Trichoderma reesei, the main filamentous fungus used for industrial cellulase production, was long considered to be asexual. The recent discovery of the mating type locus in the natural isolate QM6a and the possibility to cross this sterile female strain with a fertile natural female strain opened up a new avenue for strain optimization. We crossed the hyperproducer RutC30 with a compatible female ascospore-derived isolate of the wild-type strain CBS999.97 and analyzed about 300 offspring. A continuous distribution of secreted protein levels was observed in the progeny, confirming the involvement of several mutated loci in the hyperproductive phenotype. A bias toward MAT1-2 strains was identified for higher producers, but not directly linked to the Mating-type locus itself. Transgressive phenotypes were observed in terms of both productivity and secretome quality, with offspring that outperform their parents for three enzymatic activities. Genomic sequences of the 10 best producers highlighted the genetic diversity generated and the involvement of parental alleles in hyperproduction and fertility. IMPORTANCE

The filamentous fungus Trichoderma reesei produces cellulolytic enzymes that are essential for the hydrolysis of lignocellulosic biomass into monomerics sugars. The filamentous fungus T. reesei produces cellulolytic enzymes that are essential for the hydrolysis of lignocellulosic biomass into monomerics sugars, which can in turn be fermented to produce second-generation biofuels and bioproducts. Production performance improvement, which is essential to reduce production cost, relies on classical mutagenesis and genetic engineering techniques. Although sexual reproduction is a powerful tool for improving domesticated species, it is often difficult to apply to industrial fungi since most of them are considered asexual. In this study, we demonstrated that outbreeding is an efficient strategy to optimize T. reesei . Crossing between a natural isolate and a mutagenized strain generated a biodiverse progeny with some offspring displaying transgressive phenotype for cellulase activities.