TY - JOUR
T1 - Structure-guided engineering of Lactococcus lactis alcohol dehydrogenase LlAdhA for improved conversion of isobutyraldehyde to isobutanol
AU - Liu, Xiang
AU - Bastian, Sabine
AU - Snow, Christopher D.
AU - Brustad, Eric M.
AU - Saleski, Tatyana E.
AU - Xu, Jian-He
AU - Meinhold, Peter
AU - Arnold, Frances H.
N1 - KAUST Repository Item: Exported on 2020-10-01
Acknowledged 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.
PY - 2013/3
Y1 - 2013/3
N2 - 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.
AB - 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.
UR - http://hdl.handle.net/10754/599776
UR - https://linkinghub.elsevier.com/retrieve/pii/S0168165612006104
UR - http://www.scopus.com/inward/record.url?scp=84866839540&partnerID=8YFLogxK
U2 - 10.1016/j.jbiotec.2012.08.008
DO - 10.1016/j.jbiotec.2012.08.008
M3 - Article
C2 - 22974724
SN - 0168-1656
VL - 164
SP - 188
EP - 195
JO - Journal of Biotechnology
JF - Journal of Biotechnology
IS - 2
ER -
