WO2006088307A1

WO2006088307A1 - Rhodococcus - e. coli shuttle vector

Info

Publication number: WO2006088307A1
Application number: PCT/KR2006/000521
Authority: WO
Inventors: Joowon Rhee; Junhyeong Cho; Sanghyun Lee; Ohjin Park; Si Jae Park
Original assignee: LG Chem Ltd
Current assignee: LG Chem Ltd
Priority date: 2005-02-16
Filing date: 2006-02-15
Publication date: 2006-08-24
Anticipated expiration: 2007-08-16
Also published as: KR100738002B1; US20060263868A1; KR20060092098A; TW200641127A

Abstract

The present invention relates to a novel Rhodococcus-E. coli shuttle vector containing a replication origin of novel plasmid derived from Rhodococcus erythropolis IAM 1484 and a DNA replication origin derived from E. coli, which can replicated bidirectionally in E. coli and Rhodococcus, recombinant vector in which a target gene is operably linked to the shuttle vector, and a microorganism transformed with the recombinant vector. The shuttle vector according to the present invention has relatively small size, can be replicable in various kinds of the genus Rhodococcus microorganism and has high replication stability in a host cell. Therefore, it is useful for the cloning of various genes derived from Rhodococcus.

Description

RHODOCOCCUS-E. COLI SHUTTLE VECTOR

FIELD OF THE INVENTION

The present invention relates to a novel Rhodococcus-E. coli shuttle vector containing a replication origin of novel plasmid derived from Rhodococcus erythropolis IAM 1484 and a DNA replication origin derived from E. coli, which can replicate bidirectionally in E. coli and Rhodococcus, a recombinant vector in which a target gene is operably linked to said Rhodococcus-E. coli shuttle vector, and a microorganism transformed with the recombinant vector.

BACKGROUND OF THE RELATED ART

The genus Rhodococcus microorganism produces biosurfactant (Philp, J.C. et al, Appl. Microbiol. Biotechnol, 59:318, 2002), and antibiotics (Hu, T.L., Water ScL Technol., 47:169, 2003), etc, as well as, biodegrades steroid compounds, bioconvert xenobiotic compound and nitril compounds (US 5,135,858) and degrades acrylic acid (US 5,998,180; US application no. 11/224,314). Since it has wide metabolism activity as the above, its importance has been emphasized gradually.

In light of its importance, genetic and physiologic research of the genus Rhodococcus microorganisms are widely belong conducted (Rahman, M.T. et al., Vet. Microbiol., 94:143, 2003), however, metabolism research of the genus Rhodococcus and similar microorganisms thereof are much restricted due to absence of proper host/vector system.(Finnerty, Annu. Rev. Microbiol., 46:193, 1992). Accordingly, effective host/vector system regarding the genus Rhodococcus host are required to produce strains having high metabolism activity. US 4,952,500 teaches an E. coli-Rhodococcus shuttle vector using circular plasmid obtained from the genus Rhodococcus H 13- A. US patent No. 4,920,054 teaches a shuttle vector replicable in Rhodococcus equi, Corynebacterium, Bacillus subtilis and Stappylococcus aureus using replication origin of Rhodococcus. US 5,654,180 also teaches a shuttle vector containing replication origin derived from a replicable plasmid (pRCOOl, pRC002, pRC003, ρRC004) in Rhodococcus rhodochrous and replication origin derived from replicable plasmid (pHSG299, pHSG298, pUC19, pUC18) in E. coli. KP 1999-048213 teaches a shuttle vector containing DNA replication origin derived from pEk of E. coli, DNA replication origin derived from PCSP21197 of Rhodococcus rhodochrous, an ampicilin resistant gene as a selection marker expressed in E. coli, and a kanamycin resistant gene as a selection marker in Rhodococcus rhodochrous. Also, JP 8-056669 teaches shuttle vector which can be bidirectionally replicated in Rhodococcus and E. coli, containing DNA replication origin derived from E. coli, and DNA replication origin which can be replicated in the genus Rhodococcus derived from plasmid (pNC500, pNC 903) from a strain belongs to nocardiform bacterium.

Besides, shuttle vectors using plasmids derived from Rhodococcus fascians (Desomer et al, J. Bacteriol., 170:2401, 1998; Desomer et al, Appl. Environ. Microbiol, 56:2818, 1990), Rhodococcus erythropolis (JP 10248578; EP 757101; JP 09028379; US 5,705,386; De Mot et al., Microbiol., 146:3137, 1997), Rhodococcus rhodochrous (Kulakov et al., Plasmid, 38:61, 1997), Rhodococcus equi (Zeng st al., Plasmid, 38:180, 1997), Rhodococcus sp. (WO 89/07151; US 4,952,500; Vogt Singer st al., J. Bacteriol., 170:638, 1988; Appl. Environ. Microbiol, 64:4363, 1998) are reported.

