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The cellulolytic filamentous fungus Trichoderma reesei has a strong capability in protein synthesis and secretion and is increasingly used as a fungal chassis for the production of heterologous proteins or secondary metabolites. However, bidirectional promoters that would significantly facilitate multiple genes' expression have not been characterized in T. reesei. Herein, we show that a 767-bp intergenic region between two polyketide synthase encoding genes that were involved in the biosynthesis of the typical yellow pigment served as a bidirectional promoter in T. reesei. This region was shown to be able to drive the simultaneous expression of two fluorescence reporter genes when fused to each end. Quantitative RT-PCR analysis demonstrated that the driving strength of this bidirectional promoter from each direction reached about half of that of the commonly used promoter PgpdA. Moreover, the co-expression of two cellulase genes driven by this bidirectional promoter enabled T. reesei to produce cellulases on glucose and improved the total cellulase activities with cellulose Avicel as the carbon source. Our work identified the first bidirectional promoter in T. reesei, which would facilitate gene co-expression and find applications in synthetic biology using fungal systems. [ABSTRACT FROM AUTHOR]

Sorbicillinoids (also termed yellow pigment) are derived from either marine or terrestrial fungi, exhibit various biological activities and therefore show potential as commercial products for human or animal health. The cellulolytic filamentous fungus Trichoderma reesei is capable to biosynthesize sorbicillinoids, but the underlying regulatory mechanism is not yet completely clear. Herein, we identified a histone H3 lysine 9 (H3K9) methyltransferase, Dim5, in T. reesei. TrDIM5 deletion caused an impaired vegetative growth as well as conidiation, whereas the ∆Trdim5 strain displayed a remarkable increase in sorbicillinoid production. Post TrDIM5 deletion, the transcription of sorbicillinoid biosynthesis‐related (SOR) genes was significantly upregulated with a more open chromatin structure. Intriguingly, hardly any expression changes occurred amongst those genes located on both flanks of the SOR gene cluster. In addition, the assays provided evidence that H3K9 triple methylation (H3K9me3) modification acted as a repressive marker at the SOR gene cluster and thus directly mediated the repression of sorbicillinoid biosynthesis. Transcription factor Ypr1 activated the SOR gene cluster by antagonizing TrDim5‐mediated repression and therefore contributed to forming a relatively more open local chromatin environment, which further facilitated its binding and SOR gene expression. The results of this study will contribute to understanding the intricate regulatory network in sorbicillinoid biosynthesis and facilitate the endowment of T. reesei with preferred features for sorbicillinoid production by genetic engineering. [ABSTRACT FROM AUTHOR]

The filamentous fungus Trichoderma reesei secretes large quantities of cellulases and hemicellulases that have found wide applications in industry. Compared with extensive studies on the mechanism controlling cellulase gene expression, less is known about the regulatory mechanism behind xylanase gene expression. Herein, several putative sugar transporter encoding genes that showed significant upregulation on xylan were identified in T. reesei. Deletion of one such gene, gat1 , resulted in increased xylanase production but hardly affected cellulase induction. Further analyses demonstrated that deletion of gat1 markedly increased XYNI production at the transcriptional level and only exerted a minor effect on XYNII synthesis. In contrast, overexpressing gat1 caused a continuous decrease in xyn1 expression. Deletion of gat1 also affected the expression of xyn1 and pectinase genes when T. reesei was cultivated with galacturonic acid as the sole carbon source. Transcriptome analyses of Δ gat1 and its parental strain identified 255 differentially expressed genes that are enriched in categories of glycoside hydrolases, lipid metabolism, transporters, and transcriptional factors. The results thus implicate a repressive role of the sugar transporter GAT1 in xyn1 expression and reveal that distinct regulatory mechanisms may exist in controlling the expression of different xylanase genes in T. reesei. [ABSTRACT FROM AUTHOR]

