Background:The molecular mechanism underlying the regulation of cellulase production by T. reesei is unclear. Results: The absence of sugar transporter Stp1 enhanced cellulase gene induction whereas the absence of Crt1 abolished cellulase gene expression. Conclusion: Crt1 is essential in cellulase gene induction independent of intracellular sugar delivery. Significance: These data shed light on the mechanism by which T. reesei senses and transmits cellulose signal.

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 ␤-glucosidase BglI (CEL3a) has been shown to contribute to the efficient induction of cellulase genes. Multiple ␤-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 ␤-glucosidase displaying in vitro transglycosylation activity. We then found evidence that these two major intracellular ␤-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 ␤-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 ␤-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.

BackgroundTrichoderma 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.ResultsWe 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.ConclusionExpression 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.

bLactose (1,4-O-␤-D-galacto-pyranosyl-D-glucose) induces cellulolytic enzymes in Trichoderma reesei and is in fact one of the most important soluble carbon sources used to produce cellulases on an industrial level. The mechanism underlying the induction is, however, not fully understood. In this study, we investigated the cellular functions of the intracellular ␤-glucosidases CEL1a and CEL1b in the induction of cellulase genes by lactose in T. reesei. We demonstrated that while CEL1a and CEL1b were functionally equivalent in mediating the induction, the simultaneous absence of these intracellular ␤-glucosidases abolished cbh1 gene expression on lactose. D-Galactose restored the efficient cellulase gene induction in the ⌬cel1a strain independently of its reductive metabolism, but not in the ⌬cel1a ⌬cel1b strain. A further comparison of the transcriptional responses of the ⌬cel1a ⌬cel1b strain complemented with wild-type CEL1a or a catalytically inactive CEL1a version and the ⌬cel1a strain constitutively expressing CEL1a or the Kluyveromyces lactis ␤-galactosidase LAC4 showed that both the CEL1a protein and its glycoside hydrolytic activity were indispensable for cellulase induction by lactose. We also present evidence that intracellular ␤-glucosidase-mediated lactose induction is further conveyed to XYR1 to ensure the efficiently induced expression of cellulase genes.C ost-effective conversion of plant cell wall-derived polysaccharides holds the potential for production of an environmentally clean and renewable source of energy and platform chemicals (1). Trichoderma reesei (teleomorph Hypocrea jecorina) is well known for its high capacity to secrete large amounts of lignocellulosic enzymes that release fermentable sugars and has thus been developed into one of the most prolific industrial cellulase producers. High-yield production of the bulk of the plant cell walldegrading machinery in T. reesei is, however, dependent on induction by insoluble substrates that include cellulose, hemicellulose, and mixtures of plant polymers. Considering the ease of manipulation and the complication of separating enzymes from insoluble plant cell wall materials, soluble inducing substrates are usually preferred or required (2). Among others, the disaccharide lactose (1,4-O-␤-D-galacto-pyranosyl-D-glucose) is an important and economic soluble carbon source for cellulase production by T. reesei. However, the induced cellulase yields on lactose are usually lower than those on cellulose (3, 4). Understanding the differences in the inducing efficiency and the mode by which lactose triggers cellulase formation would be helpful for improving the performance of industrial strains.In fungi, catabolism of lactose is thought to proceed either by extracellular hydrolysis and subsequent uptake of the resulting sugar monomers or by uptake of the disaccharide followed by intracellular hydrolysis (4). For T. reesei, it has been assumed that lactose metabolism relies on the first strategy, based on several findings, including the absence of appar...

LA conduit function decreased in patients with CSFP, while contractile function increased. RA conduit function also decreased in patients with CSFP.