Enhancement of ?-amylase production by integrating and amplifying the ?-amylase gene of Bacillus amyloliquefaciens in the genome of Bacillus subtilis

Abstract

The α-amylase gene of Bacillus amyloliquefaciens was integrated into the genome of Bacillus subtilis by homologous recombination. In the first transformation step, several strains were obtained carrying the α-amylase gene as two randomly located copies. These strains produced α-amylase in the quantities comparable with that of the multicopy plasmid pKTH10, carrying the same α-amylase gene. With the plasmid system, however, the rate of the α-amylase synthesis was faster and the production phase shorter than those of the chromosomally encoded α-amylase. The two chromosomal gene copies were further multiplied either by amplification using increasing antibiotic concentration as the selective pressure or by performing a second transformation step, identical to the first integration procedure. Both methods resulted in integration strains carrying up to eight α-amylase gene copies per one genome and producing up to eightfold higher α-amylase activity than the parental strains. Six out of seven transformants, studied in more detail, were stable after growth of 42 h even without antibiotic selection. The number of the DNA and mRNA copies of the α-amylase gene was quantitavely determined by sandwich hybridization techniques, directly from culture medium.