利用定向进化提高基因工程大肠杆菌的甲醇利用能力

发布时间：2018-05-21 09:18

  本文选题：甲醇 + 核酮糖单磷酸途径　； 参考：《生物技术通报》2017年09期

【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。
[Abstract]:Methanol is an important organic chemical raw material with low price and high carbon reduction degree. It is an ideal substitute for sugar raw material in biochemical industry. However, common industrial microbial hosts such as Escherichia coli can not use methanol as a carbon source, which limits the application of methanol in the field of biological and chemical industry. Methanol dehydrogenase 3-hexose 6-phosphate synthase and 6-phosphate -3-hexose isomerase were expressed in Escherichia coli to construct a ribonose monophosphate pathway which could assimilate methanol. Using genetically engineered bacteria as the starting strain, two mutants which can grow with methanol were obtained by continuous passage and directed evolution. The mutants were screened under the pressure of methanol as carbon source. Compared with the original strain 25113 螖 frm A / p ZWM1, the growth of 25113 螖 frm A / p ZWM1 was increased by 27.6% in methanol assisted carbon source medium. The protein synthetic amino acids of 25113 螖 frm A / p ZWM1-13 strain were detected by Tracer analysis. The results showed that the ratio of C13 C in amino acids was significantly increased. The content of methionine 13 C was increased by 7.236%. Therefore, directed evolution can effectively improve the methanol utilization efficiency of genetically engineered Escherichia coli.
【作者单位】： 天津科技大学生物工程学院;中国科学院系统微生物工程重点实验室;中国科学院天津工业生物技术研究所;
【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)
【分类号】：Q789
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本文编号：1918602