Enhanced production of recombinant nattokinase in Bacillus subtilis by promoter optimization

Nattokinase (NK) is a health product for the prevention and potential control of thrombosis diseases. To explore the possibility of enhancing NK production in Bacillus subtilis by altering the promoter of NK gene (PaprN), we tested several methods. We substituted the wild-type -10 box (TACAAT) of PaprN with the consensus sequence (TATAAT) of sigma(A)-dependent promoters, mutated the original -35 box (TACTAA) to a partial consensus sequence (TACACA), and expressed aprN from two tandem promoters, respectively. The efficacies of these changes were monitored by fibrinolytic activity, SDS-PAGE, and northern blotting analyses. Fibrinolytic activity analysis showed that altering the -10 region of PaprN could increase NK production by 136%. This production is significantly higher than those reported in the literatures. Similar results were obtained in SDS-PAGE and northern blotting analyses. This engineered promoter was also able to enhance the expression of beta-glucuronidase (GUS) by 249%. Partial alteration of the -35 element could slightly improve the production of NK by 13%, while two tandem promoters just had marginal effects on the production of NK. Our study showed that alteration of -10 or -35 elements in PaprN, especially -10 element, is an effective way to enhance the production of heterologous proteins in B. subtilis.