Genetic manipulation of Bacillus amyloliquefaciens

Application of modern gene technology to strain improvement of the industrially important bacterium Bacillus amyloliquefaciens is reported. Several different plasmid constructions carrying the α-amylase gene ( amyE) from B. amyloliquefaciens were amplified in this species either extrachromosomally or intrachromosomally. The amyE gene cloned on a pUB110-derived high copy plasmid pKTH10 directed the highest yields both in rich laboratory medium and in crude industrial medium. The α-amylase activity, when compared with the parental strain, was enhanced up to 20-fold in the pKTH 10 transformant. This strain showed decreased activities for other exoenzymes, such as proteases and β-glucanase suggesting common limiting resources in the processing of these enzymes. Deletions were made in vitro in genes encoding neutral ( nprE), alkaline ( aprE) protease and β-glucanase ( bglA). The engineered genes were cloned into the thermosensitive plasmid pE194, and the resulting plasmids were used to replace the corresponding wild type chromosomal genes in B. amyloliquefaciens by integration-excision at non-permissive temperature. The double mutant deficient in the major proteases ( ΔnprEΔaprE) showed about a 2-fold further enhancement in α-amylase production in the industrial medium compared with the relevant wild type background, both when plasmid-free and when transformed with pKTH10; this strain also produced elevated levels of the chromosomally-encoded β-glucanase; pKTH10 was stably maintained both in the wild type strain and in the ΔnprEΔaprE mutant. We suggest that the higher yields in α-amylase and β-glucanase in the ΔnprEΔaprE strain are primarily due to improved access to limiting resources, and that decreased proteolytic degradation may have had a secondary role in retaining the high activity obtained.