In spite of the development of the above shuttle vectors, they are just theoretical research and development. So, till now, there is no commercially available tool which enables gene manipulation of Rhodococcus or microorganisms similar to hodococcus. Because, replicase and replication protein enabling a replication of plasmid in host are not known much.

Some proteins used in preparation of an expression vector or shuttle vector has been known (Denis-Larose et ah, Appl. Eviron. Micorbiol., 64:4363, 1998; Billington, et ah, J. Bacteriol. 180:3233, 1998; Dasen, G..H. GI:3212128; and Mendes, et ah, GI:6523480), however, the numbers and their utilities are much restricted. Although the replication stability is very important in Rhodococcus shuttle vector, there is almost no shuttle vector having the excellent replication stability. In many cases, antibiotics causing economic or safety problems in commercial utilization of microorganism must be used so that the replication stability of plasmid can be secured. However, the mechanism of maintaining for replication stability without antibiotics or related proteins thereof are not known specifically.

To solve these problems, a shuttle vector which conducts readily the cloning of other genes readily, contains strong replication proteins and has high replication stability in various hosts is required.

SUMMARY OF THE INVENTION

The present invention is devised so that the above problems are solved. An object of the present invention is to provide a shuttle vector having high replication stability without antibiotics, which enables replications in various hosts since it contains gene sequence encoding an important proteins for replication of the plasmid.

Another object of the present invention is to provide a recombinant vector in which the target gene is operably linked to the Rhodococcus -E. coli shuttle vector and the genus Rhodococcus microorganism transformed with the recombinant vector. To achieve the above objects, the present invention provides a shuttle vector containing DNA replication origin derived from E. coli, DNA replication origin derived from Rhodococcus erythropolis, an ampicilin resistant gene as a selection marker expressed in E. coli, and a kanamycin resistant gene as a selection marker in Rhodococcus.

In the present invention, said DNA replication origin from Rhodococcus erythropolis is preferably derived from plasmid of Rhodococcus erythropolis IAM 1484, and said plasmid is preferably pi AMI 484.

In the present invention, the nucleotide sequence in said shuttle vector preferably has at least 95% homology to that in pJW1484 or SEQ ID NO:1, and said shuttle vector which has at least 95% homology to that of SEQ ID NO:1 is preferably pLG1484.

The present invention also provides a recombinant vector in which the target gene is operably linked to the shuttle vector and the genus Rhodococcus microorganism transformed with said recombinant vector.

The above and other features and embodiments of the present invention will be more fully apparent from the following detailed description and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a restriction enzyme map of plasmid pIAM1484 derived from Rhodococcus erythropolis IAM 1484.

FIG. 2 is a process for preparing shuttle vector pJW1484 from plasmid pIAM1484 and pBluscript SK(-). FIG. 3 is a process for preparing the shuttle vector pLG1484 according to the present invention.

FIG. 4 is a graph showing the replication stability of preparing the shuttle vector pLG1484 according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION AND, PREFERRED

EMBODIMENT

In one aspect, the present invention is related to a novel Rhodococcus-E. coli shuttle vector replicable efficiently in Rhodococcus being used industrially as an important strain.

In the present invention, Rhodococcus-E. coli shuttle vector(pJW1484) is prepared using plasmid pIAM1484 isolated and purified from Rhodococcus erythropolis IAM1484 and plasmid pBluescript SK(-) widely used in E. coli. And, the only regions contributing to replication and stability are finally selected and shuttle vector (pLG1484) is prepared using them. Then, it is identified that the shuttle vector pLG1484 is replicable in various microorganisms and its replication stability is high.

To prepare the shuttle vector according to present invention, first, cryptic plasmid was isolated from Rhodococcus erythropolis IAM 1484. As a result of searching for restriction site of plasmid, restriction enzyme EcoBl site is discovered. So, cryptic plasmid and E. coli plasmid, pBluescript SK(-) were digested with EcoKL to clone into E. coli. And, DNA sequence of cryptic plasmid part in the cloned plasmid was determined using transposon, then, replication origin and replicase region assumed as an important region regarding replication are identified to obtain the plasmid(pJW1484) containing an incidently obtained kanamycin tolerant gene. For good transfer to a host and convenience of gene manipulation, shuttle vector plG1484 was prepared by deleting parts that are not involved in replication in pJW1484 sequence.

