# Patent

## Title
Chondroitin-producing bacterium and method of producing chondroitin

## Abstract

Chondroitin is produced by culturing a UDP-glucuronic acid-producing bacterium transfected with a kfoA gene derived from Escherichia coli K4 strain and a kfoC gene derived from Escherichia coli K4 strain and having chondroitin-producing ability. Chondroitin is collected from the bacterium.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the U.S. National Phase under 35 U.S.C. § 371 of International Application PCT/JP2008/058323, filed Apr. 24, 2008, which claims priority to U.S. Provisional Application No. 60/913,713, filed Apr. 24, 2007.


TECHNICAL FIELD
The present invention relates to a chondroitin-producing bacterium and a method of producing chondroitin.
BACKGROUND ART
Chondroitin is a polysaccharide comprising a repeated structure of disaccharides of a glucuronic acid (GlcUA) residue and a N-acetyl-D-galactosamine (GalNAc) residue (-GlcUAβ(1-3)-GalNAcβ(1-4)-; in the present specification, also referred to as a chondroitin carbohydrate backbone). Chondroitin sulfate is a polysaccharide which consists by sulfation of chondroitin.
Conventionally, chondroitin and chondroitin sulfate are extracted and purified from cartilages, organs, and the like of animals. However, in recent years, a technique for artificially synthesizing a carbohydrate backbone common to chondroitin and chondroitin sulfate has been studied because of shortage of such materials as cartilages and organs.
Chondroitin synthases, which produce chondroitin by alternately transferring GlcUA and GalNAc from their donor substrates to acceptor oligosaccharides, have been reported, and a method of producing chondroitin using the enzyme has been proposed.
J. Biol. Chem. 275(31), 24124-24129 (2000) discloses a chondroitin synthase derived from Pasteurella multocida. 
In addition, WO 2003/102193 and WO 2003/102194 disclose chondroitin synthases derived from human.
Moreover, U.S. 2003-0109693 (JP 2003-199583 A) discloses a novel chondroitin synthase (KfoC) produced by Escherichia coli K4 strain.
However, in the case of producing chondroitin by the enzymatic methods, it is necessary to prepare expensive materials such as oligosaccharides as acceptors and sugar nucleotides for donors of GlcUA and GalNAc, and thus a method of producing chondroitin using more inexpensive materials has been desired.
Escherichia coli K4 strain is known to produce a polysaccharide having a chondroitin backbone structure as a capsule. However, its structure consists of a repeating unit comprising trisaccharides of a GalNAc residue, a GlcUA residue and a fructose residue that is bound to a C3-hydroxyl group of a GlcUA residue. In addition, over 100 chemically different capsular polysaccharides have been detected in Escherichia coli. For example, Escherichia coli K5 strain produces a capsule polysaccharide K5 which has a carbohydrate backbone of heparin/heparan sulfate (J. Biol. Chem., 272(5), p 2682-2687, 1997). However, the existence of Escherichia coli which produces chondroitin itself has not been known. Therefore, a method of producing chondroitin using an Escherichia bacterium is unknown.
Although US Published Patent Application 2007-0281342 discloses a method of producing chondroitin by using a recombinant gram-positive Bacillus bacterium introduced with a chondroitin synthase gene derived from Pasteurella multocida, further development in fermentative production of chondroitin has been desired.
DISCLOSURE OF THE INVENTION
An object of the present invention is to provide a novel microorganism capable of producing chondroitin and a novel method of producing chondroitin using the microorganism.
The inventors of the present invention have made extensive studies, and as a result, have discovered that a UDP-glucuronic acid-producing bacterium such as Escherichia coli K5 strain which is introduced with kfoA and kfoC genes derived from Escherichia coli K4 strain produces chondroitin at high efficiency, thus completed the present invention.
An object of the present invention is to provide a UDP-glucuronic acid-producing bacterium, which is introduced with a kfoA gene derived from Escherichia coli K4 strain and a kfoC gene derived from Escherichia coli K4 strain and has chondroitin-producing ability.
Here, the kfoA gene derived from Escherichia coli K4 strain is preferably a gene encoding a protein selected from the group consisting of the following (A) and (B):
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2; and
(B) a protein comprising an amino acid sequence of SEQ ID NO: 2 including substitution, deletion, insertion, or addition of one or several amino acids and having UDP-glucose-4-epimerase activity.
The kfoA gene derived from Escherichia coli K4 strain is preferably a DNA selected from the group consisting of the following (a) and (b):
(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1; and
(b) a DNA that hybridizes with a DNA comprising the nucleotide sequence complementary to SEQ ID NO: 1 under stringent conditions and encodes a protein comprising UDP-glucose-4-epimerase activity.
In addition, the kfoC gene derived from Escherichia coli K4 strain is preferably a gene encoding a protein selected from the group consisting of the following (C) and (D):
(C) a protein comprising the amino acid sequence of SEQ ID NO: 4; and
(D) a protein comprising an amino acid sequence of SEQ ID NO: 4 including substitution, deletion, insertion, or addition of one or several amino acids and having chondroitin synthase activity.
In addition, the kfoC gene derived from Escherichia coli K4 strain is preferably a DNA selected from the group consisting of the following (c) and (d):
(c) a DNA comprising the nucleotide sequence of SEQ ID NO: 3; and
(d) a DNA that hybridizes with a DNA comprising the nucleotide sequence complementary to SEQ ID NO: 3 under stringent conditions and encodes a protein having chondroitin synthase activity.
In addition, the kfoC gene derived from Escherichia coli K4 strain is preferably a gene encoding a protein selected from the group consisting of the following (E) and (F):
(E) a protein comprising the amino acid sequence of SEQ ID NO: 6; and
(F) a protein comprising an amino acid sequence of SEQ ID NO: 6 including substitution, deletion, insertion, or addition of one or several amino acids and having chondroitin synthase activity.
In addition, the kfoC gene derived from Escherichia coli K4 strain is preferably a DNA selected from the group consisting of the following (e) and (f):
(e) a DNA comprising the nucleotide sequence of SEQ ID NO: 5l ; and 
(f) a DNA that hybridizes with a DNA comprising the nucleotide sequence complementary to SEQ ID NO: 5 under stringent conditions and encodes a protein having chondroitin synthase activity.
The UDP-glucronic acid-producing bacterium is preferably Escherichia coli K5 strain.
Another object of the present invention is to provide a method of producing chondroitin comprising at least the following steps (1) and (2):
(1) culturing the bacterium as described above; and
(2) collecting chondroitin from the culture.
Another object of the present invention is to provide a method of producing chondroitin sulfate comprising: producing chondroitin by the method as described above; and then sulfating the chondroitin to yield chondroitin sulfate.
Another object of the present invention is to provide a vector comprising a kfoA gene derived from Escherichia coli K4 strain and a kfoC gene derived from Escherichia coli K4 strain.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a drawing showing a structure of a vector for expressing kfoA and kfoC genes.
FIG. 2 is a drawing (photograph) showing expression of KfoA and KfoC proteins. Lane 1 shows a strain introduced with no plasmid (control), Lane 2 shows a strain introduced with pTrcHis-kfoCA, and M shows a molecular weight marker.
FIG. 3 shows the results of disaccharide composition analyses using fluorescent HPLC system for the fraction L treated with chondroitinase ABC (cABC) (A), the fraction L treated with no cABC (B), and the fraction L treated with heat-inactivated cABC (C).
FIG. 4 shows results of fluorescence disaccharide analyses for the fraction E treated with cABC (A), the fraction E treated with no cABC (B), and the fraction E treated with heat-inactivated cABC (C).
FIG. 5 shows results of fluorescence disaccharide analyses for the fraction S treated with cABC (A), the fraction S treated with no cABC (B), and the fraction S treated with heat-inactivated cABC (C).
FIG. 6 shows results of fluorescence disaccharide analyses for the chondroitin standard treated with cABC (A) and a mixture of the chondroitin standard treated with cABC and the fraction L treated with cABC (B).
FIG. 7 is a drawing (photograph) showing expression of KfoA, KfoC and KfoC proteins using (A) an anti-KfoC antibodies (EK-C) as 1st antibody, (B) an anti-KfoA antibodies (EK-A) as 1st antibody and (C) normal rabbit serum. Lane 1 and 5 show molecular marker, MagicMarkXP standard (Invitrogen), Lane 2 shows a strain introduced with pTrcHis-kfoCA, Lane 3 shows a strain introduced with pTrcHis-kfo CA and Lane 4 shows a strain introduced with pTrcHis-kfoA.



