枯草芽孢杆菌聚谷氨酸合成途径相关基因功能研究  

Functions of genes involved in polyglutamic acid synthesis in Bacillus subtilis

作　　者：周梦洁 胡汶松 胡刘秀 余春苗 王洲[1,2] 黄茜琳 黄俊宝 梁雪艳 汤俊 罗建泉 薛正莲[1,2] 刘艳[1,2] ZHOU Mengjie;HU Wensong;HU Liuxiu;YU Chunmiao;WANG Zhou;HUANG Xilin;HUANG Junbao;LIANG Xueyan;TANG Jun;LUO Jianquan;XUE Zhenglian;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;Anhui ZhangHengChun Pharmaceutical Co.,Ltd.,Wuhu 241000,Anhui,China;Institute of Process Engineering,Chinese Academy of Sciences,Beijing 100000,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 [3]安徽张恒春药业股份有限公司,安徽芜湖241000 [4]中国科学院过程工程研究所,北京100000

出　　处：《微生物学报》2023年第1期387-402,共16页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31871781,31772081);芜湖市科技计划重点项目(2020yf62);国家级大学生创新创业项目(201910363042,201810363046);安徽省大学生创新创业计划项目(S202010363255)。

摘　　要：聚谷氨酸(polyglutamic acid,PGA)作为一种天然多功能的聚合物,近年来成为研究的热点。由于很难通过化学方法合成,微生物发酵是目前生产聚谷氨酸的有效途径。【目的】从基因水平探究枯草芽孢杆菌聚谷氨酸合成途径中degS、degQ、degU、swrA、rocA、putM基因的功能,通过分子改造实现对代谢途径的调控。【方法】以枯草芽孢杆菌为出发菌株,通过对代谢途径中相关基因进行敲除或过表达,分别构建degS、degQ和degU基因缺失的重组菌,swrA、rocA和putM基因过表达的重组菌,借助菌株胞外聚谷氨酸积累的变化分析影响途径的关键节点。【结果】在摇瓶发酵条件下,重组菌Bacillus subtilis 168-swrA、Bacillus subtilis 168-rocA、Bacillus subtilis 168-putM的胞外聚谷氨酸含量分别是原始菌株的1.28倍、1.47倍和1.37倍。重组菌Bacillus subtilis 168-ΔdegS、Bacillus subtilis 168-ΔdegQ、Bacillus subtilis 168-ΔdegU的胞外聚谷氨酸含量分别是原始菌株的1.01倍、0.98倍和0.94倍。在静态培养时,BS168-ΔdegU不能形成完整的生物膜,Bacillus subtilis 168-ΔdegS、Bacillus subtilis 168-ΔdegQ、Bacillus subtilis 168-swrA、Bacillus subtilis 168-rocA和Bacillus subtilis 168-putM菌株的生物膜形成量分别是原始菌株的1.48倍、1.31倍、1.77倍、2.59倍和2.16倍,且胞外蛋白含量与生物膜的形成量呈正相关。【结论】degS、degQ和degU基因的缺失不会明显影响聚谷氨酸的合成,swrA、rocA和putM基因的过表达均能显著提升细胞合成聚谷氨酸的能力,rocA和putM基因的表达量增强能提高胞内谷氨酸的积累,从而增加聚谷氨酸的合成。Polyglutamic acid,as a natural multifunctional polymer,has become a research hotspot in recent years.Microbial fermentation is currently an effective way to produce polyglutamic acid which is difficult to be synthesized by chemical methods.[Objective]To explore the roles of degS,degQ,degU,swrA,rocA,and putM genes in the polyglutamic acid synthesis of Bacillus subtilis and realize the regulation of the synthesis pathway through molecular modification.[Methods]The genetically engineered B.subtilis strains were constructed by knocking out degS,degQ,and degU or overexpressing swrA,rocA,and putM,respectively.The key nodes in the synthesis pathway were analyzed based on the content change of extracellular polyglutamic acid secreted by the engineered strains.[Results]In shake flask culture,the extracellular polyglutamic acid contents of the recombinant strains B.subtilis 168-swrA,168-rocA,and 168-putM were 1.28,1.47,and 1.37 times that of the original strain,respectively;the extracellular polyglutamic acid contents of B.subtilis 168-ΔdegS,168-ΔdegQ,and 168-ΔdegU were 1.01,0.98 and 0.94 times that of the original strain,respectively.In static culture,B.subtilis 168-ΔdegU could not form an intact biofilm,and the biofilm formation of B.subtilis 168-ΔdegS,168-ΔdegQ,168-swrA,168-rocA,and 168-putM was 1.48,1.31,1.77,2.59,and 2.16 times that of the original strain,respectively.The extracellular protein content was positively correlated with the biofilm formation.[Conclusion]The deletion of degS,degQ,and degU did not significantly affect the synthesis of polyglutamic acid,while the overexpression of swrA,rocA,and putM significantly improved the ability of B.subtilis to synthesize polyglutamic acid.

关 键 词：枯草芽孢杆菌 聚谷氨酸 关键基因 代谢途径 群体感应调节系统 

分 类 号：Q78[生物学—分子生物学]