Cellulase production in filamentous fungus Trichoderma reesei is highly responsive to various environmental cues involving multiple positive and negative regulators. XYR1 (Xylanase regulator 1) has been identified as the key transcriptional activator of cellulase gene expression in T. reesei. However, the precise mechanism by which XYR1 achieves transcriptional activation of cellulase genes is still not fully understood. Here, we identified the TrCYC8/TUP1 complex as a novel coactivator for XYR1 in T. reesei. CYC8/TUP1 is the first identified transcriptional corepressor complex mediating repression of diverse genes in Saccharomyces cerevisiae. Knockdown of Trcyc8 or Trtup1 resulted in markedly impaired cellulase gene expression in T. reesei. We found that TrCYC8/TUP1 was recruited to cellulase gene promoters upon cellulose induction and this recruitment is dependent on XYR1. We further observed that repressed Trtup1 or Trcyc8 expression caused a strong defect in XYR1 occupancy and loss of histone H4 at cellulase gene promoters. The defects in XYR1 binding and transcriptional activation of target genes in Trtup1 or Trcyc8 repressed cells could not be overcome by XYR1 overexpression. Our results reveal a novel coactivator function for TrCYC8/TUP1 at the level of activator binding, and suggest a mechanism in which interdependent recruitment of XYR1 and TrCYC8/TUP1 to cellulase gene promoters represents an important regulatory circuit in ensuring the induced cellulase gene expression. These findings thus contribute to unveiling the intricate regulatory mechanism underlying XYR1-mediated cellulase gene activation and also provide an important clue that will help further improve cellulase production by T. reesei. Author summary: Originally identified in budding yeast, CYC8 (also known as SSN6)/TUP1 (also known as RCOA/RCO1) is one of the best studied corepressors and has served as a model for the study of similar corepressor complexes in higher eukaryotes. Besides its well-established role in transcriptional repression, CYC8/TUP1 has been reported to be involved in transcriptional activation following release from repression in S. cerevisiae. In filamentous fungi, there have been also reports on TUP1 homolog functions in specific gene repression as well as vegetative growth and asexual spore production. However, CYC8/TUP1 function in gene activation has not been described. In this study, we reveal a coactivator function for TrCYC8/TUP1 in initiating the cellulolytic response in T. reesei. TrCYC8/TUP1 itself is specifically recruited to cellulase gene promoters in an XYR1-dependent manner. We further show that TrCYC8/TUP1 contributes to the induced cellulase gene expression by improving the binding of the essential transcriptional activator XYR1. These results thus implicate a synergistic mechanism in ensuring the efficient recruitment of XYR1 and TrCYC8/TUP1 to target gene promoters. [ABSTRACT FROM AUTHOR]

The ascomycete Trichoderma reesei is a highly prolific cellulase producer. While XYR1 (Xylanase regulator 1) has been firmly established to be the master activator of cellulase gene expression in T. reesei, its precise transcriptional activation mechanism remains poorly understood. In the present study, TrGAL11, a component of the Mediator tail module, was identified as a putative interacting partner of XYR1. Deletion of Trgal11 markedly impaired the induced expression of most (hemi)cellulase genes, but not that of the major β-glucosidase encoding genes. This differential involvement of TrGAL11 in the full induction of cellulase genes was reflected by the RNA polymerase II (Pol II) recruitment on their core promoters, indicating that TrGAL11 was required for the efficient transcriptional initiation of the majority of cellulase genes. In addition, we found that TrGAL11 recruitment to cellulase gene promoters largely occurred in an XYR1-dependent manner. Although xyr1 expression was significantly tuned down without TrGAL11, the binding of XYR1 to cellulase gene promoters did not entail TrGAL11. These results indicate that TrGAL11 represents a direct in vivo target of XYR1 and may play a critical role in contributing to Mediator and the following RNA Pol II recruitment to ensure the induced cellulase gene expression. Author summary: As a model cellulolytic fungus, T. reesei is capable of rapidly producing a large quantity of (hemi)cellulases when appropriate substrates are present. This outstanding characteristic has made T. reesei a prominent producer of cellulase in industry and also a model organism for studying eukaryotic gene expression. The expression of these hydrolytic enzymes encoding genes in T. reesei is precisely regulated at a transcriptional level and controlled by a suite of transcription factors. Among others, the transcription activator XYR1 has been firmly established to be absolutely necessary for activating the expression of almost all cellulase genes. However, the precise mechanism it acts remains largely unknown. In eukaryotes, the multisubunit Mediator complex has been shown to be critical for expression of most, if not all, protein-coding genes by conveying regulatory information to the basal transcription machinery. Here, we find that XYR1 interacts with the Mediator tail module subunit, TrGAL11, which contributes to cellobiohydrolase (cbh) and endoglucanase (eg) genes but not β-glucosidase (bgl) genes expression. Thus, the induced XYR1 binding to cellulase gene promoters led to TrGAL11 and RNA Pol II recruitment to these promoters. These results show that TrGAL11 represents a direct in vivo target of XYR1 and provide evidence for not only the evolutionarily conserved function of Mediator, but also for the existence of some subtle difference in its action to mediate gene expression in different eukaryotes. [ABSTRACT FROM AUTHOR]