DESCRIPTION OF THE PREFERRED EMBODIMENTS
Hereinafter, the present invention will be described in detail.
<1> Bacterium of the Present Invention
The bacterium of the present invention is a UDP-glucuronic acid-producing bacterium introduced with kfoA and kfoC genes derived from Escherichia coli K4 strain and having chondroitin-producing ability.
The “UDP-glucuronic acid-producing bacterium” provides UDP-glucuronic acid, which is used for production of polysaccharide composed of glucuronic acid. The “UDP-glucuronic acid-producing bacterium” is not limited as long as it produces UDP-glucuronic acid, and examples thereof include a bacterium belonging to the genus Gluconacetobacter such as Gluconacetobacter hansenii and Gluconacetobacter xylinus, a bacterium belonging to the genus Rhizobium such as Rhizobium meffloti, a bacterium belonging to the genus Acetobacter such as Acetobacter xylinum, a bacterium belonging to the genus Erwinia such as Erwinia amylovora, a bacterium belonging to the genus Thiobacillus such as Thiobacillus ferrooxidans, a bacterium belonging to the genus Xylella such as Xylella fastidiosa, a bacterium belonging to the genus Sinorhizobium such as Sinorhizobium meliloti, a bacterium belonging to the genus Rhodococcus such as Rhodococcus rhodochrous, a bacterium belonging to the genus Klebsiella such as Klebsiella aerogenes, a bacterium belonging to the genus Enterobacter such as Enterobacter aerogenes, and a bacterium belonging to the genus Escherichia such as Escherichia coli. 
The “UDP-glucuronic acid-producing bacterium” is preferably a bacterium belonging to the genus Escherichia and capable of producing UDP-glucuronic acid, and more preferably a bacterium belonging to Escherichia coli and capable of producing UDP-glucuronic acid.
A specific example thereof includes Escherichia coli K5 strain. The K5 strain has been deposited at the American Type Culture Collection (ATCC: P.O. Box 1549, Manassas, Va. 20108, United States of America) under the accession number of ATCC23506 and is available from the catalogue or homepage of ATCC.
The strain to be introduced with the kfoA and kfoC genes may be a strain which is a derivative of the Escherichia coli K5 strain, and such derivative strains may be obtained by introducing a gene mutation into the Escherichia coli K5 strain or introducing a gene into the Escherichia coli K5 strain by genetic recombination. That is, one aspect of the bacterium of the present invention includes: a strain obtained by introducing kfoA and kfoC genes derived from Escherichia coli K4 strain into Escherichia coli K5 strain; a strain obtained by introducing kfoA and kfoC genes derived from Escherichia coli K4 strain into Escherichia coli K5 strain and further introducing a gene mutation; and a strain obtained by introducing kfoA and kfoC genes derived from Escherichia coli K4 strain into Escherichia coli K5 strain and further introducing another gene.
Examples of the kfoA gene derived from Escherichia coli K4 strain include a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 and a DNA comprising the nucleotide sequence of SEQ ID NO: 1.
In general, substitution, deletion, insertion, or addition in one or several amino acids that constitute a protein has no effect on the activity of the protein, in many cases, and therefore, the kfoA gene derived from Escherichia coli K4 strain may be a gene encoding a protein comprising an amino acid sequence of SEQ ID NO: 2 including substitution, deletion, insertion, or addition of one or several amino acids and having UDP-glucose-4-epimerase activity.
The phrase “one or several amino acids” as used herein refers to the number of amino acids that may cause a substitution, deletion, insertion, or addition without impairing UDP-glucose 4-epimerase activity. Specifically, the number is, for example, an integer of 1 to 20, preferably an integer of 1 to 10, more preferably an integer of 1 to 5.
Meanwhile, the kfoA gene derived from Escherichia coli K4 strain may be a gene encoding a protein comprising an amino acid sequence that is not less than 90% identical, preferably not less than 95% identical, more preferably not less than 98% identical to the entire sequence of SEQ ID NO: 2, and having UDP-glucose 4-epimerase activity.
The kfoA gene derived from Escherichia coli K4 strain may be obtained by PCR using a chromosomal DNA of Escherichia coli K4 strain as a template.
Escherichia coli K4 strain has been deposited at the American Type Culture Collection (ATCC) under the accession number of ATCC23502 and is available from the catalogue or homepage of ATCC.
The gene encoding a protein comprising an amino acid sequence of SEQ ID NO: 2 including substitution, deletion, insertion, or addition of one or several amino acids or an amino acid sequence that is not less than 90% identical to the entire sequence of SEQ ID NO: 2 and having UDP-glucose-4-epimerase activity may be obtained by modifying the nucleotide sequence of SEQ ID NO: 1 so that substitution, deletion, insertion, or addition of one or several amino acids is introduced into the amino acid sequence of SEQ ID NO: 2 by site-specific mutagenesis (Kramer, W. and Frits, H. J., Meth. in Enzymol., 154, 350(1987); Kunkel, T. A. et al., Meth. in Enzymol., 154, 367(1987)) or the like.
