A multi-plex protein expression system for production of complex enzyme formulations in Trichoderma reesei

Heterologous protein production has been challenging in the hyper-cellulolytic fungus, Trichoderma reesei as the species is known for poor transformation efficiency, low homologous recombination frequency, and marginal screening systems for the identification of successful transformants. We have app...

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Bibliographic Details
Published in:Journal of industrial microbiology & biotechnology 2022-11, Vol.49 (6), p.1
Main Authors: Subramanian, Venkataramanan, Farmer, Samuel J, Heiland, Kelsey L, Moore, Kyle T, Wall, Todd A Vander, Sun, Weiman, Chaudhari, Yogesh B, Himmel, Michael E, Decker, Stephen R
Format: Article
Language:English
Subjects:
beta-Glucosidase - metabolism
Cellulase - metabolism
Cellulose
Enzymes
Fluorescence
Foot-and-mouth disease
Gene expression
Genetics and Molecular Biology of Industrial Organisms
Hypocreales - metabolism
Peptides
Reproducibility of Results
Trichoderma - metabolism
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Summary:Heterologous protein production has been challenging in the hyper-cellulolytic fungus, Trichoderma reesei as the species is known for poor transformation efficiency, low homologous recombination frequency, and marginal screening systems for the identification of successful transformants. We have applied the 2A-peptide multi-gene expression system to co-express four proteins, which include three cellulases: a cellobiohydrolase (CBH1), an endoglucanase (EG1), and a β-D-glucosidase (BGL1), as well as the enhanced green fluorescent protein (eGFP) marker protein. We designed a new chassis vector, pTrEno-4X-2A, for this work. Expression of these cellulase enzymes was confirmed by real-time quantitative reverse transcription PCR and immunoblot analysis. The activity of each cellulase was assessed using chromogenic substrates, which confirmed the functionality of the enzymes. Expression and activity of these enzymes were proportional to the level of eGFP fluorescence, thereby validating the reliability of this screening technique. An 18-fold differencein protein expression was observed between the first and third genes within the 2A-peptide construct. The availability of this new multi-gene expression and screening tool is expected to greatly impact multi-enzyme applications, such as the production of complex commercial enzyme formulations and metabolic pathway enzymes, especially those destined for cell-free applications.
ISSN:1367-5435
1476-5535
DOI:10.1093/jimb/kuac027