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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; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
National Renewable Energy Laboratory (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Sustainable Transportation Office. Bioenergy Technologies Office
OSTI Identifier:
1618667
Alternate Identifier(s):
OSTI ID: 1344327
Report Number(s):
NREL/JA-2700-67223
Journal ID: ISSN 1754-6834; 34; PII: 710
Grant/Contract Number:  
AC36–08GO28308; AC36-08GO28308
Resource Type:
Published Article
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Name: Biotechnology for Biofuels Journal Volume: 10 Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
Springer Science + Business Media
Country of Publication:
Netherlands
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. Netherlands: N. p., 2017. Web. doi:10.1186/s13068-017-0710-7.
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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. Netherlands. https://doi.org/10.1186/s13068-017-0710-7
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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". Netherlands. https://doi.org/10.1186/s13068-017-0710-7.
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@article{osti_1618667,
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 = {Netherlands},
year = {Mon Feb 06 00:00:00 EST 2017},
month = {Mon Feb 06 00:00:00 EST 2017}
}
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Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1186/s13068-017-0710-7
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Citation Metrics:
Cited by: 25 works
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Figures / Tables:

Fig. 1 Fig. 1: Constructs used in this study. a Modified FMDV 2A peptide sequence used in this study. Red arrow represents the site of ribosomal skipping. The long blue arrow represents the peptide that gets added to the C-terminal end of the upstream protein. The short blue arrow represents the aminomore » acid (proline) that gets added to the $N$-terminal end of the downstream protein. b Bicistronic gene constructs for testing expression of Cel7A and eGFP protein in T. reesei. Cel7A and eGFP are separated by FMDV 2A peptide sequence in either orientation (left panel and right panel). Upon transcription of the bicistronic gene construct, a single mRNA transcript containing eGFP and Cel7A are produced, which upon translation gets cleaved after the glycine residue within the 2A peptide to yield two independent proteins. 21 amino acids of the 2A peptide get added to the pro-tein upstream of the peptide and one amino acid (proline) gets added to the downstream protein. Horizontal arrows and the numbers 1, 2, and 3, below the gene cassettes, represent PCR products that were used to confirm the presence of individual genes in the transformants. c Plasmid maps of the two constructs used in this study. The restriction site, SbfI, represents the site of linearization of the plasmid. Eno enolase, PGK1 phosphoglyc-erate kinase, HPT hygromycin phosphotransferase, term terminator« less
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Works referenced in this record:

A constitutive expression system for glycosyl hydrolase family 7 cellobiohydrolases in Hypocrea jecorina
journal, March 2015

  • Linger, Jeffrey G.; Taylor, Larry E.; Baker, John O.
  • Biotechnology for Biofuels, Vol. 8, Issue 1
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Internal ribosome entry sites in eukaryotic mRNA molecules
journal, July 2001

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journal, August 2011

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journal, May 2001

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journal, January 2015

Compact multi-enzyme pathways in P. pastoris
journal, January 2015

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journal, October 2011

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journal, January 2009

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journal, May 2010

Intracellular protein production in Trichoderma reesei (Hypocrea jecorina) with hydrophobin fusion technology
journal, January 2013

  • Mustalahti, Eero; Saloheimo, Markku; Joensuu, Jussi Joonas
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journal, October 2004

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journal, January 2012

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journal, January 2012

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E unum pluribus: multiple proteins from a self-processing polyprotein
journal, February 2006

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    Works referencing / citing this record:

    ATNT: an enhanced system for expression of polycistronic secondary metabolite gene clusters in Aspergillus niger
    journal, December 2017

    Synthetic fungal multifunctional cellulases for enhanced biomass conversion
    journal, January 2020

    • Brunecky, Roman; Subramanian, Venkataramanan; Yarbrough, John M.
    • Green Chemistry, Vol. 22, Issue 2
    • DOI: 10.1039/c9gc03062j

    Genetic engineering of Trichoderma reesei cellulases and their production
    journal, May 2017

    • Druzhinina, Irina S.; Kubicek, Christian P.
    • Microbial Biotechnology, Vol. 10, Issue 6
    • DOI: 10.1111/1751-7915.12726

    Polycistronic gene expression in Aspergillus niger
    journal, September 2017

    Identification of two integration sites in favor of transgene expression in Trichoderma reesei
    journal, May 2018

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      Fig. 3 (p. 6)figure
      Fig. 4 (p. 7)figure
      Fig. 5 (p. 8)figure
      Fig. 6 (p. 9)figure
      Fig. 7 (p. 10)figure
      Table 1 (p. 11)table
      Table 2 (p. 11)table
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