Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening
Abstract
Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, GH7 CBHs are a family critically important for the bioeconomy as well as a methodology for improving these and other difficult to engineer enzymes. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From ~1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially-relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorically engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55%more »
- Publication Date:
- Research Org.:
- National Renewable Energy Laboratory (NREL), Golden, CO (United States)
- Sponsoring Org.:
- USDOE; USDOE Office of Energy Efficiency and Renewable Energy (EERE), Office of Sustainable Transportation. Bioenergy Technologies Office (BETO)
- OSTI Identifier:
- 2301755
- Alternate Identifier(s):
- OSTI ID: 2316135
- Report Number(s):
- NREL/JA-2800-87398
Journal ID: ISSN 0021-9258; S002192582400125X; 105749; PII: S002192582400125X
- Grant/Contract Number:
- AC36-08GO28308
- Resource Type:
- Published Article
- Journal Name:
- Journal of Biological Chemistry
- Additional Journal Information:
- Journal Name: Journal of Biological Chemistry Journal Volume: 300 Journal Issue: 3; Journal ID: ISSN 0021-9258
- Publisher:
- Elsevier
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; cellobiohydrolase; cellulase; cellulose; enzyme improvement; genomics; protein engineering; rational design
Citation Formats
Brunecky, Roman, Knott, Brandon C., Subramanian, Venkataramanan, Linger, Jeffrey G., Beckham, Gregg T., Amore, Antonella, Taylor, II, Larry E., Vander Wall, Todd A., Lunin, Vladimir V., Zheng, Fei, Garrido, Mercedes, Schuster, Logan, Fulk, Emily M., Farmer, Samuel, Himmel, Michael E., and Decker, Stephen R. Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening. United States: N. p., 2024.
Web. doi:10.1016/j.jbc.2024.105749.
Brunecky, Roman, Knott, Brandon C., Subramanian, Venkataramanan, Linger, Jeffrey G., Beckham, Gregg T., Amore, Antonella, Taylor, II, Larry E., Vander Wall, Todd A., Lunin, Vladimir V., Zheng, Fei, Garrido, Mercedes, Schuster, Logan, Fulk, Emily M., Farmer, Samuel, Himmel, Michael E., & Decker, Stephen R. Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening. United States. https://doi.org/10.1016/j.jbc.2024.105749
Brunecky, Roman, Knott, Brandon C., Subramanian, Venkataramanan, Linger, Jeffrey G., Beckham, Gregg T., Amore, Antonella, Taylor, II, Larry E., Vander Wall, Todd A., Lunin, Vladimir V., Zheng, Fei, Garrido, Mercedes, Schuster, Logan, Fulk, Emily M., Farmer, Samuel, Himmel, Michael E., and Decker, Stephen R. Fri .
"Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening". United States. https://doi.org/10.1016/j.jbc.2024.105749.
@article{osti_2301755,
title = {Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening},
author = {Brunecky, Roman and Knott, Brandon C. and Subramanian, Venkataramanan and Linger, Jeffrey G. and Beckham, Gregg T. and Amore, Antonella and Taylor, II, Larry E. and Vander Wall, Todd A. and Lunin, Vladimir V. and Zheng, Fei and Garrido, Mercedes and Schuster, Logan and Fulk, Emily M. and Farmer, Samuel and Himmel, Michael E. and Decker, Stephen R.},
abstractNote = {Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, GH7 CBHs are a family critically important for the bioeconomy as well as a methodology for improving these and other difficult to engineer enzymes. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From ~1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially-relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorically engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55% increase over the current industrial standard T. reesei Cel7A, and 10% improvement over Aspergillus oryzae Cel7C, the best natural GH7 CBH previously identified in our laboratory.},
doi = {10.1016/j.jbc.2024.105749},
journal = {Journal of Biological Chemistry},
number = 3,
volume = 300,
place = {United States},
year = {Fri Mar 01 00:00:00 EST 2024},
month = {Fri Mar 01 00:00:00 EST 2024}
}
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