Cellodextrin transporters play an important role in cellulase production in Talaromyces pinophilus

Effective lignocellulose breakdown in filamentous fungi requires extracellular carbohydrate enzymes. Constitutively expressed spore surface CAZymes break down the insoluble complex polysaccharide into soluble oligo-, di-, and monosaccharides, which can then be sensed and transported by cell-surface sugar transporters. However, little is known about the functional and regulatory functions of sugar transporters in Talaromyces pinophilus . In this work, we identified 14 putative cellodextrin transporters (CDTs) in the strain through an in-depth phylogenetic and transcriptomic analysis of 106 putative sugar transporters. Among these, mfs1 and mfs6 were significantly upregulated under cellulase-inducing conditions. The expression of mfs1 and mfs6 responds to increasing cellobiose concentrations, indicating their involvement in cellobiose uptake. Targeted gene deletions showed that while mfs1 deletion primarily affected enzyme secretion when cellobiose was taken as the primary carbon source, loss of mfs6 impaired cellobiose uptake and significantly reduced cellulase secretion across multiple substrates. In concordance, cellulase secretion increased significantly upon separate overexpression of mfs1 and mfs6 transporters. Cellulase induction via mfs1 and mfs6 is likely modulated by CreA, as indicated by multiple CreA-binding sites in their native promoters and cbh1 promoter, enhanced expression of mfs1/mfs6 in ΔcreA and previous evidence of increased saccharification in the ΔcreA strain; therefore, we performed overexpression in a ΔcreA background. The ΔcreA - mfs1-mfs6 double overexpression strain showed no further enhancement in cellulase activities over the ΔcreA single overexpression strains, suggesting potential limitations in signal induction through transporter engineering. Together, these findings identified mfs1 / mfs6 as important players in cellulase induction, and when paired with derepression strategies, position genetically engineered T. pinophilus as a platform for cost-competitive biomass conversion and second-generation biofuels.