Abstract:Nattokinase has a variety of physiological functions and serves the treatment of cardiovascular diseases. Menaquinone-7, one of indispensable fat-soluble vitamins in the human body, can prevent diseases such as osteoporosis and Parkinson’s disease. [Objective] To enhance the co-production of nattokinase and menaquinone-7 by Bacillus subtilis, reveal the co-production mechanism in the recombinant strain, and provide new metabolic engineering strategies for the production of nattokinase and menaquinone-7. [Methods] We constructed B. subtilis 168-ΔbdhA by knocking out the 2,3-butanediol dehydrogenase gene bdhA from B. subtilis 168. RNA-seq was employed to measure the expression changes of key enzyme-coding genes in the nattokinase and menaquinone-7 synthesis pathways. [Results] Compared with B. subtilis 168, the content of 2,3-butanediol in B. subtilis 168-ΔbdhA was 2.76 g/L, which was reduced by 64.0%. The yields of nattokinase and menaquinone-7 were increased by 30.0% and 60.0%, respectively. The expression levels of genes related to central carbon metabolism, oxidative phosphorylation, and the synthesis of nattokinase and menaquinone-7 changed by RNA-seq analysis. The expression level of nattokinase negative regulator gene codY was down-regulated by 2.19-fold in the mutant. The expression of secA, tatAD, and tatC involved in protein secretion showed the down-regulation of 0.37-fold, up-regulation of 2.81-fold, and up-regulation of 0.50-fold, respectively. [Conclusion] The knockout of bdhA blocked the carbon flux of 2,3-butanediol and promoted glycerol uptake, causing more carbon fluxing to the synthesis pathways of nattokinase and menaquinone-7. The down-regulation of the negative regulator codY promoted the transcription of nattokinase. The up- and down-regulation of genes involved in protein scretion promoted extracellular secretion of menaquinone-7.
