Release of fermentable sugars from plant biomass is a limiting step in efforts to produce ethanol from renewable resources. The goal of this project is to create defined mixtures of enzymes that release hexoses and pentoses from corn stover with minimal protein loadings. We are producing hydrolytic and other enzymes by expression of Trichoderma reesei genes either in Pichia pastoris or in T. reesei itself. Initial experiments have focused on optimization of a “core six” set of enzymes (CBH1 [Cel7A], CBH2 [Cel6A], EG1 [Cel7B], xylanase 3, beta-glucosidase, beta-xylosidase – see companion poster by Banerjee et al.). However, these enzymes alone cannot achieve satisfactory yields of Glu and Xyl from corn stover. In order to produce better synthetic enzyme mixtures, we are expressing additional “accessory” enzymes, based on the secretome of Trichoderma reesei when grown on corn stover (Nagendran et al., 2009, Fung Genet Biol 46:427). To date we have expressed more than 10 accessory enzymes (including arabinofuranosidase, α-glucuronidase, Cel5A, Cel61A, Cel61B, and xylanase 2) and are testing them in all combinations together with the core set.