EP0704530B1

A kanamycin resistance gene derived from microorganisms of the genus rhodococcus

Abstract

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EP0704530B1, drawing sheet 1 — Sheet 1 of 7

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Expired 4 August 2015, 11.1 years ago.

8 claims: 1 independent, 7 dependent

1
An isolated gene conferring kanamycin resistance on a host microorganism said gene coding for the amino acid sequence of sequence No. 1.
4
The isolated gene according to any one of claims 1 to 3, wherein the host microorganism is a microorganism of the genus Rhodococcus or Escherichia coli .
5
A plasmid vector comprising a gene according to any one of claims 1 to 4 and a DNA region capable of replicating in a microorganism of the genus Rhodococcus .

Independent claims3

46 paragraphs in 4 sections, as filed

0001The present invention relates to a gene derived from microorganisms of the genus Rhodococcus and conferring kanamycin resistance on bacteria, as well as a plasmid vector containing the same.

0002Microorganisms belonging to the genus Rhodococcus are known as bacterial catalysts that hydrate or hydrolyze nitriles to the corresponding amides or acids (Japanese Patent Publication No. 4873/92 and Japanese Laid-Open Patent Publication Nos. 91189/87, 470/90 and 84198/90), and in particular, microorganisms belonging to the species Rhodococcusrhodochrous possess nitrile-hydrating activity of extremely high performance (Japanese Laid-Open Patent Publication No. 470/90).

0003Under such circumstances, one of the present inventors found cryptic plasmids in a certain strain of the species Rhodococcus rhodochrous and constructed hybrid plasmid vectors to develop a host-vector system of the genus Rhodococcus (Japanese Laid-Open Patent Publication Nos. 148685/92, 64589/93 and 68566/93).

0004For construction of a self-cloning system of higher safety, it is also necessary to develop marker genes derived from microorganisms of the genus Rhodococcus. However, only arsenious acid and cadmium resistance genes derived from microorganisms of the species Rhodococcus rhodochrous are known as such drug resistance genes (Plasmid 23, 242-247 (1990)).

0005With the aim of establishing a self-cloning system of the genus Rhodococcus, the present inventors extensively studied drug resistance genes derived from microorganisms of the genus Rhodococcus, in particular the species Rhodococcusrhodochrous, so that they found the kanamycin resistance gene of the present invention.

0006That is, the present invention relates to a gene derived from microorganisms of the genus Rhodococcus and conferring kanamycin resistance on a host microorganism, wherein said gene codes for the amino acid sequence of Sequence No. 1

0007The present invention furthermore relates to an isolated gene conferring kanamycin resistance on a host comprising the DNA sequence of Sequence No. 2 or a DNA sequence which <ul id="ul0001" list-style="none" compact="compact"><li>(a) differs from said DNA sequence due to the degeneracy of the genetic code;</li><li>(b) hybridizes with said DNA sequence or the DNA sequence of (a); or</li><li>(c) represents a fragment, allelic or other variation of the above DNA sequence.</li></ul>

0008In this context, the term "hybridization" refers to conventional hybridization conditions, preferably to stringent hybridization conditions.

0009FIG. 1 shows a restriction enzyme map of plasmid pKM001.

0010FIG. 2 shows the construction of plasmids pKM002, pKM003 and pKM004.

0011FIG. 3 shows a restriction enzyme map of plasmid pKM011.

0012As the DNA donor in the present invention, mention may be made of kanamycin mutant KM-02 (deposited as FERM BP-5137 with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan) which was obtained by spontaneous mutation of Rhodococcusrhodochrous ATCC 12674.

0013As the vectors used for cloning in the present invention, mention may be made of plasmid vectors including but not limited to E. coli vectors such as pTrc99A, pUC18, etc. and phage vectors such as λ gt11 etc. The host microorganisms include but are not limited to E. coli JM109, E. coli JM105, and Rhodococcusrhodochrous ATCC 12674.

