(176ab) Engineering and Deciphering the Effects of Spo0A and Orphan Histidine Kinases on Sporulation and Butanol Biosynthesis in Clostridium Tyrobutyricum

Hyper butyrate producing Clostridium tyrobutyricum was recently engineered to produce n-butanol at high titer, yield, and productivity. Unlike traditional ABE fermentation with solventogenic clostridia such as C. acetobutylicum, the engineered C. tyrobutyricum does not produce acetone as a byproduct, and does not rely on phase shifting from acidogenesis to solventogenesis to produce butanol. One major bottleneck in ABE fermentation is clostridial sporulation, which usually happened during the solventogenesis phase. Sporulation or the formation of spores halted cellular metabolism and solvent production in the fermentation. During the phase shift, the master sporulation regulator Spo0A is activated to initiate butanol biosynthesis while the accumulation of butanol triggers orphan histidine kinases (OHK) to phosphorylate and activate Spo0A to initiate the sporulation program. Deactivating Spo0A was proved to completely stop sporulation but also halt butanol production. Meanwhile, deactivating OHK was shown to harmonize asporulation and enhance butanol tolerance and production. Therefore, we speculated that modulating Spo0A and OHK activities could also have significant impacts on sporulation, cell metabolism, and butanol biosynthesis in C. tyrobutyricum. In this study, we first used CRISPR-Cas to knock out spo0A in the genome of C. tyrobutyricum overexpressing adhE2 and investigated the fermentation kinetics of the mutant to evaluate the effects of spo0A knockout on cell growth and butanol production in batch fermentations with and without the addition of methyl viologen (MV). Then, using computational biology tools we identified several putative OHKs and subsequently constructed OHK knockout and/or overexpression strains to prove their roles in regulating sporulation, butanol tolerance, and cell viability. In general, strains with disrupted sporulation via the knockout of spo0A or OHKs had a faster growth and up to 2.5-fold higher butanol production rate in batch fermentation. This study demonstrated that disrupting sporulation to modulate cell growth would be an effective strategy for enhancing butanol tolerance and production by engineered C. tyrobutyricum.