Analysis of global regulation of gene expression of carbon catabolite repression during the formation of cellulases by Trichoderma reesei (Hypocrea jecorina)

O ascomiceto Hypocrea jecorina (anamorfo do Trichoderma reesei) é um dos fungos celulolíticos mais bem estudados e amplamente utilizado na indústria biotecnológica, como na produção do etanol de segunda geração. O mecanismo de repressão catabólica adotado por T. reesei é mediado pelo fator de transcrição CRE1 e consiste na repressão de genes relacionados à produção de celulases quando uma fonte de carbono prontamente disponível está presente no meio. Esse trabalho tem como objetivo contribuir para o entendimento do mecanismo de repressão catabólica durante a formação de celulases, através da comparação entre a linhagem mutante de T. reesei Δcre1 com seu parental QM9414. Para isso, bibliotecas de cDNA das linhagens Δcre1 e QM9414 crescidas em 1% de celulose, 1mM de soforose e 2% de glicose foram sequenciadas por RNA-seq pela empresa LGC Genomics GmbH em Berlim/Alemanha, utilizando-se o equipamento Illumina/HiSeq2000. Os resultados do sequenciamento foram analisados pelo software de alinhamento Bowtie e pelo pacote DESeq, que faz a análise de expressão diferencial dos genes. Foram obtidas no total 264 milhões de reads que, ao serem analisadas, mostraram que 815 genes foram diferencialmente expressos no Δcre1 em relação ao QM9414 na condição celulose, 2368 em soforose e 697 em glicose, em um total de 9129 genes do genoma de T. reesei. A maioria dos genes que estavam up ou down regulados no mutante pertenciam a categorias do Gene Ontology descritas como processos metabólicos, membrana, atividade oxidorredutase, metabolismo de carboidratos e transporte. Enzimas celulolíticas, genes relacionados com o transporte de substâncias e outros fatores de transcrição foram alvo de repressão metabólica mediada por CRE1, de modo carbono-dependente. A validação dos genes diferencialmente expressos por qRT-PCR mostrou uma alta correlação entre as duas técnicas, que pode ser demonstrada por um alto coeficiente linear de Pearson (r2 = 0.94). Espera-se com esses resultados contribuir para um melhor entendimento do mecanismo de repressão catabólica em T. reesei, potencializando a aplicação desse fungo nos diversos setores biotecnológicos em que é utilizado.The ascomycete Hypocrea jecorina (anamorph of Trichoderma reesei) is one of the most well studied cellulolytic fungi and widely used in the biotechnology industry, as in the production of second generation ethanol, pulp and paper industries, textile treatments and processing of animal feed. The carbon catabolite repression mechanism adopted by T. reesei is mediated by the transcription factor CRE1 and consists in the repression of genes related to the production of cellulase when a readily available carbon source is present in the medium. This study aims to contribute to understanding the mechanism of carbon catabolite repression during the formation of cellulases, by comparing the mutant strain of T. reesei Δcre1 with its parental, QM9414. For this, the cDNA libraries of strains QM9414 and Δcre1 grown in 1% cellulose, 1 mM sophorose and 2% glucose were sequenced by RNA-seq by LGC Genomics GmbH in Berlin/Germany, using the equipment Illumina/HiSeq2000. The results of the sequencing were analyzed by the alignment software Bowtie and DEseq package, which makes the analysis of differentially expressed genes. We obtained a total of 264 million of reads that, when analyzed, showed 815 genes differentially expressed in Δcre1 in relation to the parental QM9414 on cellulose, 2368 on sophorose and 697 on glucose, for a total of 9129 genes in the genome of T. reesei. Most of genes that were up- or down-regulated in the mutant belonged to Gene Ontology categories described as metabolic processes, membrane, oxidoreductase activity, carbohydrate metabolism and transport. Cellulolytic enzymes, genes related to the transport of substances and other transcription factors were targeted for carbon catabolite repression mediated by CRE1, in a carbon source-dependent manner. Validation of differentially expressed genes by qRT-PCR showed a high correlation between the two techniques, which can be demonstrated by a high linear coefficient of Pearson (r2 = 0.94). It is hoped that these results contribute to a better understanding of the mechanism of carbon catabolite repression in T. reesei, enhancing the application of this fungus in several biotechnology sectors in which is used

Identification and characterization of a new transcription factor, homologous to Saccharomyces cerevisiae Azf1, involved in the regulation of cellulase expression in the filamentous fungus Trichoderma reesei