0014Plasmids that provide plasmid vectors constructed of the kanamycin resistance gene of the invention with a region capable of replicating in microorganisms of the genus Rhodococcus include, but are not limited to, plasmids pRCOO1, pRC002, pRC003 and pRC004. The plasmids pRCOO1, pRC002, pRC003 and pRC004 are derived from Rhodococcusrhodochrous ATCC 4276, ATCC 14349, ATCC 14348 and IFO 3338, respectively, and these plasmids are described in the aforementioned Japanese Laid-Open Patent Publication Nos. 148685/92, 64589/93 and 68566/93, respectively.

0015The present kanamycin resistance gene derived from microorganisms of the genus Rhodococcus is useful to construct vectors for microorganisms of the genus Rhodococcus, particularly vectors for self-cloning of Rhodococcusrhodochrous.

0016The present invention is described in more detail with reference to the following examples, which however are not intended to limit the scope of the present invention.

Example 1

Cloning of Kanamycin Resistance Gene from Mutant KM-02 into

E

coli

JM109

(1) Preparation of genomic DNA from KM-02 and preparation of a DNA library

0017The KM-02 strain was cultured under shaking at 30 °C in 100 ml MY medium (0.5 % polypeptone, 0.3 % Bacto-yeast extract, 0.3 % Bactomalt extract) and genomic DNA was prepared from the bacteria according to the method by Saito and Miura (Biochim. Biophys. Acta 72, 619 (1963)). A part of the resulting DNA was partially digested with restriction enzyme Sau3AI and then inserted into a BamHI site of E. coli vector pTrc99A to give a recombinant DNA library.

(2) Selection of transformants and preparation of recombinant DNA

0018The recombinant library prepared in step (1) was used to transform E. coli JM109 by the calcium chloride method, and transformants with resistance to kanamycin were selected in the following manner.

0019The transformants obtained above were plated onto LB agar medium (1 % Bacto-trypton, 0.5 % Bacto-yeast extract, 0.5 % NaCl, 1.5 % agar) containing 40 µg/ml kanamycin hydrochloride and 1 mM IPTG (isopropyl-β -thiogalactoside) and incubated overnight at 37 °C. The colonies occurring thereon were removed and applied onto the same agar medium, and their growth was confirmed.

0020Plasmid DNA was prepared from the thus obtained transformant according to the method by Birnboim and Doly (Nucleic Acids Res. 7, 1513-1523 (1979)) and designated pKM001. This plasmid was reintroduced into E. coli, and the resultant transformant with kanamycin resistance was designated JM109/pKM001 and deposited as FERM BP-5138 with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology. IPTG was required for expression of Kanamycin resistance of E.coli JM109/pKM001.

(3) A restriction enzyme map of pKM001 and location of the kanamycin resistance gene

0021A restriction enzyme map of plasmid pKM001 obtained in step (2) was prepared (FIG. 1). Thereafter, this plasmid pKM001 was used for preparing plasmids of a smaller DNA fragment. The target gene-containing region was identified by the presence or absence of the kanamycin resistance of transformants prepared in the same manner as in step (2). During this process, plasmid pKM002 (FIG. 2) was constructed.

(4) Nucleotide sequencing

0022The nucleotide sequence of the kanamycin resistance gene in plasmid pKM002 was determined using a Fluorescence Sequencer ALF II by Pharmacia (Sequence No. 3).

Example 2

Preparation of Hybrid (

E

Coli

Rhodococcus

) Plasmid Vector Carrying the Kanamycin Resistance Gene Derived from

Rhodococcus

rhodochrous

0023A hybrid plasmid vector pK4, previously constructed by one of the present inventors by ligating Rhodococcus-derived plasmid pRC004 with E. coli vector pHSG299 and deposited as FERM BP-3731 with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (Japanese Laid-Open Patent Publication Nos. 64589/93 and 68566/93), was used for preparing a 3.1 kb HindIII fragment containing the whole of pRC004 and a part of pHSG299, and the resulting fragment was ligated with the plasmid pKM002.

0024As a result, two plasmids carrying the insert in the opposite direction were obtained and designated pKM003 and pKM004, respectively (FIG. 2). These plasmids replicate in both the genus Rhodococcus and E. coli. Rhodococcusrhodochrous ATCC 12674 was transformed with these plasmids by electroporation, whereby a transformant capable of growing in MY medium containing 75µg/ml kanamycin was obtained. The plasmids obtained from the transformant were the same plasmids as those introduced. Where microorganisms of the genus Rhodococcus were used as the host, the presence of IPTG was not required for expression of kanamycin resistance.

