In-Silico Analysis and Engineering of an Aldehyde/Alcohol Dehydrogenase for Alternative Cofactor Utilization and Selective Butanol Production.

Biobutanol production by solventogenic Clostridia is limited by a low butanol titer and yield. To overcome this limitation, Clostridium tyrobutyricum was engineered to overexpress the adhE2 gene encoding a bifunctional aldehyde/alcohol dehydrogenase (AAD) for converting acetyl-CoA/butyryl-CoA to acetaldehyde/butyraldehyde and then to ethanol/butanol. In this study, we aimed to increase butanol biosynthesis in C. tyrobutyricum by engineering AAD targeting on amino acid residues in the enzyme catalytic center that could increase butanol:ethanol ratios and alter cofactor specificity. In silico mutagenesis and analysis via Rosetta analysis showed that several AAD point mutations could increase butanol production and selectivity over ethanol. We then created C. tyrobutyricum strains overexpressing various AAD mutants. Two AAD mutants, D485G and L488A, engineered to utilize NADPH as the cofactor, increased butanol production by over 100% in batch fermentation, with yields of 0.10-0.13 g/g (vs 0.05 g/g glucose for the wild-type AAD). Two additional AAD mutants, P619G and S601A_V608S_P619G, engineered for increased butanol selectivity, also gave higher butanol yields of 0.13-0.15 g/g. Butanol production further increased to 0.23 g/g when methyl viologen was added to the fermentation. This work leveraged in silico analysis to guide rational engineering of AAD with higher selectivity and activity for butanol production.