Importance of the 5' untranslated region for recombinant enzyme production in isolated Bacillus subtilis 007

The production of industrial enzymes requires an efficient expression system with a suitable host. This study investigated the isolated Bacillus subtilis 007 as a host for expressing three enzymes with potential application in the food industry. Firstly, testing the PaprE and P43 promoters and the corresponding 5' untranslated regions revealed great differences in the production of the recently discovered beta-galactosidase from Paenibacillus wnnyii. Expression controlled by the PaprE promoter yielded a significantly higher activity of 2515 mu kat/L, compared to 56 mu kat/L with the P43 promoter. Modifications on the PaprE core promoter region or the spacer, the sequence between the Shine-Dalgarno sequence and the start codon, did not improve beta-galactosidase production. Since the aprE 5' untranslated region contributes to a high mRNA stability, it was incorporated into the P43 construct to determine whether mRNA stability is responsible for the differences observed in beta-galactosidase production. Interestingly, mRNA stability was significantly improved and led to a nearly 50-fold higher beta-galactosidase production of 2756 mu kat/L. This strategy was successfully validated by the expression of two other enzymes: the cellobiose-2-epimerase from Caldicellulosiruptor saccharolyticus and the beta-glucosidase from Pyrococcus furiosus. These findings underscored the crucial role of post-transcriptional regulation and emphasized mRNA stability as a key role in recombinant enzyme production in B. subtilis 007.