The filamentous fungus Trichoderma reesei is one of the most studied cellulolytic organisms and the major producer of cellulases for industrial applications. However, undesired degradation of cellulases often happens in culture filtrates and commercial enzyme preparations. Even studies have been reported about describing proteolytic degradation of heterologous proteins in T. reesei , there are few systematic explorations concerning the extracellular proteases responsible for degradation of cellulases. In this study, the cellulase activity was observed to rapidly decrease at late cultivation stages using corn steep liquor (CSL) as the nitrogen source in T. reesei. It was discovered that this decrease may be caused by proteases. To identify the proteases, comparative secretomics was performed to analyze the concomitant proteases during the cellulase production. 12 candidate proteases from the secretome of T. reesei were identified and their encoding genes were individually deleted via homologous recombination. Furthermore, three target proteases (tre81070, tre120998, and tre123234) were simultaneously deleted by one-step genetic transformation. The triple deletion strain ΔP70 showed a 78% decrease in protease activity and a six-fold increase in cellulase activity at late fermentation stages. These results demonstrated the feasibility of improvement of cellulase production by genetically disrupting the potential protease genes to construct the T. reesei strains with low extracellular protease secretion. This dataset also provides an efficient approach for strain improvement by precise genetic engineering combined with "omics" strategy for high-production of industrial enzymes to reduce the cost of lignocellulose bioconversion. [ABSTRACT FROM AUTHOR]

Summary: Cellulase gene expression in Trichoderma reesei is highly responsive to environmental cues and is under stringent regulation by multiple transcription factors. XYR1 (Xylanase regulator 1) has been identified as the most important transcriptional activator of cellulase/hemicellulase gene expression although the precise transactivating mechanism remains largely elusive. Here we show that the activation domain of XYR1 interacts with the T. reesei homolog of the TrSNF12 subunit of SWI/SNF complex. Deletion of Trsnf12 markedly impaired the induced cellulase gene expression. Individual loss of other SWI/SNF subunits including the catalytic subunit also severely compromised cellulase gene expression and interfered with loss of histone H4 in the cbh1 and eg1 promoters upon cellulose induction. In addition, we find that the SWI/SNF occupancy on cellulase gene promoters strictly required XYR1 and TrSNF12 but TrSNF12 was dispensable for the XYR1 binding to these promoters. These data suggest a model in which XYR1 recruits SWI/SNF through direct interactions with TrSNF12 to remodel chromatin at cellulase gene promoters, thereby activating cellulase gene expression to initiate the cellulolytic response in T. reesei. [ABSTRACT FROM AUTHOR]

The stringent regulatory network of cellulase gene expression in the filamentous fungus Trichoderma reesei involves multiple transcriptional regulators. However, identification and mechanistic investigation of these regulators are still insufficient. Here, we identified a novel transcriptional regulator, CLP1, a plant homeo domain (PHD) Protein that participates in regulating T. reesei cellulase gene expression. Phylogenetic analyses demonstrated that CLP1 homologs are widely distributed in filamentous fungi including Trichoderma , Penicillium , Fusarium , Neurospora , and Aspergillus species. We demonstrated that CLP1 is a nuclear protein and lack of CLP1 significantly impaired the induced expression of cellulase genes. ChIP experiments showed CLP1 binding to the cellulase gene promoters specifically under cellulose conditions and compromised XYR1 occupancy on the same promoters in the absence of CLP1 at the early induction stage. XYR1 overexpression fully rescued the defect in cellulase production but not the defect in conidia formation in the clp1 null mutant. Further analysis showed that the PHD is required for the CLP1 appropriate subcellular localization as well as the induced cellulase gene expression and conidiation. Taken together, these data demonstrated an important role of CLP1 in the regulation of cellulase and xylanase gene expression in T. reesei. [ABSTRACT FROM AUTHOR]

