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

Biobutanol production by solventogenic <i>Clostridia</i> is limited by a low butanol titer and yield. To overcome this limitation, <i>Clostridium tyrobutyricum</i> was engineered to overexpress the <i>adh</i>E2 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 <i>C. tyrobutyricum</i> by engineering AAD targeting on amino acid residues in the enzyme catalytic center that could increase butanol:ethanol ratios and alter cofactor specificity. <i>In silico</i> mutagenesis and analysis via Rosetta analysis showed that several AAD point mutations could increase butanol production and selectivity over ethanol. We then created <i>C. tyrobutyricum</i> 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 <i>in silico</i> analysis to guide rational engineering of AAD with higher selectivity and activity for butanol production.