8-07: Engineering Neurospora crassa for increased production of lignocellulose-derived lipids

A major limiting factor of heterotrophic lipid production is the high production cost attributed to the feedstock. While lignocellulosic biomass as a feedstock for biofuels production has been highlighted as a viable option due to its abundance and low value, major challenges remain in cost-effectively obtaining required fermentable simple sugars. We propose to overcome this limitation using the cellulolytic filamentous fungus, Neurospora crassa, as a whole-cell biocatalyst to directly convert lignocellulose to lipid. In this study we demonstrate enhanced lipid production and accumulation in N. crassa by redirecting carbon flux away from glycogen biosynthesis and towards fatty acid biosynthesis, as well as by relieving feedback inhibition by fatty acyl-CoA esters in acyl-CoA synthetase mutant strains. In only two mutations we were able to triple lipid content of N. crassa by redirecting carbon flux, thus engineering an oleaginous variant. Additionally, we demonstrate the feasibility N. crassa as a whole-cell biocatalyst for lipid production directly from non-detoxified dilute acid pretreated Miscanthus slurry.