The gene may also be obtained by: screening a DNA encoding a protein having UDP-glucose 4-epimerase activity by hybridization with a DNA comprising the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or a partial sequence thereof under stringent conditions.
The term “stringent conditions” refers to conditions under which so-called specific hybrid is formed and non-specific hybrid is not formed (see Sambrook, J. et al., Molecular Cloning A Laboratory Manual, second Edition, Cold Spring Harbor Laboratory Press (1989), etc.). Specific examples of the “stringent conditions” include conditions for hybridization at 42° C. in a solution containing 50% formamide, 4×SSC, 50 mM HEPES (pH 7.0), 10× Denhardt's solution, 100 μg/ml salmon sperm DNA and washing with 2×SSC, 0.1% SDS solution at room temperature and then 0.1×SSC, 0.1% SDS solution at 60° C.
Whether the gene obtained as described above encodes a protein having UDP-glucose 4-epimerase activity can be judged by introducing the resultant gene into an appropriate host to express the protein and then measuring the UDP-glucose 4-epimerase activity in accordance with the method described in J. Biol. Chem., 277(24), p 21567-21575, 2002.
Examples of the kfoC gene derived from Escherichia coli K4 strain include a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 4 and a DNA comprising the nucleotide sequence of SEQ ID NO: 3. Examples of the kfoC gene derived from Escherichia coli K4 strain also include a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 6 and a DNA comprising the nucleotide sequence of SEQ ID NO: 5.
The kfoC gene derived from Escherichia coli K4 strain may be a gene encoding a protein comprising an amino acid sequence of SEQ ID NO: 4 or 6 including substitution, deletion, insertion, or addition of one or several amino acids and having chondroitin synthase activity.
The kfoC gene derived from Escherichia coli K4 strain may be a gene encoding a protein comprising an amino acid sequence that is not less than 90% identical, preferably not less than 95% identical, more preferably not less than 98% identical to the entire sequence of SEQ ID NO: 4 or 6 and having chondroitin synthase activity.
The term “chondroitin synthase activity” as used herein refers to an activity to alternately transfer GlcUA from a GlcUA donor or GalNAc from a GalNAc donor to the non-reduced end of a sugar chain.
The kfoC gene derived from Escherichia coli K4 strain may be obtained by PCR using a chromosomal DNA of Escherichia coli K4 strain as a template. Furthermore, the kfoC gene encoding the amino acid sequence of SEQ ID NO: 6 such as a DNA comprising the nucleotide sequence of SEQ ID NO: 5 may be obtained according to the method described in WO2007/145197.
The gene encoding a protein comprising an amino acid sequence of SEQ ID NO: 4 or 6 including substitution, deletion, insertion, or addition of one or several amino acids or an amino acid sequence that is not less than 90% identical to the entire sequence of SEQ ID NO: 4 or 6 and having chondroitin synthase activity may be obtained by modifying the nucleotide sequence of SEQ ID NO: 3 or 5 so that substitution, deletion, insertion, or addition of one or several amino acids is introduced into the amino acid sequence of SEQ ID NO: 4 or 6 by site-specific mutagenesis or the like.
The gene may also be obtained by: screening a DNA encoding a protein having chondroitin synthase activity by hybridization with a DNA comprising the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 3 or 5 or a partial sequence thereof under stringent conditions.
The definitions of the terms “one or several” and “stringent conditions” are the same as described above.
Whether the gene obtained as described above encodes a protein having chondroitin synthase activity can be judged by introducing the resultant gene into an appropriate host to express the protein and then measuring the chondroitin synthase activity in accordance with the method described in U.S. 2003-0109693 (JP 2003-199583 A).
The bacterium of the present invention can be obtained by introducing a kfoA gene derived from Escherichia coli K4 strain and a kfoC gene derived from Escherichia coli K4 strain into a UDP-glucuronic acid-producing bacterium as described above.
The term “introduction” used in the present invention includes introduction of both of the genes into a UDP-glucuronic acid-producing bacterium using a vector such as a plasmid or a bacteriophage and introduction of both of the genes on a chromosome of the UDP-glucuronic acid-producing bacterium by homologous recombination or the like.
The vector such as a plasmid or a bacteriophage to be used herein is not particularly limited as long as they can be used for gene introduction into Escherichia coli, and examples thereof include pTrcHis (Invitrogen Corporation), pET vector (Novagen), and pGEX vector (Amersham Pharmacia).
