High level food-grade expression of maltogenic amylase in Bacillus subtilis through genomic integration and comA overexpression

Abstract

Maltogenic amylase AmyM can improve softness retention and extend shelf life of baked foods, while the low copy number of genomic integration and the limited, non-universal enhancement provided by existing heterologous protein synthesis-associated genes are the main constraints on achieving high food-grade expression levels of AmyM. In this study, we constructed a food-grade Bacillus subtilis strain that efficiently expressed AmyM by genomic multicopy integration and synthesis enhancer genes overexpression. Specifically, amyM (encoding AmyM) was sequentially integrated into 7 different sites of B. subtilis WS9C genome, yielding strain WS9C7. Then, transcriptome analysis of strains WS9C1 and WS9C7 was performed, and results showed that genes involved in iron ion homeostasis and amino acid metabolism were significantly changed. Twenty-six significant differentially expressed genes were chosen to be modified, and results showed that 9 genes had positive effect on AmyM expression. The best one, encoding the quorum-sensing regulator ComA, improved AmyM expression level by 1.55-fold reaching 10847 U/mL, which is currently the highest reported AmyM activity, and has been a novel modification target for higher recombinant expression.