EP0704530A2

A kanamycin resistance gene derived from microorganisms of the genus rhodococcus

Abstract

The present invention relates to a DNA derived from microorganisms of the genus Rhodococcus and conferring kanamycin resistance on hosts with a DNA sequence coding for the amino acid sequence of Sequence No. 1 or a polypeptide containing a partial sequence thereof. The kanamycin resistance gene of the present invention is useful to construct vectors for microorganisms of the genus Rhodococcus, particularly vectors for self-cloning of Rhodococcusrhodochrous.

EP0704530A2, drawing sheet 1 — Sheet 1 of 10

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9 claims: 2 independent, 7 dependent

1
A gene derived from a microorganism of the genus Rhodococcus and conferring kanamycin resistance on a host, said gene coding for the amino acid sequence of Sequence No. 1 or a polypeptide containing a partial sequence thereof.
4
The 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 .
9
Use of the gene of any one of claims 1 to 4 as a marker for the construction of a self-cloning system.

Independent claims4

45 paragraphs in 3 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 Rhodococcusrhodochrous 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 Rhodococcusrhodochrous 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 Rhodococcus rhodochrous, 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 hosts, wherein said gene codes for the amino acid sequence of Sequence No. 1 or a polypeptide containing a partial sequence thereof.

0007The present invention furthermore relates to a 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, whether said variation results in changes in the polypeptide sequence or not.</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 plasmid 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 in 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 pRC001, pRC002, pRC003 and pRC004. The plasmids pRC001, 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 % Bacto-malt 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) Preparation of transformants and selection 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 96 NaCl, 1.5 % agar) containing 40 µ g/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.

0020A plasmid DNA was prepared from the thus obtained transformant according to the method by Birnboim and Doly (Nucleic Acid 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 by Fluorescence Sequencer ALF II produced 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 75µ g/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 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).<img file="EP0704530A2_D0001.tif" /><img file="EP0704530A2_D0002.tif" /><img file="EP0704530A2_D0003.tif" /><img file="EP0704530A2_D0004.tif" /><img file="EP0704530A2_D0005.tif" /><img file="EP0704530A2_D0006.tif" />

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Citations
Document	Relation	Office	Cited during
US7416859B2	Cited by	United States of America	Applicant
US11214815B2	Cited by	United States of America	Applicant
US6949362B2	Cited by	United States of America	Applicant
US8871503B2	Cited by	United States of America	Applicant
EP0502476A2	Cites	European Patent Office (EPO)	Search report
JPH02470A	Cites	Japan	Applicant
JPH0284198A	Cites	Japan	Applicant
JPH04148685A	Cites	Japan	Applicant
JPH044873A	Cites	Japan	Applicant
JPH0564589A	Cites	Japan	Applicant
JPH0564589A	Cites	Japan	Applicant
JPH0568566A	Cites	Japan	Applicant
JPS6291189A	Cites	Japan	Applicant
PLASMID, vol. 23, 1990, pages 242 - 247	Non-patent	–	Applicant
NUCLEIC ACID RES., vol. 7, 1979, pages 1513 - 1523	Non-patent	–	Applicant

13 members in 7 offices; this record represents the family

This recordMember, by publication datePriority claim

Priority claims4

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
EP0704530A2This record	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
EP0704530B1	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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JM109

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