In introduction of a kfoA gene derived from Escherichia coli K4 strain and a kfoC gene derived from Escherichia coli K4 strain, native promoters of the genes may be used, but it is preferable to use a promoter that is potent in Escherichia coli, such as lac promoter, trp promoter, trc promoter, PR promoter, or lacUV promoter.
The kfoA gene and kfoC gene derived from Escherichia coli K4 strain may be introduced one by one or simultaneously introduced using a single vector. It is more preferable to use a vector such as a plasmid or a bacteriophage carrying both of the kfoA and kfoC genes because introduction and expression of the genes can be easily performed.
The kfoA and kfoC genes derived form Escherichia coli K4 strain may be introduced so that they are expressed as fusion proteins with other peptides.
Examples of the other peptides include polyhistidine tag and GST (glutathione-S-transferase) tag. Expression of the gene products as fusion proteins is preferable because detection (confirmation of expression) of the gene products can be easily performed.
Introduction of a gene may be performed by a known transformation method. Examples of the method include the electroporation method, the DEAE-dextran method, and the calcium phosphate method.
Introduction of a gene can be confirmed by: a method of detecting a gene, such as RT-PCR or Northern blotting; a method of peculiarly detecting expression of a recombinant protein, such as Western blotting; and an activity measurement method as described above.
If the kfoA gene derived from Escherichia coli K4 strain and the kfoC gene derived from Escherichia coli K4 strain introduced work in a UDP-glucuronic acid-producing bacterium, the bacterium acquires the ability to produce chondroitin as a capsular polysaccharide.
<2> Method of Producing Chondroitin
The method of producing chondroitin of the present invention comprises at least the following steps (1) and (2).
(1) culturing a bacterium of the present invention.
(2) collecting chondroitin from the culture.
The culture may be performed by a general method for culturing bacteria belonging to the genus Escherichia. 
The culture medium is not particularly limited as long as it can be used for culture of bacteria belonging to the genus Escherichia, and preferable examples thereof include LB medium (Luria-Bertani medium) (containing 10.0 g of Bacto-tryptone, 5.0 g of Bacto-yeast extract, and 5.0 g of NaCl per liter) and CYG medium (2.0% casamino acid, 0.5% yeast extract, and 0.2% glucose, adjust pH to 7.0 before autoclaving). In the case where a gene is introduced using a vector containing an antibiotics-resistant gene, the medium preferably contains an antibiotics corresponding to the gene.
Culture conditions are not particularly limited as long as bacteria belonging to the genus Escherichia can grow, and in order to produce chondroitin at high efficiency, culture is preferably performed at 20 to 40° C. for 8 to 72 hours.
In order to produce chondroitin at high efficiency, a bacterium may be precultured in a plate or liquid medium.
The method of collecting chondroitin from culture is not particularly limited as long as chondroitin can be collected, and examples thereof include the method as described in the Examples shown below. Specifically, harvested recombinant cells are suspended into PBS (phosphate-buffered saline) or the like; treating the cells with lysozyme, DNaseI, and proteinase K; and removing proteins. Chondroitin can be detected by, for example, treating the fraction with chondroitinase and performing a disaccharide composition analysis.
<3> Method of Producing Chondroitin Sulfate
Chondroitin sulfate can be produced by further sulfating chondroitin prepared from the recombinant bacteria disclosed in this invention.
The sulfation may be performed by a known method of sulfating glycosaminoglycan, which is not particularly limited, and examples thereof include the method described in JP 61-47701 A.
In addition, the sulfation may be performed using an enzyme for transferring a sulfate group to chondroitin (sulfotransferase). Under existence of 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as a sulfate donor, sulfotransferases can produce chondroitin sulfate by reacting to chondroitin produced by the recombinant bacteria disclosed in this invention. Examples of a known sulfotransferase for transferring a sulfate group to chondroitin include chondroitin 6-O-sulfate group transferase (J. Biol. Chem., 275 (28), 21075-21080 (2000)) and galactosaminoglycan 4-sulfate group transferase (JP 2000-4877 A).
EXAMPLES
Hereinafter, the present invention will be described in detail by referring to the examples.
Example 1
Preparation of KfoC/KfoA Co-Expression Vector
<Amplification of kfoA Gene>
PCR was performed using a chromosomal DNA of E. coli K4 strain as a template according to the method described in J. Biol. Chem., Vol. 277, Issue 24, 21567-21575, and the resultant PCR product was inserted into pTrcHis (histidine fusion protein expression vector, Invitrogen), to thereby yield pTrcHis-kfoA.
Primers used are as follows:








