Wataru Ogasawara

A new Zn(II)2Cys6-type transcription factor BglR regulates β-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 β-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 β-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 β-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.

Construction of a recombinant Trichoderma reesei strain expressing Aspergillus aculeatus β-glucosidase 1 for efficient biomass conversion.

To develop a Trichoderma reesei strain appropriate for the saccharification of pretreated cellulosic biomass, a recombinant T. reesei strain, X3AB1, was constructed that expressed an Aspergillus aculeatus β‐glucosidase 1 with high specific activity under the control of the xyn3 promoter. The culture supernatant from T. reesei X3AB1 grown on 1% Avicel as a carbon source had 63‐ and 25‐fold higher β‐glucosidase activity against cellobiose compared to that of the parent strain PC‐3‐7 and that of the T. reesei recombinant strain expressing an endogenous β‐glucosidase I, respectively. Further, the xylanase activity was 30% lower than that of PC‐3‐7 due to the absence of xyn3. X3AB1 grown on 1% Avicel‐0.5% xylan medium produced 2.3‐ and 3.3‐fold more xylanase and β‐xylosidase, respectively, than X3AB1 grown on 1% Avicel. The supernatant from X3AB1 grown on Avicel and xylan saccharified NaOH‐pretreated rice straw efficiently at a low enzyme dose, indicating that the strain has good potential for use in cellulosic biomass conversion processes. Biotechnol. Bioeng. 2012;109: 92–99.

Application of Trichoderma reesei Cellulase and Xylanase Promoters through Homologous Recombination for Enhanced Production of Extracellular β-Glucosidase I

One of the limiting factors for the application of Trichoderma reesei to degrade cellulosic biomass is its low β-glucosidase activity, required to convert cellobiose to glucose. The egl3 and the xyn3 promoters were used to express β-glucosidase 1 gene bgl1 through homologous recombination to improve the cellulose degradation ability of T. reesei. The recombinant strains expressing β-glucosidase 1 (BGLI) under the control of either the egl3 or the xyn3 promoter had 4.0 and 7.5 fold higher β-glucosidase activity than the native strain, which compares well to the finding that in wild-type T. reesei PC-3-7, the levels of egl3 and xyn3 mRNA expression were 6.0 and 12 fold higher respectively than that of bgl1. Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis of proteins secreted by the recombinant strains demonstrated that BGLI was overproduced. The increase in the transcription of bgl1 and the concomitant elevated level of BGLI in these recombinant strains were sufficient to degrade the cellobiose and cellotriose formed during the degradation of pretreated cedar powder.

Evaluation and characterization of Trichoderma reesei cellulase and xylanase promoters

Comprehensive analyses on promoters of four cellulase and one xylanase genes of Trichoderma reesei were performed expressing a single reporter uidA from Escherichia coli to construct highly functional cellulase-overproducing strains. GUS amount expressed under each promoter correlated entirely with each mRNA amount, suggesting that GUS production was controlled at the transcriptional level. The uidA transcript levels were much lower than the native gene mRNAs, but they were produced in proportion to the mRNA of native cellulase and xylanase genes driven by the same promoters except for the cbh2 promoter. Cellulose-degrading activity and protein amount was reduced in cbh1 and cbh2 disruptant mutants compared to the wild-type T. reesei PC-3-7 and other uidA transformants. The cbh1 disruptant strain was observed to produce more CBH II, EG I, EG III, and xylanases than native PC-3-7 and the other uidA transformants with the same amounts of protein in SDS-PAGE gels. This observation was further analyzed by measuring mRNA levels of cellulase and xylanase genes in the disruptants using quantitative real-time PCR. In the Pcbh1-gus, mRNA levels for cbh2 and egl1 genes were higher than those in native T. reesei PC-3-7 and all other disruptant strains. The cbh2 disruptant strain had the highest amount of cbh1 mRNA among the strains tested. Homologous integration of uidA at the egl1, egl3, and xyn3 loci was also found to cause a slight increased level of cbh1 mRNA, whereas mRNA levels for egl1, egl3, and xyn3 in all the disruptants were similar to those of T. reesei PC-3-7.

