(265c) Increased Biobutanol Production in Clostridium Tyrobutyricum through Cofactor Regeneration and Butyrate Reassimilation

Butanol is seen as a next-generation biofuel because of its promise as a direct replacement for gasoline in internal combustion engines. C. tyrobutyricum has been engineered to produce n-butanol by introducing a heterologous aldehyde-alcohol dehydrogenase (AAD) encoded by adhE2. However, butanol biosynthesis in the engineered C. tyrobutyricum strain is limited by the available NADH cofactor pool and the co-production of butyrate. In this study, we further engineered C. tyrobutyricum to express phosphite dehydrogenase (PTDH) for NADH cofactor regeneration and carboxylic acid reductase (CAR) for butyrate reduction to butyraldehyde, which can be further reduced to butanol by AAD. The mutant showed a five-fold increase in butanol yield from glucose when compared to the strain expressing adhE2 alone. The codon-optimized PTDH expressed in C. tyrobutyricum resulted in a high butanol yield of 0.27 g/g glucose in batch fermentation. Interestingly, no acetate nor ethanol was produced in this mutant strain. Expression of a heterologous CAR gene was enhanced by also expressing tRNAs rare to C. tyrobutyricum, resulting in the mutant capable of reducing butyrate to butyraldehyde and butanol. The mutant strain also produced more ethanol, which could be attributed to the activity of CAR on acetate, resulting in acetaldehyde that further reduced to ethanol by AAD. With further optimization, the engineered C. tyrobutyricum strain can achieve a high butanol yield of greater than 0.4 g/g glucose with minimal acid by-products (acetate and butyrate).