title: Structure-guided engineering of Lactococcus lactis alcohol dehydrogenase LlAdhA for improved conversion of isobutyraldehyde to isobutanol.
abstract: 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 1.9Å and 2.5Å 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.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Xiang
authors.initials: X
authors.lastname: Liu
authors.forename: Sabine
authors.initials: S
authors.lastname: Bastian
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Snow
authors.forename: Eric M
authors.initials: EM
authors.lastname: Brustad
authors.forename: Tatyana E
authors.initials: TE
authors.lastname: Saleski
authors.forename: Jian-He
authors.initials: JH
authors.lastname: Xu
authors.forename: Peter
authors.initials: P
authors.lastname: Meinhold
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000440:Butanols / Q000378:metabolism*; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000447:Aldehydes; D001426:Bacterial Proteins; D000440:Butanols; D011994:Recombinant Proteins; C040507:isobutyl alcohol; C017439:isobutyraldehyde; D000429:Alcohol Oxidoreductases