利用定向进化提高基因工程大肠杆菌的甲醇利用能力

doi:10.13560/j.cnki.biotech.bull.1985.2017-0369

生物技术通报 ›› 2017, Vol. 33 ›› Issue (9): 101-109.doi: 10.13560/j.cnki.biotech.bull.1985.2017-0369

利用定向进化提高基因工程大肠杆菌的甲醇利用能力

王晓璐¹,²,³,王钰²,³,刘娇²,³,郑平²,³,路福平¹

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

收稿日期:2017-05-08 出版日期:2017-09-01 发布日期:2017-09-15
作者简介:王晓璐,女,硕士研究生,研究方向：轻工技术与工程；E-mail：616300688@qq.com
基金资助:
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）

Enhanced Methanol Utilization in Genetically Engineered Escherichia coli by Directed Evolution

WANG Xiao-lu¹,²,³,WANG Yu²,³,LIU Jiao²,³,ZHENG Ping²,³,LU Fu-ping¹

1. School of Biological Engineering,Tianjin University of Science and Technology,Tianjin 300457；
2. Key Laboratory of Systems Microbial Biotechnology,Chinese Academy of Sciences,Tianjin 300308；
3. Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308

Received:2017-05-08 Published:2017-09-01 Online:2017-09-15

摘要/Abstract

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,25113ΔfrmA/pZWM1-13号突变株较原始菌株25113ΔfrmA/pZWM1菌体生长量增加27.6%。对25113ΔfrmA/pZWM1-13号突变株进行13C示踪分析检测蛋白质合成氨基酸,结果表明氨基酸中13C比例有明显提高。其中,甲硫氨酸13C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。

关键词: 甲醇, 核酮糖单磷酸途径, 大肠杆菌, 定向进化

Abstract: Methanol is an important organic chemical raw material as well as an attractive alternative of sugar for biochemical industry because 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 genetically engineered E. coli as a starting strain,the random mutation occurred by continuous passage and direct evolution. Then the mutants were screened using methanol as the carbon source,and 2 mutants that grew well on methanol were obtained. When methanol used as an accessory carbon source,biomass of the mutant 25113ΔfrmA/pZWM1-13 increased by 27.6% compared to the starting strain 25113ΔfrmA/pZWM1. 13C labeling of proteinogenic amino acids detection showed that 13C labeled amino acids increased obviously in the mutant 25113ΔfrmA/pZWM1-13. Among them,13C labeled methionine increased by 7.236%. Therefore,directed evolution improved methanol utilization efficiency of the genetically engineered E. coli.

Key words: methanol, ribulose monophosphate pathway, Escherichia coli, directed evolution

王晓璐, 王钰,刘娇,郑平,路福平. 利用定向进化提高基因工程大肠杆菌的甲醇利用能力[J]. 生物技术通报, 2017, 33(9): 101-109.

WANG Xiao-lu, WANG Yu, LIU Jiao,ZHENG Ping,LU Fu-ping. Enhanced Methanol Utilization in Genetically Engineered Escherichia coli by Directed Evolution[J]. Biotechnology Bulletin, 2017, 33(9): 101-109.

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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

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