Expressing heterologous cellulases in oleaginous yeast Yarrowia lipolytica: bottlenecks and opportunities

Yarrowia lipolytica was evaluated for its potential to express cellulolytic enzymes in order to develop a direct microbial conversion (DMC) platform for producing hydrocarbon fuels from cellulosic substrates. Y. lipolytica is an oleaginous yeast known to intracellularlly accumulate lipids and is capable of metabolizing glucose and xylose to produce lipids, but lacks cellulolytic enzymes to break down biomass.  To reduce costly separated production of cellulases, we investigated the expression of the key cellulase CBHI in Y. lipolytica.  Initial efforts in expressing T. reesei CBH I lead to a successful expression of the protein but with limited enzymatic activity.  However, the heterologous expression of chimeric Talaromyces emersonii CBH I catalytic domain-Trichoderma reesei linker-CBM demonstrated remarkably improved enzymatic activity.  The purified chimeric TeCBH I-TrLinker-TrCBM showed specific Avicelase activity comparable to that of native CBHI produced by T. reesei.  In parallel, we successfully expressed CBH II and endoglucanase II (EGII) proteins in Yarrowia, and characterized the individual target cellulases.  To test their cellulose-degrading capability, we measured the enzymatic hydrolysis of cellulosic substrates, both in vitro and in vivo, by different combinations of Yarrowia transformants and their expressed cellulases.  More importantly, the bottlenecks and opportunities for co-expressing these cellulase genes in Yarrowia to enable utilization of cellulolytic substrates were explored, which revealed that multiple factors (including the promoters and secretion) were involved in the control of the cellulase expression.  This work lays a foundation for further engineering of Yarrowia to become cellulolytic and therefore capable of utilizing cellulolosic substrates and converting them to biofuels.