The impact of a single-nucleotide mutation of bgl2 on cellulase induction in a Trichoderma reesei mutant.

BACKGROUND: The filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) produces increased cellulase expression when grown on cellulose or its derivatives as a sole carbon source. It has been believed that ß-glucosidases of T. reesei not only metabolize cellobiose but also contribute in the production of inducers of cellulase gene expression by their transglycosylation activity. The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer. The comparative genomics analysis of PC-3-7 and its parent revealed a single-nucleotide mutation within the bgl2 gene encoding intracellular ß-glucosidase II (BGLII/Cel1a), giving rise to an amino acid substitution in PC-3-7, which could potentially account for the enhanced cellulase expression when these strains are cultivated on cellulose and cellobiose. RESULTS: To analyze the effects of the BGLII mutation in cellulase induction, we constructed both a bgl2 revertant and a disruptant. Enzymatic analysis of the transformant lysates showed that the strain expressing mutant BGLII exhibited weakened cellobiose hydrolytic activity, but produced some transglycosylation products, suggesting that the SNP in bgl2 strongly diminished cellobiase activity, but did not result in complete loss of function of BGLII. The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked ß-1,4, ß-1,3, or a mixture of both. PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates. Furthermore, the effect of this bgl2 mutation was reproduced in the common strain QM9414 in which the transformants showed cellulase production comparable to that of PC-3-7. CONCLUSION: We conclude that BGLII plays an important role in cellulase induction in T. reesei and that the bgl2 mutation in PC-3-7 brought about enhanced cellulase expression on cellobiose. The results of the investigation using PC-3-7 suggested that other mutation(s) in PC-3-7 could also contribute to cellulase induction. Further investigation is essential to unravel the mechanism responsible for cellulase induction in T. reesei.

Hyphal surface architecture and cell morphology of Trichoderma reesei.

The filamentous fungus Trichoderma reesei which is known to secrete high amounts of cellulolytic enzymes was found to produce a massive amount of fibrous material at the outer surface of the cell wall as observed by ultrahigh-resolution low-voltage scanning electron microscopy. Using transmission electron microscopy, the cell wall ornamentation of the hyper-cellulosic mutant PC-3-7 was found to be less massive and much thinner than for QM9414. A significant amount of fibrous material was produced in Avicel-grown cultures that were less abundant in glucose-grown cultures and Avicel was occasionally found entangled within the cell wall-associated fibrous layer.

A new Zn(II)(2)Cys(6)-type transcription factor BglR regulates ß-glucosidase expression in Trichoderma reesei.

BglR (PI: 52368, beta-glucosidaseregulator) was identified as a new transcription factor that up-regulates expression of specific genes encoding ß-glucosidases. Based on a comparative genomic analysis to verify SNPs between Trichoderma reesei mutant PC-3-7 and its parent KDG-12, 19 were confirmed. One of the SNPs was found to cause a missense mutation close to the end of the DNA-binding region of BglR that turned out to be a Zn(II)(2)Cys(6)-type fungal-specific transcription factor. BglR was found to share little homologous to amyR of Aspergillus oryzae that is commonly considered a key regulator of starch degradation. A mutant lacking the bglr gene as well as the PC-3-7 mutant exhibited elevated cellulase production during growth on cellobiose. Reversion of the SNP missence mutation within bglr to the wild-type allele resulted in reduced cellulase production. Expression of specific ß-glucosidase genes in a bglr gene disruptant was repressed with the mutant exhibiting little ability to hydrolyze cellobiose during early log phase even when induced. Thus, one of the functions of BglR is to up-regulate specific ß-glucosidase genes (with the exception of bgl1, which is seemingly under the direct control of Xyr1). The glucose produced then triggers carbon catabolite repression in cellobiose culture.