Unravelling molecular mechanisms of fungal hypersecretion

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The goal of this proposal is to elucidate the molecular mechanism that underlie fungal hypersecretion of proteins. Filamentous fungi are used for commercial production of enzymes due to their robustness and capacity to secrete proteins. The industrial enzymes market ranges in billions USD. Genetic and genomic analysis revealed that filamentous fungi possess all the key components of the secretion machinery found in yeast. However, the mechanisms that confer filamentous fungi with a higher propensity to secrete proteins than yeast remain largely unknown. I propose to use forward and reverse genetics coupled with whole-genome sequencing to identify the molecular underpinnings of hypersecretion in Aspergillus niger. In addition to being the most commonly used cell factory for the production of extracellular enzymes and organic acids, A. niger is the first and only fungal genome, fully curated and annotated by human curators. Elucidation of the mechanisms responsible for hypersecretion will be done as following. 1) Generating and identifying hypersecretion mutations. Random mutagenesis remains a powerful tool for strain improvement as it can readily elicit both loss-of-function and gain-of-function mutations. To identity which mutations favor a protein hypersecretion phenotype we will use a top-down and a bottom-up approach. Specifically, we will randomly mutagenize A. niger and screen for total protein hypersecretion. Mutations will be identified by genome sequencing. Although random mutagenesis can affect any part of the metabolism, we will focus on mutations predicted to be involved in hyphal morphology, the secretory pathway and regulatory functions. 2) Establishing the role of previously identified mutations in hypersecretion. Previous characterization of hypersecretion mutant strains of filamentous fungi has uncovered numerous mutations. Genes predicted to be involved in regulatory functions, hyphal morphology and secretion machinery are candidates for a hypersecretion phenotype. However, very few of these mutations have experimental confirmation of being involved in hypersecretion. Orthologues of these genes in A. niger will be deleted (loss of function) or replaced (gain of function) using the genome editing method CRISPR/Cas9, each mutant will be tested phenotypically. 3) Characterization of the combinatorial effect of hypersecretion mutations and validation of the mutations. The hypersecretion phenotype is expected to be the result of a combined action of multiple mutations. The mutations identified from approach 1 and 2 will be pooled and transformed into A. niger resulting in a genomic library of mutants that will help identifying the combinatorial effect of the mutations. Understanding the secretion machinery in filamentous fungi is crucial to engineer fungal host strains to reduce enzyme production cost for industrial and environmental applications.