Molecular cloning of a dual-promoter system for the expression of extracellular α-amylase and neutral protease in Bacillus subtilis PY79

The genus Bacillus has extensively been exploited for the production of biopharmaceutical proteins and industrial enzymes as naturally secreted products.Recent studies in Bacillus suggested that combining two strong promoters could potentially enhance protein yields.In this study we aimed to construct Bacillus subtilis PY79 that secretes -amylase and neutral protease under the control of either one or two promoters and evaluate their secreted enzyme's activities.First, plasmids pDGM1 and pDGM2 containing PspoVG and PamyL-PspoVG promoter system, respectively, were constructed.Genes encoding -amylase (amyE) from B. subtilis PY79 and neutral protease (nprE) from Bacillus amyloliquefaciens were then inserted to the constructed plasmids, after which they were transformed into B. subtilis PY79.Effects of promoter systems on the expression of these two genes were evaluated through the activity of enzymes secreted in the growth medium.As a result, after 48 hours, the recombinant carrying the PamyL-PspoVG dual promoter provided the highest enzyme activity, 2.6-fold higher than the control B. subtilis PY79 for the expression of both extracellular enzymes.At the same time, the folds increased in enzyme activity of B. subtilis PY79 strain bearing the PspoVG single promoter was 1.3 and 1.9 for -amylase and neutral protease, respectively.The result of this study consolidated the rational design of dual-promoter systems to enhance the gene expression in B. subtilis.In particular, the recombinant B. subtilis strain carrying the PamyL-PspoVG dual promoter holds promising potentials for larger scale enzyme production applications in pharmaceutical biotechnology.