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

Abstract

BackgroundThe 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 β-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 β-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.ResultsTo 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 β-1,4, β-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.ConclusionWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0420-y) contains supplementary material, which is available to authorized users.

Deciphering the molecular mechanisms behind cellulase production in Trichoderma reesei, the hyper-cellulolytic filamentous fungus

“…26) Recently, we reported that PC-3-7's enhanced cellulase-producing ability is related to the SNPs in cre1, bglr and bgl2. 17,26,27) This enhanced cellulase-producing ability, if combined with an efficient saccharification machinery, could aid in an efficient bioconversion of cellulosic biomass into biofuels. The second-generation biofuel ethanol produced from lignocellulosic biomass offers a promising alternative and environmentally friendly energy due to its cost benefits, abundance, and renewability.…”

Engineering of Trichoderma reesei for enhanced degradation of lignocellulosic biomass by truncation of the cellulase activator ACE3

“…Many mutations with essential roles in cellulase hyperproduction still need to be identified. Several reports have identified some mutagenic events by comparative genomic screening, especially in known TFs, which provided a better understanding of the regulation in such hyper-producers of cellulase and have resulted in the construction of more efficient cellulase-producing strains through genetic engineering [5,13,14].…”

“…This may result in an explanation to the underlying mechanism of cellulase induction by ACE3. Meanwhile, two specific mutations of the PC-3-7 bgl2 and bglr genes were proven to enhance cellulase expression [13,24]. These functional mutations originated from classical random mutagenesis by scanning the overproducing mutants for improved cellulase production.…”

“…T. reesei strain PC-3-7 [10], derived from QM9414 through several rounds of mutagenesis, is a cellulase hyper-producing mutant that exhibits twice as much cellulase activity as QM9414 [11]. This has led to the identification of a large number of mutagenic events that contribute to the improvements of cellulase in the QM9414 and NG14 branch [5,[12][13][14]. However, most of the mutation sites are found in non-coding regions of the genome during classical mutagenesis, which makes it hard to understand their role in cellulase hyper-production.…”

“…Genetic engineering is a feasible strategy to increase cellulase production, for example by manipulating transcription factors (TFs) to regulate gene expression [1,16]. The bgl2 gene encoding intracellular β-glucosidase II (BGLII/Cel1a) is mutated in PC-3-7; this mutation reduces the cellobiose metabolic rate, resulting in an indirect release from carbon catabolite repression of cellulase expression by glucose, which enhances cellulase expression [13]. The engineering of TFs involved in transcription of cellulase genes has been developed to enhance the production of cellulase in filamentous fungi [5,17].…”