservative substitution is a mutation in which a substitution occurs between Phe, Trp and Tyr, in the case where the substitution site is an aromatic amino acid, the substitution occurs between Leu, Ile and Val, in the case where the substitution site is a hydrophobic amino acid, the substitution occurs between Gln and Asn, in the case where the substitution site is a polar amino acid, the substitution occurs between Lys, Arg and His, in the case where the substitution site is a basic amino acid, the substitution occurs between Asp and Glu, in the case where the substitution site is an acidic amino acid, and the substitution occurs between Ser and Thr, in the case where the substitution site is an amino acid having hydroxyl groups. Specific examples of substitutions considered as a conservative substitution include the substitution of Ala for Ser or Thr; replacing Arg with Gln, His or Lys; replacing Asn with Glu, Gln, Lys, His or Asp; replacing Asp with Asn, Glu or Gln; replacing Cys with Ser or Ala; replacing Gln with Asn, Glu, Lys, His, Asp or Arg; replacing Glu with Gly, Asn, Gln, Lys or Asp; replacing Gly with Pro; replacing His with Asn, Lys, Gln, Arg or Tyr; replacing Ile with Leu, Met, Val or Phe; replacing Leu with Ile, Met, Val or Phe; replacing Lys with Asn, Glu, Gln, His or Arg; replacing Met with Ile, Leu, Val or Phe; replacing Phe with Trp, Tyr, Met, Ile or Leu; replacing Ser with Thr or Ala; replacing Thr with Ser or Ala; replacing Trp with Phe or Tyr; substitution of Tyr with His, Phe or Trp; and substitution of Val with Met, Ile or Leu. In addition, the substitutions, deletions, insertions, additions of amino acids as described above, their inversions, etc. include substitutions, deletions, insertions and additions, inversions, etc. that are caused by mutations (mutants or variants) occurring naturally, such as mutations based on individual differences or species differences in the organism from which the gene is derived.

[0039] Furthermore, provided that their original functions are retained, each of the genes in (1) to (3) above may be genes encoding a protein having 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identity with the entire amino acid sequence.

[0040] Furthermore, provided that their original functions are maintained, each of the genes in (1) to (3) above may be a DNA that hybridizes, under stringent conditions, with a probe that can be obtained from known gene sequences, such as a sequence complementary to all or part of a nucleotide sequence. The term "stringent conditions" refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Examples thereof include conditions under which DNAs having identity with each other at a higher level, such as DNAs having 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identity with each other, hybridize with each other, and DNAs having identity with each other at a lower level do not hybridize with each other; or conditions that are the washing conditions for normal Southern hybridization, in which the washing is carried out once, preferably two to three times, at a salt concentration and temperature corresponding to 60 degrees C, 1 x SSC and 0.1% SDS, preferably 60 degrees C, 0.1 x SSC and 0.1% SDS, and more preferably 68 degrees C, 0.1 x SSC and 0.1% SDS.

[0041] The probe used for the above hybridization may be a portion of a sequence complementary to each gene. Such a probe can be obtained by PCR using an oligonucleotide obtained based on a known gene sequence as a primer and using a DNA fragment containing each of the genes in (1) to (3) above as a template. For example, a DNA fragment having a length of about 300 bp can be used as a probe. In a case where a DNA fragment having a length of about 300 bp is used as a probe, examples of washing conditions in the hybridization include conditions of 50 degrees C, 2 x SSC, and 0.1% SDS.

[0042] Furthermore, since codon degeneracy differs depending on the host, each of the genes in (1) - (3) above may be genes obtained by replacing any codon with an equivalent codon, provided that their original functions are retained. For example, the genes in Tables 1 - 3 can be modified to have optimal codons depending on the codon usage frequency for the host used.

[0043] Examples of the treatments for mutagenesis include a method of treating in vitro a DNA molecule having a nucleotide sequence of each of the genes in (1) to (3) above using hydroxylamine or the like; a method of treating microorganisms carrying each of the genes in (1) to (3) above using X-ray irradiation, ultraviolet irradiation, or a mutagen such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS), and the like.

[0044] Genetic modification to increase gene expression levels

The phrase "increasing gene expression" means that the expression of the gene is increased, compared to the expression for an unmodified strain. Examples of one aspect of increasing gene expression include an aspect in which the expression of the gene is preferably increased by 1.5 times or more, more preferably increased by 2 times or more, and even more preferably increased by 3 times or more, compared to the expression for an unmodified strain.

[0045] In addition, the phrase "gene expression is increased" means not only an increase in the expression of a target gene in a strain in which the target gene is originally expressed, but also means that the target gene is expressed in a strain in which the target gene is not originally expressed. That is, the phrase "gene expression is increased" includes, for example, a case in which the target gene is introduced into a strain that does not have the target gene and the target gene is expressed therein. In addition, the phrase "gene expression is increased" is also designated as the phrases "gene expression is enhanced" and "gene expression is increased".

[0046] An increase in gene expression can be achieved, for example, by increasing the number of copies of the gene. An increase in the number of copies of the gene can be achieved by introducing the gene into the host chromosome. The introduction of the gene into the chromosome can be accomplished using, for example, homologous recombination (Miller I, J.H. Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Only one copy of the gene can be introduced, or two or more copies can be introduced.

[0047] For example, multiple copies of a gene can be introduced into a chromosome by performing homologous recombination while targeting a sequence having multiple copies on the chromosome. Examples of a sequence having multiple copies on a chromosome include a repetitive DNA sequence (repetitive DNA) and inverted repeats present at both ends of a transposon.

[0048] Alternatively, homologous recombination can be carried out while targeting a corresponding sequence on a chromosome, such as a gene that is not necessary for the production of a target substance. Homologous recombination can be carried out, for example, by a method using linear DNA, a method using a plasmid containing a temperature-sensitive origin of replication, a method using a plasmid capable of conjugative transfer, a method using a suicide vector lacking an origin of replication that functions in the host, or a transduction method using a phage. In addition, a gene can also be randomly introduced into a chromosome using a transposon or Mini-Mu (JP-A-H2-109985).

[0049] Whether a target gene has been introduced into a chromosome can be checked by Southern hybridization using a probe having a sequence complementary to all or part of the gene, PCR using a primer designed based on the gene sequence, or the like.

[0050] In addition, the copy number of a gene can also be increased by introducing a vector containing the gene into a host. For example, it is possible to increase the copy number of a gene by constructing an expression vector for the gene, by ligating a DNA fragment containing the target gene with a vector that functions in the host, and transforming the host using the expression vector. The DNA fragment containing the target gene can be obtained, for example, by PCR using the genomic DNA of a microorganism having the target gene as a template. The transformation method is not particularly limited, and a generally known method can be used.

[0051] As the vector, a vector that can autonomously replicate in a host cell can be used. The vector is preferably a multi-copy vector. In addition, the vector preferably has a marker such as an antibiotic resistance gene or other gene described in the literature [Karl Friehs, Plasmid Copy Number and Plasmid Stability, Adv Biochem Engin/Biotechnol 86: 47-82 (2004)] for selecting a transformant. In addition, the vector may have a promoter or terminator for expressing the inserted gene. Examples of the vector include a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, cosmids, phagemids, and the like.

[0052] Specific examples of vectors capable of autonomous replication in bacteria from the Enterobacteriaceae , such as Escherichia coli , include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, and pSTV29 (all from Takara Bio Inc.), pACYC184, pMW219, pMW118, and pMW119 (all from Nippon Gene), pTrc99A (Pharmacia), pPROK vector (Clontech), pKK233-2 (Clontech), pET vector (Novagen), pQE vector (Qiagen), and the broad host range vector RSF1010.

[0053] In the case of introducing a gene, it is sufficient that the gene is maintained in a bacterium of the genus Escherichia having the genetic modification of the present invention. Specifically, it is sufficient that the gene is introduced so that it is expressed under the control of a promoter sequence that functions in the bacterium of the present invention. The promoter may be a promoter derived from the host or a heterologous promoter. The promoter may be the own promoter of the gene to be introduced or a promoter of other genes. As the promoter, for example, a stronger promoter, which will be described later, can be used.

[0054] A terminator for terminating transcription may be located downstream of a gene. The terminator is not particularly limited as long as it functions in the bacterium of the present invention. The terminator may be a host-derived terminator or a heterologous terminator. The terminator may be a terminator specific to the gene to be introduced, or may be a terminator from other genes. Specific examples of terminators include the T7 terminator, the T4 terminator, the fd phage terminator, the tet terminator, and the trpA terminator.

[0055] Vectors, promoters and terminators that can be used in various microorganisms are described in detail, for example, in “Basic Lecture 8 on Microbiology, Gene Engineering, KYORITSU SHUPPAN, 1987”, and can be used.

