A stable plasmid vector and control of its copy number in Bacillus brevis 47, a protein-producing bacterium.
A low-copy-number plasmid vector, pHY481, was constructed by combining a macrolide resistance gene of a Staphylococcus aureus plasmid with a cryptic plasmid found in a Bacillus brevis strain isolated from soil. The plasmid introduced into B. brevis 47, an extensively investigated protein-producing bacterium, was maintained very stably in the absence of selective antibiotics. A Bacillus megaterium alpha-amylase gene subcloned into pHY481 was retained much more stably in B. brevis 47 than one subcloned into a plasmid of S. aureus origin. B. brevis 47 mutants were also isolated in which the copy number of pHY481 was amplified about 10-fold. The copy number of pHY481 with the inserted amylase gene also increased in the mutants. As a result, a severalfold-higher amount of the enzyme was produced in the mutants compared with that produced in wild-type B. brevis 47. Thus, the plasmid vector constructed here and the copy-number mutants of B. brevis 47 are useful for cloning foreign genes and performing genetic engineering in the protein-producing bacterium.
Yamagata
H
H
Nakagawa
K
K
Tsukagoshi
N
N
Udaka
S
S
D000681:Amylases / Q000096:biosynthesis; D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D005822:Genetic Vectors; D009154:Mutation; D010957:Plasmids
D000681:Amylases