Glycosylation Isoforms of Heterologous Fungal Cellobiohydrolases (CBH1) Determined by "Top-Down" High Resolution/High Accuracy

A critical step in the lignocellulosic biofuels pipeline is the extraction of fermentable sugars from plant biomass. Extracellular fungal enzymes are currently the most effective way of deconstructing biomass, but their efficiency needs to be improved. One of the most important of the cellulose-degrading enzymes is cellobiohydrolase1 (CBH1, Cel7A). The Walton lab is studying the natural variation among CBH1 enzymes across all sequenced genomes with the ultimate goal of improving its enzymatic properties relevant to biomass conversion. We have expressed 24 genes, reflecting the entire evolutionary space of CBH1, in the fungal host, Trichoderma reesei, and are in the process of characterizing their enzymological properties. The goal of this proposal is to take advantage of the advanced mass spectrometric capacity at EMSL to analyze the glycosylation and other posttranslational modifications (PTMs) that the heterologous enzymes undergo in T. reesei. In particular, the proposed research will use “top down” proteomics to characterize in detail the proteoforms (isoforms) of heterologous CBH1 enzymes. A major ultimate goal is to understand what effects the observed PTMs have on critical enzymatic properties such as specific activity, pH and temperature optima, and cooperativity with other enzymes. The expected results will not only be relevant to the discovery of superior forms of CBH1 but also more generally to a better understanding of the limitations of expressing heterologous proteins in filamentous fungi.