[0056] Furthermore, in a case where two or more genes are introduced, it is sufficient that each gene is maintained in the bacterium of the present invention in a manner suitable for expression. For example, each gene of all may be maintained in a separate expression vector, or all may be maintained on a chromosome. Furthermore, each gene may be separately maintained on a plurality of expression vectors, or may be separately maintained on one or a plurality of expression vectors and on a chromosome. Furthermore, two or more genes may form an operon and be introduced. Examples of "cases where two or more genes are introduced" include a case of introducing genes respectively encoding two or more enzymes, a case of introducing genes respectively encoding two or more subunits that form one enzyme, and combinations thereof.

[0057] The gene to be introduced is not particularly limited as long as it encodes a protein that functions in the host. The gene to be introduced may be a gene derived from the host or a heterologous gene. The gene to be introduced can be obtained, for example, by PCR using primers designed based on the nucleotide sequence of the gene and using genomic DNA from an organism having the gene or a plasmid or the like carrying the gene as a template. In addition, the gene to be introduced may be entirely synthesized based on, for example, the nucleotide sequence of the gene [Gene, 60 (1), 115-127 (1987)].

[0058] In addition, an increase in gene expression can be achieved by improving the efficiency of gene transcription. An improvement in the efficiency of gene transcription can be achieved, for example, by replacing the gene promoter on the chromosome with a stronger promoter. A "stronger promoter" refers to a promoter that enhances gene transcription, compared to a natural wild-type promoter.

[0059] Examples of “stronger promoters” include known high expression promoters such as the uspA promoter, the T7 promoter, the trp promoter, the lac promoter, the thr promoter, the tac promoter, the trc promoter, the tet promoter, the araBAD promoter, the rpoH promoter, the PR promoter, and the PL promoter.

[0060] In addition, as a stronger promoter, a conventional promoter of a highly active type can be obtained using various reporter genes. For example, the activity of the promoter can be increased by moving the -35 and -10 regions in the promoter region closer to the consensus sequence (WO2000/18935).

[0061] Examples of highly active type promoters include various tac-like promoters (Katashkina JI et al. Russian Federation Patent Application 2006134574) and the pnlp8 promoter (WO2010/027045). Methods for assessing promoter strength and examples of strong promoters are described in known literature [Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995), etc.].

[0062] In addition, an increase in the expression level of a gene can be achieved by improving the efficiency of gene translation. An improvement in the efficiency of gene translation can be achieved, for example, by replacing the Shine-Dalgarno (SD) sequence (also called the ribosome binding site (RBS)) of a gene on a chromosome with a stronger SD sequence.

[0063] "Stronger SD sequence" refers to an SD sequence for which mRNA translation is improved, compared to the natural wild-type SD sequence. Examples of a stronger SD sequence include the RBS of gene 10 from phage T7 (Olins P.O. et al, Gene, 1988, 73, 227-235). In addition, it is known that substitution, insertion or deletion of several nucleotides in the spacer region between the RBS and the initiation codon, in particular in the sequence immediately upstream of the initiation codon (5'-UTR), significantly affects the stability and translation efficiency of mRNA, and thus the translation efficiency of the gene can be improved by modifying them.

[0064] According to the present invention, regions influencing gene expression, such as a promoter, an SD sequence, and a spacer region between the RBS and the start codon, are also collectively referred to as "expression control regions". The expression control regions can be determined using gene search software such as vector or GENETYX for searching promoters. Modification of these expression control regions can be carried out, for example, by a method using a temperature-sensitive vector or a Red-directed integration method (WO2005/010175).

[0065] Improvement of gene translation efficiency can also be achieved, for example, by modifying codons. Specifically, for example, in the case where heterologous expression of a gene is carried out, etc., the efficiency of gene translation can be improved by replacing rare codons present in the gene with synonymous codons used more frequently.

[0066] The codon substitution can be carried out, for example, by a site-directed mutagenesis method in which a target mutation is introduced into a target site in DNA. Examples of site-directed mutagenesis methods include a method using PCR [Higuchi, R., 61, in PCR technology, Erlich, H.A. Eds., Stockton press (1989); Carter, P., Meth. In Enzymol., 154, 382 (1987)], and a method using phage [Kramer, W. and Frits, H.J., Meth. In Enzymol., 154, 350 (1987); Kunkel, T.A. et al., Meth. In Enzymol., 154, 367 (1987)]. Alternatively, the gene fragment in which the codons are substituted can be entirely synthesized. The frequency of codon usage in various organisms is described in the “codon usage database” [http://www.kazusa.or.jp/codon; Nakamura, Y. et al, Nucl. Acids Res., 28, 292 (2000)].

[0067] In addition, gene expression can also be increased by amplifying a regulator that increases gene expression, or by deleting or weakening a regulator that decreases gene expression. Such methods for increasing gene expression as described above can be used alone or can be used in any combination.

[0068] An increase in gene expression can be tested, for example, by testing an increase in the transcription level of the gene or by testing an increase in the amount of protein expressed from the gene. In addition, an increase in gene expression can be tested, for example, by testing an increase in the activity of the protein expressed from the gene.

[0069] The increase in the transcription level of a gene can be checked by comparing the amount of mRNA transcribed from the gene with that of an unmodified strain such as a wild-type strain or a parental strain. Examples of methods for estimating the amount of mRNA include Northern hybridization, RT-PCR, and the like [Sambrook, J., et al., Molecular Cloning A Laboratory Manual/Third Edition, Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001]. The increase in the amount of mRNA refers to, for example, a case in which the amount of mRNA is preferably increased by 1.5 times or more, more preferably increased by 2 times or more, and even more preferably increased by 3 times or more, compared with that of the unmodified strain.

[0070] The increase in the amount of protein can be checked, for example, by Western blotting using an antibody. The increase in the amount of protein refers to, for example, a case in which the amount of protein is preferably increased by 1.5 times or more, more preferably increased by 2 times or more, and even more preferably increased by 3 times or more, compared with the amount in the unmodified strain.

[0071] The increase in protein activity can be verified, for example, by measuring the protein activity. The increase in protein activity refers to, for example, a case in which the protein activity is preferably increased by 1.5 times or more, more preferably increased by 2 times or more, and even more preferably increased by 3 times or more, compared with the activity in the unmodified strain.

[0072] The above-described methods for increasing gene expression can be used to enhance the expression of each of the genes (1) and (2) above.

[0073] The genetic modification that increases the expression of the kpsS gene is preferably at least one of a modification of the expression control regions of the kpsS gene and a genetic modification that increases the copy number. The kpsFEDUCS gene is present as a group of heparosan production genes, but as will be described later in Examples, the inventors of the present invention have found that the effect of particularly improving the production of heparosan is obtained by increasing the expression of only the kpsS gene among them. Accordingly, as a genetic modification that increases the expression of the kpsS gene, a genetic modification for increasing the copy number of the kpsS gene is particularly preferable.

[0074] While the bacterium of the present invention has a genetic modification that increases the expression of the kpsS gene, the bacterium of the present invention preferably does not have a genetic modification that increases the expression of the kpsC gene, more preferably does not have a genetic modification that increases the expression of the kpsC gene and at least one gene selected from the kpsF, kpsE, kpsD and kpsU genes, and most preferably does not have a genetic modification that increases the expression of all of the kpsC, kpsF, kpsE, kpsD and kpsU genes.

[0075] A genetic modification that increases the expression of at least one gene selected from the kfiA, kfiB, kfiC and kfiD genes is preferably at least one of modifying the expression control regions of at least one gene selected from the kfiA, kfiB, kfiC and kfiD genes and increasing the copy number of the genes. The kfiA, kfiB, kfiC and kfiD genes constitute an operon as shown in FIG. 1. A genetic modification that enhances the entire operon constituted by the kfiA, kfiB, kfiC and kfiD genes is preferred, and modifying the expression control region of the kfiA, kfiB, kfiC and kfiD genes is more preferred.

[0076] A genetic modification that causes loss of gene function

Examples of genetic modifications that cause loss of function of the yhbJ gene from (3) above include genetic modification in which the function of the protein encoded by the portion corresponding to yhbJ is reduced or completely stopped by modifying the DNA encoding the portion corresponding to yhbJ in the genomic DNA of a bacterium of the genus Escherichia as a host.

[0077] In the method of the present invention, the form of modification to be added to the DNA encoding the part corresponding to yhbJ is not particularly limited, as long as the function of the protein encoded by the part corresponding to yhbJ is reduced or completely stopped, and a known method can be appropriately used.

[0078] Examples of forms that reduce or completely abolish the function of the protein encoded by the portion corresponding to yhbJ include any of the following modifications (a) to (c):

(a) All or part of the DNA encoding the portion corresponding to yhbJ is deleted,

(b) One or more substitutions, deletions or additions are introduced into the DNA encoding the portion corresponding to yhbJ, and

(c) The DNA coding portion corresponding to yhbJ is replaced with a DNA sequence having less than 80% identity to the DNA sequence before modification.

[0079] Examples of loss of function of the yhbJ gene include a case in which the activity of the yhbJ gene is preferably 20% or less, more preferably 10% or less, and even more preferably 5% or less, compared with that of the unmodified strain. The activity of yhbJ can be checked by checking the expression level of glmS by a Northern blot method, a Western blot method, or the like [Kalamorz F. et al, (2007) “Feedback control of glucosamine-6-phosphate synthase GlmS expression depends on the small RNA GlmZ and involves the novel protein YhbJ in Escherichia coli”. Mol Microbiol. 65 (6):1518-33].

