Li Ang, Wen Zhiqiang, Fang Dahui, Lu Minrui, Ma Yuheng, Xie Qian, Jin Mingjie
School of Environmental and Biological Engineering, Nanjing University of Science & Technology, Nanjing 210094, China.
School of Environmental and Biological Engineering, Nanjing University of Science & Technology, Nanjing 210094, China.
Bioresour Technol. 2020 Jun;305:123066. doi: 10.1016/j.biortech.2020.123066. Epub 2020 Feb 21.
Clostridium diolis can efficiently utilize various inexpensive, renewable resources such as crude glycerol and lignocellulosic biomass hydrolysate to produce bulk chemicals and fuels. However, its study has been impeded by the lack of efficient plasmids electro-transformation techniques. In this study, an efficient electroporation protocol for C. diolis was developed and two replicons functional in C. diolis were identified. After optimizing parameters, the electro-transformation efficiency was enhanced from 5 to 692 transformants/ug DNA. Moreover, metabolic engineering of C. diolis was performed as proof of concept for the first time. By simply overexpressing heterologous genes based on the replicable plasmids, the strain was engineered to improve productions of diol (1,3-propanediol) and n-alcohol (butanol), and to enable butyl acetate synthesis in vivo, respectively under different culture conditions. This work represented a milestone of breeding C. diolis using metabolic engineering, and paved the way for studying C. diolis on the molecular level.
双孢梭菌能够高效利用各种廉价的可再生资源，如粗甘油和木质纤维素生物质水解物，以生产大宗化学品和燃料。然而，由于缺乏高效的质粒电转化技术，其研究受到了阻碍。在本研究中，开发了一种针对双孢梭菌的高效电穿孔方案，并鉴定了两种在双孢梭菌中具有功能的复制子。优化参数后，电转化效率从每微克DNA产生5个转化子提高到了692个转化子。此外，首次对双孢梭菌进行了代谢工程改造作为概念验证。通过基于可复制质粒简单地过表达异源基因，分别在不同培养条件下对该菌株进行工程改造，以提高二醇（1,3-丙二醇）和正醇（丁醇）的产量，并使其能够在体内合成乙酸丁酯。这项工作代表了利用代谢工程培育双孢梭菌的一个里程碑，并为在分子水平上研究双孢梭菌铺平了道路。
