(250d) Engineering Aldehyde/Alcohol Dehydrogenases for Enhanced n-Butanol Production in Clostridium Tyrobutyricum

Butanol derived from acetone-butanol-ethanol (ABE) fermentation (i.e., biobutanol) has potential to replace gasoline as fuel for automobiles powered by internal combustion engines. The roadblocks preventing this transition are the inherent limitations of the ABE fermentation process, such as butanol toxicity, expensive feedstocks, and high energy input requirements for butanol recovery. The high cost of butanol recovery can be lessened by increasing the selectivity of the biobutanol fermentation. The Yang lab has developed a strain of C. tyrobutyricum which is resistant to butanol toxicity and capable of achieving high butanol titers by overexpressing a heterologous aldehyde-alcohol dehydrogenase (AAD) enzyme adhE2. The adhE2 enzyme catalyzes the production of both ethanol and butanol from substrates acetaldehyde and butyraldehyde, respectively. Following a rational design approach, we have engineered the adhE2 enzyme to be more selective and productive in producing butanol. We have additionally achieved further yields by engineering this enzyme with cofactor promiscuity; it can utilize both NADH and NADPH. Initial protein designs were determined in-silico using the Rosetta Software Suite, where Rosetta enzyme design was used to mutate residues in the active site of the enzyme to enhance enzyme selectivity for the butyraldehyde substrate and to enable usage of NADH. These enzyme mutants were then evaluated in-vivo and in-vitro through serum bottle fermentations and enzyme assays. This work has resulted in a strain of C. tyrobutyricum whose titer is 2.5 times that of the strain which uses the wild-type adhE2 enzyme.