K4A-SP:



5′-CGGGATCCCGATGAGTATTCTTAATCAAGC-3′
(SEQ ID NO: 7)


and


 


K4A-AS:


5′-GGAATTCCGGCCAGTCTACATGTTTATCAC-3′
(SEQ ID NO: 8)






<Amplification of kfoC Gene>

PCR was performed using a chromosomal DNA of E. coli K4 strain as a template according to the method described in JP 2003-199583 A, and the resultant PCR product was inserted into pTrcHis, to thereby yield pTrcHis-kfoC.
Primers used are as follows:







(SEQ ID NO: 9)








K4C-SP:
5′-CGGGATCCCGATGAGTATTCTTAATCAAGC-3′


and


 







(SEQ ID NO: 10)








K4C-AS:
5′-GGAATTCCGGCCAGTCTACATGTTTATCAC-3′.






<Preparation of pTrcHis-kfoCA>

pTrcHis-kfoCA was prepared from the above-mentioned pTrcHis-kfoA and pTrcHis-kfoC as follows.
First, the NotI-SalI site was introduced into the C-terminal sequence of pTrcHis-kfoC by PCR. PCR was performed using the primers shown below to amplify the full length of pTrcHis-kfoC from the C-terminal side of kfoC, and the resultant PCR product was self-ligated, to thereby yield a plasmid pTrcHis-kfoC-NotI-SalI.
Primers used are as follows:







(SEQ ID NO: 11)









NotSal-pTrcHisC-rev:



5′-GCGGCCGCAAAACAGCCAAGCTTCGAATTC-3′



and



 







(SEQ ID NO: 12)









NotSal-pTrcHisC-for:



