Targeted deletion of genes encoding extracellular enzymes in Bacillus licheniformis and the impact on the secretion...

The general secretory pathway is routinely concerned with a multitude of extracellular enzymes. By eliminating obstructive competitors the export machinery may transport larger quantities of remaining proteins under circumstances in which the secretion machinery is fully loaded. Hence, in this study, genes encoding efficiently expressed but dispensable exoenzymes were knocked out in Bacillus licheniformis MD1. Single, double, and triple mutants with deletions of celA, chiA, and amyB, respectively, were generated via in vivo recombination by making use of a vector with a temperature sensitive origin of replication. Overexpression of a heterologous amylase gene on a multi-copy plasmid, a common scenario in biotechnological processes, resulted in an articulate reduction of chromosomally encoded extracellular enzyme activities indicating that the secretion machinery works to capacity in such transformants. Deletion mutants with the expression plasmid displayed enhanced amylase activities compared to the strain with the wild type genetic background. In addition, the chromosomally encoded protease activity was clearly higher in transformants with deletions.