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Published December 15, 2012 | Supplemental Material + Accepted Version
Journal Article Open

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 1.9 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 K_M for isobutyraldehyde is ∼17-fold lower and catalytic efficiency (k_(cat)/K_M) 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.

Additional Information

© 2012 Elsevier B.V. Received 29 March 2012. Received in revised form 14 August 2012. Accepted 20 August 2012. Available online 3 September 2012. This research was sponsored by the Army Research Laboratory and was accomplished under Cooperative Agreement Number W911NF-09-2-0022. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of the Army Research Laboratory or the U.S. Government. The U.S. Government is authorized to reproduce and distribute reprints for Government purposes notwithstanding any copyright notation herein. X.L. received support from the China Scholarship Council (CSC), E.M.B. was supported by a Ruth L. Kirschstein National Research Service Award (1F32-GM087102) from the National Institutes of Health, and C.D.S. was supported by a research fellowship (KUS-F1-028-03) from King Abdullah University of Science and Technology (KAUST). The Molecular Observatory is supported by the Gordon and Betty Moore Foundation, the Beckman Institute and the Sanofi-Aventis Bioengineering Research Program at Caltech. Author contributions: X.L., S.B., C.D.S., P.M. and F.H.A. were responsible for study concept and design, analysis and interpretation of data, and preparation of manuscript. X.L., S.B., E.B., C.D.S., T.S. and J.-H.X. were responsible for acquisition of data. Conflict of interest: F.H.A. and P.M. are co-founders and shareholders of Gevo, Inc.

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Accepted Version - nihms405119.pdf

Supplemental Material - mmc1.docx

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Additional details

Created:
August 22, 2023
Modified:
October 24, 2023