[0080] A bacterium of the genus Escherichia

The bacterium belonging to the genus Escherichia is not specifically limited, and examples thereof include the bacterium classified in the genus Escherichia , according to the classification known to experts in microbiology. Examples of bacteria belonging to the genus Escherichia include bacteria described in the literature [Backmann, BJ 1996. Derivations and Genotypes of some mutant derivatives of Escherichia coli K-12, p. 2460-2488. Table 1. In F. D. Neidhardt (ed.), Escherichia coli and Salmonella Cellular and Molecular Biology/Second Edition, American Society for Microbiology Press, Washington, DC].

[0081] Examples of bacteria belonging to the genus Escherichia include Escherichia coli . Examples of Escherichia coli include Escherichia coli strains K-12, such as strain W3110 (ATCC 27325) and strain MG1655 (ATCC 47076); Escherichia coli strain K5 (ATCC 23506); Escherichia coli strains B, such as strain BL21 (DE3); Escherichia coli strain Nissle 1917 (DSM 6601); and derivative strains thereof.

[0082] These strains can be ordered, for example, from the American Type Culture Collection (address: 12301 Parklawn Drive, Rockville, Maryland 20852, P.O. Box 1549, Manassas, VA 20108, United States of America). That is, a registration number is assigned to each strain, and the strains can be ordered using this registration number (link https://www.atcc.org/). The registration number corresponding to each strain is described in the catalog of the American Type Culture Collection. In addition, strain BL21 (DE3) is available, for example, from Life Technologies (product number C6000-03).

[0083] The bacterium of the present invention may originally have the ability to produce heparosan, or may be modified to have the ability to produce heparosan. The bacterium having the ability to produce heparosan can be obtained, for example, by imparting the ability to produce heparosan to the above-mentioned bacterium.

[0084] The ability to produce heparosan can be imparted by introducing a gene encoding a protein involved in the production of heparosan, with reference to Metabolic Engineering 14 (2012) 521-527, Carbohydrate Research 360 (2012) 19-24, U.S. Patent No. 9975928, and the like. Examples of proteins involved in the production of heparosan include glycosyltransferase and a heparosan efflux carrier protein. In the present invention, one kind of gene may be introduced, or two or more kinds of genes may be introduced. The introduction of the gene may be carried out in the same manner as in the above-described method for increasing the copy number of a gene.

[0085] Method for producing heparosan

The method for producing heparosan according to the present invention, comprising culturing the bacterium according to the present invention in a medium for producing and accumulating heparosan in the medium. The method for producing heparosan according to the present invention may further comprise, if necessary, collecting heparosan from the medium.

[0086] The medium used is not particularly limited as long as the bacterium of the present invention can grow and heparosan is produced and accumulated. As the medium, for example, a normal medium used for culturing bacteria can be used. Examples of the medium include LB medium (Luria-Bertani medium), however, examples are not limited thereto. As the medium, it is possible to use, for example, a medium containing a component selected from a carbon source, a nitrogen source, a phosphate source, a sulfur source, and other various organic components and inorganic components, as necessary. A person skilled in the art can appropriately set the type and concentration of the components of the medium.

[0087] The carbon source is not particularly limited as long as it can be used by the bacterium of the present invention such that heparosan can be produced. Examples of the carbon source include sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, molasses, starch hydrolysate and biomass hydrolysate, organic acids such as acetic acid, fumaric acid, citric acid, succinic acid and malic acid, alcohols such as glycerol, crude glycerol and ethanol, and fatty acids. As the carbon source, one kind of carbon source may be used, or two or more kinds of carbon sources may be combined.

[0088] Examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen sources such as peptone, yeast extract, meat extract and soy protein degradation products, ammonia and urea. As the nitrogen source, one kind of nitrogen source may be used, or two or more kinds of nitrogen sources may be combined.

[0089] Examples of phosphate sources include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphoric acid polymers such as pyrophosphoric acid. As the phosphate source, one kind of phosphate source may be used, or two or more kinds of phosphate sources may be combined.

[0090] Examples of sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates and sulfites, and sulfur-containing amino acids such as cysteine, cystine and glutathione. As the sulfur source, one kind of sulfur source may be used, or two or more kinds of sulfur sources may be combined.

[0091] Specific examples of various other organic components and inorganic components include inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium and calcium; vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide and vitamin B12; amino acids; nucleic acids; organic components containing them such as peptone, casamino acid, yeast extract and soy protein degradation products. As the various other organic components and inorganic components, one kind of component may be used, or two or more kinds of components may be used in combination.

[0092] In addition, in the case where an auxotrophic mutant requiring an amino acid or the like for growth is used, it is preferable to supplement a nutrient required for the medium. In addition, when a gene is introduced using a vector carrying an antibiotic resistance gene, it is preferable to add an appropriate antibiotic to the medium.

[0093] The culture conditions are not particularly limited as long as the bacterium of the present invention can grow and heparosan is produced and accumulated. The culture can be carried out, for example, under normal conditions used for culturing bacteria. A person skilled in the art can appropriately set the culture conditions.

[0094] The culturing can be carried out aerobically, for example, by means of an aeration culture or a shake culture using a liquid medium. The culturing temperature can be, for example, 30 degrees C to 37 degrees C. The culturing period can be, for example, 16 to 72 hours. The culturing can be carried out by means of a batch culturing, a fed-batch culturing, a continuous culturing, or a combination thereof. In addition, the culturing can be divided into a preliminary culturing and a main culturing. The preliminary culturing can be carried out using, for example, a plate medium or a liquid medium.

[0095] By culturing the bacterium of the present invention as described above, heparosan accumulates in the medium.

[0096] The method for collecting heparosan from the culture solution is not particularly limited as long as heparosan can be collected. Examples of the methods for collecting heparosan from the culture solution include the methods described in the Examples. Specifically, for example, the culture supernatant can be separated from the culture solution, and then heparosan in the supernatant can be precipitated by precipitation with ethanol. The amount of ethanol added can be, for example, 2.5 to 3.5 times the amount of the liquid supernatant. For the precipitation of heparosan, not only ethanol but also an organic solvent optionally miscible with water can be used.

[0097] Examples of organic solvents include ethanol, methanol, n-propanol, isopropanol, n-butanol, t-butanol, sec-butanol, propylene glycol, acetonitrile, acetone, DMF, DMSO, N-methylpyrrolidone, pyridine, 1,2-dimethoxyethane, 1,4-dioxane, and THF.

[0098]

Further examples of a method for collecting heparosan from a culture solution include a method comprising purifying heparosan by targeting Kdo present at the end of heparosan.

[0099] The precipitated heparosan can be dissolved, for example, in water that is twice the amount of the original supernatant. The collected heparosan can contain components such as bacterial cells, media components, water, and bacterial metabolic by-products in addition to heparosan. Heparosan can be purified to a desired degree. The purity of heparosan can be, for example, 30% (w/w) or more, 50% (w/w) or more, 70% (w/w) or more, 80% (w/w) or more, 90% (w/w) or more, or 95% (w/w) or more.

[0100] Detection and quantification of heparosan can be carried out by a known method. Specifically, for example, heparosan can be detected and quantified by a method using carbazole, as will be described later in Examples. The method using carbazole is a method widely used as a method for quantifying uronic acid, and it is possible to detect and quantify heparosan by thermally reacting heparosan with carbazole in the presence of sulfuric acid, and measuring the absorbance at 530 nm by forming a colored substance [Bitter T. and Muir H.M., (1962) "A modified uronic acid carbazole reaction". Analytical Biochemistry, 4 (4):330-334]. In addition, for example, heparosan can be detected and quantified by treating heparosan with heparinase III, which is an enzyme that degrades heparosan, and analyzing the composition of disaccharides.

[0101] EXAMPLES

Examples are shown below, but the present invention is not limited to the following examples.

Example 1

[0102] Construction of a strain with a gene deletion

(a) Construction of a marker gene fragment for gene deletion

A DNA fragment containing the chloramphenicol resistance gene (cat) and the levansucrase gene (sacB), used as marker genes for the deletion of Escherichia coli genes using homologous recombination, was obtained as follows.

[0103] PCR was performed using synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 1 and 2 as a primer group and using plasmid pHSG396 (Takara Bio Inc.) as a template, thereby obtaining a DNA fragment containing the cat gene. PCR was performed using PrimeSTAR Max DNA polymerase (Takara Bio Inc.) as described in the user manual. In addition, PCR was performed using synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 3 and 4 as a primer group and using pMOB3 (derived from ATCC 77282) as a template, thereby obtaining a DNA fragment containing the sacB gene.

