Biofuels Production projects
Engineering Filamentous Fungi for Increased Lipid Production and Secretion
The majority of biodiesel produced in the United States is derived from oilseed crops such as soybean and rapeseed. The acreage of these crops required to displace a significant fraction of diesel is beyond current sustainable production capacity. New technologies are required to make biodiesel a sustainable, renewable fuel option. Heterotrophic lipid production has the potential to replace the lipids produced by oilseed crops. Two major challenges limit the economic feasibility of this biodiesel production: simple sugars are required as a feedstock, and lipid recovery is challenging. This project uses a celluloytic filamentous fungus, Neurospora crassa, to produce and secrete lipids from a lignocellulosic feedstock. The ultimate goal of this project is to develop an approach for the redesign of any cellulolytic fungus, to reveal targets for genetic manipulation with the ultimate objective of rationally designing industrially relevant celluloytlc fungi for production and secretion of lipids (started in 2012).
Strategies are necessary for engineering biosynthetic pathways to maximize lignocellulose-derived lipid yields and productivity. We took a multi-gene approach to increasing lignocellulose-derived lipid biosynthesis in the model filamentous fungus Neurospora crassa. We first demonstrated that redirecting carbon flux away from glycogen and towards fatty acid biosynthesis in a previously identified enhanced biosynthetic strain of N. crassa yields a 2-fold increase in lipid accumulation over the control. We then further improved the cellulose degrading potential of this double deletion strain by knocking out the carbon catabolite regulation transcription factor to create the triple deletion strain Δacs-3; Δgsy-1; Δcre-1, and achieved 4-fold increased lipid accumulation over the control growing on an inhibitor-free model cellulose medium. The Δcre-1, however, is not beneficial for lipid accumulation from pretreated lignocellulose. We also constructed the strain Δacs-3; Δgsy-1, which can accumulate 1.3 g/L of lipid directly from dilute-acid pretreated Miscanthus.
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 lipids. In this project so far, we have demonstrated 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. In addition, we demonstrated the feasibility of N. crassa as a whole-cell biocatalyst for lipid production directly from non-detoxified dilute-acid pretreated Miscanthus slurry.