Takanori Furukawa

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.

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.

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.

Sterol Biosynthesis and Azole Tolerance Is Governed by the Opposing Actions of SrbA and the CCAAT Binding Complex.

Azole drugs selectively target fungal sterol biosynthesis and are critical to our antifungal therapeutic arsenal. However, resistance to this class of drugs, particularly in the major human mould pathogen Aspergillus fumigatus, is emerging and reaching levels that have prompted some to suggest that there is a realistic probability that they will be lost for clinical use. The dominating class of pan-azole resistant isolates is characterized by the presence of a tandem repeat of at least 34 bases (TR34) within the promoter of cyp51A, the gene encoding the azole drug target sterol C14-demethylase. Here we demonstrate that the repeat sequence in TR34 is bound by both the sterol regulatory element binding protein (SREBP) SrbA, and the CCAAT binding complex (CBC). We show that the CBC acts complementary to SrbA as a negative regulator of ergosterol biosynthesis and show that lack of CBC activity results in increased sterol levels via transcriptional derepression of multiple ergosterol biosynthetic genes including those coding for HMG-CoA-synthase, HMG-CoA-reductase and sterol C14-demethylase. In agreement with these findings, inactivation of the CBC increased tolerance to different classes of drugs targeting ergosterol biosynthesis including the azoles, allylamines (terbinafine) and statins (simvastatin). We reveal that a clinically relevant mutation in HapE (P88L) significantly impairs the binding affinity of the CBC to its target site. We identify that the mechanism underpinning TR34 driven overexpression of cyp51A results from duplication of SrbA but not CBC binding sites and show that deletion of the 34 mer results in lack of cyp51A expression and increased azole susceptibility similar to a cyp51A null mutant. Finally we show that strains lacking a functional CBC are severely attenuated for pathogenicity in a pulmonary and systemic model of aspergillosis.

Microbial enzyme systems for lignin degradation and their transcriptional regulation

Lignocellulosic biomass is the most abundant renewable resource in nature and has received considerable attention as one of the most promising alternatives to oil resources for the provision of energy and certain raw materials. The phenolic polymer lignin is the second most abundant constituent of this biomass resource and has been shown to have the potential to be converted into industrially important aromatic chemicals after degradation. However, due to its chemical and structural nature, it exhibits high resistance toward mechanical, chemical, and biological degradation, and this causes a major obstacle for achieving efficient conversion of lignocellulosic biomass. In nature, lignin-degrading microorganisms have evolved unique extracellular enzyme systems to decompose lignin using radical mediated oxidative reactions. These microorganisms produce a set of different combinations of enzymes with multiple isozymes and isoforms by responding to various environmental stimuli such as nutrient availability, oxygen concentration and temperature, which are thought to enable effective decomposition of the lignin in lignocellulosic biomass. In this review, we present an overview of the microbial ligninolytic enzyme systems including general molecular aspects, structural features, and systematic differences in each microorganism. We also describe the gene expression pattern and the transcriptional regulation mechanisms of each ligninolytic enzyme with current data.

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

The filamentous fungus Trichoderma reesei is a potent cellulase producer and the best-studied cellulolytic fungus. A lot of investigations not only on glycoside hydrolases produced by T. reesei, but also on the machinery controlling gene expression of these enzyme have made this fungus a model organism for cellulolytic fungi. We have investigated the T. reesei strain including mutants developed in Japan in detail to understand the molecular mechanisms that control the cellulase gene expression, the biochemical and morphological aspects that could favor this phenotype, and have attempted to generate novel strains that may be appropriate for industrial use. Subsequently, we developed recombinant strains by combination of these insights and the heterologous-efficient saccharifing enzymes. Resulting enzyme preparations were highly effective for saccharification of various biomass. In this review, we present some of the salient findings from the recent biochemical, morphological, and molecular analyses of this remarkable cellulase hyper-producing fungus. Graphical abstract For Trichoderma reesei, transcriptional regulation of cellulolytic enzyme genes, comparative genomics and morphological insights, and applications of them were summarized.

Engineering of the Trichoderma reesei xylanase3 promoter for efficient enzyme expression

The GH10 xylanase XYNIII is expressed in the hyper-cellulase-producing mutant PC-3-7, but not in the standard strain QM9414 of Trichoderma reesei. The GH11 xylanase gene xyn1 is induced by cellulosic and xylanosic carbon sources while xyn3 is induced only by cellulosic carbon sources in the PC-3-7 strain. In this study, we constructed a modified xyn3 promoter in which we replaced the cis-acting region of the xyn3 promoter by the cis-acting region of the xyn1 promoter. The resulting xyn3 chimeric promoter exhibited improved inductivity against cellulosic carbon over the wild-type promoter and acquired inductivity against xylanosic carbon. Furthermore, PC-3-7 expressing the heterologous β-glycosidase gene, Aspergillus aculeatus bgl1, under the control of the xyn3 chimeric promoter, showed enhanced saccharification ability through increased cellobiase activity. We also show that the xyn3 chimeric promoter is also functional in the QM9414 strain. Our results indicate that the xyn3 chimeric promoter is very efficient for enzyme expression.