The inventors examined a host range of pLG1484 for many kinds of the genus Rhodococcus strains and replication stability from Rhodococcus erythropolis LG 12 to identify that the shuttle vector of present invention contained strong replication proteins and had high replication stability in various hosts.

The shuttle vector according to the present invention is useful to introduce a target gene into the genus Rhodococcus microorganism. Therefore, in another aspect, the present invention is related to a recombinant vector in which the target gene is operbly linked to the shuttle vector and the genus Rhodococcus microorganism transformed with said recombinant vector.

The introduction of the target gene into the shuttle vector according to the present invention is achieved by the conventional method. To facilitate the introduction of the target gene into the shuttle vector, synthetic oligonucleotide adaptor or linker according to method can be used.

Also, to increase the expression of the target gene, expression control sequence can be added. In the present invention, "expression control sequence" means DNA sequence essential for the expression of encoding sequence linked operably in a certain host. This control sequence contains a promoter for transcription, arbitrary operator sequence for the control of transcription, a sequence encoding proper mRNA ribosome binding region, and a control sequence of termination of transcription and translation.

Nucleic acid is linked operably to other nucleic acids in case of arrangement of functional relationship. This can be a gene and control sequence(s) linked by the method enabling the gene expression when proper target gene is combined with control sequence(s). For example, when DNA for a pre-sequence or secretion leader is expressed as pre-protein participating in the secretion of polypeptide, it is linked operably to DNA for a polypeptide; and, if a promoter or an enhancer effects the transcription of sequence, it is linked operably to encoding sequence; or, if ribosome binding region effects the transcription of sequence, it is operably linked to an encoding sequence; or, if a ribosome binding region is arranged to facilitate the translation, it is operably linked to an encoding sequence. Generally, "operably linked" means that linked DNA sequence is in contact and, in case of secretion leader, it is in contact and exists in reading frame. However, an enhancer does not need to be in contact. The linkage of these sequences is conducted by ligation in convenient restriction enzyme site. If restriction enzyme site does not exist, synthetic oligonucleotide adaptor or linker is used by conventional method.

The recombinant vector in which the shuttle vector and a target gene according to present invention are operably linked, can be transformed with a proper host cell by method. Preferred host cell is the genus Rhodococcus bacterium. In the present invention the genus Rhodococcus bacteria that can be used are not particularly limited, but, it can be generally selected from the group consisting of Rhodococcus coprophilus, R. equi, R. erythropolis, R. fascians, R. globerula, R. rhodnii, R. rhodochrous, R. ruber and R. rubrum.

Examples

Hereinafter, the present invention will be described in more detail by examples. It is to be understood, however, that these examples are given to more fully describe the present invention and are not construed to limit the present invention.

Example 1: Isolation of cryptic plasmid derived from Rhodococcus erythropolis IAM1484 In this example, the cryptic plasmid was isolated from Rhodococcus erythropolis IAM1484 {Rhodococcus erythropolis IAM1484; ACTC 15961). First, 5ml of Rhodococcus erythropolis IAM 1484 strain was cultured in YEPD medium (bactopeptone 2%, yeast extracts 1%, glucose 2%) in test tube at 30°C and 200rpm overnight, then cell pellet was obtained by centrirugation of cultures. The obtained cell pellet was washed twice with 500 μl of TE buffer (Tris 1OmM, EDTA ImM, pH 8) to obtain pellet by centrifugation. The pellet was suspended with 500 βi of TE buffer, and lmg of lysozyme was added and suspended sufficiently, and then it was kept at 37^°C for lhr to weaken the cell wall, followed by isolating and purifying plasmid (pIAM1484; FIG. 1) by the process disclosed in Molecular Cloning, Cold Spring Harbor Laboratory, 1989.

Example 2: Cloning of cryptic plasmid

To simplify the handling of cryptic plasmid, pIAM1484 isolated in Example 1 was combined with replication origin (ori) in E. coli and pBluescript SK(-) having an ampicilin resistant gene (FIG. 2). The precise location replication origin of cryptic plasmid was not known, however, one EcoKl restriction enzyme site existed in the present invention, so that one linear plasmid could be secured in the case of digesting cryptic plasmid with EcoRI restriction enzyme. And, it was ligated with pBluescript SK(-) plasmid which was digested with EcoRl restriction enzyme and was dephosphorylated. Then, about 8kb of plasmid was obtained by introduction of E. coli.

