WO2005103262A1

WO2005103262A1 - A novel diol dehydratase gene cluster in listeria genus

Info

Publication number: WO2005103262A1
Application number: PCT/KR2004/000901
Authority: WO
Inventors: Young-Hoon Park; Kwang-Myung Cho; Seong-Uk Kang
Original assignee: Cj Corp.
Priority date: 2004-04-20
Filing date: 2004-04-20
Publication date: 2005-11-03

Abstract

This invention provides polynucleotide sequences coding diol dehydratase set forth as SEQ ID NO.1 or SEQ ID NO.6. this invention also provides vector and host cell comprising the polynucleotide.

Description

A NOVEL DIOL DEHYDRATASE GENE CLUSTER IN LISTERIA GENUS

Technical Field The present invention relates to a diol dehydratase gene cluster, a vector and a host cell including the same.

Background Art 1 ,3-propanediol is a monomer utilized for the production of polyester fibers, polyurethanes, and cyclic compounds, and the like, Currently, various chemical synthesis processes of 1 ,3-propanediol have been well known. For example, 1 ,3-propanediol may be produced by catalytic reaction of ethylene oxide, phosphine, water, carbon monoxide, hydrogen, and acid; liquid-phase hydration of acrolein followed by reduction; or catalytic reaction of hydrocarbon such as glycerol, carbon monoxide, and hydrogen in the presence of a metal catalyst of Group VIII of the periodic table. However, the above chemical synthesis processes are costly and produce waste streams containing environmental pollutants. Meanwhile, fermentation of glycerol to 1 ,3-propandiol has also been known. Examples of bacteria that can produce 1 ,3-propanediol include Citrobacter sp., Closthdium sp., Enterobacter sp., Ilyobacter sp., Klebsiella sp., Lactobacillus sp., and Pelobactersp. Glycerol is subjected to an oxidative or reductive pathway in these bacteria. In the oxidative pathway of glycerol, first, glycerol is converted to dihydroxyacetone (DHA) by glycerol dehydrogenase. DHA is converted to dihydroxyacetone-phosphate (DHAP) by ATP-dependent kinase. DHAP is used in glycolysis pathway. In the reductive pathway of glycerol, first, glycerol is isomerized or dehydrated to 3-hydroxypropionaldehyde (3-HPA) by glycerol dehydratase. 3-HPA is converted to 1 ,3-propanediol by 1 ,3-propanediol:NAD+oxidoreductase. At this time, 1 ,3-propanediol is not further metabolized. In Klebsiella pneumoniae, other enzymes involved in the oxidative pathway as well as the above two enzymes involved in the reductive pathway are coordinately regulated. The above-described four enzyme systems are functionally linked, and thus, the conversion of glycerol to 1 ,3-propanediol is dependent on the presence of a reductant that is fed into the pathway from DHA to DHAP. In this regard, to overcome disadvantages due to interdependence of these enzyme systems, recombinant bacteria containing a foreign gene that participates in the production of 1 ,3-propanediol were developed. For example, U.S. Patent No. 6,136,576 discloses a method for producing 1 ,3-propanediol using a recombinant microorganism including at least nucleic acid encoding a dehydratase activity and a nucleic acid encoding a protein X.

According to the method, the recombinant microorganism is cultured in the presence of at least one carbon source capable of being converted to 1 ,3 propanediol and under conditions suitable for the production of 1 ,3 propanediol. Here, the gene encoding the protein X is isolated from a glycerol dehydratase gene cluster of an organism selected from the group of genera consisting of Klebsiella and Citrobacter or a diol dehydratase gene cluster of an organism selected from the group of genera consisting of Klebsiella, Clostridium, and Salmonella. The recombinant microorganism produces 1 ,3-propanediol in a higher level, relative to a microorganism containing no the nucleic acid encoding the protein X. The protein X has no enzymatic activity. The carbon source is selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and a one-carbon substrate. However, until now, there have been no patents or documents about enzymes derived from the strains of the genus Listeria that participate in the production of 1 ,3-propanediol. There have been also no reports about nucleotide sequences of genes encoding the enzymes. Therefore, while searching for enzymes able to produce 1 ,3-propanediol from the strains of the genus Listeria, the present inventors found a novel gene cluster expressing a diol dehydratase activity and participating in the production of

1 ,3-propanedioS and completed the present invention. Disclosure of the Invention The present invention provides a diol dehydratase gene cluster derived from the genus Listeria, a vector and a host cell including the same.

Brief Description of the Drawings FIG. 1 is a genetic map of a pSE-380 vector containing a diol dehydratase gene cluster derived from Listeria innocua. FIG. 2 is a genetic map of a pSE-380 vector containing a diol dehydratase gene cluster derived from Listeria monocytogenes. FIG. 3 is a genetic map of a pSE-380 vector containing a diol dehydratase gene cluster derived from Klebsiella pneumoniae.

