(176ac) Modulating Aging By Knocking out the Autolysin Gene Lytb in Clostridium Tyrobutyricum Increased Cell Chronological Lifespan and n-Butanol Production in Fermentation

Clostridium tyrobutyricum, a Gram-positive, strictly anaerobic acidogenic bacterium, has attracted high interest as a powerful workhorse for butyrate and n-butanol production. However, aging in C. tyrobutyricum is one of the most concerning issues because aging would decrease cell viability (vitality), reduce productivity, and shorten the production duration in fermentation, hindering industrial fermentation process scale up for butyrate and n-butanol production. Upon aging, cell autolysis occurs, which decreases cell growth and viable cell density in fermentation, resulting in poor growth-dependent fermentation performance with significantly reduced productivity. The goal of this study was to reduce aging and increase the chronological lifespan (CLS) of C. tyrobutyricum by knocking out its autolysins encoded by genes such as lytB, an essential gene responsible for vegetative peptidoglycan cell wall hydrolysis. We hypothesized that lytB knockout may inhibit autolysis and thus increase the chronological lifespan of C. tyrobutyricum and its fermentation performance. In this study, we used CRISPR-Cas genome editing to knock out lytB and successfully constructed C. tyrobutyricum ΔlytB::adhE2 mutant strain for n-butanol production. The effects of lytB knockout on cell viability and CLS were evaluated in the stationary phase of batch fermentation. Compared to the control strain, ΔlytB::adhE2 mutant strain with reduced autolysis had extended CLS, especially for cells in the stationary phase, and higher butanol production. The ΔlytB::adhE2 mutant strain also showed higher tolerance to butanol and biomass hydrolysate inhibitors. Thus, the knockout of lytB autolysin gene in C. tyrobutyricum was effective in increasing CLS and butanol production in fermentation.