5′-ACGCGTCGACGGCGGATGAGAGAAGATTTTCA-3′






Then, Primers (NotI-RBS-KfoA-N: 5′-GCGGCCGCAAAATTAAAGAGGTATATATTAATGTATCGA-3′ (SEQ ID NO: 13) and SalI-KfoA-C: 5′-GTCGACCTCTCATCCGCCAAAACA-3′ (SEQ ID NO: 14)) were designed at the upstream of the ribosome binding site (RBS) and the C-terminal region of kfoA of pTrcHis-kfoA, respectively and used for PCR using pTrcHis-kfoA as a template. Then, the resultant PCR product was inserted into pCR4-TOPO (Invitrogen), to thereby yield pCR4-TOPO-kfoA.
An insert containing kfoA was excised from pCR4-TOPO-kfoA with NotI-SalI and introduced into the NotI-SalI site of pTrcHis-kfoC-NotI-SalI, and the resultant plasmid was named pTrcHis-kfoCA (FIG. 1).
Example 2
Co-Expression of the Recombinant KfoC and KfoA
The expression vector, pTrcHis-kfoCA, was introduced into Escherichia coli K5 strain by electroporation (cell 100 μL, 200 Ω, 25 μF, 2.5 kV, cuvette 0.1 cm), to thereby yield E. coli K5/pTrcHis-kfoCA strain. Colonies were transferred into LB medium supplemented with 100 ppm ampicillin, and then the seed culture was incubated at 37° C. overnight. One mL of the seed culture was transferred to 20 mL of CYG medium supplemented with 100 ppm ampicillin. The recombinant bacteria were cultured at 37° C. for 3 hours (OD600=1.8, 1.7-2.0), and then to the culture was added IPTG at a final concentration of 1.0 mM. The resulting culture was cultivated at 37° C. for further 5 hours to induce expression of the recombinant proteins.
Bacterial cells were harvested from 900 μL of the culture by centrifugation. The obtained cells were suspended in 90 μL of Laemmli buffer (×1). After heating in a boiling water bath for 10 min, 10 μL of the supernatant was subjected to SDS-PAGE in a 5/20% gradient gel and transferred to a PVDF membrane, followed by Western analysis to detect expression of recombinant proteins. Anti-Penta-His-HRP (QIAGEN) diluted to 1/2,000 was used for detection of recombinant histidine fusion proteins. As a result, expressions of the recombinant proteins KfoA and KfoC were confirmed (FIG. 2).
Example 3
Preparation of Polysaccharide and its Detection by Disaccharide Analysis
<Preparation of Bacterial Cells>
E. coli K5/pTrcHis-kfoCA strain was cultured in LB medium supplemented with 100 ppm ampicillin as a seed culture. The seed culture (750 μL) was transferred into CYG medium supplemented with 100 ppm ampicillin (15 mL), and then it was cultured at 37° C. for 3 hours. To the culture was added IPTG (final concentration: 1 mM), and cultivation was further performed for 5 hours.
The bacterial cells were harvested by centrifugation.
<Preparation of Culture Supernatant>
The seed culture (1 mL) was transferred into CYG medium supplemented with 100 ppm ampicillin (20 mL), and then it was cultured at 37° C. for 3 hours. To the culture was added IPTG (final concentration: 0.1 mM), and cultivation was further performed for 5 hours. The culture supernatant was harvested by centrifugation.
<Preparation of Polysaccharide Fraction>
Polysaccharides were prepared from the recombinant cells and culture supernatant by the following three methods.
Preparation 1) The obtained bacterial described above were resuspended in PBS and treated with lysozyme and DNaseI at 37° C. for 1 hour, followed by treatment with proteinase K at 37° C. for 1 hour. Then, the enzymes were subjected to heat inactivation, and proteins were removed by precipitation with 70% ammonium sulfate. The resulting supernatant fraction was dialyzed against 10 mM Tris-HCl (pH 8.0) to yield Sample L.
Preparation 2) The obtained bacterial described above were treated with the agents for purifying plasmid: P1 (containing lysozyme), P2, and P3 (QIAGEN) sequentially, according to the manufacturer's instructions. After harvesting by centrifugation, the supernatant was subjected to ethanol precipitation. The precipitates were harvested by centrifugation and resuspended in 100 μL of sterilized water. The suspension was treated with DNaseI at 37° C. for 1 hour to digest DNAs, followed by treatment with proteinase K at 37° C. for 1 hour. Then, the enzymes were subjected to heat inactivation, and proteins were removed by precipitation with 70% ammonium sulfate. The resulting supernatant fraction was dialyzed against 10 mM Tris-HCl (pH 8.0) to yield Sample E.
Preparation 3) The culture supernatant (20 mL) was evaporated to dryness. The dried materials were dissolved into 2 ml of distilled water. The solution was dialyzed against 10 mM Tris-HCl (pH 8.0) to yield Sample S.
<Detection of Chondroitin by Disaccharide Analysis>
Samples L, E and S (100 μL each) were treated with cABC (chondroitinase ABC; manufactured by Seikagaku Corporation) in 50 mM Tris-HCl buffer (pH 8.0) containing 50 mM NaOAc at 37° C. overnight.
As controls, Samples L, E and S treated with no cABC and Samples L, E and S treated with heat-inactivated cABC were prepared. Ultrafiltration was performed to remove macromolecules with sizes equal to or more than 10,000, and the samples were analyzed by disaccharide composition analysis using a fluorescent HPLC system (J Biol Chem. 2000 Jul. 21;275(29):2269-2275.) (FIGS. 3, 4 and 5). Senshu Pak Decosil C22 (4.6 I.D.×150 mm) was used as a separation column in the HPLC system, and excitation and emission wavelength of the fluorescence detector were set to 346 nm and 410 nm, respectively.
