基于RNA-seq分析bdhA敲除对枯草芽孢杆菌产纳豆激酶和MK-7的影响

RNA-seq reveals the effects of bdhA knockout on co-production of nattokinase and menaquinone-7 by Bacillus subtilis

摘要:

纳豆激酶(nattokinase, NK)具有多种生理功能，是治疗心血管疾病的理想药物。甲萘醌-7 (menaquinone-7, MK-7)是人体不可缺少的脂溶性维生素之一，可预防骨质疏松和帕金森等疾病。【目的】提高枯草芽孢杆菌中NK和MK-7共同生产的产量，揭示重组菌中共同生产NK和MK-7的机理，为MK-7和NK的生成提供新的代谢工程策略。【方法】以枯草芽孢杆菌为出发菌株，敲除2,3-丁二醇脱氢酶基因(bdhA)，构建一株能增加NK和MK-7共同生产的枯草芽孢杆菌(Bacillus subtilis) 168-ΔbdhA。利用RNA-seq分析NK和MK-7合成途径关键酶编码基因的变化，总结NK和MK-7共同生产的机制。【结果】与原始菌株相比，Bacillus subtilis 168-ΔbdhA中2,3-丁二醇含量降低64.0%，为2.76 g/L。NK和MK-7的产量较原始菌株提高30.0%和60.0%。RNA-seq分析表明，中心碳代谢、氧化磷酸化和NK及MK-7合成等过程相关的基因表达存在差异。NK负调控因子codY下调2.19倍。在蛋白质分泌途径中，secA下调0.37倍，tatAD和tatC分别上调2.81倍和0.50倍。【结论】bdhA的敲除阻断了2,3-丁二醇的碳通量，促进甘油的吸收，碳通量更多地流向NK和MK-7的合成途径。负调控因子codY的下调促进NK转录，蛋白转运相关途径基因的上下调促进MK-7的胞外分泌，从而实现其产量的增加。

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.