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

作　　者：黄茜琳 黄俊宝 高旭丽 罗雅妮 陶伟[1] 郭明雨 刘永圆 吴晶 吴超[1] 薛正莲[1,2] 陈宇 刘艳[1,2] HUANG Xilin;HUANG Junbao;GAO Xuli;LUO Yani;TAO Wei;GUO Mingyu;LIU Yongyuan;WU Jing;WU Chao;XUE Zhenglian;CHEN Yu;LIU Yan(College of Biology and Food Engineering,Anhui Polytechnic University,Wuhu 241000,Anhui,China;Anhui Engineering Laboratory for Industrial Microbiology Molecular Breeding,Wuhu 241000,Anhui,China)

机构地区：[1]安徽工程大学生物与食品工程学院,安徽芜湖241000 [2]安徽省工业微生物分子育种工程实验室,安徽芜湖241000

出　　处：《微生物学报》2024年第7期2394-2406,共13页Acta Microbiologica Sinica

基　　金：国家自然科学基金(32372295);安徽省高校杰出青年科研项目(2023AH020013);安徽省大学生创新创业计划(202310363254)。

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。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.

关 键 词：枯草芽孢杆菌 纳豆激酶 甲萘醌-7 2 3-丁二醇脱氢酶 RNA-SEQ 

分 类 号：Q789[生物学—分子生物学] Q939.9