Phylogenetics-based identification and characterization of a superior 2,3-butanediol dehydrogenase for Zymomonas mobilis expression
Abstract
Abstract Background Zymomonas mobilis has recently been shown to be capable of producing the valuable platform biochemical, 2,3-butanediol (2,3-BDO). Despite this capability, the production of high titers of 2,3-BDO is restricted by several physiological parameters. One such bottleneck involves the conversion of acetoin to 2,3-BDO, a step catalyzed by 2,3-butanediol dehydrogenase (Bdh). Several Bdh enzymes have been successfully expressed in Z. mobilis, although a highly active enzyme is yet to be identified for expression in this host. Here, we report the application of a phylogenetic approach to identify and characterize a superior Bdh, followed by validation of its structural attributes using a mutagenesis approach. Results Of the 11 distinct bdh genes that were expressed in Z. mobilis, crude extracts expressing Serratia marcescens Bdh ( Sm Bdh) were found to have the highest activity (8.89 µmol/min/mg), when compared to other Bdh enzymes (0.34–2.87 µmol/min/mg). The Sm Bdh crystal structure was determined through crystallization with cofactor (NAD + ) and substrate (acetoin) molecules bound in the active site. Active Sm Bdh was shown to be a tetramer with the active site populated by a Gln247 residue contributed by the diagonally opposite subunit. Sm Bdh showed a more extensive supporting hydrogen-bond network in comparison tomore »
- Authors:
- Publication Date:
- Research Org.:
- National Renewable Energy Laboratory (NREL), Golden, CO (United States)
- Sponsoring Org.:
- USDOE Office of Energy Efficiency and Renewable Energy (EERE)
- OSTI Identifier:
- 1708965
- Alternate Identifier(s):
- OSTI ID: 1726035
- Report Number(s):
- NREL-JA-2700-77185
Journal ID: ISSN 1754-6834; 186; PII: 1820
- Grant/Contract Number:
- AC36-08GO28308
- Resource Type:
- Published Article
- Journal Name:
- Biotechnology for Biofuels
- Additional Journal Information:
- Journal Name: Biotechnology for Biofuels Journal Volume: 13 Journal Issue: 1; Journal ID: ISSN 1754-6834
- Publisher:
- BioMed Central
- Country of Publication:
- Netherlands
- Language:
- English
- Subject:
- 09 BIOMASS FUELS; butanediol dehydrogenase; Serratia marcescens; acetoin; 2,3-butanediol; crystallography; phylogenetics
Citation Formats
Subramanian, Venkataramanan, Lunin, Vladimir V., Farmer, Samuel J., Alahuhta, Markus, Moore, Kyle T., Ho, Angela, Chaudhari, Yogesh B., Zhang, Min, Himmel, Michael E., and Decker, Stephen R. Phylogenetics-based identification and characterization of a superior 2,3-butanediol dehydrogenase for Zymomonas mobilis expression. Netherlands: N. p., 2020.
Web. doi:10.1186/s13068-020-01820-x.
Subramanian, Venkataramanan, Lunin, Vladimir V., Farmer, Samuel J., Alahuhta, Markus, Moore, Kyle T., Ho, Angela, Chaudhari, Yogesh B., Zhang, Min, Himmel, Michael E., & Decker, Stephen R. Phylogenetics-based identification and characterization of a superior 2,3-butanediol dehydrogenase for Zymomonas mobilis expression. Netherlands. https://doi.org/10.1186/s13068-020-01820-x
Subramanian, Venkataramanan, Lunin, Vladimir V., Farmer, Samuel J., Alahuhta, Markus, Moore, Kyle T., Ho, Angela, Chaudhari, Yogesh B., Zhang, Min, Himmel, Michael E., and Decker, Stephen R. Tue .
"Phylogenetics-based identification and characterization of a superior 2,3-butanediol dehydrogenase for Zymomonas mobilis expression". Netherlands. https://doi.org/10.1186/s13068-020-01820-x.
@article{osti_1708965,
title = {Phylogenetics-based identification and characterization of a superior 2,3-butanediol dehydrogenase for Zymomonas mobilis expression},
author = {Subramanian, Venkataramanan and Lunin, Vladimir V. and Farmer, Samuel J. and Alahuhta, Markus and Moore, Kyle T. and Ho, Angela and Chaudhari, Yogesh B. and Zhang, Min and Himmel, Michael E. and Decker, Stephen R.},
abstractNote = {Abstract Background Zymomonas mobilis has recently been shown to be capable of producing the valuable platform biochemical, 2,3-butanediol (2,3-BDO). Despite this capability, the production of high titers of 2,3-BDO is restricted by several physiological parameters. One such bottleneck involves the conversion of acetoin to 2,3-BDO, a step catalyzed by 2,3-butanediol dehydrogenase (Bdh). Several Bdh enzymes have been successfully expressed in Z. mobilis, although a highly active enzyme is yet to be identified for expression in this host. Here, we report the application of a phylogenetic approach to identify and characterize a superior Bdh, followed by validation of its structural attributes using a mutagenesis approach. Results Of the 11 distinct bdh genes that were expressed in Z. mobilis, crude extracts expressing Serratia marcescens Bdh ( Sm Bdh) were found to have the highest activity (8.89 µmol/min/mg), when compared to other Bdh enzymes (0.34–2.87 µmol/min/mg). The Sm Bdh crystal structure was determined through crystallization with cofactor (NAD + ) and substrate (acetoin) molecules bound in the active site. Active Sm Bdh was shown to be a tetramer with the active site populated by a Gln247 residue contributed by the diagonally opposite subunit. Sm Bdh showed a more extensive supporting hydrogen-bond network in comparison to the other well-studied Bdh enzymes, which enables improved substrate positioning and substrate specificity. This protein also contains a short α6 helix, which provides more efficient entry and exit of molecules from the active site, thereby contributing to enhanced substrate turnover. Extending the α6 helix to mimic the lower activity Enterobacter cloacae ( Ec Bdh) enzyme resulted in reduction of Sm Bdh function to nearly 3% of the total activity. In great contrast, reduction of the corresponding α6 helix of the Ec Bdh to mimic the Sm Bdh structure resulted in ~ 70% increase in its activity. Conclusions This study has demonstrated that Sm Bdh is superior to other Bdhs for expression in Z. mobilis for 2,3-BDO production. Sm Bdh possesses unique structural features that confer biochemical advantage to this protein. While coordinated active site formation is a unique structural characteristic of this tetrameric complex, the smaller α6 helix and extended hydrogen network contribute towards improved activity and substrate promiscuity of the enzyme.},
doi = {10.1186/s13068-020-01820-x},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 13,
place = {Netherlands},
year = {Tue Nov 10 00:00:00 EST 2020},
month = {Tue Nov 10 00:00:00 EST 2020}
}
https://doi.org/10.1186/s13068-020-01820-x
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