[0104] After purifying the DNA fragment containing the cat gene and the DNA fragment containing the sacB gene, the DNA fragments were cut using SalI. Phenol/chloroform treatment and ethanol precipitation were performed, and the two fragments were mixed at an equimolar ratio and ligated using a DNA Ligation Kit ver.2 (Takara Bio Inc.). The ligation reaction solution was subjected to phenol/chloroform treatment and purified by ethanol precipitation, and PCR was performed using the product thus obtained as a template, and using synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 5 and 6 as a group of primers. The obtained amplified DNA was purified using the Qiaquick PCR product purification kit (Qiagen), and thus a DNA fragment (cat-sacB fragment) containing the cat gene and the sacB gene was obtained.

[0105] (b) Construction of a yhbJ-deficient strain

The first PCR was performed using synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 7 and 8 and synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 9 and 10 as primer sets, and using genomic DNA of Escherichia coli strain Nissle 1917 [DSM 6601, Mutaflor (Pharma-Zentrale GmbH), hereinafter referred to as the Nissle strain] obtained by a conventional method as a template. As a result, DNA fragments containing 1,000 bp upstream of the region close to the start codon of the yhbJ gene and approximately 1,000 bp downstream of the region close to the stop codon of the yhbJ gene, respectively, were obtained. PCR was performed using PrimeSTAR Max DNA polymerase (Takara Bio Inc.) as described in the user manual.

[0106] The second PCR was performed using, as a template, a mixture obtained by mixing the amplified product purified using the QIAquick PCR Product Purification Kit (manufactured by Qiagen) and the cat-sacB fragment at an equimolar ratio. PCR was performed using PrimeSTAR GXL DNA polymerase (Takara Bio Inc.) according to the description in the user manual. For a group of primers, synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 7 and 10 were used. The amplified product was subjected to agarose gel electrophoresis to separate a DNA fragment of approximately 4.6 kb, and thus a DNA fragment containing the peripheral region of yhbJ into which the cat-sacB fragment was inserted was obtained.

[0107] In addition, the first PCR was performed using synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 7 and 11 and synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 12 and 10 as primer groups, and using the genomic DNA of Escherichia coli strain Nissle as a template. As a result, DNA fragments containing 1,000 bp upstream of a region close to the start codon of the yhbJ gene and approximately 1,000 bp downstream of a region close to the stop codon of the yhbJ gene were obtained, respectively.

[0108] The second PCR was performed using a mixture obtained by mixing the purified amplified product at an equimolar ratio as a template. For a group of primers, synthetic DNA consisting of the nucleotide sequences shown in 7 and 10 was used. The amplified product was subjected to agarose gel electrophoresis to separate a DNA fragment of approximately 2.0 kb, and thus a DNA fragment containing the peripheral region of yhbJ of the defective yhbJ gene was obtained.

[0109] Then, Escherichia coli of the Nissle strain (Nissle/pKD46 strain) retaining pKD46 was cultured in LB medium [10 g/L bactotryptone (manufactured by Difco), 5 g/L yeast extract (manufactured by Difco), and 5 g/L sodium chloride] in the presence of 15 g/L L-arabinose and 100 mg/L ampicillin. The pKD46 plasmid has a lambda-Red recombinase gene, and thus, the expression of the gene can be induced by L-arabinose. Accordingly, when Escherichia coli retaining pKD46, which was grown in the presence of L-arabinose, is transformed using linear DNA, homologous recombination occurs at a high frequency. In addition, since pKD46 has a temperature-sensitive origin of replication, the plasmid can be easily removed by growing pKD46 at 42 degrees C. Competent cells of the Nissle/pKD46 strain were prepared, and a DNA fragment containing the peripheral region of yhbJ into which the cat-sacB fragment obtained above was inserted was introduced into them by electroporation.

[0110] The resulting transformant was plated on LB agar medium containing 15 mg/L chloramphenicol and 100 mg/L ampicillin (LB+chloramphenicol+ampicillin), and cultured, and chloramphenicol-resistant colonies were selected. Since the strain in which homologous recombination occurred is resistant to chloramphenicol and sensitive to sucrose, the selected colonies were replicated on LB agar medium containing 10% sucrose and 100 mg/L ampicillin and LB+chloramphenicol+ampicillin (LB+sucrose+ampicillin), and thus a strain having resistance to chloramphenicol and sensitivity to sucrose was selected.

[0111] Colony PCR was performed for the selected strain using synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 13 and 10 as a primer group, and it was verified that the cat-sacB fragment was inserted at the position of the yhbJ gene. The strain in which the cat-sacB fragment was inserted at the position of the yhbJ gene was cultured in the same manner as described above to obtain competent cells, and the DNA fragment obtained above and containing the peripheral region of the yhbJ defective yhbJ gene was introduced into them by electroporation.

[0112] The resulting transformant was cultured in LB+sucrose agar medium, and sucrose-resistant colonies were selected. Since the strain subjected to homologous recombination does not contain the cat-sacB fragment and is thus sensitive to chloramphenicol and resistant to sucrose, the selected colonies were replicated in LB+chloramphenicol agar medium and in LB+sucrose agar medium, and thus a strain having sensitivity to chloramphenicol and resistance to sucrose was obtained.

[0113] Colony PCR was performed for the selected strain using synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 13 and 10 as a primer group, and it was verified that the yhbJ gene was deleted. The strain in which the yhbJ gene was verified to be deleted was plated on LB agar medium and cultured at 42 degrees C, and then, a strain having sensitivity to ampicillin, that is, a strain from which pKD46 was deleted, was selected.

[0114] A strain deficient in the yhbJ gene was obtained as described above and designated as Escherichia coli strain NY.

Example 2

[0115] Construction of a strain with an enhanced gene

A strain having a replaced promoter region of the kfiA gene was constructed by the following method. The first PCR was performed using synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 14 and 15 and synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 16 and 17 as primer groups, and using genomic DNA from Escherichia coli strain Nissle obtained by a conventional method as a template. As a result, DNA fragments containing 1,000 bp upstream of about 100 bp upstream of the initiation codon of the kfiA gene and about 1,000 bp downstream of a region close to the initiation codon of the kfiA gene were obtained, respectively.

[0116] The second PCR was performed using, as a template, a mixture obtained by mixing the amplified product purified using the QIAquick PCR product purification kit (manufactured by Qiagen) and the cat-sacB fragment at an equimolar ratio. For a group of primers, synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 14 and 17 were used. The amplified product was subjected to agarose gel electrophoresis to separate a DNA fragment of approximately 4.6 kb, and thus a DNA fragment containing the peripheral promoter region of the kfiA gene into which the cat-sacB fragment was inserted was obtained.

[0117] The first PCR was performed using synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 14 and 18 and synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 19 and 17 as primer groups, and using the genomic DNA of Escherichia coli strain Nissle obtained by a conventional method as a template. As a result, DNA fragments containing 1,000 bp upstream of approximately 100 bp upstream of the initiation codon of the kfiA gene and approximately 1,000 bp downstream of a region close to the initiation codon of the kfiA gene were obtained, respectively. In addition, PCR was performed using synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 20 and 21 as a primer group and using the genomic DNA of Escherichia coli strain W (ATCC 9637) obtained by a conventional method as a template, and thus a DNA fragment of approximately 300 bp containing the uspA promoter was obtained.

[0118] A second PCR was performed using a mixture obtained by mixing the purified amplified products in an equimolar ratio as a template. For a group of primers, synthetic DNAs consisting of the nucleotide sequences shown in 14 and 17 were used. The amplified product was subjected to agarose gel electrophoresis to separate a DNA fragment of approximately 2.3 kb, and thus a DNA fragment deficient in the kfiA promoter region from the peripheral promoter region of kfiA but containing the uspA promoter instead was obtained.

[0119] Then, Escherichia coli strain Nissle (strain Nissle/pKD46) maintaining the plasmid pKD46 containing the gene encoding gamma-Red recombinase and Escherichia coli strain NY (strain NY/pKD46) constructed in Example 1 were cultured in the presence of 15 g/L L-arabinose and 100 mg/L ampicillin. Competent cells of both strains were obtained, and a DNA fragment containing the peripheral promoter region of kfiA into which the cat-sacB fragment obtained above was inserted was introduced into them by electroporation.

[0120] The resulting transformant was cultured in LB+chloramphenicol+ampicillin agar medium, and chloramphenicol-resistant colonies were selected. A strain having resistance to chloramphenicol and sensitivity to sucrose was then selected from the selected colonies.

[0121] Colony PCR was performed for the selected strain using synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 22 and 17 as a group of primers, and it was verified that the cat-sacB fragment was inserted into the promoter region of kfiA.

[0122] The strain in which the cat-sacB fragment was inserted into the kfiA promoter region was cultured in the same manner as described above to obtain competent cells, and the DNA fragment obtained above and deficient in the kfiA promoter region from the peripheral promoter region of kfiA but containing the uspA promoter instead was introduced into them by electroporation.

[0123] The resulting transformant was cultured in LB+sucrose agar medium, and colonies with sucrose resistance were selected. A strain having sensitivity to chloramphenicol and resistance to sucrose was then selected from the selected colonies.

