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 language | English (US) |
|---|---|
| Pages (from-to) | 188-195 |
| Number of pages | 8 |
| Journal | Journal of Biotechnology |
| Volume | 164 |
| Issue number | 2 |
| DOIs | |
| State | Published - Mar 2013 |
| Externally published | Yes |
Bibliographical note
KAUST Repository Item: Exported on 2020-10-01Acknowledged KAUST grant number(s): KUS-F1-028-03
Acknowledgements: 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.
This publication acknowledges KAUST support, but has no KAUST affiliated authors.
