Structure-Guided Engineering of Lactococcus lactis Alcohol Dehydrogenase LlAdhA for Improved Conversion of Isobutyraldehyde to Isobutanol

摘要

We have determined the X-ray crystal structures of the NADH-dependent alcohol dehydrogenase LlAdhA from Lactococcus lactis and its laboratory-evolved variant LlAdhA^RE1 at 1.9 Å and 2.5 Å resolution, respectively. LlAdhA^RE1, which contains three amino acid mutations (Y50F, I212T, and L264V), was engineered to increase the microbial production of isobutanol (2-methylpropan-1-ol) from isobutyraldehyde (2-methylpropanal). Structural comparison of LlAdhA and LlAdhA^RE1 indicates that the enhanced activity on isobutyraldehyde stems from increases in the protein’s active site size, hydrophobicity, and substrate access. Further structure-guided mutagenesis generated a quadruple mutant (Y50F/N110S/I212T/L264V), whose KM for isobutyraldehyde is ~17-fold lower and catalytic efficiency (kcat/KM) is ~160-fold higher than wild-type LlAdhA. Combining detailed structural information and directed evolution, we have achieved significant improvements in non-native alcohol dehydrogenase activity that will facilitate the production of next-generation fuels such as isobutanol from renewable resources.