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

Journal Article · Mon Feb 06 00:00:00 EST 2017 · Biotechnology for Biofuels

DOI:https://doi.org/10.1186/s13068-017-0710-7· OSTI ID:1618667

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.

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.

• Research Organization: National Renewable Energy Laboratory (NREL), Golden, CO (United States)
• Sponsoring Organization: USDOE Office of Energy Efficiency and Renewable Energy (EERE), Sustainable Transportation Office. Bioenergy Technologies Office
• Grant/Contract Number: AC36–08GO28308; AC36-08GO28308
• OSTI ID: 1618667
• Alternate ID(s): OSTI ID: 1344327
• Report Number(s): NREL/JA-2700-67223; 34; PII: 710
• Journal Information: Biotechnology for Biofuels, Journal Name: Biotechnology for Biofuels Vol. 10 Journal Issue: 1; ISSN 1754-6834
• Publisher: Springer Science + Business Media
• Country of Publication: Netherlands
• Language: English

Related Subjects

09 BIOMASS FUELS
Trichoderma reesei
foot-and-mouth disease virus (FMDV) 2A peptide
protein expression
cellobiohydrolase
fungus
biomass hydrolysis
green fluorescence protein