Example 3; Preparation of pJW1484

The whole sequence of cryptic plasmid was determined using EZ::TN™ <KAN-2>

Insertion Kits of Epicentri corp. as disclosed in the prior art (Goryshin, LY. and

Reznikoff, W.S., J. Biol. Chem., 273: 7367, 1998). The colonies in which transposon was added at 500bp intervals in cryptic plasmid region were selected, and they were transformed into Rhodococcus erythropolis LG 12, respectively. Then, important region for replication was assumed using success or failure of transformation.

As a result, colony which could not be transformed was plasmids in which transposon is added at 300bp, 800bp, and 1300bp, respectively from start region of cryptic plasmid. By Blast search, 706~1877bp region of l~2000bp region was assumed as a binding protein region and replicase replicating plasmid directly and l~706bp region was identified as replication origin. The plasmid in which transposon containing a kanamycin tolerant gene is located at 2kb from EcoRl site of cryptic plasmid was isolated, and this is named pJW1484 (FIG. 2).

1 βg of isolated pJW1484 was introduced to Rhodococcus erythropolis LG 12 (KCTC 18102P) using cuvet for electroporation, having lmm gap at 1250 Volt, 25 μ¥ in Gene Pulser II System of Biorad.

Example 4: Preparation of pLG1484

To minimize the size of a shuttle vector, sequences unrelated to replication of E. coli and Rhodococcus was deleted among sequences of pJW1484 plasmid prepared in Example 3, and the assumed region related to replication and antibiotics resistance in Rhodococcus was selected. In other words, PCR was preformed using pJW1484 as a template with primers, SEQ ID NOs 2 and 3 to obtain about 3.3kb of DNA PCR products. The 5' of the primer was modified with phosphate. Cryptic forward (forward primer) (SEQ ID NO: 2): 5-P-GACACATTTCGACCGAAGGACATC-3 Kan reverse (reverse primer) (SEQ ID NO: 3): 5-P-CACGGTTGATGAGAGCTTTGTTGTAG-S Also, PCR was performed using pJW1484 as a template with primers, SEQ ID NOs 4 and 5 to obtain the region related to replication and antibiotic tolerance in E. coli . As a result, 2.8kb of DNA fragment was obtained. M13 forward RC (forward primer) (SEQ ID: 4): 5-ACTGGCCGTCGTTTTAC-3

M13 reverse RC (reverse primer) (SEQ ID: 5): 5-CATGGTCATAGCTGTTTCC-3

Each PCR product was blunt-ended using T4 DNA polymerase, ligated using T4 DNA ligase, and introduced to E. coli. The cloned 6153bp of plasmid was named pLG1484 (FIG. 3) and deposited to international deposition institute, KCTC on January 21, 2005. DNA sequence of pLG1484 is depicted in SEQ ID NO: 1.

Example 5: Replication stability of pLG1484

After Rhodococcus erythropolis LG 12 cells containing pLG1484 prepared in Example 4 was cultured in YEPD medium containing 40 μg/mL of kanamycin until late exponential phase, the cells in 1 :100 dilution were inoculated in 5OmL of liquid YEPD medium without antibiotics and cultured at 30 ^°C . 24 hours later, the cells were again diluted lOOtimes and inoculated in 5OmL of liquid YEPD medium without anitobiotics. As repeated, cells of each step are plated on solid YEPD medium without antibiotics, and generation time is measured by colony counting.

After 100 colnies produced from the solid media were inoculated in solid YEPD medium without antibiotics and cultured for 24hours, each colony was inoculated in solid YEPD medium containing 40 μglmL of kanamycin. Then, the replication stability of pLG1484 was examined by measuring the number of kanamycin- sensitive colonies among the 100 colonies (FIG. 4). As a result, as illustrated in FIG. 4, Rhodococcus erythropolis LG 12 cells containing pLG1484 according to the present invention showed excellent replication stability without selection by antibiotics. About 0.22% of whole cells per one reproduction in medium excluding antibiotics were cells without shuttle vector pLG1484. As compared to the report that the pMVS301 plasmid in Rhodococcus sp. AS-50-1 (Vogt Singer & Finnerty, J. Bacteriol, 170:638, 1998) and ρK4 plasmid in Rhdococcus rhodochrous ATCC 1267 AiProc. Natl. Acad. ScL USA. 93(20): 10572, 1996) were deactivated at the rate of 1-1.5%, it was identified that pLG1484 according to the present invention has high replication stability.

