竹虫肠道微生物源纤维素酶基因在运动发酵单胞菌中的异源表达  

Heterologous Expression of Cellulase Gene from the Gut of Omphisa fuscidentalis in Zymomonas mobilis

作　　者：谭琼 吴波 何燕 夏薇 胡国全 何明雄 王彦伟 TAN Qiong;WU Bo;HE Yan;XIA Wei;HU Guoquan;HE Mingxiong;WANG Yanwei(Biogas Institute of Ministry of Agriculture and Rural Affairs,Biomass Energy Technology Research Center,Chengdu 610041,China;Graduate School of Chinese Academy of Agricultural Sciences,Beijing 100081,China;Chengdu University of Technology,Chengdu 610041,China;Key Laboratory of Development and Application of Rural Renewable Energy,Ministry of Agriculture and Rural Affairs,Chengdu 610041,China)

机构地区：[1]农业农村部成都沼气科学研究所,生物质能技术研究中心,成都610041 [2]中国农业科学院研究生院,北京100081 [3]成都理工大学,成都610041 [4]农业农村部农村可再生能源开发与利用重点实验室,成都610041

出　　处：《中国沼气》2023年第5期24-33,共10页China Biogas

基　　金：中国农业科学院科技创新工程(CAAS-ASTIP-2016-BIOMA);中央级公益性科研院所基本科研业务费专项(1610012021001_03202);四川省科技计划项目(2022YFS0508);青海科技厅科技成果转化专项项目(2022-NK-124);成都市科技计划项目(2022-YF05-00753-SN);国家成都农业科技中心地方财政专项(NASC2023TD06)。

摘　　要：运动发酵单胞菌(Zymomonas mobilis,Z.mobilis)是生产燃料乙醇的传统优势菌株,也是秸秆燃料乙醇研究方向的热点。秸秆为底物具有难降解性的特性,在实际生产应用过程中往往需要额外投加酶制剂,导致菌株发酵效率降低,发酵成本增高。为缓解这一问题,通过引入外源纤维素酶基因,在Z.mobilis内构建纤维素酶的分泌途径,让发酵菌株能够水解利用纤维素。研究利用竹虫肠道内微生物含有丰富的纤维素酶基因,连接到质粒转化菌株后并检测其在大肠杆菌和Z.mobilis的异源表达。结果显示,研究构建了21株重组菌ZMS912,胞外酶活显示,ZMS912-GH44酶活最高为192.56 U·mL^(-1),ZMS912-GH44C酶活为139.19 U·mL^(-1),ZMS912-GH28为138.7 U·mL^(-1),分别比出发菌株提高了11倍、7.7倍和7.6倍。在菌株内构建分泌纤维素酶途径以降低生产中加酶量是一个可行的降低成本的方法。Zymomonas mobilis has been widely used in the research of straw fuel ethanol because of dominant strain for fuel ethanol production.However,it will lead to high production costs in the actual production application process,since cellulose is difficult to degrade and additional enzyme preparations are required.In this study,a secretion pathway for cellulase production was constructed in the fermentation strain to alleviate the cost pressure in the production process,and the microorganisms in the gut of Omphisa fuscidentalis contain abundant cellulase genes,which can provide the source of enzyme genes for the transformation of strains.The microbial-derived cellulase genes of bamboo worms were analyzed by bioinformatics.Using ZM's own promoter terminator and signal peptide,the gene expression box was connected and shuttle plasmid was constructed to express heterologous genes in E.coli and Z.mobilis.The results showed that the subcellular localization of 6 foreign genes showed that GH5 was an intracellular protein.GH6,GH10,GH26,GH28 and GH44 are secreted proteins.Extracellular enzyme activity showed that the highest enzyme activity of ZMS912-GH44 was 192.56 U·mL^(-1),that of ZMS912-GH44C was 139.19 U·mL^(-1)and that of ZMS912-GH28 was 138.7 U·mL^(-1).Constructing cellulase secretion pathway in strain to reduce the amount of enzyme added in production is a feasible method to reduce cost.Z.mobilis has the potential to construct CBP strains and secrete cellulose.

关 键 词：运动发酵单胞菌 纤维素酶基因 信号肽 竹虫 酶活 

分 类 号：S216.4[农业科学—农业机械化工程] X712[农业科学—农业工程]