As a result, only in the cases of the samples treated with cABC, specific peaks were detected. The disaccharide composition profile of each sample was closely corresponding to the profile of the sample obtained by treating the chondroitin standard with cABC as shown in FIG. 6(A). Meanwhile, in the case where the chondroitin standard treated with cABC was mixed with Sample L treated with cABC, only one peak was detected, and thus Sample L was confirmed to be containing chondroitin (FIG. 6(B)). In the case of Sample E and S, the same results were obtained (data not shown).
Example 4
Preparation of KfoC/KfoA co-expression vector
<Amplification of kfoC Gene>
According to the method described in WO2007/145197, a DNA encoding the N-terminal-truncated KfoC comprising the nucleotide sequence of SEQ ID NO: 5 (kfoC gene) was cloned. The kfoC gene was inserted into pTrcHis to obtain pTrcHis-kfoC.
<Preparation of pTrcHis-kfoCA>
The plasmid pTrcHis-kfoCA prepared in the Example 1 was cleaved using restriction enzymes SphI and SmaI.
DNA fragment (2.9 kDa) was obtained by digestion of the plasmid pTrcHis-kfoC using the same restriction enzymes. According to the conventional method, the DNA fragment was inserted into the SphI-SmaI site of the cleaved plasmid, and then the resulting plasmid was named pTrcHis-kfoCA.
Example 5
Preparation of Anti-KfoA and Anti-KfoC Antibodies
<Preparation of Anti-KfoA Antibodies>
Oligopeptide comprising the peptide sequence of SEQ ID No: 15 (CIVSR RDGDI AESWS SPEKA NK, Purity: >70%) was synthesized, and 4 mg of the oligopeptide was conjugated with KLH (keyhole limpet hemocyanin) by conventional method. After preparing the antigen solution by mixing with complete adjuvant, immunization was carried out 5 times in the interval for 2 weeks. The whole blood was collected, after antibody titre is confirmed. From each rabbit blood, anti-KfoA serum was obtained 48 mL (Sample A) and 60 mL (Sample B), respectively. After purification using Protein A column, IgG fraction EK-A was obtained 32 ml (IgG concentration: 5.30 mg/mL) from Sample A. <Preparation of Anti-KfoC Antibodies>
Oligopeptide comprising the peptide sequence of SEQ ID No: 16 (CQEPP GKENE TDRAA GK, Purity: >70%) was synthesized, and 4 mg of the oligopeptide was conjugated with KLH (keyhole limpet hemocyanin) by conventional method. After preparing the antigen solution by mixing with complete adjuvant, immunization was carried out 5 times in the interval for 2 weeks. The whole blood was collected, after antibody titre is confirmed. From each rabbit blood, anti-KfoA serum was obtained 65 mL (Sample A) and 39 mL (Sample B), respectively. After purification using Protein A column, IgG fraction EK-A was obtained 39 ml (IgG concentration: 4.41 mg/mL) from Sample A.
Example 6
In the same manner as in Example 2, the plasmid pTrcHis-kfo CA was introduced into the E. coli K5 strain to obtain E. coli K5/pTrcHis-kfoCA strain, and the recombinant strain was cultured to evaluate the expression of the recombinant proteins.
The seed culture grown in LB medium supplemented with 100 ppm ampicillin (0.5 mL) was transferred into CYG medium supplemented with 100 ppm ampicillin (10 mL), and then the culture was carried out at 37° C. for 1.5 hours. After adding IPTG (final concentration: 1 mM), the cultivation was continued for further 4 hours. The bacterial cells were harvested from 900 μL of the culture and suspended into 90 μL of Laemmli buffer (×1). After heating in a boiling water bath for 10 min, 10 μL of the supernatant was subjected to SDS-PAGE in a 7.5% gel and transferred to a PVDF membrane, followed by Western analysis to detect expression of recombinant proteins. Anti-KfoA antibodies (EK-A) and anti-KfoC antibodies (EK-C) diluted to 1/1,000 was used as 1st antibody for detection of recombinant proteins KfoA, KfoC and KfoC. Anti-Rabbit Immunoglobulins HRP (DAKO,#PO448) was used as 2nd antibody. In addition, the same examinations using the culture of E. coli K5/pTrcHis-kfoCA and E. coli K5/pTrcHis-kfoA were carried out to compare the expression level of recombinant proteins. As a result, there was no the great difference on expression level of KfoA and KfoC(KfoC) between E. coli K5/pTrcHis-kfoCA and E. coli K5/pTrcHis-kfoA (FIG. 7).
Example 7
Preparation of Polysaccharide and its Detection by Disaccharide Analysis
<Preparation of Bacterial Cell>
In the same manner as in Example 3, bacterial cells and supernatant from the culture of E. coli K5/pTrcHis-kfoCA, E. coli K5/pTrcHis-kfoCA and E. coli K5/pTrcHis-kfoA strains were prepared after autoclaving at 121° C. for 5 min.
<Preparation of Polysaccharide Fraction>
The supernatant (1 mL) was dialyzed against running tap water overnight, and then dialyzed Solution was lyophilized. The lyophilized materials were dissolved into 100 μL of 50 mM Tris-HCl (pH8.0) to use as a polysaccharide fraction.
<Detection of Chondroitin by Disaccharide Analysis>
In the same manner as in Example 3, the obtained fraction was analyzed by the disaccharide composition analysis. Chondroitin standard solution (200 μg/mL and 100 μg/mL) was analyzed by the fluorescent HPLC to make the calibration curve of chondroitin. The relationship between chondroitin concentration and peak area of unsaturated double sugar, Di-OS, was shown in Equation-1.

