(29r) Disrupting Sporulation in Engineered C. Tyrobutyricum Strain Showed Increased Butanol Production and More Stable Bioprocess

Sporulation is a biological process that clostridial species adapted to overcome environmental stresses. Traditional ABE fermentation by C. acetobutylicum and C. beijerinckii often suffered from unstable process caused by sporulation – as butanol accumulates, spores are formed, and the metabolic process is gradually halted. Thus, sporulation is considered one bottleneck for higher butanol production and stable long-term bioprocess. Comparative genomics showed that asporulating and high butanol producing C. acetobutylicum strain JB200 has one single base deletion that truncated the coding region of orphan histidine kinase cac3319. Knocking out cac3319 in wild type C. acetobutylicum showed promising butanol titer and zero spores are formed, and, later, knocking out other orphan histidine kinases – cac0323, cac0903, cac0437, cac2730 showed various effects on butanol production and sporulation. On the other hand, ABE fermentation requires more unit operations and energy to separate acetone. Recently, Engineered C. tyrobutyricum strain showed hyper butanol producing capability without acetone produced as byproduct. To investigate the effect of sporulation on this engineered strain’s butanol producing capability and long-term process stability, master sporulation regulating machinery spo0A was knocked out and its fermentation kinetics was studied. Further, orphan histidine kinases cac3319, cac0323, cac0903, cac0437, cac2730 were screened on C. tyrobutyricum genome and three corresponding genes were identified - CTK_RS0210, CTK_RS06310 and CTK_RS08940. TypeII-B endogenous CRISPR system was utilized to knock out these genes and their fermentation kinetics are studied. Lastly, the best performing mutant is continuously cultured in a gas-stripping coupled fibrous bed bioreactor to test its process longevity and butanol producing performance. Our result showed that sporulation disrupted engineered C. tyrobutyricum strain is a long-term process-stable and extra-hyper butanol producing strain.