A Multi-Plex Protein Expression System for Production of Complex Enzyme Formulations in Trichoderma reesei: Article No. kuac027

Venkataramanan Subramanian, Samuel Farmer, Kelsey Heiland, Kyle Moore, Todd Vander Wall, Weiman Sun, Yogesh Chaudhari, Michael Himmel, Stephen Decker

Research output: Contribution to journalArticlepeer-review

Abstract

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 ..beta..-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 difference in 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.
Original languageAmerican English
Number of pages12
JournalJournal of Industrial Microbiology and Biotechnology
Volume49
Issue number6
DOIs
StatePublished - 2022

NREL Publication Number

  • NREL/JA-2700-83909

Keywords

  • AZCL-HE-cellulose
  • beta D-glucosidase
  • cellobiohydrolase
  • cellulase activity
  • endoglucanese
  • foot-and-mouth disease virus 2A-peptide
  • protein expression
  • Trichoderma reesei

Fingerprint

Dive into the research topics of 'A Multi-Plex Protein Expression System for Production of Complex Enzyme Formulations in Trichoderma reesei: Article No. kuac027'. Together they form a unique fingerprint.

Cite this