Temperature sensitive plasmid for coryneform bacteria

Abstract

A coryneform bacterium in which a DNA fragment is incorporated into its chromosome is prepared by (a) obtaining a recombinant plasmid through ligation of a DNA fragment having a sequence homologous to a gene present on a chromosome of a coryneform bacterium to a plasmid that has a wild-type replication control region segment of a particular nucleotide sequence including a mutation and is autonomously replicable in a coryneform bacterium cell at a culture temperature lower than 31° C. but not autonomously replicable in the cell at a temperature of 31° C. or higher, (b) introducing the recombinant plasmid into the coryneform bacterium cell, (c) culturing the bacterium at a temperature of 31° C. or higher, (d) causing homologous recombination between the DNA fragment and the gene present on the chromosome of the coryneform bacterium and having a sequence homologous to the DNA fragment, and (e) selecting a coryneform bacterium in which the DNA fragment is incorporated into its chromosome. According to the present invention, there is provided a method for efficiently modifying genetic traits of a host in a short period of time by obtaining a temperature sensitive plasmid from a plasmid not exhibiting homology with already reported temperature sensitive plasmids or not exhibiting incompatibility therewith.