Example 6: Host range of pLG1484

Rhodococcus globerulla (KCCM 40036), Rhodococcus rhodochrous (KCCM 40120), Rhdococcus equi (KCCM 12541) and Rhodococcus rubber (KCCM 41053) obtained from KCCM (Korea Culture Center of Microorganism) were transformed with pLG1484, respectively, so that host range of Rhodococcus-E. coli shuttle vector pLG1484 was identified. After each cell was cultured, mixed with 1 μg of recombinant plasmid pLG1484 and conducted transformation using cuvet for electroporation, having lmm gap in the condition of 1250 Volt, 25 μ¥. The transformant was determined by acquisition of resistance to 50 βg/mL of kanamycin.

Table 1

As a result, as illustrated in Table 1, pLG1484 according to the present invention has broad host range because it can be replicated in Rhdococcus equi, Rhodococcus globerulla, and Rhodococcus erythropolis etc. whose genealogy are in the far distance. Transformation efficiency of Rhodococcus equi was 7 times greater than that of Rhodococcus erythropolis, and that of Rhodococcus globerulla was half of that of Rhodococcus erythropolis. And there was no transformation in case of Rhodococcus Rhodochrous and Rhodococcus Rubber. From these results, it is identified that the shuttle vector pLG1484 according to the present invention has broad host range compared with pAN12 (WO 02/055709 A2), pDA71 (Dabbs E.R. et al, Plasmid, 23:42, 1990), and pFAJ2600 (Demot et al, Microbiol, 146:3137, 1997) which are already developed shuttle vectors for Rhodococcus.

While the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

INDUSTRIAL APPLICABILITY

As described more in detail, the present invention has an effect to provide a shuttle vector which can replicate bidirectionally in E. coli and Rhodococcus, and a recombinant vector in which the target gene is operably linked to the shuttle vector, and a microorganism transformed with the recombinant vector. The shuttle vector according to the present invention has many advantages upon gene manipulation due to relatively small size and contains strong replication proteins. Also, it secures stability in fermentation process or biotransformation process because it can be replicated sufficiently in medium without antibiotics due to its high replication stability in Rhodococcus. In addition, it has a broad host range for many kinds of the genus Rhodococcus, so it is useful for the cloning of target genes derived from the genus Rhodococcus and various cells.

Claims

THE CLAIMSWhat is claimed is:

1. A Rhodococcus-E. coli shuttle vector comprising a DNA replication origin derived from Rhodococcus erythropolis, a DNA replication origin derived from E. coli, an ampicilin resistant gene as a selection marker expressed in E. coli, and a kanamycin resistant gene as a selection marker in the genus Rhodococcus.

2. The Rhodococcus-E. coli vector according to claim 1, wherein the DNA replication origin derived from Rhodococcus erythropolis is derived from plasmid of Rhodococcus erythropolis IAM1484.

3. The Rhodococcus-E. coli shuttle vector according to claim 2, wherein the plasmid is pIAM 1484.

4. The Rhodococcus-E. coli shuttle vector according to claim 1, wherein the shuttle vector is pJW1484.

5. The Rhodococcus-E. coli shuttle vector according to claim 1, wherein the shuttle vector having at least 95% homology to a nucleotide sequence depicted in SEQ ID NO:1.

6. The Rhodococcus-E. coli shuttle vector according to claim 5, wherein the shuttle vector is pLG1484.

7. A recombinant vector, in which a target gene is operably linked to the pJW1484 shuttle vector of claim 4.

8. A microorganism transformed with the recombinant vector of claim 7.

9. The transformed microorganism according to claim 8, wherein the microorganism is the genus Rhodococcus.

10. The transformed microorganism according to claim 9, wherein the genus Rhodococcus microorganism is selected from the group consisting of Rhodocoaccus coprophilus, R. equi, R. erythropolis, R. fascians, R. globerula, R. rhodnii, R. rhodochrous, R. ruber and R. rubrum.

11. A recombinant vector, in which a target gene is operably linked to the pLG1484 shuttle vector of claim 6.

12. A microorganism transformed with the recombinant vector of claim 11.

13. The transformed microorganism according to claim 12, wherein the microorganism is the genus Rhodococcus.

14. The transformed microorganism according to claim 13, wherein the genus Rhodococcus microorganism is selected from the group consisting of Rhodococcus coprophilus, R. equi, R. erythropolis, R. fascians, R. globerula, R. rhodnii, R. rhodochrous, R. ruber and R. rubrum.