Role of four major cellulases in triggering of cellulase gene expression by cellulose in Trichoderma reesei

Abstract

The relative contributions of four major cellulases of Trichoderma reesei (1,4-␤-D-glucan cellobiohydrolase I [CBH I], CBH II, endo-1,4-␤-D-glucanase I [EG I], and EG II) to the generation of the cellulase inducer from cellulose were studied with isogenic strains in which the corresponding genes (cbh1, cbh2, egl1, and egl2) had been deleted by insertion of the Aspergillus nidulans amdS marker gene. During growth on lactose (a soluble carbon source provoking cellulase gene expression), these strains showed no significant alterations in their ability to express the respective other cellulase genes, with the exception of the strain containing ⌬cbh1, which exhibited an increased steady-state level of cbh2 mRNA. On crystalline cellulose as the only carbon source, however, significant differences were apparent: strains in which cbh2 and egl2, respectively, had been deleted showed no expression of the other cellulase genes, whereas strains carrying the cbh1 or egl1 deletion showed these transcripts. The ⌬cbh1-containing strain also showed enhanced cbh2 mRNA levels under these conditions. A strain in which both cbh1 and cbh2 had been deleted, however, was unable to initiate growth on cellulose. Addition of 2 mM sophorose, a putative inducer of cellulase gene expression, to such cultures induced the transcription of egl1 and egl2 and restored the ability to grow on cellulose. We conclude that CBH II and EG II are of major importance for the efficient formation of the inducer from cellulose in T. reesei and that removal of both cellobiohydrolases renders T. reesei unable to attack crystalline cellulose.Cellulose, a linear, essentially insoluble ␤-1,4-glucosidically linked homopolymer with a size of about 8,000 to 12,000 glucose units, is used as an energy source by numerous and diverse microorganisms, including fungi and bacteria, which produce functionally complete cellulase enzyme systems. Among the best characterized of these systems are the inducible cellulases of the saprophytic fungus Trichoderma reesei. They consist of at least two 1,4-␤-D-glucan cellobiohydrolases (CBH I and II; EC 3.2.1.91), four endo-1,4-␤-D-glucanases [EG I, II, III, and V; 1,3-(1,3;1,4)-␤-D-glucan 3(4) glucanohydrolase; EC 3.2.1.4], and two 1,4-␤-D-glucosidases (BGL I and II; EC 3.2.1.21), which are formed adaptively in the presence of cellulose to synergistically cooperate in its degradation.The mechanism by which an insoluble substrate triggers the formation of enzymes for its degradation in the fungus has been a matter of speculation for three decades. We have previously demonstrated the presence of cellulases bound to the conidial surface of T. reesei and have shown that they are essential for growth on cellulose as the sole carbon source (10, 17). Evidence for a major role of one of these cellulases, CBH II, to enable the fungus to start growth on cellulose has also been presented (12,21). We interpreted these data as showing that CBH II releases small amounts of cellobiose, which may, either directly or after further conversion (7, 14), ...

Systems analysis of plant cell wall degradation by the model filamentous fungus Neurospora crassa

“…crassa cellobiohydrolase(I) (CBHI) is the most highly produced extracellular protein during growth on Avicel or Miscanthus, and deletion of this gene causes the most severe growth deficiencies on cellulosic substrates. By contrast, in H. jecorina, deletion of cbhII caused the most severe phenotype (23)(24)(25). In N. crassa, deletion of cellobiohydrolase(II) also causes growth deficiencies on cellulosic substrates, but to a much lesser extent than CBH(I), suggesting that exoglucanase activity in N. crassa is predominantly from CBH(I) and that endoglucanases and other CBHs do not compensate for the loss of CBH(I).…”

Regulation of gene expression in industrial fungi: Trichoderma

“…Instead, elevated levels of CBHII (approximately twice that of CBHI) were observed (Kubicek et al 1993b). That CBHI and CBHII act as initiators of growth of Trichoderma on cellulose was further supported by the findings that conidia from a cbh1-and cbh2-negative strain were almost unable to initiate growth on cellulose (Seiboth et al 1997). Addition of the cellulase-inducer sophorose to a culture of this deletion strain induced transcription of remaining cellulases such as egl1 and egl2 and restored the ability to attack cellulose.…”

Cellotriose and Cellotetraose as Inducers of the Genes Encoding Cellobiohydrolases in the Basidiomycete Phanerochaete chrysosporium

“…Antibody competition and antisense RNA experiments have led to the proposal that low basal levels of cellulases (mainly Cel7A and Cel6A) are necessary for further induction (5,12). Analysis of CBH gene expression by using cellulase deletion mutants strongly suggested that basal expression of Cel6A and GH family 5 EG (Cel5A) is indispensable for the formation of the inducer(s) of CBHs from cellulose (37,38).…”