Developing Clostridium diolis as a biorefinery chassis by genetic manipulation.

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.