17-22: Genetic engineering of Neurospora crassa for direct sugar aldonates production from cellulose

Tuesday, May 1, 2012
Napoleon Ballroom C-D, 3rd fl (Sheraton New Orleans)
Edyta Szewczyk1, Weihua Wu1, Takao Kasuga2 and Zhiliang Fan1, (1)Department of Biological and Agricultural Engineering, University of California, Davis, CA, (2)Department of Plant Pathology, United States Department of Agriculture - Agricultural Research Service, University of California, Davis, CA
Starting from cellulosic feedstock, the conventional gluconic acid and isobutanol production process involves five steps: pretreatment, cellulase production, enzymatic hydrolysis to make sugars, aerobic fermentation to produce gluconic acid from cellulose hydrolysate, and anaerobic fermentation to produce isobutanol from sugars. Cellulase production or purchase represents a substantial portion of processing costs. In a proposed route, cellulase production, enzymatic hydrolysis, and aerobic fermentation will be consolidated into a single step — producing cellooligosaccharide aldonates (COAs) from pretreated cellulose by an engineered cellulolytic fungus in an aerobic fermentation step — and isobutanol and gluconate will be produced from COAs in a subsequent anaerobic fermentation step. Hence, the proposed process has only three steps: pretreatment, aerobic fermentation, and anaerobic fermentation. Compared to the conventional process, the proposed process is more consolidated and requires neither a dedicated process for cellulase production nor the exogenous addition of cellulase, both of which will result in lower processing costs.

To divert carbon flow toward cellooligosaccharide aldonates, the fungus Neurospora crassa was modified by deleting six of seven copies of the bgl genes encoding β-glucosidase (BGL) by genetic crossing. To further genetically modify the strain, a self-excising marker employing the β-rec/six site-specific recombination system established previously in Aspergillus fumigatus, but never before validated in N. crassa,was adopted to obtain multiple targeted deletions. Further improvement of the strain involves deleting the specific repressor ace1, as well as the global regulator of carbon catabolite repression cre1, to improve both cellulase and cellobiose dehydrogenase (CDH) production.

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