Engineering the filamentous fungus Trichoderma reesei for hydrocarbon production from lignocellulosic biomass

Direct microbial conversion (DMC) offers a number of advantages for consolidating the processing units for biofuel production, thus increasing production efficiency and lowering overall process costs. Significant research advances have been made recently in the successful expression of cellobiohydrolases from ethanol-producing yeast (Ilmen et al., 2011).  Fermentation using the cellobiohydrolases and other cellulase-expressing yeast has shown that much lower enzyme loadings are required for fermenting solid biomass substrates. We are working to extend DMC to a “biomass to hydrocarbon production system" as proof of concept.  T. reesei is an industrially important cellulolytic filamentous fungus. Because of T. reesei's capacity to secrete large amounts of cellulases and hemi-cellulases, we reasoned that engineering this organism to produce hydrocarbons or intermediates would generate an effective DMC strain of T. reesei.   In general, engineering non-cellulolytic strains to secrete sufficient titers of cellulases has been difficult in the case of fuel producing organisms.  Since we have developed a transformation system for T. reesei at NREL, we initiated work to engineer this strain for hydrocarbon production by introducing genes encoding hydrocarbon synthases that can produce well-established hydrocarbon molecules from the isoprenoid pathway intermediate naturally produced by this strain.  Among the three different hydrocarbon synthase genes we introduced, only one of the genes is successfully expressed and the resulting strain produced hydrocarbon from glucose.  In addition, we demonstrated that hydrocarbons can also be produced from engineered T. reesei from D-xylose, L-arabinose and cellulose.