Cellulase gene expression in the model cellulolytic fungus Trichoderma reesei is supposed to be controlled by an intricate regulatory network involving multiple transcription factors. Here, we identified a novel transcriptional repressor of cellulase gene expression, Rce1. Disruption of the rce1 gene not only facilitated the induced expression of cellulase genes but also led to a significant delay in terminating the induction process. However, Rce1 did not participate in Cre1-mediated catabolite repression. Electrophoretic mobility shift (EMSA) and DNase I footprinting assays in combination with chromatin immunoprecipitation (ChIP) demonstrated that Rce1 could bind directly to a cbh1 (cellobiohydrolase 1-encoding) gene promoter region containing a cluster of Xyr1 binding sites. Furthermore, competitive binding assays revealed that Rce1 antagonized Xyr1 from binding to the cbh1 promoter. These results indicate that intricate interactions exist between a variety of transcription factors to ensure tight and energy-efficient regulation of cellulase gene expression in T. reesei. This study also provides important clues regarding increased cellulase production in T. reesei. [ABSTRACT FROM AUTHOR]

Gcn5 is a well-established histone acetyltransferase involved in chromatin modification by catalyzing the acetylation of specific lysine residues within the N-terminal tails of the core histones. To assess the role of chromatin remodeling in the transcriptional response of cellulolytic Trichoderma reesei to the changes of environmental conditions, we identified the T. reesei ortholog of Saccharomyces cerevisiae Gcn5 by sequence alignment and functional analysis. Heterologous expression of TrGcn5 in S. cerevisiae gcn5Δ strain restored the growth defect under nutrient limitation as well as stresses. In contrast, mutant TrGcn5 with site-directed changes of residues critical for Gcn5 histone acetyltransferase activity could not complement the growth defect. The T. reesei gcn5Δ mutant strain displayed a strongly decreased growth rate and dramatic morphological changes including misshapen hyphal cells and abolished conidiation. Moreover, the induced expression of cellulase genes was severely impaired in the gcn5Δ T. reesei with acetylation of K9 and K14 of histone H3 in the cellulase gene promoter dramatically affected in the absence of TrGcn5. The results indicate that TrGcn5 plays a critical role in filamentous growth, morphogenesis, and transcriptional activation of specific genes including cellulase encoding genes. [ABSTRACT FROM AUTHOR]

Background: Trichoderma reesei is a primary lignocellulosic enzyme producer in industry. However, the mechanisms underlying cellulase synthesis as well as other physiological processes are insufficiently understood partly due to the sophisticated process for its genetic manipulation. Target gene knockdown by RNA interference (RNAi) is a powerful tool for genetic research and biotechnology in eukaryotes including filamentous fungi. Previously reported RNAi system in T. reesei was either uncontrollable or only applicable in certain nutrition state. Results: In the present study, we incorporated the copper-responsive tcu1 promoter into an RNAi-mediated silencing system to develop a controllable RNAi-mediated silencing system in T. reesei. As the proof-of-concept, a prototrophic pyr4 gene, highly expressed cel7a and xyr1 genes induced by Avicel and a fab1 gene, whose knockout has proved to be intractable, were successfully knocked down in the absence of copper when the respective RNAi fragment was expressed. Importantly, the phenotype of RNAi strains was shown to be reversed easily to mimic the complementation for excluding any unwanted effects resulted from the random integration of the hpRNA cassette by adding copper in the media. Thus, this controllable RNAi-mediated silencing system can be turned on and turned off only depending on the absence and presence of copper ions in the media, respectively, and not on the nutritional states. Conclusions: The copper-controlled RNA interference system represents an effective tool for reversible silencing of target genes in T. reesei. This reported strategy to conditionally knock down or turn off genes will contribute to our understanding of T. reesei gene functions, especially those that are difficult to be knocked out due to various reasons. In addition, this simple and cost-effective method holds great potential for the application in synthetic biology and genetic engineering of T. reesei. [ABSTRACT FROM AUTHOR]