Best mode for carrying out the Invention The present invention provides a polynucleotide including a nucleotide sequence encoding a diol dehydratase activity as set forth in SEQ ID NO: 1 or 6. The polynucleotide including the nucleotide sequence of SEQ ID NO: 1 is a gene cluster encoding a diol dehydratase activity derived from Listeria innocua. The gene cluster encodes polypeptides including amino acid sequences as set forth in SEQ ID NOS: 2, 3, 4, and 5. The polynucleotide including the nucleotide sequence of SEQ ID NO: 6 is a gene cluster encoding a diol dehydratase activity derived from Listeria monocytogenes. The gene cluster encodes polypeptides including amino acid sequences as set forth in SEQ ID NOS: 7, 8, 9, and 10. The term "diol dehydratase" indicates an enzymatic activity that catalyzes the conversion of a glycerol molecule to 3-hydroxypropionaldehyde. The present invention also provides a vector containing the polynucleotide including the nucleotide sequence of SEQ ID NO: 1 or 6. The vector is not particularly limited provided that a polynucleotide of an expected length is delivered into a host cell. Preferably, the vector is a vector that can be independently propagated in Escherichia sp. For example, the vector may be phage, plasmid, or cosmid. Examples of the phage and cosmid vectors include pWE15, M13, λ EMBL3, λ- EMBL4,

Λ. FIXII, DASHII, λ ZAPII, λ. gt11 , Charon4A, and Charon21A. Examples of the plasmid vector include pSE series, pBR series, pUC series, pBluescriptll series, pGEM series, pTZ series, and pET series. The present invention also provides a transformed host cell obtained by transforming a host cell with the vector containing the polynucleotide including the nucleotide sequence of SEQ ID NO: 1 or 6. The host cell is not particularly limited and may be selected from yeasts and bacteria. The bacteria may be gram-positive bacteria or gram-negative bacteria Preferably, the transformed host cell is Escherichia sp. and more preferably is E. coli KCCM-10468 and 10469 that have been deposited in the Korean Culture Center of Microorganisms (KCCM) on February 19, 2003 (deposition numbers: KCCM-10468 and 10469). The transformed cell may be manufactured by a conventional transformation method with the vector [Sambrook, J. et al., 2th (1989), Cold Spring Harbor Laboratory Press]. The present invention also provides a method for producing 1 ,3-propanediol, which includes: culturing the host cell transformed with the vector containing the polynucleotide including the nucleotide sequence of SEQ ID NO: 1 or 6 and recovering 1 ,3-propanediol from the culture. Novel gene clusters encoding a diol dehydratase activity according to the present invention were identified from the total genome sequences of Listeria innocua (KCTC 3586) and Listeria monocytogenes (KCTC 3569) by a homology search against the known nucleotide sequences of glycerol dehydratase gene clusters of Klebsiella pneumoniae, Clostridium pasteurianum, and Salmonella. The homology search was performed using BLAST program. Hereinafter, the present invention will be described more specifically by Examples. However, the following Examples are provided only for illustrations and thus the present invention is not limited -o or by them. Example 1 : Cloning of gene cluster of the total nucleotide seguence of Listeria innocua homologous to known diol dehydratase genes In this Example, a gene cluster of the total nucleotide sequence of Listeria innocua homologous to known diol dehydratase genes was searched using BLAST program and cloned. As a result, a gene cluster as presented in Table 1 below was obtained.

Table 1 : Results of Homology Search

*R.F: Reactivating Factor

Primers (SEQ ID NOS: 11 and 12) for amplification of the diol dehydratase gene cluster were designed using the search results. The diol dehydratase gene cluster was amplified and cloned as the follows. First, Listeria innocua (KCTC 3586) was cultured in a Brain heart infusion broth medium (Difco) at 37 ^°C for 16 hours and 100 βg of a chromosome was recovered using a chromosome recovery kit (Qiagen). Polymerase chain reaction (PCR) was performed using the recovered chromosome as a template and the primer pair as set forth in SEQ ID NOS: 11 and 12 to amplify and isolate a gene fragment of interest. The isolated gene fragment was cloned using topocloning kit (Invitrogen, America).

Example 2: Cloning of gene cluster of the total nucleotide seguence of Listeria monocytogenes homologous to known diol dehydratase genes In this Example, a gene cluster of the total nucleotide sequence of Listeria monocytogenes homologous to known diol dehydratase genes was searched using BLAST program and cloned. As a result, a gene cluster as presented in Table 2 below was obtained.

