Screening for genes involved in cellulase regulation by expression under the control of a novel constitutive promoter in Trichoderma reesei

Abstract

• We characterized the upstream region of cfe1 as promising promoter without upstream open reading frames (uORFs) or introns. • The cfe1 promoter enabled up to 400 fold overexpression. • We analyzed transcriptomic data with support from machine learning to select target genes. • We report three genes affecting cellulase regulation. The filamentous ascomycete Trichoderma reesei is a biotechnological workhorse used in the production of homologous and heterologous proteins for diverse applications including biofuel production, textile finishing and food additives. This fungus uses a complex adaptation machinery to regulate its cellulases in response to environmental conditions. Detailed understanding of this regulation allows for improvement of enzyme production using the strong enzyme gene promotors. Here, we selected six genes with characteristic transcript levels associated with cellulase production to be tested for their enzyme regulatory function. Machine learning for inference of a gene regulatory network (GRN) was applied to support the association of these genes with cellulase induction. Additionally, we screened available transcriptomic data for genes with strong constitutive transcript levels and selected the promoter of the gene cfe1 , whose transcript levels were above those of tef1 and cDNA1 and near those of cbh1 , for gene overexpression testing. Using this promoter, we explored the relevance to cellulose degradation efficiency of three transporters, two ferric reductases and one gene of unknown function, which were overexpressed in T. reesei grown on cellulose. This promoter enabled up to 400 fold overexpression and yielded transcript levels above those of tef1 or cDNA1. We provide evidence of effects of a ferric reductase, an ammonium permease and a gene of unknown function on the regulatory machinery of cellulase expression in T. reesei . In summary we identified the cfe1 promoter, a novel constitutive promoter with promising efficiency, as well as three genes relevant to cellulase regulation in T. reesei .