Structure-guided engineering of Lactococcus lactis alcohol dehydrogenase LlAdhA for improved conversion of isobutyraldehyde to isobutanol

Xiang Liu, Sabine Bastian, Christopher D. Snow, Eric M. Brustad, Tatyana E. Saleski, Jian-He Xu, Peter Meinhold, Frances H. Arnold

Research output: Contribution to journalArticlepeer-review

27 Scopus citations

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.
Original languageEnglish (US)
Pages (from-to)188-195
Number of pages8
JournalJournal of Biotechnology
Volume164
Issue number2
DOIs
StatePublished - Mar 2013
Externally publishedYes

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