Example 3

Construction of a Vector for Microorganisms of the Genus

Rhodococcus

0025The hybrid plasmid vector pKM004 was cleaved with restriction enzyme KpnI to give a 4.3 kb KpnI fragment which was then self-ligated and introduced into Rhodococcusrhodochrous ATCC 12674 by electroporation. The resulting transformant showed the same degree of kanamycin resistance as did the transformant of Example 2. From this transformant, a plasmid was obtained and designated pKM011 (FIG. 3).

SEQUENCE TABLE

0026<ul id="ul0002" list-style="none" compact="compact"><li>Sequence No: 1</li><li>Length: 171</li><li>Sequence Type: amino acid</li><li>Topology: linear</li><li>Nature: protein</li><li>Origin <ul id="ul0003" list-style="none" compact="compact"><li>Microorganism: Rhodococcusrhodochrous</li><li>Strain: KM-02</li></ul></li><li>Sequence:<img file="EP0704530B1_D0001.tif" /></li><li>Sequence No: 2</li><li>Length: 516</li><li>Nature: nucleic acid</li><li>Strand Form: double-stranded</li><li>Topology: linear</li><li>Origin <ul id="ul0004" list-style="none" compact="compact"><li>Microorganism: Rhodococcusrhodochrous</li><li>Strain: KM-02</li></ul></li><li>Sequence:<img file="EP0704530B1_D0002.tif" /></li><li>Sequence No: 3</li><li>Length: 748</li><li>Sequence Type: nucleic Acid</li><li>Strand Form: double-stranded</li><li>Topology: linear</li><li>Origin <ul id="ul0005" list-style="none" compact="compact"><li>Microorganism: Rhodococcusrhodochrous</li><li>Strain: KM-02</li></ul></li><li>Sequence:<img file="EP0704530B1_D0003.tif" /></li></ul>

Contents4

Every citation, both ways

Citations
Document	Relation	Office
EP0502476A	Cites	European Patent Office (EPO)
BERG, DE ET AL.: "Transposition of R factor genes to bacteriophage lambda." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, U.S.A., vol. 72, no. 9, September 1975, pages 3628-3632, XP002043593	Non-patent	–
HASHIMOTO Y ET AL: "Development of a host-vector system in a Rhodococcus strain and its use for expression of the cloned nitrile hydratase gene cluster." JOURNAL OF GENERAL MICRIBIOLOGY, vol. 138, May 1992, pages 1003-1010, XP002043211	Non-patent	–
DABBS E. R. ET AL.: "Nocardioform arsenic resistance plasmids and construction of Rhodococcus cloning vectors" PLASMID, vol. 23, no. 3, May 1990, pages 242-247, XP002043212	Non-patent	–

13 members in 7 offices; this record represents the family

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Priority claims
Document	Office	Kind	Date
20158294	Japan	–	1994-08-04
20158294	Japan	A	1994-08-04
JP19940201582	–	–	–
20158294	–	–	–

Members13

Family members, oldest first
Document	Office	Kind
JPH0838184A	Japan	A	1996-02-13
KR960007778A	Republic of Korea	A	1996-03-22
EP0704530A2	European Patent Office (EPO)	A2	1996-04-03
CN1133342A	China	A	1996-10-16
EP0704530A3	European Patent Office (EPO)	A3	1997-12-29
US5776771A	United States of America	A	1998-07-07
TW338069B	Taiwan Province of China	B	1998-08-11
JP3235934B2	Japan	B2	2001-12-04
CN1080308C	China	C	2002-03-06
KR100343424B1	Republic of Korea	B1	2002-11-14
EP0704530B1This record	European Patent Office (EPO)	B1	2003-10-29
DE69532024D1	Germany	D1	2003-12-04
DE69532024T2	Germany	T2	2004-06-24

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2015-08-14	Patent ceasedCeasedPL	PL	CH
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A kanamycin resistance gene derived from microorganisms of the genus rhodococcus

Abstract

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E

JM109

E

SEQUENCE TABLE

Priority claims4

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