[Chondroitin;μg/mL of supernatant]=(0.000591×[Peak area]+1.219)/[concentration rate] (r2=0.9984)  (Equation-1)

The peak agreed with unsaturated disaccharide (Di-OS) was detected in the samples prepared from the E. coli K5/pTrcHis-kfo CA and E. coli K5/pTrcHis-kfoCA (Data not shown). Table 1 shows the result of chondroitin concentration calculated using the Equation-1 in the recombinant cultures.
Table 1 Comparison of chondroitin content in the culture of the recombinant strains.
















Chondroitin


E. coli K5
Concentration
Peak area
[μg/mL]


















K5/pTrcHis-kfoA
10
10,187.4
0.7


K5/pTrcHis-kfoCA
10
505,032.3
30.0


K5/pTrcHis-kfo CA
10
888,677.6
52.6









By comparing chondroitin productivity between E. coli K5/pTrcHis-kfoCA and E. coli K5/pTrcHis-kfoCA, the recombinant strain E. coli K5/pTrcHis-kfoCA is more suitable for producing chondroitin by fermentation. This result suggested that chondroitin production could be enhanced by using the kfoC gene which encodes N-terminal-truncated KfoC.
INDUSTRIAL APPLICABILITY
By using the bacterium of the present invention, chondroitin and chondroitin sulfate can be produced at high efficiency and at low cost.

## Claims

1. An isolated UDP-glucuronic acid-producing bacterium, which is introduced with a kfoA gene derived from the Escherichia coli K4 strain and a kfoC gene derived from Escherichia coli K4 strain, wherein said bacterium has chondroitin-producing ability, and wherein the kfoA gene encodes a protein selected from the group consisting of (A) and (B), and wherein the kfoC gene encodes a protein selected from the group consisting of (C), (D), (E) and (F):
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2;
(B) a protein comprising the amino acid sequence of SEQ ID NO: 2 comprising substitution, deletion, insertion, or addition of 1-20 amino acids and having UDP-glucose-4-epimerase activity;
(C) a protein comprising the amino acid sequence of SEQ ID NO: 4;
(D) a protein comprising the amino acid sequence of SEQ ID NO: 4 comprising substitution, deletion, insertion, or addition of 1-20 amino acids and having chondroitin synthase activity;
(E) a protein comprising the amino acid sequence of SEQ ID NO: 6; and
(F) a protein comprising the amino acid sequence of SEQ ID NO: 6 comprising substitution, deletion, insertion, or addition of 1-20 amino acids and having chondroitin synthase activity.

2. The bacterium according to claim 1, wherein the kfoA gene is a DNA selected from the group consisting of (a) and (b), and wherein the kfoC gene is a DNA selected from the group consisting of (c), (d), (e) and (f), and wherein stringent conditions are conditions for hybridization at 42° C. in a solution containing 50% formamide, 4×SSC, 50 mM HEPES (pH 7.0), 10×Denhardt's solution, 100 μg/ml salmon sperm DNA and washing with 2×SSC, 0.1% SDS solution at room temperature and then 0.1×SSC, 0.1% SDS solution at 60° C.:
(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1;
(b) a DNA that hybridizes with a DNA comprising the nucleotide sequence complementary to SEQ ID NO: 1 under stringent conditions and encodes a protein having UDP-glucose-4-epimerase activity;
(c) a DNA comprising the nucleotide sequence of SEQ ID NO: 3;
(d) a DNA that hybridizes with a DNA comprising the nucleotide sequence complementary to SEQ ID NO: 3 under stringent conditions and encodes a protein having chondroitin synthase activity;
(e) a DNA comprising the nucleotide sequence of SEQ ID NO: 5; and
(f) a DNA that hybridizes with a DNA comprising the nucleotide sequence complementary to SEQ ID NO: 5 under stringent conditions and encodes a protein having chondroitin synthase activity.

3. The bacterium according to claim 1, wherein said bacterium is Escherichia coli K5 strain.

4. A method of producing chondroitin comprising at least the following steps (1) and (2):
(1) culturing the bacterium according to claims 1; and
(2) collecting chondroitin from the culture.

5. A method of producing chondroitin sulfate comprising: producing chondroitin by the method according to claim 4; and sulfating the chondroitin to yield chondroitin sulfate.

6. A vector comprising a kfoA gene derived from Escherichia coli K4 strain and a kfoC gene derived from Escherichia coli K4 strain, wherein the kfoA gene is a DNA selected from the group consisting of (a) and (b), and wherein the kfoC gene is a DNA selected from the group consisting of (c), (d), (e) and (f), and wherein stringent conditions are conditions for hybridization at 42° C. in a solution containing 50% formamide, 4×SSC, 50 mM HEPES (pH 7.0), 10×Denhardt's solution, 100 μg/ml salmon sperm DNA and washing with 2×SSC, 0.1% SDS solution at room temperature and then 0.1×SSC, 0.1% SDS solution at 60° C.:
(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1;
(b) a DNA that hybridizes with a DNA comprising the nucleotide sequence complementary to SEQ ID NO: 1 under stringent conditions and encodes a protein having UDP-glucose-4-epimerase activity;
(c) a DNA comprising the nucleotide sequence of SEQ ID NO: 3;
(d) a DNA that hybridizes with a DNA comprising the nucleotide sequence complementary to SEQ ID NO: 3 under stringent conditions and encodes a protein having chondroitin synthase activity;
(e) a DNA comprising the nucleotide sequence of SEQ ID NO: 5; and
(f) a DNA that hybridizes with a DNA comprising the nucleotide sequence complementary to SEQ ID NO: 5 under stringent conditions and encodes a protein having chondroitin synthase activity.