Differential Involvement of β-Glucosidases from Hypocrea jecorina in Rapid Induction of Cellulase Genes by Cellulose and Cellobiose

Abstract

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.

Two Major Facilitator Superfamily Sugar Transporters from Trichoderma reesei and Their Roles in Induction of Cellulase Biosynthesis

“…Equal amounts of mycelia were transferred to a fresh medium containing the respective carbon sources after being washed twice with medium without carbon source, and incubation was continued for the indicated time period. When sophorose (1 mM) was used as an inducer, resting cell cultivations were performed as described previously (12).…”

“…Transformation of T. reesei was carried out essentially as described previously (12). Transformants were selected on minimal medium either for uridine prototroph or for resistance to hygromycin (120 g/ml).…”

“…Total RNA was extracted with the TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Southern hybridization and Northern analysis were performed with the digoxigenin nonradioactive system from Roche Applied Science as described previously (12). Relative transcription levels were analyzed semiquantitatively by densitometry using ImageJ software.…”

“…Detection of cellobiohydrolase CBH1 was performed by immunoblotting using a polyclonal antibody raised against amino acids (426 -446) of CBH1 as described previously (12).…”

Deciphering the Effect of the Different N-Glycosylation Sites on the Secretion, Activity, and Stability of Cellobiohydrolase I from Trichoderma reesei

“…DNA fragments with ϳ1.5 kb of 5= and 3= flanking sequences of cbh1 were amplified from T. reesei genomic DNA and successively inserted into pUCpyr4 to obtain pUCcbh1pyr4, which was further digested by SalI and EcoRI to generate the 6-kb deletion cassette. This 6-kb fragment was subsequently used to transform T. reesei TU-6 as previously described (31). Transformants were selected on minimal medium for uridine prototrophy.…”