The glucose transporter is an important player in cell metabolism that mediates the intracellular uptake of glucose. Here, we characterized the glucose transporter Stp1 from the filamentous fungus Trichoderma reesei. The individual substitution of several conserved residues for Ala in Stp1 corresponding to those interacting with D-glucose in the xylose/H+ symporter XylE inflicted contrasting effects on its ability to support the growth of an hxt-null yeast on glucose. The targeted change of Phe 50, proximal to the substrate-binding site, was also found to exert a profound effect on the activity of Stp1. In contrast with the charged residues, the substitution of Phe 50 with either the hydrophilic residues Asn and Gln or the small residues Gly and Ala significantly enhanced the transport of glucose and its fluorescent analogue, 2-NBDG. On the other hand, a variant with the three substitutions I115F, F199I and P214L displayed remarkably improved activity on glucose and 2-NBDG transport. Further analysis indicated that the combined mutations of Ile 115 and Pro 214, positioned on the lateral surface of the Stp1 N-domain, fully accounted for the enhanced transport activity. These results provide insight into the structural basis for glucose uptake in fungal sugar transporters. [ABSTRACT FROM AUTHOR]

[Display omitted] • TLL-T3 was achieved via directed evolution of the commonly used lipase TLL. • TLL-T3 displayed significantly enhanced acid tolerance. • Usage of TLL-T3 led to a high biodiesel yield up to 90% (w/w) from acidified oil. • Production of TLL-T3 in Trichoderma reesei in a 20 L fermenter reached ∼ 9.6 g/L. • TLL-T3 production was further increased by ∼ 30 % after fermentation optimization. Lipases catalyze the synthesis of biodiesel, which is an important renewable alternative energy source. Cost-efficient conversion of waste acidified oil to biodiesel entails acid-tolerant lipases which have not been extensively studied. This study showed that the commonly used Thermomyces lanuginosus lipase TLL displayed a weak acid tolerance and an unsatisfactory performance in biodiesel production from acidified oil. Directed evolution of TLL identified one TLL-T3 variant with three residue substitutions (A69S/V150P/N222G). TLL-T3 displayed significantly enhanced acid tolerance, and its application in acidified oil treatment led to a biodiesel yield up to 90 % (w/w). A scaled-up production of TLL-T3 in Trichoderma reesei was further achieved and the highest extracellular lipase activity reached 16,123 U/mL after fermentation optimization. These results provide new insights into structural adaptation to acid tolerance by lipases and show that TLL-T3 holds great potential in commercial biodiesel production from waste acidified oil. [ABSTRACT FROM AUTHOR]

[Display omitted] • The DBD of Xyr1 and the TAD of Tmac1 were fused to construct XT. • XT enabled improved (hemi)cellulase production on glucose in Trichoderma reesei. • XT elevated the abundance of hemicellulases in the secretome. • The produced enzyme cocktail improved the saccharification of pretreated corn stover. To reduce the high cost of (hemi)cellulase production in lignocellulose biorefining, it is important to develop strategies to enhance enzyme productivity from economic and also readily manipulatable carbon sources. In this study, an artificial transcription factor XT was designed by fusing the DNA binding domain of Xyr1 to the transactivation domain of Tmac1. When overexpressed in Trichoderma reesei QM9414 Δ xyr1 , the XT recombinant strain (OEXT) greatly improved (hemi)cellulase production on repressing glucose compared with QM9414 on Avicel with 1.7- and 8.2-fold increases in pNPCase and xylanase activity, respectively. Both activities were even higher (0.9- and 33.8-fold higher, respectively) than the recombinant strain similarly overexpressing Xyr1. The dramatically enhanced xylanase activities in OEXT resulted from the elevated expression of various hemicellulases in the secretome. Moreover, the enzyme cocktail from OEXT improved the saccharification efficiency toward corn stover by 60% compared with enzymes from QM9414 with equal volume loading. [ABSTRACT FROM AUTHOR]