O fungo Trichoderma reesei é conhecido por sua elevada capacidade de secreção de enzimas celulolíticas que atuam degradando o polímero de celulose em moléculas de glicose, sendo este um ponto chave na produção do etanol de segunda geração. O presente trabalho tem por objetivo contribuir para o entendimento dos mecanismos moleculares envolvidos no processo de desconstrução da biomassa por T. reesei através da identificação de novos fatores de transcrição associados a este processo. Para isso, foi realizado um screening de 14 fatores de transcrição potencialmente envolvidos com a degradação de biomassa e identificação do gene Tr103275, homólogo ao regulador Azf1 de Saccharomyces cerevisiae. Posteriormente, foi feita a construção de um mutante com deleção do gene azf1 em T. reesei. Durante o crescimento em celulose e bagaço de cana, o gene azf1 foi altamente expresso em T. reesei, ao passo que glicose reprimiu a expressão gênica desse fator de transcrição. A ausência de azf1 afetou o crescimento e a esporulação em T. reesei, uma vez que a linhagem mutante produziu esporos menores e em quantidade reduzida em relação ao parental. Além disso, a proteína TrAzf1 atua como um regulador positivo da expressão de enzimas holocelulolíticas, considerando que as os genes que codificam as CBHs, EGs, BGs e xyn4 foram down regulados no mutante Δazf1 em celulose e bagaço de cana. O sítio de ligação para TrAzf1 foi predito nesse trabalho através de análise de bioinformática. Para avaliar a interação de TrAzf1 com esse sítio de ligação no DNA, o motivo dedo de zinco de Azf1 recombinante foi obtido por expressão heteróloga em E. coli e utilizado nas análises de SPR. No entanto, a validação da interação fator de transcrição-DNA por SPR não foi conclusiva e outros experimentos devem ser realizados para validar o motivo de ligação de TrAzf1 em T. reesei. O papel de Azf1 na regulação da degradação da biomassa vegetal foi descrito, pela primeira vez, nesse trabalho. Esse fator de transcrição é um alvo em potencial para manipulação genética de T. reesei, visando o melhoramento da produção de enzimas holocelulolíticas por essa espécie.The fungus Trichoderma reesei is known for its high capacity in secrete cellulolytic enzymes that act in the degradation of the cellulose polymer into glucose molecules, being a key point in the production of second generation ethanol. The present work aims to contribute to the understanding of the molecular mechanisms involved in the process of biomass deconstruction in T. reesei through the identification of new transcription factors associated to this process. For this, we screened 14 transcription factors potentially involved in the degradation of biomass and identified the gene Tr103275, homologous to the Azf1 regulator of Saccharomyces cerevisiae. Posteriorly, a mutant strain with deletion of azf1 was obtained. During growth in cellulose and sugarcane bagasse, azf1 was highly induced in T. reesei, while glucose repressed the gene expression of this transcription factor. The absence of azf1 affected growth and sporulation in T. reesei, since the mutant strain produced less and smaller spores relative to the parental strain. In addition, we have shown that TrAzf1 acts as a positive regulator of the expression of holocellulolytic enzymes, whereas the genes coding for CBHs, EGs, BGs and xyn4 were down-regulated in the Δazf1 mutant in cellulose and sugarcane bagasse. The binding site for TrAzf1 was predicted in this work through bioinformatics analysis. To evaluate the interaction of TrAzf1 with this DNA binding site, the recombinant Azf1 zinc finger motif was obtained by heterologous expression in E. coli and used in the SPR analyzes. However, validation of the transcription factor-DNA interaction by SPR was not conclusive and other approaches should be performed to validate the binding motif of TrAzf1 in T. reesei. The role of Azf1 in regulating the degradation of plant biomass was described for the first time in this work. This transcription factor is a potential target for the genetic manipulation of T. reesei, aiming to improve the production of holocellulolytic enzymes by this species

Schematic representation of the approach used for motif discovery in <i>T. reesei</i>.

<p>Three sets of co-regulated genes were retrieved from RNA-seq experiments of <i>T. reesei</i> cells grown on cellulose, sophorose or glucose <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099366#pone.0099366-DosSantosCastro1" target="_blank">[45]</a>. Only up-regulated genes encoding for putative TFs were selected. A fourth set of genes are 22 cellulases analysed through RT-PCR experiments <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099366#pone.0099366-Castro1" target="_blank">[46]</a>. A 1.5 kb promoter region of each gene from the four groups was retrieved from the complete genome sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099366#pone.0099366-Martinez1" target="_blank">[49]</a> and used for motif discovery using MEME. From the resulting identified motifs, those sharing similarities with the reported binding consensus for XYR1 and CRE1 were selected for further analysis.</p

Deciphering the <i>Cis</i>-Regulatory Elements for XYR1 and CRE1 Regulators in <i>Trichoderma reesei</i>

