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Significance Lignocellulose-based biorefinery relies on plant cell wall degrading enzymes. Current genome editing methods can create fungal enzyme hypersecreter strains by design. However, the identification of candidate genes for targeted engineering of this trait remains a bottleneck, and the necessity of specific inducer molecules further complicates production. By the resequencing of a classical hypersecretingNeurospora crassa mutant, we uncovered that mutation of a gene encoding an F-box protein (exo -1 ) causes inducer-independent hypersecretion of amylases, invertase, and pectinases. Systems biology and genetic studies of Δexo -1 shed light on the regulation of enzyme secretion in filamentous fungi, allowing targeted reverse engineering of industrially employed fungi, such asMyceliophthora thermophila , demonstrating the power of classical mutants in combination with modern sequencing and omics technologies.

The F-box protein gene exo - 1 is a target for reverse engineering enzyme hypersecretion in filamentous fungi