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Title: A constitutive expression system for glycosyl hydrolase family 7 cellobiohydrolases in Hypocrea jecorina

Abstract

One of the primary industrial-scale cellulase producers is the ascomycete fungus, Hypocrea jecorina, which produces and secretes large quantities of diverse cellulolytic enzymes. Perhaps the single most important biomass degrading enzyme is cellobiohydrolase I (cbh1or Cel7A) due to its enzymatic proficiency in cellulose depolymerization. However, production of Cel7A with native-like properties from heterologous expression systems has proven difficult. In this study, we develop a protein expression system in H. jecorina (Trichoderma reesei) useful for production and secretion of heterologous cellobiohydrolases from glycosyl hydrolase family 7. Building upon previous work in heterologous protein expression in filamentous fungi, we have integrated a native constitutive enolase promoter with the native cbh1 signal sequence. The results are the following: The constitutive eno promoter driving the expression of Cel7A allows growth on glucose and results in repression of the native cellulase system, severely reducing background endo- and other cellulase activity and greatly simplifying purification of the recombinant protein. Coupling this system to a Δcbh1 strain of H. jecorina ensures that only the recombinant Cel7A protein is produced. Two distinct transformant colony morphologies were observed and correlated with high and null protein production. Production levels in ‘fast’ transformants are roughly equivalent to those in the nativemore » QM6a strain of H. jecorina, typically in the range of 10 to 30 mg/L when grown in continuous stirred-tank fermenters. ‘Slow’ transformants showed no evidence of Cel7A production. Specific activity of the purified recombinant Cel7A protein is equivalent to that of native protein when assayed on pretreated corn stover, as is the thermal stability and glycosylation level. Purified Cel7A produced from growth on glucose demonstrated remarkably consistent specific activity. Purified Cel7A from the same strain grown on lactose demonstrated significantly higher variability in activity. In conclusion, he elimination of background cellulase induction provides much more consistent measured specific activity compared to a traditional cbh1 promoter system induced with lactose. This expression system provides a powerful tool for the expression and comparison of mutant and/or phylogenetically diverse cellobiohydrolases in the industrially relevant cellulase production host H. jecorina.« less

Authors:
 [1];  [2];  [2];  [2];  [2];  [2];  [2];  [2]
  1. National Renewable Energy Lab. (NREL), Golden, CO (United States). National Bioenergy Center
  2. National Renewable Energy Lab. (NREL), Golden, CO (United States). Biosciences Center
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B)
OSTI Identifier:
1220747
Report Number(s):
NREL/JA-5100-64253
Journal ID: ISSN 1754-6834
Grant/Contract Number:  
AC36-08GO28308
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Volume: 8; Journal ID: ISSN 1754-6834
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; 59 BASIC BIOLOGICAL SCIENCES; hypocrea jecorina; trichoderma reesei; cellobiohydrolase; cellulase expression; fungal molecular biology; biomass hydrolysis

Citation Formats

Linger, Jeffrey G., Taylor, II, Larry E., Baker, John O., Vander Wall, Todd, Hobdey, Sarah E., Podkaminer, Kara, Himmel, Michael E., and Decker, Stephen R. A constitutive expression system for glycosyl hydrolase family 7 cellobiohydrolases in Hypocrea jecorina. United States: N. p., 2015. Web. doi:10.1186/s13068-015-0230-2.
Linger, Jeffrey G., Taylor, II, Larry E., Baker, John O., Vander Wall, Todd, Hobdey, Sarah E., Podkaminer, Kara, Himmel, Michael E., & Decker, Stephen R. A constitutive expression system for glycosyl hydrolase family 7 cellobiohydrolases in Hypocrea jecorina. United States. doi:10.1186/s13068-015-0230-2.
Linger, Jeffrey G., Taylor, II, Larry E., Baker, John O., Vander Wall, Todd, Hobdey, Sarah E., Podkaminer, Kara, Himmel, Michael E., and Decker, Stephen R. Wed . "A constitutive expression system for glycosyl hydrolase family 7 cellobiohydrolases in Hypocrea jecorina". United States. doi:10.1186/s13068-015-0230-2. https://www.osti.gov/servlets/purl/1220747.
@article{osti_1220747,
title = {A constitutive expression system for glycosyl hydrolase family 7 cellobiohydrolases in Hypocrea jecorina},
author = {Linger, Jeffrey G. and Taylor, II, Larry E. and Baker, John O. and Vander Wall, Todd and Hobdey, Sarah E. and Podkaminer, Kara and Himmel, Michael E. and Decker, Stephen R.},
abstractNote = {One of the primary industrial-scale cellulase producers is the ascomycete fungus, Hypocrea jecorina, which produces and secretes large quantities of diverse cellulolytic enzymes. Perhaps the single most important biomass degrading enzyme is cellobiohydrolase I (cbh1or Cel7A) due to its enzymatic proficiency in cellulose depolymerization. However, production of Cel7A with native-like properties from heterologous expression systems has proven difficult. In this study, we develop a protein expression system in H. jecorina (Trichoderma reesei) useful for production and secretion of heterologous cellobiohydrolases from glycosyl hydrolase family 7. Building upon previous work in heterologous protein expression in filamentous fungi, we have integrated a native constitutive enolase promoter with the native cbh1 signal sequence. The results are the following: The constitutive eno promoter driving the expression of Cel7A allows growth on glucose and results in repression of the native cellulase system, severely reducing background endo- and other cellulase activity and greatly simplifying purification of the recombinant protein. Coupling this system to a Δcbh1 strain of H. jecorina ensures that only the recombinant Cel7A protein is produced. Two distinct transformant colony morphologies were observed and correlated with high and null protein production. Production levels in ‘fast’ transformants are roughly equivalent to those in the native QM6a strain of H. jecorina, typically in the range of 10 to 30 mg/L when grown in continuous stirred-tank fermenters. ‘Slow’ transformants showed no evidence of Cel7A production. Specific activity of the purified recombinant Cel7A protein is equivalent to that of native protein when assayed on pretreated corn stover, as is the thermal stability and glycosylation level. Purified Cel7A produced from growth on glucose demonstrated remarkably consistent specific activity. Purified Cel7A from the same strain grown on lactose demonstrated significantly higher variability in activity. In conclusion, he elimination of background cellulase induction provides much more consistent measured specific activity compared to a traditional cbh1 promoter system induced with lactose. This expression system provides a powerful tool for the expression and comparison of mutant and/or phylogenetically diverse cellobiohydrolases in the industrially relevant cellulase production host H. jecorina.},
doi = {10.1186/s13068-015-0230-2},
journal = {Biotechnology for Biofuels},
number = ,
volume = 8,
place = {United States},
year = {Wed Mar 18 00:00:00 EDT 2015},
month = {Wed Mar 18 00:00:00 EDT 2015}
}

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