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

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 1,3PD 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.