Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-03-08
2001-10-16
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S252320, C435S320100, C536S023100, C536S023700
Reexamination Certificate
active
06303383
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel temperature sensitive plasmid for coryneform bacteria. This plasmid can be utilized for modifying a chromosomal gene of coryneform bacteria, which are used for the production of useful substances such as amino acids by fermentation, to change their genetic traits. Thus, the plasmid can be utilized for breeding of microorganisms useful for the production of amino acids by fermentation and so forth.
2. Related Art
There has already been reported an attempt to alter a genetic trait of coryneform bacteria by intentionally modifying a particular gene on their chromosomes, or by stably incorporating a gene of a defined copy number into the chromosomes, thereby utilizing such coryneform bacteria for the production of useful substances such as amino acids by fermentation [Japanese Patent Publication Laid-open (Kokai) No. 5-7491]. This utilizes a plasmid in which a replication control region of the plasmid DNA enabling autonomous replication of the plasmid is modified to be a replication control region having temperature sensitive mutation, which makes the replication impossible when the culture temperature is elevated. However, when such a plasmid containing a temperature sensitive replication control region is used in coryneform bacteria harboring another plasmid having a wild-type replication control region from which the temperature sensitive replication control region has been derived, homologous recombination may be caused between the plasmids. Thus, a phenomenon that the plasmid harbored by the transformant no longer has the temperature sensitive replication control region has been observed. Similarly, when it is intended to modify a gene on a chromosome of coryneform bacteria using such a plasmid, a phenomenon that this plasmid is eliminated due to incompatibility in the process of breeding has also been observed if a host coryneform bacterium harbors a plasmid which has a replication control region of the same origin as the aforementioned plasmid.
SUMMARY OF THE INVENTION
An object of the present invention is to obtain a temperature sensitive plasmid not exhibiting homology with already reported temperature sensitive plasmids or not exhibiting incompatibility therewith, thereby providing a method for efficiently modifying genetic traits of a host in a short period of time.
The inventors of the present invention actively studied in order to achieve the aforementioned object. As a result, they successfully obtained a plasmid containing a mutation that permitted its autonomous replication at a low temperature but did not permit its autonomous replication at an elevated temperature from a plasmid pAM330 extracted from
Brevibacterium lactofermentum
ATCC13869 [Japanese Patent Publication Laid-open (Kokai) No. 58-67699; Miwa, K. et al., Agric. Biol. Chem., 48, 2901 (1984)]. Thus, they accomplished the present invention.
That is, the present invention provides a plasmid containing a temperature sensitive replication control region and a marker gene, wherein the sensitive replication control region is derived from a plasmid pAM330 harbored by
Brevibacterium lactofermentum
ATCC13869 and allows the plasmid to replicate autonomously at a low temperature but does not allow the plasmid to replicate autonomously at an elevated temperature in coryneform bacteria within a temperature range in which the bacteria can grow.
The aforementioned marker gene is preferably an antibiotic resistance gene derived from a bacterium belonging to the genus Streptococcus, and specific examples thereof include a kanamycin resistance gene, tetracycline resistance gene, spectinomycin resistance and so forth.
In a preferred embodiment of the aforementioned plasmid, the plasmid further contains a replication control region that enables autonomous replication of the plasmid in Escherichia bacteria.
The present invention also provides a temperature sensitive replication control region, which is a replication control region included in the nucleotide sequence of SEQ ID NO: 17 and derived from a plasmid pAM330 harbored by
Brevibacterium lactofermentum
ATCC13869, contains one or more mutations selected from a mutation for substitution of T for C at the nucleotide number 1255, mutation for substitution of T for C at the nucleotide number 1534, mutation for substitution of A for G at the nucleotide number 1866, mutation for substitution of A for G at the nucleotide number 2058, mutation for substitution of T for C at the nucleotide number 2187, and mutation for substitution of A for G at the nucleotide number 3193 in the nucleotide sequence, and allows autonomous replication at a low temperature but does not allow autonomous replication at an elevated temperature within a temperature range in which coryneform bacteria can grow.
The present invention further provides a method for creating a coryneform bacterium in which a DNA fragment is incorporated into its chromosome, which comprises the following steps of:
(a) introducing a recombinant plasmid obtained by ligating a DNA fragment having a sequence homologous to a DNA sequence present on a chromosome of a coryneform bacterium to the aforementioned plasmid into a coryneform bacterium cell,
(b) culturing the bacterium at a temperature at which the plasmid is autonomously replicable to cause homologous recombination between the DNA fragment and the DNA sequence having a sequence homologous to the DNA fragment present on the chromosome of the coryneform bacterium, and
(c) selecting a bacterium in which the DNA fragment is incorporated into the chromosome together with the plasmid.
In a preferred embodiment of the aforementioned method, the method further comprises the following steps of:
(d) culturing the bacterium to cause homologous recombination between the DNA fragment incorporated into the chromosome and a DNA sequence which has a sequence homologous to the DNA fragment and originally exists on the chromosome of the coryneform bacterium,
(e) culturing the bacterium at an elevated temperature to eliminate the DNA sequence which originally exists on the chromosome and the plasmid from the chromosome, and
(f) selecting a bacterium in which the DNA sequence on the chromosome is replaced with the DNA fragment.
The term “temperature sensitive replication control region” used for the present invention refers to a replication control region which makes a plasmid autonomously replicable, and has a mutation which permits autonomous replication of a plasmid containing the region at a certain temperature, but makes autonomous replication of the plasmid impossible at a temperature higher than that temperature. Further, a plasmid having a temperature sensitive replication control region is referred to as a temperature sensitive plasmid.
The present invention provides a novel temperature sensitive plasmid derived from coryneform bacteria. Because the plasmid can exist together with another conventionally known plasmid in a coryneform bacterial cell, it is useful for breeding of microorganisms harboring such a plasmid and so forth.
REFERENCES:
patent: 4778762 (1988-10-01), Miwa et al.
patent: 4954441 (1990-09-01), Katsumata et al.
patent: 5616480 (1997-04-01), Sugimoto et al.
patent: 5756347 (1998-05-01), Sugimoto et al.
patent: 0 093 611 (1983-11-01), None
S. Ankri, et al., Medline, 12 pages, “Electrotransformation of Highly DNA-Restrictive Corynebacteria with Synthetic DNA”, Jan. 22, 1999.
Miwa et al. Gene 39:281-286, 1985.*
Morinaga et al. Agric. Biol. Chem. 51:93-100, 1987.
Kanno Sohei
Kimura Eiichiro
Matsui Kazuhiko
Nakamatsu Tsuyoshi
Nakamura Jun
Ajinomoto Co. Inc.
Gansheroff Lisa
Ketter James
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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