Structure-guided engineering of Lactococcus lactis alcohol dehydrogenase LlAdhA for improved conversion of isobutyraldehyde to isobutanol
Highlights
► The crystal structures of L. lactis alcohol dehydrogenase LlAdhA and its variant were determined at high resolution. ► The structure helped guide engineering of a new variant with lower KM and higher catalytic efficiency. ► Increases in active site size, hydrophobicity, and substrate access lead to enhanced activity on isobutyraldehyde.
Introduction
Alcohol dehydrogenases (ADH, EC 1.1.1.1) are oxidoreductases that catalyze the reversible oxidation of a wide range of alcohols to the corresponding aldehydes or ketones using nicotinamide adenine dinucleotides (NAD(P)H) as coenzymes. Ubiquitous in bacteria, yeast, plants, and mammals, ADHs can be divided into three classes based on size (Reid and Fewson, 1994): short-chain ADHs (c.a. 250 residues), long-chain, iron-activated ADHs (c.a. 385 residues), and medium-chain, zinc-dependent ADHs (c.a. 350 residues) which also belong to the medium-chain dehydrogenase/reductase (MDR) super-family. Several MDR-ADHs have been structurally characterized, including horse liver ADH (HLADH, Eklund et al., 1976, Eklund and Ramaswamy, 2008) and yeast Saccharomyces cerevisiae ADH (YADH, Leskovac et al., 2002). MDR-ADHs are either dimeric or tetrameric. Dimeric ADHs are usually found in plants and mammals, whereas tetrameric ADHs are mostly found in bacteria and yeast. The MDR-ADH catalytic mechanism was established through studies of HLADH (Ramaswamy et al., 1994, Agarwal et al., 2000) and supplemented by studies of related MDR-ADHs (Eklund and Ramaswamy, 2008, Bakera et al., 2009).
ADHs play important roles in numerous natural and engineered metabolic pathways. The latter includes the work of Liao and coworkers, who engineered Ehrlich and valine biosynthetic pathways to produce isobutanol, a next-generation biofuel, in E. coli (Atsumi et al., 2008). Liao's isobutanol pathway diverts 2-ketoisovalerate, a valine precursor, to isobutanol by over-expression of a 2-ketoisovalerate decarboxylase and an ADH. The ADH catalyzes the final step, conversion of isobutyraldehyde to isobutanol. This pathway can be used to produce isobutanol in a variety of microorganisms including E. coli (Atsumi et al., 2008, Atsumi et al., 2009, Atsumi et al., 2010, Cann and Liao, 2008, Shen and Liao, 2008, Connor and Liao, 2009, Savrasova et al., 2011, Baez et al., 2011), Corynebacterium glutamicum (Smith et al., 2010, Blombach et al., 2011), Bacillus subtilis (Li et al., 2011), and Clostridium cellulolyticum (Higashide et al., 2011). Atsumi and coworkers reported that the NADH-dependent AdhA from Lactococcus lactis (LlAdhA) functions in this pathway, as does an NADPH-dependent homologue, YqhD, that is native to E. coli (Atsumi et al., 2010). Although the KM of YqhD toward isobutyraldehyde is 5-fold lower than that of LlAdhA, its preference for NADPH over NADH generates an overall cofactor imbalance in the biosynthetic pathway. To relieve the cofactor imbalance and improve isobutanol production, Bastian and coworkers in this laboratory used directed evolution by sequential rounds of random mutagenesis and screening to enhance L1AdhA activity on isobutyraldehyde (Bastian et al., 2011). The resulting ADH variant, LlAdhARE1, contained three amino acid substitutions (Y50F, I212T, and L264V) that led to a ∼7-fold decrease in KM and a ∼30-fold increase in catalytic efficiency (kcat/KM) over wild-type LlAdhA. Inclusion of LlAdhARE1 in the isobutanol biosynthetic pathway in place of wild-type LlAdhA increased anaerobic isobutanol titer from 8.4 g/L to 13.4 g/L in a 24-h fermentation (Bastian et al., 2011).
To better understand the activity enhancements in LlAdhARE1 and to guide further mutagenesis to decrease the KM for isobutyraldehyde, a non-native substrate of LlAdhA, we determined the crystal structures of LlAdhA and LlAdhARE1. We have used the structures to further improve LlAdhARE1 for this pathway and provide insights into how these improvements were achieved.
Section snippets
General
Strains, plasmids and primers are listed in Tables S1 and S2, Supplemental Information. Biological media were purchased from Research Products International (Mt. Prospect, IL, USA), NADH from Codexis, Inc. (Redwood City, CA, USA), aldehydes from Sigma–Aldrich (St. Louis, MO, USA), oligonucleotides from Integrated DNA Technologies (San Diego, CA, USA), DNA polymerases, restriction enzymes, and T4 ligase from New England Biolabs (Ipswich, MA, USA). DNA sequencing was performed by Laragen (Los
Crystal structures of LlAdhA and variant LlAdhARE1
We determined the crystal structures of LlAdhA and variant LlAdhARE1 to understand the basis of the activity increase in the variant and to obtain guidance for further enzyme engineering. This work provides the first structure of an ADH from L. lactis. The crystallographic statistics for LlAdhA and LlAdhARE1 data collection and refinement are shown in Table 1. Both crystal structures were observed in space group C2221 with two molecules per asymmetric unit. Size exclusion chromatography
Conclusion
Two new crystal structures of ADHs from the medium-chain dehydrogenase/reductase superfamily, LlAdhA and its evolved variant LlAdhARE1, were determined at high resolution. Using structure-guided saturation mutagenesis, we were able to further engineer LlAdhA to obtain an overall ∼17-fold decrease in KM and a ∼160-fold increase in catalytic efficiency toward isobutyraldehyde. Structural analysis and substrate profiling showed that the increased activity is accompanied by enlargement and
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.
Acknowledgments
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
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- 1
Current address: Chemical and Biological Engineering, Colorado State University, 1370 Campus Delivery, Fort Collins, CO 80523, USA.
- 2
Current address: Department of Chemistry, University of North Carolina at Chapel Hill, CB3290, Chapel Hill, NC 27599, USA.