[0124] Colony PCR was performed for the selected strain using synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 22 and 17 as a primer group, and it was verified that the uspA promoter was inserted into the kfiA promoter region. The strain in which the uspA promoter was verified to be inserted into the kfiA promoter region was plated on LB agar medium and cultured at 42 degrees C, and then a strain having sensitivity to ampicillin, that is, a strain from which pKD46 was deleted, was selected.

[0125] Strains in which the uspA promoter was inserted into the kfiA promoter region as described above were obtained and, respectively, designated as the NA strain and the NYA strain of Escherichia coli .

Example 3

[0126] Testing of Heparosan Production Using NY Strain, NA Strain and NYA Strain

(a) Cultivation of heparosan-producing strain

The yhbJ-deficient mutant NY strain obtained in Example 1, the kfiA promoter exchange strain NA and the kfiA promoter exchange strain NYA deficient in yhbJ obtained in Example 2, and the Nissle strain, which was the parent strain, were cultured at 30°C for 24 hours on an LB agar medium, and respectively inoculated into a 2 L baffle flask containing 330 mL of a preculture medium [10 g/L soybean peptide (Hinute AM; manufactured by Fuji Oil Co., Ltd.), 5 g/L sodium chloride, 5 g/L yeast extract powder (AY-80; manufactured by Asahi Food and Healthcare Co., Ltd.), adjusted with sodium hydroxide so that the pH became pH 7.2], and cultured at 30°C. degrees C for 18 hours.

[0127] In a tank fermenter containing 760 ml of a primary culture medium [in which 20 g/L glucose, 13.5 g/L potassium dihydrogen phosphate, 4 g/L diammonium phosphate, 1.7 g/L citric acid, 1.7 g/L magnesium sulfate heptahydrate, 10 mg/L thiamine hydrochloride and 10 ml/L trace element solution were adjusted using 5 mol/L sodium hydroxide so that the pH became pH 6.7, and glucose and magnesium sulfate heptahydrate were added separately after sterilization by autoclaving (at 120 degrees C for 20 minutes)], 40 ml of the resulting preculture was inoculated and cultured at 37 degrees C for 72 hours with a stirring speed of 800 rpm and an aeration speed of 1.5 l/min.

[0128] The trace element solution refers to a solution in which 10 g/L of ferrous sulfate heptahydrate, 2 g/L of calcium chloride, 2.2 g/L of zinc sulfate heptahydrate, 0.5 g/L of manganese sulfate tetrahydrate, 1 g/L of copper sulfate pentahydrate, 0.1 g/L of hexammonium heptamolybdate tetrahydrate, and 0.02 g/L of sodium tetraborate decahydrate were dissolved in 5 mol/L of hydrochloric acid.

[0129] From the time when the glucose concentration in the culture solution became 0 g/L (0 hour) to 72 hours, a feeding solution [500 g/L glucose, 33.6 g/L potassium dihydrogen phosphate, 14.3 g/L magnesium sulfate, 0.4 g/L thiamine hydrochloride, and 14.3 ml/L trace element solution] was added at 7.0 ml/hour. The amount of the feeding solution added at the end of the culture was approximately 450 ml.

[0130] (b) Partial purification of heparosan from a culture solution of a heparosan-producing strain After incubating the culture solution diluted 10-fold with distilled water at 100 degrees C for 30 minutes, the bacterial cells were removed from the culture solution by centrifugation, and 200 microliters of the resulting supernatant were transferred to a 1.5 ml Eppendorf tube. After adding and mixing 40 microliters of a 0.5 mol/L sodium sulfate aqueous solution, 400 microliters of a 10 g/L hexadecylpyridinium chloride aqueous solution were added and mixed by inversion, and the mixture was allowed to stand at 37 degrees C for 1 hour.

[0131] The solution was centrifuged to form a precipitate, and the supernatant was removed. After washing the precipitate with distilled water, 100 microliters of a solution [an aqueous solution containing 0.5 mol/L sodium chloride and 4% (v/v) ethanol] was added to dissolve the precipitate. After further allowing it to stand at 4 degrees C overnight, 900 microliters of a 0.25 mol/L sodium chloride aqueous solution was added and stirred, and thus a crude heparosan solution was obtained. A zero control was also prepared by treating 200 microliters of the main culture medium in the same manner.

[0132] (c) Measurement of the amount of accumulated heparosan by the carbazole-sulfuric acid method

While 20 microliters of crude heparosan solution was cooled on ice, 100 microliters of sulfuric acid solution [a solution in which 9.5 g/L sodium tetraborate decahydrate was dissolved in concentrated sulfuric acid] was added and mixed, followed by incubation at 100 degrees C for 10 minutes. While the solution was again cooled on ice, 4 microliters of carbazole solution [a solution in which 1.25 g/L carbazole was dissolved in 100% ethanol] was added thereto and mixed, and incubated at 100 degrees C for 15 minutes.

[0133] After re-cooling the solution on ice, the temperature was returned to room temperature, and the absorbance at 530 nm was measured using a microplate reader. Using 0, 0.1, and 0.2 g/L sodium glucuronate monohydrate for calibration curves, calibration curves were obtained with the concentration (g/L) of glucuronic acid as the horizontal axis and the absorbance at 530 nm as the vertical axis.

[0134] The concentration of heparosan was calculated according to the calculation expression described below. When the desired concentration of heparosan is H (g/L), the obtained calibration curve is y=ax+b, the measurement value of A530 for the crude heparosan sample is h, the zero value of the measurement A530 is k, and the final dilution factor is n. 0.5387 denotes the content of glucuronic acid in heparosan, and 216/234 denotes the content of sodium glucuronate in sodium glucuronate monohydrate.

[0135]

[Mathematical formula 1]

[0136] (d) Results of measuring the amount of accumulated heparosan

Table 1 shows the amount of heparosan accumulated when measured by the above method.

[0137]

Table 1 Strain Amount of accumulated heparosan (g/l) Nissle 3.9 NY 9.9 NA 5.5 NYA 12.5

[0138] As shown in Table 1, the amount of heparosan accumulated in the yhbJ-deficient NY strain was more than twice that of the parental Nissle strain. Furthermore, even in the NA strain, in which the kfiA promoter was replaced by the uspA promoter, the amount of heparosan accumulated was increased compared to that of the parental Nissle strain. For the NYA strain, in which these two mutations were combined, an even higher yield of heparosan production was shown.

Example 4

[0139] Construction of a plasmid expressing a gene involved in heparosan production

A PCR reaction was performed using the chromosomal DNA of Escherichia coli strain Nissle as a template, and using synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 23 and 24 as a group of primers, and thus a DNA fragment of approximately 3.3 kb containing regions of the kpsC and kpsS genes was obtained (hereinafter referred to as the amplified fragment of the kpsCS gene).

[0140] A PCR reaction was performed using the pMW118 vector as a template, and using synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 25 and 26 as a group of primers, thereby obtaining a linear DNA fragment of pMW118 of approximately 4 kb. In addition, a PCR reaction was performed using the chromosomal DNA of Escherichia coli strain W (ATCC 9637) as a template, and using synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 20 and 21 as a group of primers, thereby obtaining a DNA fragment of approximately 300 bp containing the uspA promoter region.

[0141] The linear DNA fragment of pMW118, the DNA fragment containing the uspA promoter region, and the amplified fragment of the kpsCS gene obtained as above were mixed and ligated using the In-Fusion HD Cloning Kit (Takara Bio Inc.).

[0142] Escherichia coli strain DH5 alpha was transformed using the obtained ligated DNA, and transformants were selected using ampicillin resistance as an indicator. A plasmid was isolated from the transformant according to a known method, and a PCR reaction was performed using synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 27 and 24 as a primer group, and thus it was verified that a gene expression plasmid was obtained, which was designated as pMW118-kpsCS.

[0143] Furthermore, a PCR reaction was performed using the chromosomal DNA of Escherichia coli strain Nissle as a template, and using synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 28 and 24 and synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 29 and 24 as primer groups, and thereby, respectively, a DNA fragment of approximately 1.2 kb containing the kpsS gene region (hereinafter referred to as an amplified kpsS gene fragment) and a DNA fragment of approximately 7.9 kb containing the kpsF, kpsE, kpsD, kpsU, kpsC and kpsS gene regions (hereinafter referred to as an amplified kpsFEDUCS gene fragment) were obtained.

[0144] Furthermore, a PCR reaction was performed using the plasmid pMW118-kpsCS obtained above as a template and using synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 25 and 21 as a group of primers, and thus a linear DNA fragment of pMW118 of approximately 4.3 kb containing the uspA promoter sequence was obtained.

[0145] The linear DNA fragment of pMW118 containing the uspA promoter sequence and the amplified fragment of the kpsS gene obtained above were mixed and ligated using the In-Fusion HD Cloning Kit (Takara Bio Inc.). In the same manner, the linear DNA fragment of pMW118 containing the uspA promoter sequence and the amplified fragment of the kpsFEDUCS gene were mixed and ligated.

