食品与发酵工业 >

2023 , Vol. 49 >Issue 19: 44 - 52

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.035036

研究报告

代谢工程改造大肠杆菌生产氢咕啉酸

  • 董宁 ,
  • 房欢 ,
  • 赵莹 ,
  • 姜平涛 ,
  • 赵江华 ,
  • 张同存 ,
  • 张大伟
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  • 1(天津科技大学 生物工程学院,天津,300457)
    2(中国科学院天津工业生物技术研究所,低碳合成工程生物学重点实验室,天津,300308)
    3(国家合成生物技术创新中心,天津,300308)
    4(中国科学院大学,北京,300192)
    5(工业发酵微生物教育部重点实验室,天津市工业微生物重点实验室,天津科技大学生物工程学院,天津,300457)
第一作者:硕士研究生(张同存教授和张大伟研究员为共同通信作者,E-mail:tony@tust.edu.cn;zhang_dw@tib.cas.cn)

收稿日期: 2023-02-06

  修回日期: 2023-03-08

  网络出版日期: 2023-11-01

基金资助

国家重点研发计划项目(2021YFC2100700);国家自然科学基金项目(22178372,22208367);中国科学院青年创新促进会项目(2020182)

收起

Metabolic engineering of Escherichia coli for hydrogenobyrinic acid production

  • DONG Ning ,
  • FANG Huan ,
  • ZHAO Ying ,
  • JIANG Pingtao ,
  • ZHAO Jianghua ,
  • ZHANG Tongcun ,
  • ZHANG Dawei
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  • 1(College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China)
    2(Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China)
    3(National Center of Technology Innovation for Synthetic Biology, Tianjin 300308, China)
    4(University of Chinese Academy of Sciences, Beijing 300192, China)
    5(Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Lab of Industrial Microbiology, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China)

Received date: 2023-02-06

  Revised date: 2023-03-08

  Online published: 2023-11-01

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摘要

维生素B12是唯一含有金属离子的维生素,其合成途径尤为复杂。大肠杆菌工程菌是生成维生素B12的新菌种。氢咕啉酸作为维生素B12合成途径重要的稳定中间体,通过代谢工程提高大肠杆菌氢咕啉酸的产量具有重要意义。经过不同宿主的对比发现BW25113(DE3)是生产氢咕啉酸的合适宿主。利用CRISPR/Cas9介导的基因组编辑技术将氢咕啉酸生物合成基因在染色体上表达,氢咕啉酸产量提高12倍,达到6.38 mg/L。增加前体尿卟啉原III供应,氢咕啉酸产量再次提高,达到11.61 mg/L。敲除ackA-pta对氢咕啉酸产量提高没有效果。经过引入运动发酵假单胞菌来源的Entner-Doudoroff途径提高还原型烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NADPH)水平,1株无抗菌株的氢咕啉酸产量提高到14.60 mg/L。该研究为大肠杆菌高效生产维生素B12奠定了基础。

关键词： 氢咕啉酸; 大肠杆菌; 适配筛选; 代谢工程; Entner-Doudoroff途径

本文引用格式

董宁 , 房欢 , 赵莹 , 姜平涛 , 赵江华 , 张同存 , 张大伟 . 代谢工程改造大肠杆菌生产氢咕啉酸[J]. 食品与发酵工业, 2023 , 49(19) : 44 -52 . DOI: 10.13995/j.cnki.11-1802/ts.035036

Abstract

Vitamin B12 is the only vitamin containing metal ion and its biosynthetic pathway is extremely complex. Engineered Escherichia coli is a new strain for vitamin B12 production. Hydrogenobyrinic acid is an important stable intermediate of vitamin B12 biosynthetic pathway, so it is of great significance to increase the titer of E. coli hydrogenobyrinic acid via metabolic engineering. BW25113 (DE3) was found to be the suitable host for hydrogenobyrinic acid production via comparing various hosts. Hydrogenobyrinic acid titer was increased 12-fold to 6.38 mg/L after expressing the genes involved in hydrogenobyrinic acid biosynthesis on the chromosome via CRISPR/Cas9 mediated genome editing. When the supply of precursor uroporphyrinogen III was increased, the hydrogenobyrinic acid titer was increased further to 11.61 mg/L. Knocking out ackA-pta had no effect on increasing the hydrogenobyrinic acid titer. The hydrogenobyrinic acid titer of an antibiotic-free strain was increased finally to 14.60 mg/L via introducing the Entner-Doudoroff pathway from Zymomonas mobilis to improve NADPH level. The study laid the foundation for efficient production of vitamin B12 in E. coli.

Key words： hydrogenobyrinic acid; Escherichia coli; adaptation screening; metabolic engineering; Entner-Doudoroff pathway

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