<div><p>In this work, we report the <i>in silico</i> identification of the <i>cis</i>-regulatory elements for XYR1 and CRE1 proteins in the filamentous fungus <i>Trichoderma reesei</i>, two regulators that play a central role in the expression of cellulase genes. Using four datasets of condition-dependent genes from RNA-seq and RT-qPCR experiments, we performed unsupervised motif discovery and found two short motifs resembling the proposed binding consensus for XYR1 and CRE1. Using these motifs, we analysed the presence and arrangement of putative <i>cis</i>-regulatory elements recognized by both regulators and found that shortly spaced sites were more associated with XYR1- and CRE1-dependent promoters than single, high-score sites. Furthermore, the approach used here allowed the identification of the previously reported XYR1-binding sites from <i>cel7a</i> and <i>xyn1</i> promoters, and we also mapped the potential target sequence for this regulator at the <i>cel6a</i> promoter that has been suggested but not identified previously. Additionally, seven other promoters (for <i>cel7b</i>, <i>cel61a</i>, <i>cel61b</i>, <i>cel3c</i>, <i>cel3d</i>, <i>xyn3</i> and <i>swo</i> genes) presented a putative XYR1-binding site, and strong sites for CRE1 were found at the <i>xyr1</i> and <i>cel7b</i> promoters. Using the <i>cis</i>-regulatory architectures nearly defined for XYR1 and CRE1, we performed genome-wide identification of potential targets for direct regulation by both proteins and important differences on their functional regulons were elucidated. Finally, we performed binding site mapping on the promoters of differentially expressed genes found in <i>T. reesei</i> mutant strains lacking <i>xyr1</i> or <i>cre1</i> and found that indirect regulation plays a key role on their signalling pathways. Taken together, the data provided here sheds new light on the mechanisms for signal integration mediated by XYR1 and CRE1 at cellulase promoters.</p></div

Search for repeated XYR1 and CRE1 binding sites on different datasets.

<p><b>A</b>) In the XYR1, both inverted and everted sites were considered and only sites within a distance between 8 and 30 bp were taken. <b>B</b>) Representation of repeated binding sites at the cellulase promoters and at the genome scale. The y-axis (fold) represents the number of sites identified relative to the number of promoters from the datasets. The enrichment group represents the rate between sites per promoters from the cellulase promoters and the corresponding valued from the genome group. Grey shaded region highlight the score with higher enrichment. <b>C</b>) For the prediction of CRE1-binding sites, both inverted and direct repeats spaced between 5 to 30 bp were considered. <b>D</b>) Representation of sites per promoters and the enrichment at the glucose dataset vs. the genome, calculated as in <b>B</b>.</p

Search for single XYR1 and CRE1 binding sites on different promoter datasets.

<p>For the analysis, only the best site was retrieved for each studied promoter. <b>A</b>) Distribution of XYR1-binding sites score for all genes from the <i>T. reesei</i> genome. <b>B</b>) Score of XYR1-binding sites at the 22 cellulase promoters. <b>C</b>) Distribution of CRE1-bindind sites scores at the genome scale. <b>D</b>) Scores of CRE1-bindind sites found at the 18 promoters from the glucose dataset.</p

Defining the categories of genes potentially regulated directly by XYR1 and CRE1.

<p>Percentages of genes belonging to each functional category for both regulons are shown. Red triangles indicate functional categories enriched in the XYR1 regulon, while grey triangles point to those more abundant on the CRE1 regulon.</p

Motifs identified in the cellulose and glucose regulated genes.

<p><b>A)</b> The two motifs identified in the promoter dataset of 22 cellulases that resemble the XYR1 consensus (5′-GGCWWW-3′) are shown. <b>B)</b> Combination of the Cell-M1 and Cell-M2 motifs to create the XYR1 consensus used to search for XYR1 binding sites in <i>T. reesei</i>. <b>C)</b> The two motifs identified using the promoters of TFs up regulated under glucose growth that share similarity to the proposed CRE1-binding consensus (5′-SYGGRG-3′) are shown. <b>D)</b> Representation of the consensus resulting from the combination of Glu-M1 and Glu-M2 motifs.</p

Identification of XYR1 and CRE1-binding sites at target promoters.

<p><b>A</b>) Representation of the binding sites found at the promoters of 10 cellulase encoding-genes and at promoters of the <i>xyr1</i> gene. Each circle represent a binding site formed by repeats of the core sequences recognized by the two regulators. <b>B</b>) Zoom in at the promoter region of <i>cel6a</i> gene, showing the CAE (vertical red bar), the TATA-box (vertical blue bar), the nucleosome −1 binding region (horizontal grey rectangle) and the nearly identified binding site for XYR1, labelled as UAS_XYR1. The DNA sequences of each regulatory element are shown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099366#pone.0099366-Zeilinger1" target="_blank">[26]</a>. Shown positions are relative to the start codon (ATG) of the <i>cel6a</i> gene.</p