Analysis of the saccharification capability of high-functional cellulase JN11 for various pretreated biomasses through a comparison with commercially available counterparts

Although the capabilities of Trichoderma reesei cellulases have been greatly improved, these enzymes are still too costly for commercial use. The aim of this research was to assess the biomass saccharification capability of JN11, a recombinant cellulase, compared with that of the commercially available cellulases Accellerase 1500 and Cellic CTec. The activities of JN11, Accellerase 1500, and Cellic CTec were compared by using various types of cellulosic biomass, including rice straw, Erianthus, eucalyptus, and Japanese cedar. JN11 had higher saccharification capability for rice straw, Erianthus, eucalyptus, and Japanese cedar compared with the commercial cellulases. The JN11 saccharification of cellulosic biomasses, including hemicellulose (NaOH-pretreated biomasses), resulted in high glucose and xylose yields because of the high xylanase/xylosidase activity of JN11. Moreover, even JN11 saccharification of hemicellulose-free biomasses (sulfuric acid-, hydrothermally, and steam exploded-pretreated biomasses) resulted in high glucose yields. The cellulase activity of JN11, however, was comparable to that of its commercial counterparts. These findings indicate that the saccharification ability of cellulase is unrelated to its cellulase activity when measured against Avicel, CMC, pNP-lactoside, and other substrates. JN11 showed high activity for all types of pretreated cellulosic biomasses, indicating its usefulness for saccharification of various cellulosic biomasses.

Ethanol production from high cellulose concentration by the basidiomycete fungus Flammulina velutipes

Ethanol production by Flammulina velutipes from high substrate concentrations was evaluated. F. velutipes produces approximately 40-60 g l(-1) ethanol from 15% (w/v) D-glucose, D-fructose, D-mannose, sucrose, maltose, and cellobiose, with the highest conversion rate of 83% observed using cellobiose as a carbon source. We also attempted to assess direct ethanol fermentation from sugarcane bagasse cellulose (SCBC) by F. velutipes. The hydrolysis rate of 15% (w/v) SCBC with commercial cellulase was approximately 20%. In contrast, F. velutipes was able to produce a significant amount of ethanol from 15% SCBC with the production of β-glucosidase, cellobohydrolase, and cellulase, although the addition of a small amount of commercial cellulase to the culture was required for the conversion. When 9 mg g(-1) biomass of commercial cellulase was added to cultures, 0.36 g of ethanol was produced from 1 g of cellulose, corresponding to an ethanol conversion rate of 69.6%. These results indicate that F. velutipes would be useful for consolidated bioprocessing of lignocellulosic biomass to bioethanol.

Identification of major facilitator transporters involved in cellulase production during lactose culture of Trichoderma reesei PC-3-7

Although lactose is a preferred cellulase inducer in the industrial production of cellulase by Trichoderma reesei, the mechanism of induction is not fully understood. Because sugar transporters might be involved at an early step of induction by oligosaccharides, we sought permeases associated with cellulase induction by lactose. Two such MFS sugar transporters in the T. reesei hyper-cellulolytic PC-3-7 strain, an industrial cellulase producer developed in Japan, were identified in a screening for lactose permeases. Disruption of the genes encoding these two transporters resulted in decreased lactose uptake and delayed growth in lactose culture. Further, the deletion strains produced less cellulase when cultivated on lactose. No substantial differences were observed in cellulase production when PC-3-7 was cultivated in cellulose-based medium. The present work provides evidence that these transporters are critical for cellulase production in lactose culture.

Single Nucleotide Polymorphism Analysis of a Trichoderma reesei Hyper-Cellulolytic Mutant Developed in Japan

The ascomycete Trichoderma reesei is known as one of the most prolific producers of plant biomass-degrading enzymes. While several mutant strains have been developed by mutagenesis to improve enzyme productivity for a variety of industrial applications, little is known about the mechanical basis of these improvements. A genomic sequence comparison of mutant and wild-type strains was undertaken to provide new insights in this regard. We identified a number of single-nucleotide polymorphisms (SNPs) after sequencing the genome of a hyper-cellulolytic T. reesei strain, PC-3-7, with a next-generation sequencer. Of these, the SNP detected in cre1, the carbon catabolite repressor gene, was found to be responsible for increased cellulase production. Further comparative genomic analysis enabled the identification of an SNP that correlated well with high cellulase production in a T. reesei mutant. These results provide a better understanding of the genetic changes induced by classical mutagenesis and how they correlate with desirable phenotypes in filamentous fungi.

Multicopy integration and expression of heterologous genes in the oleaginous yeast, Lipomyces starkeyi

The oleaginous yeast, Lipomyces starkeyi, is an excellent lipid producer with great industrial potential. However, methods for molecular breeding have not been established for L. starkeyi. We describe the development of a system for targeted rDNA integration of multiple copies of a gene into L. starkeyi genome by spheroplast–polyethylene glycol transformation.

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

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.