[0146] Escherichia coli strain DH5 alpha was transformed using each of the obtained ligated DNAs, and transformants were selected using ampicillin resistance as an indicator. A plasmid was isolated from the transformant according to a known method, and a PCR reaction was performed using synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 20 and 24 as a primer group for a plasmid into which the amplified fragment of the kpsS gene was ligated, and using synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 27 and 30 and synthetic DNAs consisting of the nucleotide sequences shown in SEQ ID NOs: 31 and 32 as a primer group for a plasmid into which the amplified fragment of the kpsFEDUCS gene was ligated. Accordingly, it was verified that plasmids expressing genes were obtained, which were respectively designated as pMW118-kpsS and pMW118-kpsFEDUCS.

Example 5

[0147] Testing of heparosan production using a strain that retains a plasmid for gene expression - 1

The plasmids pMW118 and pMW118-PuspA-kpsS, pMW118-PuspA-kpsCS, and pMW118-PuspA-kpsFEDUCS obtained in Example 4, respectively, were transformed into the NY strain obtained in Example 1. The resulting transformants were respectively designated as the NY/pMW118 strain, the NY/pMW118-PuspA-kpsS strain, the NY/pMW118-PuspA-kpsCS strain, and the NY/pMW118-PuspA-kpsFEDUCS strain.

[0148] The transformants obtained above were inoculated into a large tube containing 5 ml of LB medium containing 100 mg/l ampicillin and cultured at 37 degrees C for 15 hours. A 1% culture solution was inoculated into a test tube containing 5 ml of R medium containing 100 mg/L ampicillin [in which 20 g/L glucose, 13.5 g/L potassium dihydrogen phosphate, 4 g/L diammonium phosphate, 1.7 g/L citric acid, 1 g/L magnesium sulfate heptahydrate, 10 mg/L thiamine hydrochloride and 10 ml/L trace element solution were adjusted using 5 mol/L sodium hydroxide so that the pH became pH 6.8, and glucose and magnesium sulfate heptahydrate were added separately after sterilization by autoclaving (at 120 degrees C for 20 minutes)], and cultured at 37 degrees C for 24 hours. In addition, the same operation was carried out for the NY strain under conditions without ampicillin.

[0149] The obtained culture solution was treated by the method described in Example 3, and the amount of heparosan accumulated in the culture solution was measured. The results are shown in Table 2.

[0150]

Table 2 Strain Amount of accumulated heparosan (g/l) NY 192 NY/pMW118 220 NY/pMW118-PuspA-kpsS 349 NY/pMW118-PuspA-kpsCS 328 NY/pMW118-PuspA-kpsFEDUCS 348

[0151] As shown in Table 2, in the NY/pMW118-PuspA-kpsS strain expressing kpsS, the amount of accumulated heparosan was clearly increased, compared with that in the parental NY strain and the NY/pMW118 strain retaining only the pMW118 vector.

[0152] However, based on the result that the NY/pMW118-PuspA-kpsCS strain expressing kpsC and kpsS and the NY/pMW118-PuspA-kpsFEDUCS strain expressing kpsF, kpsE, kpsD, kpsU, kpsC and kpsS showed almost the same heparosan production level as the NY/pMW118-PuspA-kpsS strain, it was found that the effect of improving the heparosan production ability was sufficiently obtained by enhancing the expression of only kpsS in kpsFEDUCS, which is a group of heparosan production genes.

Example 6

[0153]

Testing of heparosan production using a strain retaining a plasmid for gene expression - 2

The plasmids pMW118, pMW118-PuspA-kpsS, pMW118-PuspA-kpsCS, and pMW118-PuspA-kpsFEDUCS obtained in Example 4 were transformed into the wild-type Escherichia coli Nissle strain, respectively. The resulting transformants were designated as strain N/pMW118, strain N/pMW118-PuspA-kpsS, strain N/pMW118-PuspA-kpsCS, and strain N/pMW118-PuspA-kpsFEDUCS, respectively.

[0154]

The transformants obtained above were inoculated into a large tube containing 5 ml of LB medium containing 100 mg/l ampicillin and cultured at 37 degrees C for 15 hours. 1% culture solution was inoculated into a test tube containing 5 ml of R medium containing 100 mg/L ampicillin [in which 20 g/L glucose, 13.5 g/L potassium dihydrogen phosphate, 4 g/L diammonium phosphate, 1.7 g/L citric acid, 1 g/L magnesium sulfate heptahydrate, 10 mg/L thiamine hydrochloride and 10 ml/L trace element solution were adjusted using 5 mol/L sodium hydroxide so that the pH became pH 6.8, and glucose and magnesium sulfate heptahydrate were added separately after sterilization by autoclaving (at 120 degrees C for 20 minutes)], and cultured at 37 degrees C for 24 hours. In addition, the same operation was performed for the wild-type Nissle strain under ampicillin-free conditions.

[0155]

The obtained culture solution was processed by the method described in Example 3, and the amount of heparosan accumulated in the culture solution was measured. The results are shown in Table 3.

[0156]

Table 3 Strain Amount of accumulated heparosan (Ratio when wild type strain Nissle is 1) Wild-type strain Nissle 1.0 N/pMW118 0.9 N/pMW118-PuspA-kpsS 3.3 N/pMW118-PuspA-kpsCS 1.7 N/pMW118-PuspA-kpsFEDUCS 1.8

[0157]

As shown in Table 3, in the N/pMW118-PuspA-kpsS strain expressing kpsS, the amount of heparosan accumulated was increased threefold or more compared with that in the parental wild-type strain Nissle and the N/pMW118 strain retaining only the pMW118 vector.

[0158] However, in the N/pMW118-PuspA-kpsCS strain expressing kpsC and kpsS and in the N/pMW118-PuspA-kpsFEDUCS strain expressing kpsF, kpsE, kpsD, kpsU, kpsC and kpsS, the titer was improved by at most 1.8 times, compared with the parental strain. In this regard, it was found that the effect of improving the heparosan producing ability was sufficiently obtained by enhancing the expression of only kpsS among the kpsFEDUCS group of heparosan producing genes, and that the heparosan producing ability became higher when the expression of kpsC and kpsFEDU was not enhanced, instead of being enhanced.

[0159] While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to those skilled in the art that various changes and modifications can be made thereto without departing from the spirit and scope thereof. The entire contents of all references cited in this specification are hereby incorporated by reference. This application is based on International Application No. PCT/JP2020/015384, filed April 3, 2020, the entire contents of which are hereby incorporated by reference.

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LIST OF SEQUENCES

<110> Rensselaer Polytechnic Institute

Otsuka Pharmaceutical Factory, Inc

KYOWA HAKKO BIO CO.,LTD.

<120> A method for producing heparosan and a bacterium from the genus Escherichia, which has

ability to produce heparosan

<130> W528319

<160> 38

<170> PatentIn version 3.5

<210> 1

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 1

aatcgggatc cgcggccgca gaggcggttt gcgtattgga gc 42

<210> 2

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 2

ggatttgact acgggcctaa agtcgacaga ataaataaat cctggtgtcc c 51

<210> 3

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 3

acggagcatg cgcggccgct caaaatcggt ggagctgcat ga 42

<210> 4

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 4

gggacaccag gatttattta ttctgtcgac tttaggcccg tagtctgcaa atcc 54

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 5

cacttattca ggcgtagcac c 21

<210> 6

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 6

atcggcattt tctttgcgtt ttta 24

<210> 7

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 7

gaagtaccga gagtctttat cc 22

<210> 8

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 8

ctacgcctga ataagtggta ctgtttctcc tcacaacg 38

<210> 9

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 9

gcaaagaaaa tgccgatccg tcaagcaaac tgttgaaatc 40

<210> 10

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 10

tcgtttccat aaccgctctt gc 22

<210> 11

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 11

caacagtttg cttgacgggt actgtttctc ctcacaacg 39

<210> 12

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 12

gtgaggagaa acagtacccg tcaagcaaac tgttgaaatc 40

<210> 13

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 13

gaaattaatc gcggcggaaa accc 24

<210> 14

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 14

gtatagatga gtataaaatg tatc 24

<210> 15

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 15

ctacgcctga ataagtggtt gattagaatg actccgcac 39

<210> 16

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 16

gcaaagaaaa tgccgatatg attgttgcaa atatgtc 37

<210> 17

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 17

ggtggcgtta tgtgataata from 21

<210> 18

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 18

caccacaaaa gcggttgttg attagaatga ctccgcac 38

<210> 19

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 19

tggaaggagt aacactatga ttgttgcaaa tatgtc 36

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 20

aaccgctttt gtggtgacca 20

<210> 21

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 21

agtgttactc cttccataaa g 21

<210> 22

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 22

gaaagagttt tgtgatgtgg cg 22

<210> 23

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 23

ctttatggaa ggagtaacac tatgattggc atttactcgc ctgg 44

<210> 24

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 24

gtaaaacgac ggccagtgcc ttaataataa accgcattaa cctg 44

<210> 25

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 25

ggcactggcc gtcgttttac aacgtc 26

<210> 26

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 26

gcgctttctc atagctcacg ctg 23

<210> 27

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 27

caccgaactg agatacctac agc 23

<210> 28

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 28

ctttatggaa ggagtaacac tatgcaaggt aatgcactaa ccg 43

<210> 29

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 29

caactttatg gaaggagtaa cactatgtct gaaagacatt tacc 44

<210> 30

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 30

gttgcgcgtc atgatggctt tcc 23

<210> 31

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 31

gaagtttggg tggcaacaga cgatc 25

<210> 32

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized oligonucleotide for primer

