Evaluating expression and catalytic activity of anaerobic fungal fibrolytic enzymes native topiromyces sp E2 inSaccharomyces cerevisiae

Abstract

The development of efficient methods to convert plant material (lignocellulose) to fermentable sugars is a promising avenue towards the development of renewable biofuels. Anaerobic fungi that reside within the digestive tract of large herbivores are among the most efficient and robust digesters of lignocellulosic material known in nature. Despite the powerful cellulose degrading capacity of gut fungi, remarkably little is known about the cellulolytic enzymes from these organisms due to the difficulties associated with their isolation and culture. We have cloned five such saccharolytic enzymes (bglA, cel48A, celpin, xylA, and xylB) from a cDNA library prepared from the anaerobic fungus Piromyces sp E2 for expression and extracellular secretion in the model eukaryote Saccharomyces cerevisiae. All enzymes were readily produced, but only low-molecular weight proteins (xylA and xylB) were secreted efficiently into the extracellular medium. Piromyces sp E2 enzymes were posttranslationally modified in S. cerevisiae according to their sequence prediction, suggesting compatibility between the two systems, yet not all of the recombinant proteins were catalytically active. Activation of cellular stress mechanisms in the secretory pathway suggest enzyme misfolding during the production of cel48A and bglA, which likely limits activity. However, bglA, exhibited moderate reactivity against pNPG when produced in S. cerevisiae. Furthermore, since we aim to understand how the cellulolytic enzymes of anaerobic fungi are produced in their native system, we have implemented methods to isolate and culture anaerobic fungi obtained from the digestive tract of a horse. © 2011 American Institute of Chemical Engineers Environ Prog, 2012