甘露醇磷酸化酶基因的敲除对D-甘露醇合成的影响

doi:10.13560/j.cnki.biotech.bull.1985.2018-1039

生物技术通报 ›› 2019, Vol. 35 ›› Issue (5): 118-124.doi: 10.13560/j.cnki.biotech.bull.1985.2018-1039

甘露醇磷酸化酶基因的敲除对D-甘露醇合成的影响

赵雅童, 何光明, 瓮茹茹, 石爱琴, 路福平, 李玉

省部共建食品营养与安全国家重点实验室工业微生物教育部重点实验室天津科技大学生物工程学院,天津 300457

收稿日期:2018-12-07 出版日期:2019-05-26 发布日期:2019-05-23
作者简介:赵雅童,硕士研究生,研究方向：酶工程;E-mail：1350554816@qq.com
基金资助:
国家重点研发计划（2016YFD0400803）

Effect of Mannitol Phosphorylase Gene Knockout on D-mannitol Synthesis

ZHA0 Ya-tong, HE Guang-ming, WENG Ru-ru, SHI Ai-qin, LU Fu-ping, LI Yu

State Key Laboratory of Food Nutrition and Safety,Key Laboratory of Industrial Microbiology,Ministry of Education,College of Biotechnology,Tianjin University of Science & Technology,Tianjin 300457

Received:2018-12-07 Published:2019-05-26 Online:2019-05-23

摘要/Abstract

摘要： 为了探究甘露醇分解利用途径对甘露醇合成的影响,在重组菌株R₁（K-12/pTrc99a-mdh）的基础上,通过CRISPR/Cas9敲除E.coli PTS系统中的cmtA、cmtB、mtlA基因,阻断了E. coli K-12中甘露醇的分解途径,获得了重组菌株R₃（K-12/∆cmtA∆cmtBmdh⁺）和R₅（K-12/∆cmtA∆cmtB∆mtlAmdh⁺）。与出发菌株R₁相比,R₃的生长速率没有降低,而R₅的生长速率明显下降。并且R₅在以甘露醇为唯一碳源的培养基上已无法生长,说明cmtA、cmtB、mtlA三个基因全部敲除后,菌株已无法再利用甘露醇作为碳源进行生长。最后,构建的重组菌株R₅,测得MDH酶活力为258 U/mL,用高效液相色谱对胞外产物进行检测,可检测到少量的甘露醇,为进一步探究大肠杆菌合成甘露醇的调控机制奠定基础。

关键词: 基因敲除, 甘露醇, 甘露醇脱氢酶, NADH

Abstract: In order to explore the effect of mannitol decomposition and utilization pathways on mannitol synthesis,the cmtA,cmtB and mtlA genes in Escherichia. coli PTS system were knocked out by CRISPR/Cas9 on the basis of recombinant strain R1（K-12/pTrc99a-mdh）,which blocked the decomposition pathway of mannitol in E. coli K-12,and the recombinant strain R₃（K-12/∆cmtA∆cmtBmdh⁺）and R₅（K-12/∆cmtA∆cmtB∆mtlAmdh⁺）were obtained. Compared with the original strain R₁,the growth rate of R₃ did not decrease,but that of R₅ decreased significantly. Moreover,R₅ did not grow on the medium with mannitol as the sole carbon source,indicating that the strain could no longer use mannitol as the carbon source after all the three genes cmtA,cmtB and mtlA were knocked out. Finally,the recombinant strain R₅ was constructed,the activity of MDH enzyme was 258 U/mL,and a small amount of mannitol was detected by high performance liquid chromatography,which laid a foundation for further exploring the regulation mechanism of mannitol synthesis in E. coli.

Key words: gene knockout, mannitol, mannitol dehydrogenase, NADH

赵雅童, 何光明, 瓮茹茹, 石爱琴, 路福平, 李玉. 甘露醇磷酸化酶基因的敲除对D-甘露醇合成的影响[J]. 生物技术通报, 2019, 35(5): 118-124.

ZHA0 Ya-tong, HE Guang-ming, WENG Ru-ru, SHI Ai-qin, LU Fu-ping, LI Yu. Effect of Mannitol Phosphorylase Gene Knockout on D-mannitol Synthesis[J]. Biotechnology Bulletin, 2019, 35(5): 118-124.

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甘露醇磷酸化酶基因的敲除对D-甘露醇合成的影响

Effect of Mannitol Phosphorylase Gene Knockout on D-mannitol Synthesis

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