<400> 32

cagggttttc ccagtcacga cgttg 25

<210> 33

<211> 1170

<212> DNA

<213> Escherichia coli

<400> 33

atgcaaggta atgcactaac cgttttatta tccggtaaaa aatatctgct attgcagggg 60

ccaatgggac cctttttcag tgatgttgcc gagtggctag agtcattagg tcgtaacgct 120

gtgaatgttg tattcaacgg tggggatcgt ttttactgcc gccatcgaca atacctagct 180

tactaccaga caccgaaaga gtttcccgga tggttacggg atctccaccg gcaatatgac 240

tttgacacaa tcctctgctt tggcgactgc cgcccattgc ataaagaagc aaaacgctgg 300

gcaaagtcga aagggatccg cttcctggca tttgaagaag gatatttacg cccgcaattt 360

attaccgttg aagaaggcgg agtgaacgca tattcatcgc taccgcgcga tccggatttt 420

tatcgtaagt taccagatat gcctacgccg cacgttgaga acttaaaacc ttcaacgatg 480

aaacgtatag gccatgctat gtggtattac ctgatgggct ggcattaccg tcatgagttt 540

cctcgctacc gccaccacaa atcattttcc ccctggtatg aagcacgttg ctgggttcgt 600

gcatactggc gcaagcaact ttacaaggta acacagcgta aggtattacc gaggttaatg 660

aacgaactgg accagcgtta ttatcttgct gttttgcagg tgtataacga tagccagatt 720

cgtaaccaca gcagttataa cgatgtgcgt gactatatta atgaagtcat gtactcattt 780

tcgcgtaaag cgccgaaaga aagttatttg gtgatcaaac atcatccgat ggatcgtggt 840

cacagactct atcgaccatt aattaaacgg ttgagtaagg aatatggctt aggtgagcga 900

atcctttatg tgcacgatct cccgatgccg gaattattac gccatgcaaa agcggtggtg 960

acgattaaca gtacggcggg gatctctgcg ctgattcata acaaaccact caaagtgatg 1020

ggcaatgccc tgtacgacat caagggcttg acgtatcaag ggcatttgca ccagttctgg 1080

caggctgatt ttaaaccaga tatgaaactg tttaagaagt ttcgtgggta tttattggtg 1140

aagacgcagg ttaatgcggt ttattattaa 1170

<210> 34

<211> 717

<212> DNA

<213> Escherichia coli

<400> 34

atgattgttg caaatatgtc atcataccca cctcgaaaaa aagagttggt gcattctata 60

caaagtttac atgctcaagt agataaaatt aatctttgcc tgaatgagtt tgaagaaatt 120

cctgaggaat tagatggttt ttcaaaatta aatccagtta ttccagataa agattataag 180

gatgtgggca aatttatatt tccttgcgct aaaaatgata tgatcgtact tacagatgat 240

gatattattt accctcccga ttatgtagaa aaaatgctca atttttataa ttcctttgca 300

atattcaatt gcattgttgg gattcatggc tgtatataca tagatgcatt tgatggagat 360

cagtctaaaa gaaaagtatt ttcatttact caagggctat tgcgaccgag agttgtaaat 420

caattaggta cagggactgt ttttcttaag gcagatcaat taccatcttt aaaatatatg 480

gatggttctc aacgattcgt cgatgttaga ttttctcgct atatgttaga gaatgaaatt 540

ggtatgatat gtgttcccag agaaaaaaac tggctaagag aggtctcatc aggttcaatg 600

gaaggacttt ggaacacatt tacaaaaaaa tggcctttag acatcataaa agaaacacaa 660

gcaatcgcag gatattcaaa acttaacctc gaattagtgt ataatgtgga agggtaa 717

<210> 35

<211> 1689

<212> DNA

<213> Escherichia coli

<400> 35

atgaataaat tagtgctagt cggacatcct ggctcaaagt atcagatagt tgaacatttt 60

ttgaaagaaa ttggcatgaa ctcaccaaat tattctacaa gtaataaaat ttccccagaa 120

tatatcaccg cttcattatg tcaattttat caaacaccag aagttaatga tgtagtagat 180

gagagagaat tctcagctgt tcaagtctca accatgtggg atagcatggt tcttgaacta 240

atgatgaaca atctaaataa caaactttgg gggtggggcag atccatctat aatatttttt 300

cttgattttt ggaaaaatat agataaaagc ataaaattca tcatgatata tgatcaccct 360

aaatataatt taatgcgttc agtaaataat gcccctctct ctttaaatat aaataatagt 420

gtagataact ggattgcata taataaaaga ttgcttgatt tttttttgga gaataaagaa 480

cgatgtgtgt tgattaattt tgaggcgttt caaagcaata agaaaaatat tataaagcca 540

ttgagtaata ttataaaaat agataatcta atgtctgcgc attacaaaaa ttcaatattg 600

tttgatgtgg ttgagaataa tgattataca aaatcaaatg aaattgccct gcttgaaaaa 660

tatacaactt tattttcttt aagtgcaaat gagactgaaa ttacatttaa tgatacaaag 720

gttagtgagt acttagtatc tgaattaata aaagaaagaa ccgaggttct gaagctttat 780

aatgagttac aagcctatgc aaacctacct tatatagaaa catcgaaaga taacgtttcg 840

gctgaggctg cattatggga ggtagtcgaa gagagaaatt ctatcttcaa tattgtatct 900

catttggtgc aagagtcaaa aaagaaggat gcagatattg aattgactaa atctatattt 960

aagaaaagac aatttttatt attgaacagg attaatgagc taaaaaaaga aaaggaagag 1020

gtaattaaac tttcaaaaat aaatcacaac gatgttgtga gacaagaaaa atatccagat 1080

gatattgaaa aaaaaataaa tgacatacag aaatatgaag aagagataag cgaaaaagaa 1140

tcaaaactca ctcaggcaat atcagaaaaa gaacagattt taaaacaatt gcataaatat 1200

gaagaagaga taagcgaaaa agaatcaaaa ctcactcagg caatatcaga aaaagaacag 1260

attttaaaac aattgcatat agtgcaagag cagttggaac actattttat agaaaatcag 1320

gaaattaaaa agaaacttcc acctgtgcta tatggagcag ctgagcagat aaaacaagag 1380

ttaggttatc gacttggtta tattatagtc tcgtattcta aatccctcaa ggggattatt 1440

accatgccat ttgcacttat ccgtgagtgt gtttttgaaa aaaaacgtaa gaagagttat 1500

ggcgttgatg tgccactcta tttatatgct gatgctgata aggctgaaag agttaagaaa 1560

catttatctt atcaattagg gcaggctatt atctccagtg ctaattcgat atttggattc 1620

attacccttc catttaagtt aattgttgtt gtttataaat atagggagagc taaaatcaag 1680

ggctgttaa 1689

<210> 36

<211> 1563

<212> DNA

<213> Escherichia coli

<400> 36

atgaacgcag aatatataaa tttagttgaa cgtaaaaaga aattagggac aaatattggt 60

gctcttgatt ttttattatc aattcataag gagaaagttg atcttcaaca taaaaactcg 120

cctttaaaag gtaacgataa ccttattcac aaaagaataa acgaatacga caatgtactt 180

gaactatcta agaatgtatc agctcagaat tctggcaatg agttttctta tttattggga 240

tatgcagatt ctcttagaaa agttggtatg ttggatactt atattaaaat tgtttgttat 300

ctaacaattc aatctcgtta ttttaaaaat ggcgaacgag ttaagctttt tgaacatata 360

agtaacgctc tacggtattc aaggagtgat tttctcatta atcttatttt tgaacgatat 420

atcgaatata taaaccatct aaaattgtcg cccaaacaaa aagattttta tttttgtacg 480

aagttttcaa aatttcatga ttatactaaa aatggatata aatatttagc atttgataat 540

caagccgatg cagggtatgg cctgacttta ttattaaatg caaacgatga tatgcaagat 600

agttataatc tactccctga gcaagaactt tttatttgta atgctgtaat agataatatg 660

aatatttata ggagtcaatt taacaaatgt ctacgaaaat acgatttatc agaaataact 720

gatatatacc caaataaaat tatattgcaa ggaattaagt tcgataagaa aaaaaatgtt 780

tatggaaaag atcttgttag tataataatg tcagtattca attcagaaga tactattgca 840

tactcattac attcattgtt gaatcaaaca tatgaaaata ttgaaattct cgtgtgcgat 900

gattgttcat cggacaaaag ccttgaaata attaagagca tagcttattc tgattcaaga 960

gtgaaagtat atagctcacg aaaaaaccaa ggcccttata atataagaaa tgagctaata 1020

aaaaaagcac acggtaattt catcaccttt caagatgcag atgatctttc tcatccggag 1080

agaatacaaa gacaagttga ggttcttcgc aataataagg ctgtaatctg tatggctaac 1140

tggatccgtg ttgcgtcaaa tggaaaaatt caattcttct atgatgataa agccacaaga 1200

atgtctgttg tatcgtcaat gataaaaaaa gatatttttg cgacagttgg tggctataga 1260

caatctttaa ttggtgcaga tacggagttt tatgaaacag taataatgcg ttatgggcga 1320

gaaagtattg taagattact gcagccattg atattggggt tatggggaga ctccggactt 1380

accaggaata aaggaacaga agctctacct gatggatata tatcacaatc tcgaagagaa 1440

tatagtgata tcgcggcaag acaacgagtg ttagggaaaa gtatcgtaag tgataaagat 1500

gtacgtggtt tattatctcg ctatggtttg tttaaagatg tatcaggaat aattgaacaa 1560

tag 1563

<210> 37

<211> 1179

<212> DNA

<213> Escherichia coli

<400> 37

atgttcggaa cactaaaaat aactgtttca ggcgctggtt acgttgggct ttcaaatgga 60

attctaatgg ctcaaaatca tgaagtggtt gcatttgata cccatcaaaa aaaagttgac 120

ttacttaatg ataaactctc tcctatagag gataaggaaa ttgaaaatta tctttcaact 180

aaaatactta attttcgcgc aactactaac aaatatgaag cctataaaaa tgccaattac 240

gttattattg ctacaccaac gaattatgac ccaggttcaa attactttga tacatcaagc 300

gttgaagctg tcattcgtga cgtaacggaa atcaacccaa acgcaattat ggtggttaaa 360

