Post-Translational Modification and Subcellular Trafficking Effects on Trichoderma reesei Cellulase Production, Secretion, Stability, and Efficacy Post-Translational Modification and Subcellular Trafficking Effects on Trichoderma reesei Cellulase Product
EMSL Project ID
49365
Abstract
We propose that Post-Translation Modification (PTM) of cellulases, especially glycosylation and N-terminal capping, has significant effects on glycoside hydrolase performance; as well as productivity and secretory efficiency. We further hypothesize that these changes are caused by several strain-specific factors, such as carbon source-induced changes in cellulase-modifying enzymes, subcellular organelle alteration, and regulation of cellulase induction. The subcellular cellulase trafficking and post-translation processing of native and engineered industrial T. reesei fungal strains are largely unknown. Differences in post-translational modification (PTM) appear to have a significant impact on production levels, secretion efficacy, and intrinsic activity for cellulases from T. reesei. These differences in PTM are readily apparent in various mutational strains that have mutated regulatory control, altered Golgi and endoplasmic reticulum structure, and heterologous cellulase genes [2, 3]. The objective of the proposed research is to identify variations in the cellulase-processing proteome (glycosyl transferase, endoglycosidase - glycan trimming, glutamyl cyclase, secretion, and regulatory elements) and correlate these differences with productivity, glycosylation patterns, and subcellular trafficking through native and altered Golgi/ER. Correlation with differences in intrinsic cellulase activity as a function of PTM will point the way towards more effective cellulase secretomes (exoproteomes) and more efficient enzyme production.
1. Determine differences in glycan modification of Cel7A for selected T. reesei strains and as a function of growth conditions.
2. Determine differences of glycan processing enzyme levels and ratios between different strains as a function of growth conditions.
3. Measure protease levels and ratios as a function of growth conditions and culture time course for three selected strains.
4. Track Cel7A through intracellular processing in different key T. reesei strains.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2016-10-01
End Date
2018-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Co-Investigator(s)
Team Members
Related Publications
Subramanian V., K.T. Moore, L.A. Schuster, Y. Chaudhari, S. Farmer, D. Hu, and E.L. Bredeweg, et al. 2017. "Factors influencing bicistronic protein expression in the cellulolytic fungus Trichoderma reesei." Biotechnology for Biofuels. PNNL-SA-130384. [Unpublished]