C. tyrobutyricum, a naturally hyper butyrate producer, has been engineered to produce butyrate, butanol, and hydrogen from renewable biomass. However, its sporulation program has not been well investigated. The objective of this study is to investigate how Spo0A and orphan histidine kinases (OHK) mediate C. tyrobutyricum sporulation and to examine its potential to enhance butanol and butyrate production. We first constructed Spo0A and OHK knockout and over expression mutants and examined the extent of spore formation using sporulation assay and microscopy with endospore staining. Second, butanol, butyrate and salt tolerance assay were conducted to evaluate whether enhanced stress tolerance could be achieved. Next, mutants were investigated for their batch fermentation performance in butyrate titer, productivity and yield. Finally, the aldehyde-alcohol dehydrogenase (adhE2) from C. acetobutylicum was integrated on the knockout mutant’s genome to create Dspo0A::adhE2 and DOHK::adhE2 strains, which were investigated for their fermentation performance (butanol titer, productivity and yield). Our results showed that asporulation strains with Spo0A or OHK knockout had higher butyrate and butanol production due to enhanced stress tolerance, providing a solution for more economical biobutanol production. In addition, a hyper-sporulating strain overexpressing Spo0A was also identified for potential applications in food, health and gut microbiota areas.
