Proper perception of the extracellular insoluble cellulose is key to initiating the rapid synthesis of cellulases by cellulolytic Trichoderma reesei. Uptake of soluble oligosaccharides derived from cellulose hydrolysis represents a potential point of control in the induced cascade. In this study, we identified a major facilitator superfamily sugar transporter Stp1 capable of transporting cellobiose by reconstructing a cellobiose assimilation system in Saccharomyces cerevisiae. The absence of Stp1 in T. reesei resulted in differential cellulolytic response to Avicel versus cellobiose. Transcriptional profiling revealed a different expression profile in the Δstp1 strain from that of wild-type strain in response to Avicel and demonstrated that Stp1 somehow repressed induction of the bulk of major cellulase and hemicellulose genes. Two other putative major facilitator superfamily sugar transporters were, however, up-regulated in the profiling. Deletion of one of them identified Crt1 that was required for growth and enzymatic activity on cellulose or lactose, but was not required for growth or hemicellulase activity on xylan. The essential role of Crt1 in cellulase induction did not seem to rely on its transporting activity because the overall uptake of cellobiose or sophorose by T. reesei was not compromised in the absence of Crt1. Phylogenetic analysis revealed that orthologs of Crt1 exist in the genomes of many filamentous ascomycete fungi capable of degrading cellulose. These data thus shed new light on the mechanism by which T. reesei senses and transmits the cellulose signal and offers potential strategies for strain improvement.

Appropriate perception of cellulose outside the cell by transforming it into an intracellular signal ensures the rapid production of cellulases by cellulolytic Hypocrea jecorina . The major extracellular β-glucosidase BglI (CEL3a) has been shown to contribute to the efficient induction of cellulase genes. Multiple β-glucosidases belonging to glycosyl hydrolase (GH) family 3 and 1, however, exist in H. jecorina . Here we demonstrated that CEL1b, like CEL1a, was an intracellular β-glucosidase displaying in vitro transglycosylation activity. We then found evidence that these two major intracellular β-glucosidases were involved in the rapid induction of cellulase genes by insoluble cellulose. Deletion of cel1a and cel1b significantly compromised the efficient gene expression of the major cellulase gene, cbh1 . Simultaneous absence of BglI, CEL1a, and CEL1b caused the induction of the cellulase gene by cellulose to further deteriorate. The induction defect, however, was not observed with cellobiose. The absence of the three β-glucosidases, rather, facilitated the induced synthesis of cellulase on cellobiose. Furthermore, addition of cellobiose restored the productive induction on cellulose in the deletion strains. The results indicate that the three β-glucosidases may not participate in transforming cellobiose beyond hydrolysis to provoke cellulase formation in H. jecorina . They may otherwise contribute to the accumulation of cellobiose from cellulose as inducing signals.

Trichoderma reesei represents an important workhorse for industrial production of cellulases as well as other proteins. The large-scale production is usually performed in a substrate-inducing manner achieved by a fine-tuned cooperation of a suite of transcription factors. Their production and subsequent analysis are, however, often either difficult to manipulate or complicated by the concomitant production of other inducible proteins. Alternatives to control gene expression independent of the nutritional state are thus preferred in some cases to facilitate not only biochemical studies of proteins but also genetic engineering of the producer. We identified a copper transporter encoding gene tcu1 (jgi:Trire2:52315) in T. reesei, the transcription of which was highly responsive to copper availability. Whereas excess copper repressed the expression of tcu1 from T. reesei, eliminating copper addition in the medium resulted in a high-level transcription of tcu1. The usefulness of the system was further illustrated by the high-level expression of specific cellulases driven by the tcu1 promoter in T. reesei when cultivated on D-glucose or glycerol as the sole carbon source. A recombinant T. reesei strain, which overexpressed the main transcription activator of hydrolases (xylanase regulator 1) under the control of tcu1 promoter, was found to be relieved from the carbon catabolite repression and thus displayed a constitutive cellulase expression. Moreover, the amount and activities of cellulases produced by this strain on glycerol or glucose fully recapitulated those of the parental strain produced on Avicel. Expression of T. reesei tcu1 gene was tightly controlled by copper availability, and a homologous protein expression system was developed based on this promoter. Deregulation of XYR1 (xylanase regulator 1) mediated by the tcu1 promoter not only overcame the carbon catabolite repression of cellulases but also resulted in their full expression even on the non-inducing carbon sources.