Table 2: Results of Homology Search

*R.F: Reactivating Factor

Primers (SEQ ID NOS: 13 and 14) for amplification of the diol dehydratase gene cluster were designed using the search results. The diol dehydratase gene cluster was amplified and cloned as the follows. First, Listeria monocytogenes (KCTC 3569) was cultured in a Brain heart infusion broth medium (Difco) at 37 ^"C for 16 hours and 100 g of a chromosome was recovered using a chromosome recovery kit (Qiagen). PCR was performed using the recovered chromosome as a template and the primer pair as set forth in SEQ ID NOS:13 and 14 to amplify and isolate a gene fragment of interest. The isolated gene fragment was cloned using topocloning kit (Invitrogen, America). Example 3: Cloning of gene cluster of the total nucleotide seguence of Klebsiella pneumoniae homologous to known diol dehydratase genes To construct a standard vector for diol dehydratase activity measurement, a gene fragment of interest was recovered, amplification isolated, and cloned in the same manner as in Examples 1 and 2 except that 200 βg of the chromosome of Klebsiella pneumoniae cultured according to Cameron method was amplified by PCR using a set of primers as set forth in SEQ ID NOS: 15 and 16. Example 4: Construction of recombinant vector (A) and insertion of the recombinant vector (A) into E. coli In order to induce insertion into an E.coli expression vector, pSE-380, the cloned gene fragment of Example 1 were digested with restriction enzymes, BamHI and Sad, followed by electrophoresis. Then, a novel gene fragment of 5.2 kb was recovered using a gel recovery kit (Qiagen). The recovered gene fragment was inserted between restriction sites, BamHI and Sad, of the pSE-380 and ligated using T4 ligase to construct a recombinant vector (A). The recombinant vector (A) thus constructed is shown in FIG. 1. After a host cell, E.coli DH5α , was transformed with the recombinant vector (A), the transformed E.coli cell was designated as E.coli DH5α /pCJP-015 and then deposited in the Korean Culture Center of Microorganisms (KCCM) on February 19, 2003 (accession number: KCCM-10468).

Example 5: Construction of recombinant vector (B) and insertion of the recombinant vector (B) into E. coli A recombinant vector (B) was constructed in the same manner as in Example 4 except that the cloned gene fragment of Example 2 digested with restriction enzymes, Kpnl and Sad, was inserted between restriction sites, Kpnl and Sad, of the pSE-380. The recombinant vector (B) thus constructed is shown in FIG. 2. After a host cell, E.coli DH5α , was transformed with the recombinant vector (B), the transformed E.coli cell was designated as E.coli DH5α /pCJP-015 and then deposited in the KCCM on February 19, 2003 (accession number: KCCM-10469).

Example 6: Construction of recombinant vector (C) and insertion of the recombinant vector (C) into E.coli A recombinant vector (C) was constructed in the same manner as in Examples 4 and 5 except that the cloned gene fragment of Example 3 digested with restriction enzymes, Nhel and Sad, was inserted between restriction sites, Nhe. and Ss , o the pSE-380. The recombinant vector (C) thus constructed is shown in FIG. 3. A host cell, E.coli DH5α , was transformed with the recombinant vector (C).

Example 7: Evaluation of activities of diol dehvdratases The transformed E. coli cells of Examples 4 through 6 and an E. coli cell (as control) transformed with a non-recombinant natural pSE-380 vector were cultured in 5 ml LB media containing a 100 μg ampicillin at 37 °C at 200 rpm for one day. The cultures were again inoculated on the same media for 4 hours until O.D. (600 nm) reached 0.1. After the addition of 1 mM of IPTG, the resultant cultures were centrifuged at 50 rpm for 4 hours and recovered. Proteins were separated from the IPTG-induced cultures using a protein recovery kit (B-PER kit; catalogue no. 500-006, Pierce) and quantified using a protein assay kit (catalogue no. 500-006, Bio-Rad). The activities of diol dehydratases among the quantified proteins were measured by MBTH method (Biotechnology and Bioengineering, 1998, Vol. 59, No. 5, pp.544-552). The diol dehydratase activities of the transformed E. coli cells of Examples 4 through 6 and the E. coli control cell are presented in Table 3 below.

Table 3: Activities of diol dehydratases

Industrial Applicability The polynucleotide encoding a diol dehydratase activity of the present invention is a novel gene cluster derived from Listeria innocua or monocytogenes and exhibits excellent expression characteristics relative to a known cio. dehydratase gene cluster.

INDICATIONS RELATING TO DEPOSITED MICROORGANISM O OTHERBIOLOGICA MATERIAL

A. The indications made below relate Io the deposited microorganism or other biological material referred to in the description on page 4 , line 3 B mEN -DTCATION OF DEPOSIT Further deposits are identified on an additional sheet | | Name of depositary institution KCCM (Korean Culture Center of Microorganisms) Address of depositary institution (Including postal code and country) 361-221 , Yuπm B/D, Honje 1 , Sudaemun, Seoul, 120-091 , Republic of Korea

Date of deposit Accession Number 19 February 2003 KCCM-10468 C ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet j- |

D DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (If the indications are not for all designated States)

E SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the International Bureau later (speajy the general nature of the indications e g "Accession Number of Deposit")

For receiving Office use only For International Bureau use only | I ThiB sheet was received with the international application | I This sheet was received by the International Bureau on Authorized officer Authorized officer

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OROTHERBIOLOGICAL MATERIAL (PCT Rule 13iι_t)

For receiving Office use only For International Bureau use only | | This sheet was received with the intemafaonal application | I This sheet was received by the International Bureau on Authonzed officer Authorized officer

Claims

What is claimed is: 1. A polynucleotide comprising a nucleotide sequence encoding a diol dehydratase activity as set forth in SEQ ID NO: 1 or 6.

2. A vector comprising the polynucleotide of claim 1.

3. A host cell transformed with the vector of claim 2.

4. The host cell of claim 3, which is Escherichia sp.

5. The host cell of claim 4, which is E. coli KCCM-10468 or E. coli KCCM-10469.