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

Liu, Xiang and Bastian, Sabine and Snow, Christopher D. and Brustad, Eric M. and Saleski, Tatyana E. and Xu, Jian-He and Meinhold, Peter and Arnold, Frances H. (2012) Structure-guided engineering of Lactococcus lactis alcohol dehydrogenase LlAdhA for improved conversion of isobutyraldehyde to isobutanol. Journal of Biotechnology, 164 (2). pp. 188-195. ISSN 0168-1656. PMCID PMC3542407. doi:10.1016/j.jbiotec.2012.08.008.

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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.

Item Type:Article
Related URLs:
URLURL TypeDescription CentralArticle
Snow, Christopher D.0000-0002-7690-3519
Brustad, Eric M.0000-0002-7031-5433
Arnold, Frances H.0000-0002-4027-364X
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.
Funding AgencyGrant Number
Army Research LaboratoryW911NF-09-2-0022
China Scholarship CouncillUNSPECIFIED
NIH Predoctoral Fellowship1F32-GM087102
King Abdullah University of Science and Technology (KAUST)KUS-F1-028-03
Gordon and Betty Moore FoundationUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Caltech Sanofi-Aventis Bioengineering Research ProgramUNSPECIFIED
Subject Keywords:Alcohol dehydrogenase; Crystal structure; Site-saturation mutagenesis; Directed evolution; Isobutyraldehyde; Biofuel
Issue or Number:2
PubMed Central ID:PMC3542407
Record Number:CaltechAUTHORS:20130611-082534229
Persistent URL:
Official Citation:Xiang Liu, Sabine Bastian, Christopher D. Snow, Eric M. Brustad, Tatyana E. Saleski, Jian-He Xu, Peter Meinhold, Frances H. Arnold, Structure-guided engineering of Lactococcus lactis alcohol dehydrogenase LlAdhA for improved conversion of isobutyraldehyde to isobutanol, Journal of Biotechnology, Volume 164, Issue 2, 15 December 2012, Pages 188-195, ISSN 0168-1656, 10.1016/j.jbiotec.2012.08.008. (
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:38883
Deposited By: Ruth Sustaita
Deposited On:11 Jun 2013 15:53
Last Modified:09 Nov 2021 23:40

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