tctacggtcc cagtaggttt cacaaaaaca attaaagaac atttaggtat taataatatt 420

atcttctctc cagaattttt acgagaagga agagccctat acgataatct ccatccatct 480

cgcattatta tcggtgaatg ttctgaacgg gcagaacgtt tggcagtgtt atttcaggaa 540

ggagcgatta aacaaaatat acccgtttta tttacagatt ctacggaagc ggaagcgatt 600

aagttatttt caaatactta tttggctatg cgagttgcat tttttaatga attggatagt 660

tacgcagaaa gttttggtct gaatacgcgt cagattattg acggtgtttg tttggatccg 720

cgcattggta attactacaa taatccttct tttggttatg gtggctactg tttgccaaaa 780

gataccaagc aattattagc caactatcag tctgttccga ataaacttat atctgcaatt 840

gttgatgcta accgtacacg taaggacttt atcactaatg ttattttgaa acatagacca 900

caagttgtgg gggtttatcg tttgattatg aaaagtggtt cagataattt tagagattct 960

tctattcttg gtattataaa gcgtatcaag aaaaaaggcg tgaaagtaat tatttatgag 1020

ccgcttattt ctggagatac attctttaac tcacctttgg aacgggagct ggcgatcttt 1080

aaagggaaag ctgatattat tatcactaac cgaatgtcag aggagttgaa cgatgtggtc 1140

gacaaagtct atagtcgcga tttgtttaaa tgtgactaa 1179

<210> 38

<211> 855

<212> DNA

<213> Escherichia coli

<400> 38

atggtactga tgatcgtcag cggacgttca ggttcaggta aatctgtcgc cctgcgtgcg 60

ctggaagata tgggttttta ctgcgtggat aaccttcccg tagtgttgtt acccgatctg 120

gctcgaactc tggccgatcg agagatttct gccgccgtca gcattgatgt tcgtaatatg 180

ccggagtcac cagaaatatt cgaacaggcg atgagtaacc tgcctgacgc tttctcaccg 240

caactactgt tcctggatgc cgaccgtaat accttaattc gtcgttacag tgacacgcgc 300

cgactgcatc cgctttccag caaaaacctg tcgctggaaa gtgctatcga caaagaaagc 360

gatttgctgg agcctctgcg ttcgcgagcg gatctgattg tcgacacctc agaaatgtcc 420

gttcacgagc tggcagaaat gctgcgtacc cgtctgctgg gtaaacgtga acgtgaactg 480

accatggtct ttgagtcttt cggcttcaaa cacggtatcc ctatcgatgc agattacgtc 540

tttgacgtgc gcttcttgcc gaacccgcac tgggatccga aactgcgtcc aatgacaggt 600

cttgataaac ctgtcgccgc gttcctcgac cgccacacag aagtacacaa ttttatctac 660

cagacgcgaa gctatcttga gctatggtta cctatgctgg aaaccaacaa ccgtagctac 720

ctgacggtcg ccattggttg taccggcggg aagcaccgtt cggtgtatat tgcagagcaa 780

ctggcagact acttccgctc gcgcggtaaa aacgtccagt cacgccatcg gacgctggaa 840

aaacgtaaac catga 855

<---

Claims (23)

1. A method for producing heparosan, comprising: culturing a bacterium of the genus Escherichia , which: has the following genetic modification (1) and produces heparosan, in a heparosan production medium: (1) genetic modification that increases the expression of the kpsS gene compared to the expression in unmodified bacteria; and does not have the following genetic modification (4): (4) genetic modification that increases the expression of the kpsC gene compared to the expression in unmodified bacteria. 2. The method for producing heparosan according to claim 1, wherein the bacterium from the genus Escherichia additionally has at least one of the following genetic modifications (2) and (3): (2) a genetic modification that increases the expression of at least one gene selected from the kfiA gene, the kfiB gene, the kfiC gene and the kfiD gene, compared to the expression of an unmodified bacterium, and (3) genetic modification causing loss of function of the yhbJ gene. 3. The method for producing heparosan according to claim 1 or 2, wherein the genetic modification (1) is at least one of modifying the expression control region of the kpsS gene and increasing the copy number of the kpsS gene. 4. The method for producing heparosan according to claim 2 or 3, wherein the genetic modification (2) is at least one of modifying the expression control region of at least one gene selected from the kfiA gene, the kfiB gene, the kfiC gene and the kfiD gene, and increasing the copy number of at least one gene selected from the kfiA gene, the kfiB gene, the kfiC gene and the kfiD gene. 5. A method for producing heparosan according to any one of claims 2 to 4, wherein the genetic modification (3) is a deletion of the yhbJ gene. 6. A method for producing heparosan according to any one of claims 1-5, wherein the bacterium from the genus Escherichia is Escherichia coli . 7. The method for producing heparosan according to any one of claims 1 to 6, wherein the kpsS gene is a DNA comprising the nucleotide sequence shown in SEQ ID NO: 33, or a DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 33, and increasing the production of heparosan by a bacterium of the genus Escherichia that produces heparosan when the expression level is increased in the bacterium. 8. A method for producing heparosan according to any one of claims. 2-7, wherein the kfiA gene is a DNA comprising a nucleotide sequence shown in SEQ ID NO: 34 or a DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 34 and increasing the production of heparosan by a bacterium of the genus Escherichia that produces heparosan when the expression level is increased in the bacterium, the kfiB gene is a DNA comprising a nucleotide sequence shown in SEQ ID NO: 35 or a DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 35 and increasing the production of heparosan by a bacterium of the genus Escherichia that produces heparosan when the expression level is increased in the bacterium, the kfiC gene is a DNA comprising a nucleotide sequence shown in SEQ ID NO: 36, or a DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 36 and increasing the production of heparosan by a bacterium of the genus Escherichia that produces heparosan when the expression level is increased in the bacterium, and the kfiD gene is a DNA comprising the nucleotide sequence shown in SEQ ID NO: 37, or a DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 37 and increasing the production of heparosan by a bacterium of the genus Escherichia that produces heparosan when the expression level is increased in the bacterium. 9. The method for producing heparosan according to any one of claims 2 to 8, wherein the yhbJ gene is a DNA comprising the nucleotide sequence shown in SEQ ID NO: 38, or a DNA comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 38, and increasing the production of heparosan by a bacterium of the genus Escherichia that produces heparosan when the expression level is reduced in the bacterium. 10. A bacterium of the genus Escherichia that produces heparosan and has the following genetic modification (1): (1) genetic modification that increases the expression of the kpsS gene compared to the expression in unmodified bacteria; and does not have the following genetic modification (4): (4) genetic modification that increases the expression of the kpsC gene compared to the expression in unmodified bacteria. 11. A bacterium from the genus Escherichia according to claim 10, additionally having at least one of the following genetic modifications (2) and (3): (2) a genetic modification that increases the expression of at least one gene selected from the kfiA gene, the kfiB gene, the kfiC gene and the kfiD gene compared to the expression of an unmodified bacterium, and (3) genetic modification causing loss of function of the yhbJ gene.