Improving tolerance and 1,3-propanediol production of Clostridium butyricum using physical mutagenesis, adaptive evolution and genome shuffling

Abstract

Bioconversion efficiency of glycerol to 1,3-propanediol (1,3-PD) by Clostridium butyricum is bottlenecked by its low tolerance to various stressors, especially glycerol as the substrate, 1,3-PD as the end product, and butyric acid as a by-product, which eventually decreases 1,3-PD yield. This study aimed at improving the tolerance and 1,3-PD production capability of C. butyricum using random mutagenesis and evolutionary techniques. Mutagenesis of wild strain by atmospheric room temperature plasma (ARTP) provided the first population with maximum tolerance to 160 g/L glycerol, while microbial microdroplet culture system (MMC)-mediated adaptive laboratory evolution (ALE) generated the second population with tolerance to 100 g/L 1,3-PD. Subsequently, genome shuffling of both populations yielded a final strain, GJH-418, which generated 60.12 g/L1,3-PD with a productivity of 1.72 g/L/h. The transcript analysis of the mutant and wild strains revealed the possible involvement of 8 genes in high tolerance and high 1,3-PD production through either up- or down-regulation.

Keywords: Biosynthesis; Microbial microdroplet culture system; Microbial tolerance; Random mutagenesis; Waste valorization.