Development of a stationary phase-specific autoinducible expression system in Bacillus subtilis

Bacillus thuringiensis produces crystal proteins (Cry) that account for up to 25% of the dry cell weight during the stationary phase. The high-level expression and stationary phase-specific autoinduction of the cry gene led to development of a cry promoter-based Bacillus expression system. Among the various cry promoters, cry3Aa promoter was selected by comparing the lacZ expression levels in Bacillus subtilis. An extracellular enzyme cellulase was highly upregulated during the stationary phase while under control of the cry3Aa promoter. Improvement of the cry3Aa promoter was obtained by modification of the promoter sequence. Specifically, a 5-fold increase in lacZ expression was obtained by changing both the −35 and −10 boxes of the cry3Aa promoter to the consensus sequence of the σA-dependent promoter of B. subtilis. The modified cry3Aa promoter produced a significantly higher yield of AprE, which suggests that the promoter may be useful for high-level protein expression in B. subtilis.