A combined top-down and bottom-up glycoproteome analysis of O-glycoform diversity of the secretome of the lignocellulose degrading fungus Neurospora crassa
EMSL Project ID
48631
Abstract
Enzymatic hydrolysis of (ligno)cellulose by cellulases is key for the production of second generation biofuels which represent a long-standing leading theme in the field of sustainable energy. Filamentous fungi are long-known and powerful cellulase producers. Filamentous fungi, particularly Trichoderma reesei, are the most efficient sources of large amounts of industrial enzymes. However, the enzymes produced by T. reesei and other industrial fungi are not optimal for lignocellulose depolymerization and the enzymes they produce are rapidly inactivated under industrial depolymerization conditions through a variety of mechanisms, including lignin binding. This inefficency of the enzymatic destruction of (ligno)cellulose is a big hurdle in making biofuels economically viable. Recent research has suggested that glycosylation of cellulases is a possible key factor. It is thought to influence productive and unproductive binding behavior of the enzymes, as well as their stability, and activity. Supported by recent molecular dynamics studies, we hypothesize that glycosylation of these enzymes is pivotal in preventing nonproductive enzyme absorption to lignin, which significantly hinders efficient biofuel production from lignocellulose. Unfortunately, little is known about cellulase glycosylation, and the secreted glycoproteome of filamentous fungi is complex. Deciphering how filamentous fungi glycosylate their proteins, and also understanding the influence of glycosylation is hence required to advance our understanding of these processes. We here propose to use the well-characterized filamentous fungus Neurospora crassa, which is closely related to T. reesei, to study O-glycosylation of cellulases using a combined top-down and bottom-up glycoproteomic approach. This requires high resolution in order to unravel the complex glycoproteome secreted by the fungus. The high mass resolution MS available at the EMSL is perfectly suited to elucidate this complex system.
N. crassa serves as a model system for filamentous fungi and a powerful genetic tool set is available, including a nearly comprehensive set of gene knockouts. Moreover, its lignocellulolytic secretome is thoroughly characterized and a solid knowledge base of its secretome on various lignocellulosic materials is available. However, nothing is known about the glycoproteome. In this project, we want to establish a comprehensive understanding of the response of N. crassa's glycoproteome when grown on different lignocellulosic substrates, especially lignin. This is of great significance, since inactivation of cellulases by lignin is a considerable problem in industrial enzymatic depolymerization of cellulose. Furthermore, identifying key enzymes involved in glycosylation is planned.
We anticipate that understanding glycosylation of cellulases will eventually render it possible to first, increase the high-level expression of heterologous proteins in industrial filamentous fungi by glyco-engineering of their pattern of glycosylation, to conform to the structural requirements of the secretory system (eg., via proteins such as calnexin); and second, to modify the surface of proteins in order to minimize binding of lignin, or other destabilizing factors. Increased stability will allow enzyme recycling, thereby greatly reducing enzyme costs, thus opening up new strategies to make biofuel production from lignocellulosic feedstock economic.
Project Details
Project type
Special Science
Start Date
2014-12-23
End Date
2016-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Co-Investigator(s)
Team Members