Dissecting Cellulase Hypersecretion in Neurospora crassa

Fungi are the major decomposers of lignocellulosic material in the natural world, and our ability to harness the enzymes that fungi use for degrading plant biomass in their environment have revolutionized the food, fuel, textile and pharmaceutical industries. These enzymes are used to alter naturally occurring polymers from plant materials into everyday products like corn starch based sweeteners, renewable cellulosic biofuels, better performing clothing detergents, or pharmaceuticals. What makes many of these industries feasible is the high capacity of secretion that is inherent to many filamentous fungal organisms. While yeast strains are able to secrete up to 3 g/L of human serum albumin, filamentous hosts have been shown to secrete upwards of 100g/L of cellulase proteins into the supernatant. Thus, many of these industries are fundamentally interested in generating a high producing strain of their protein of interest, and generating a highly productive filamentous fungal host could provide a potential source for these secreted proteins.Unfortunately, the efficient production of well folded and functional protein becomes the major cost limitation of many of these industries. While the essential components of the secretory pathway in fungi are well studied, much less is publicly known about how to engineer a filamentous fungal host for more efficient protein production. There are many reasons for this: (1) known hypersecreting strains have poor genetic amenability, (2) these strains have very few established tools for determining causal alleles and (3), the intellectual property culture that revolves around many of these industrial strains prevents the dissemination of much of this knowledge. In order to address our knowledge gap in filamentous fungal hyper-production, we perform five tasks in this work, (1) we establish Neurospora crassa as a filamentous host for protein production through an iterative mutagenesis process using a quantitative screen to select for the highest cellulase hypersecreters, (2) we use bulk segregant DNA pooling and next-gen sequencing to determine the causative locus, (3) we identify the functional knockout of a conserved flippase as one of the reasons for transcriptionally-independent hypersecretion phenotype, (4), we characterize the flippase mutant for mechanistic alterations to the secretion pathway that may be contributing to hypersecretion, and (5) we determine functional signal peptides for the initial step of secretion of a heterologously produced protein.Finally, we propose further lines of inquiry using the established tools for studying cellulase secretion and regulation in a model filamentous fungal host. This may lead to the identification of additional conserved alleles for future engineering studies of protein expression. This work takes both a discovery-based and a directed approach to study fungal hypersecretion with the ultimate goal of understanding the biology of protein secretion in a filamentous fungus. This may uncover new strategies to rationally engineer fungal organisms for more efficient protein production that may significantly benefit society by the development carbon neutral fuels and more sustainable industrial processes.