Engineering the Filamentous Fungus Neurospora crassa for Lipid Production From Lignocellulosic Biomass

Microbially produced triacylglycerol (TAG) is a potential feedstock for the production of biodiesel, but its commercialization will require high yields from low-cost renewable feedstocks such as lignocellulose. The present study employs a multi-gene approach to increasing TAG biosynthesis in the filamentous fungus Neurospora crassa. We demonstrate the redirection of carbon flux from glycogen biosynthesis towards fatty acid biosynthesis in a glycogen synthase deletion strain (gsy-1). Furthermore, combining gsy-1 with an enhanced TAG biosynthetic strain (acyl-Coenzyme A synthase; acs-3) of N. crassa yielded a twofold increase in total fatty acid accumulation over the control strain. The cellulose degrading potential of this double deletion strain was improved by deleting of the carbon catabolite regulation transcription factor (cre-1) to create the triple deletion strain acs-3 cre-1; gsy-1. This strain exhibited early and increased cellulase expression, as well as fourfold increased total fatty acid accumulation over the control on inhibitor-free model cellulose medium. The cre-1 mutation, however, was not beneficial for total fatty acid accumulation from pretreated lignocellulose. Conversion of dilute-acid pretreated Miscanthus to TAG was maximum in the constructed strain acs-3; gsy-1, which accumulated 2.3-fold more total fatty acid than the wild-type control strain, corresponding to a total fatty acid yield of 37.9mg/g dry untreated Miscanthus. Biotechnol. Bioeng. 2014;111: 1097-1107. (c) 2014 Wiley Periodicals, Inc.