skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: A versatile 2A peptide-based bicistronic protein expressing platform for the industrial cellulase producing fungus, Trichoderma reesei

Abstract

Here, the industrial workhorse fungus, Trichoderma reesei, is typically exploited for its ability to produce cellulase enzymes, whereas use of this fungus for over-expression of other proteins (homologous and heterologous) is still very limited. Identifying transformants expressing target protein is a tedious task due to low transformation efficiency, combined with highly variable expression levels between transformants. Routine methods for identification include PCR-based analysis, western blotting, or crude activity screening, all of which are time-consuming techniques. To simplify this screening, we have adapted the 2A peptide system from the foot-and-mouth disease virus (FMDV) to T. reesei to express a readily screenable marker protein that is co-translated with a target protein. The 2A peptide sequence allows multiple independent genes to be transcribed as a single mRNA. Upon translation, the 2A peptide sequence causes a 'ribosomal skip' generating two (or more) independent gene products. When the 2A peptide is translated, the 'skip' occurs between its two C-terminal amino acids (glycine and proline), resulting in the addition of extra amino acids on the C terminus of the upstream protein and a single proline addition to the N terminus of the downstream protein. To test this approach, we have cloned two heterologous proteins on eithermore » side of a modified 2A peptide, a secreted cellobiohydrolase enzyme (Cel7A from Penicillium funiculosum) as our target protein, and an intracellular enhanced green fluorescent protein (eGFP) as our marker protein. Using straightforward monitoring of eGFP expression, we have shown that we can efficiently monitor the expression of the target Cel7A protein.« less

Authors:
ORCiD logo [1];  [1];  [1];  [1];  [1];  [1];  [1];  [1];  [1]
  1. National Renewable Energy Lab. (NREL), Golden, CO (United States)
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:
1344327
Report Number(s):
NREL/JA-2700-67223
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: 10; Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; Trichoderma reesei; foot-and-mouth disease virus (FMDV) 2A peptide; protein expression; cellobiohydrolase; fungus; biomass hydrolysis; green fluorescence protein

Citation Formats

Subramanian, Venkataramanan, Schuster, Logan A., Moore, Kyle T., Taylor, II, Larry E., Baker, John O., Vander Wall, Todd A., Linger, Jeffrey G., Himmel, Michael E., and Decker, Stephen R. A versatile 2A peptide-based bicistronic protein expressing platform for the industrial cellulase producing fungus, Trichoderma reesei. United States: N. p., 2017. Web. doi:10.1186/s13068-017-0710-7.
Subramanian, Venkataramanan, Schuster, Logan A., Moore, Kyle T., Taylor, II, Larry E., Baker, John O., Vander Wall, Todd A., Linger, Jeffrey G., Himmel, Michael E., & Decker, Stephen R. A versatile 2A peptide-based bicistronic protein expressing platform for the industrial cellulase producing fungus, Trichoderma reesei. United States. doi:10.1186/s13068-017-0710-7.
Subramanian, Venkataramanan, Schuster, Logan A., Moore, Kyle T., Taylor, II, Larry E., Baker, John O., Vander Wall, Todd A., Linger, Jeffrey G., Himmel, Michael E., and Decker, Stephen R. Mon . "A versatile 2A peptide-based bicistronic protein expressing platform for the industrial cellulase producing fungus, Trichoderma reesei". United States. doi:10.1186/s13068-017-0710-7. https://www.osti.gov/servlets/purl/1344327.
@article{osti_1344327,
title = {A versatile 2A peptide-based bicistronic protein expressing platform for the industrial cellulase producing fungus, Trichoderma reesei},
author = {Subramanian, Venkataramanan and Schuster, Logan A. and Moore, Kyle T. and Taylor, II, Larry E. and Baker, John O. and Vander Wall, Todd A. and Linger, Jeffrey G. and Himmel, Michael E. and Decker, Stephen R.},
abstractNote = {Here, the industrial workhorse fungus, Trichoderma reesei, is typically exploited for its ability to produce cellulase enzymes, whereas use of this fungus for over-expression of other proteins (homologous and heterologous) is still very limited. Identifying transformants expressing target protein is a tedious task due to low transformation efficiency, combined with highly variable expression levels between transformants. Routine methods for identification include PCR-based analysis, western blotting, or crude activity screening, all of which are time-consuming techniques. To simplify this screening, we have adapted the 2A peptide system from the foot-and-mouth disease virus (FMDV) to T. reesei to express a readily screenable marker protein that is co-translated with a target protein. The 2A peptide sequence allows multiple independent genes to be transcribed as a single mRNA. Upon translation, the 2A peptide sequence causes a 'ribosomal skip' generating two (or more) independent gene products. When the 2A peptide is translated, the 'skip' occurs between its two C-terminal amino acids (glycine and proline), resulting in the addition of extra amino acids on the C terminus of the upstream protein and a single proline addition to the N terminus of the downstream protein. To test this approach, we have cloned two heterologous proteins on either side of a modified 2A peptide, a secreted cellobiohydrolase enzyme (Cel7A from Penicillium funiculosum) as our target protein, and an intracellular enhanced green fluorescent protein (eGFP) as our marker protein. Using straightforward monitoring of eGFP expression, we have shown that we can efficiently monitor the expression of the target Cel7A protein.},
doi = {10.1186/s13068-017-0710-7},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 10,
place = {United States},
year = {Mon Feb 06 00:00:00 EST 2017},
month = {Mon Feb 06 00:00:00 EST 2017}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 2 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Dilute-Sulfuric Acid Pretreatment of Corn Stover in Pilot-Scale Reactor: Investigation of Yields, Kinetics, and Enzymatic Digestibilities of Solids
journal, January 2003

  • Schell, Daniel J.; Farmer, Jody; Newman, Millie
  • Applied Biochemistry and Biotechnology, Vol. 105, Issue 1-3, p. 69-86
  • DOI: 10.1385/ABAB:105:1-3:69

Trichoderma reesei RUT-C30 - thirty years of strain improvement
journal, October 2011