利用定向进化提高基因工程大肠杆菌的甲醇利用能力  

Enhanced Methanol Utilization in Genetically Engineered Escherichia coli by Directed Evolution

作　　者：王晓璐 王钰[2,3] 刘娇 郑平[2,3] 路福平[1] WANG Xiao-lu WANG Yu LIU Jiao ZHENG Ping LU Fu-ping(School of Biological Engineering, Tianjin University of Science and Technology, Tianjin 300457 Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308 Tianjin Institute of lndustrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国科学院系统微生物工程重点实验室,天津300308 [3]中国科学院天津工业生物技术研究所,天津300308

出　　处：《生物技术通报》2017年第9期101-109,共9页Biotechnology Bulletin

基　　金：国家自然科学基金青年项目(31700044);中国科学院重点部署研究项目(ZDRW-ZS-2016-2)

摘　　要：甲醇是一种重要的有机化工原料,价格低廉,碳还原度高,在生物化工中是糖质原料的理想替代原料。但是常用的工业微生物宿主如大肠杆菌不能利用甲醇作为碳源,这限制了甲醇在生物化工领域的应用。通过表达甲醇脱氢酶、3-己酮糖-6-磷酸合成酶和6-磷酸-3-己酮糖异构酶在大肠杆菌中构建可同化甲醇的核酮糖单磷酸途径。以基因工程菌作为出发菌株,通过连续传代和定向进化引入随机突变,突变株进行以甲醇为碳源的压力筛选,得到了2株可以利用甲醇生长的突变株。在以甲醇为辅助碳源的培养基中,25113Δfrm A/p ZWM1-13号突变株较原始菌株25113Δfrm A/p ZWM1菌体生长量增加27.6%。对25113Δfrm A/p ZWM1-13号突变株进行^(13)C示踪分析检测蛋白质合成氨基酸,结果表明氨基酸中^(13)C比例有明显提高。其中,甲硫氨酸^(13)C标记含量增加7.236%。因此,定向进化有效地提高了基因工程大肠杆菌的甲醇利用效率。Methanol is an important organic chemical raw material as well as an attractive alternative of sugar for biochemical industrybecause of its cost advantage and higher degree of carbon reduction. However,commonly used industrial microorganisms such as Escherichia coli cannot utilize methanol as a carbon source,which has become a bottleneck for the application of methanol in bio-production processes.In this study,a methanol utilization pathway（ribulose monophosphate pathway）was constructed in E. coli using an engineering geneticallyengineered E. coli as a starting strain,the random mutation occurred by continuous passage and direct evolution. Then the mutants werescreened using methanol as the carbon source,and 2 mutants that grew well on methanol were obtained. When methanol used as an accessorycarbon source,biomass of the mutant 25113Δfrm A/p ZWM1-13 increased by 27.6% compared to the starting strain 25113Δfrm A/p ZWM1. 13 Clabeling of proteinogenic amino acids detection showed that ^13C labeled amino acids increased obviously in the mutant 25113Δfrm A/p ZWM1-13. Among them,^13C labeled methionine increased by 7.236%. Therefore,directed evolution improved methanol utilization efficiency of thegenetically engineered E. coli.

关 键 词：甲醇 核酮糖单磷酸途径 大肠杆菌 定向进化 

分 类 号：Q789[生物学—分子生物学]