Identification and optimization of genes potentially related to protein expression for enhancing α-amylase production in Bacillus subtilis

Bacillus subtilis, as a model microorganism with a clear background, has the advantages of strong secretion ability and a generally recognized as safe status. Although the production of heterologous proteins is increasing with the development of biotechnology, the expression level of many heterologous proteins could not meet the requirements for industrial application. Here, to further enhance the production of α-amylase from Bacillus stearothermophilus (AmyS) which is industrially important due to its wide application, 12 potential expression-related genes were selected due to upregulation in high production strain and, respectively, overexpressed to evaluate their function in the expression of AmyS in B. subtilis 1A976. The highest enzyme activity was obtained by overexpression of ponA (1.58-fold), which was the major penicillin-binding protein in cell wall synthesis. In addition, the sources and expression level of ponA were investigated. Subsequently, to exert the superb secretion ability of B. subtilis WS9, a host strain with excellent expression capability, these identified enhancers were, respectively, investigated in this strain. Due to the extremely low transformation efficiency of B. subtilis WS9, many attempts were taken to improve transformation efficiency of B. subtilis WS9. As a decisive regulator of the competent cell formation, comK was integrated into the genome of B. subtilis WS9, named as WS9C. The easy-transformable WS9C highly facilitated the subsequent genetic manipulation. Integration of ponA also increased the production of AmyS (1.37-fold) in WS9C. On this basis, combinatorial overexpression of ponA with other five screened genes liaH, oppA, secA, prsA, and ltaS was performed, respectively, and the most suitable combination was overexpression of ponA combined with ltaS, which facilitated the AmyS activity (1.53-fold). Finally, the highest enzyme activity of recombinant strain reached 2901.6 U/mL. This study provided many manipulated targets for improving the production of recombinant proteins and laid foundations for functional annotation of genes in B. subtilis.