In Vivo Footprinting Analysis in Trichoderma reesei.

The in vivo footprinting method identifies protein-targeted DNA regions under different conditions such as carbon sources. Dimethyl sulfate (DMS) generates methylated purine bases at DNA sites which are not bound by proteins or transcription factors. The DNA is cleaved by HCl, and the resulting DNA fragments are 5'-end [6-FAM]-labeled by a linker-mediated PCR (LM-PCR). Fluorescent fragments are separated and analyzed on a capillary sequencer, followed by automated data analysis using the software tool ivFAST.

Identification of the citrate exporter Cex1 of Yarrowia lipolytica.

Yarrowia lipolytica is a yeast with many talents, one of them being the production of citric acid. Although the citrate biosynthesis is well studied, little is known about the transport mechanism by which citrate is exported. To gain better insight into this mechanism, we set out to identify a transporter involved in citrate export of Y. lipolytica. A total of five proteins were selected for analysis based on their similarity to a known citrate exporter, but neither a citrate transport activity nor any other phenotypic function could be attributed to them. Differential gene expression analysis of two strains with a distinct citrate productivity revealed another three putative transporters, one of which is YALI0D20196p. Disrupting YALI0D20196g in Y. lipolytica abolished citrate production, while extrachromosomal expression enhanced citrate production 5.2-fold in a low producing wildtype. Furthermore, heterologous expression of YALI0D20196p in the non-citrate secreting yeast Saccharomyces cerevisiae facilitated citrate export. Likewise, expression of YALI0D20196p complemented the ability to secrete citrate in an export-deficient strain of Aspergillus niger, confirming a citrate export function of YALI0D20196p. This report on the identification of the first citrate exporter in Y. lipolytica, termed Cex1, represents a valuable starting point for further investigations of the complex transport processes in yeasts.

Engineering of the citrate exporter protein enables high citric acid production in Aspergillus niger.

Aspergillus niger was engineered using a gene responsible for citric acid transport, which has a significant impact on citric acid secretion when overexpressed. The transport gene was identified by a homology search using an itaconic acid transporter from Ustilago maydis as template. The encoding homologous protein CexA belongs to the major facilitator superfamily subclass DHA1 and members of this family work as drug-H+ antiporter. The disruption of this gene completely abolishes citric acid secretion, which indicates that this protein is the main citric acid transporter in A. niger. In the disruption strain, the metabolism is re-routed mainly to oxalic acid, which is a known by-product during citric acid production. The gene can be heterologously expressed in Saccharomyces cerevisiae, which leads to the secretion of citric acid during the growth on glucose. These results confirm the functionality of CexA as the main transporter for citric acid of A. niger. Overexpression of cexA leads to a significant increase in secreted citric acid. Thereby, striking differences between a strong constitutive expression system using pmbfA as a promoter and an inducible expression system using ptet-on can be observed. The inducible system significantly outcompetes the constitutive expression system yielding up to 109 g/L citric acid, which is 5 times higher compared to the parental wild-type strain and 3 times higher compared to the constitutive expression system. These results demonstrate the importance of the cellular transport system for an efficient production of metabolites. By overexpressing a single gene, it is possible to significantly improve the citric acid secretion capability of a moderately producing parental strain.

Truncation of the transcriptional repressor protein Cre1 in Trichoderma reesei Rut-C30 turns it into an activator.

BACKGROUND: The filamentous fungus Trichoderma reesei (T. reesei) is a natural producer of cellulolytic and xylanolytic enzymes and is therefore industrially used. Many industries require high amounts of enzymes, in particular cellulases. Strain improvement strategies by random mutagenesis yielded the industrial ancestor strain Rut-C30. A key property of Rut-C30 is the partial release from carbon catabolite repression caused by a truncation of the repressor Cre1 (Cre1-96). In the T. reesei wild-type strain a full cre1 deletion leads to pleiotropic effects and strong growth impairment, while the truncated cre1-96 enhances cellulolytic activity without the effect of growth deficiencies. However, it is still unclear which function Cre1-96 has in Rut-C30. RESULTS: In this study, we deleted and constitutively expressed cre1-96 in Rut-C30. We found that the presence of Cre1-96 in Rut-C30 is crucial for its cellulolytic and xylanolytic performance under inducing conditions. In the case of the constitutively expressed Cre1-96, the cellulase activity could further be improved approximately twofold. The deletion of cre1-96 led to growth deficiencies and morphological abnormalities. An in silico domain prediction revealed that Cre1-96 has all necessary properties that a classic transactivator needs. Consequently, we investigated the cellular localization of Cre1-96 by fluorescence microscopy using an eYFP-tag. Cre1-96 is localized in the fungal nuclei under both, inducing and repressing conditions. Furthermore, chromatin immunoprecipitation revealed an enrichment of Cre1-96 in the upstream regulatory region of the main transactivator of cellulases and xylanases, Xyr1. Interestingly, transcript levels of cre1-96 show the same patterns as the ones of xyr1 under inducing conditions. CONCLUSIONS: The findings suggest that the truncation turns Cre1 into an activating regulator, which primarily exerts its role by approaching the upstream regulatory region of xyr1. The conversion of repressor proteins to potential activators in other biotechnologically used filamentous fungi can be applied to increase their enzyme production capacities.

Identification of the Main Regulator Responsible for Synthesis of the Typical Yellow Pigment Produced by Trichoderma reesei.

The industrially used ascomycete Trichoderma reesei secretes a typical yellow pigment during cultivation, while other Trichoderma species do not. A comparative genomic analysis suggested that a putative secondary metabolism cluster, containing two polyketide-synthase encoding genes, is responsible for the yellow pigment synthesis. This cluster is conserved in a set of rather distantly related fungi, including Acremonium chrysogenum and Penicillium chrysogenum In an attempt to silence the cluster in T. reesei, two genes of the cluster encoding transcription factors were individually deleted. For a complete genetic proof-of-function, the genes were reinserted into the genomes of the respective deletion strains. The deletion of the first transcription factor (termed yellow pigment regulator 1 [Ypr1]) resulted in the full abolishment of the yellow pigment formation and the expression of most genes of this cluster. A comparative high-pressure liquid chromatography (HPLC) analysis of supernatants of the ypr1 deletion and its parent strain suggested the presence of several yellow compounds in T. reesei that are all derived from the same cluster. A subsequent gas chromatography/mass spectrometry analysis strongly indicated the presence of sorbicillin in the major HPLC peak. The presence of the second transcription factor, termed yellow pigment regulator 2 (Ypr2), reduces the yellow pigment formation and the expression of most cluster genes, including the gene encoding the activator Ypr1. IMPORTANCE: Trichoderma reesei is used for industry-scale production of carbohydrate-active enzymes. During growth, it secretes a typical yellow pigment. This is not favorable for industrial enzyme production because it makes the downstream process more complicated and thus increases operating costs. In this study, we demonstrate which regulators influence the synthesis of the yellow pigment. Based on these data, we also provide indication as to which genes are under the control of these regulators and are finally responsible for the biosynthesis of the yellow pigment. These genes are organized in a cluster that is also found in other industrially relevant fungi, such as the two antibiotic producers Penicillium chrysogenum and Acremonium chrysogenum The targeted manipulation of a secondary metabolism cluster is an important option for any biotechnologically applied microorganism.

The Relation Between Promoter Chromatin Status, Xyr1 and Cellulase Ex-pression in Trichoderma reesei.

The ascomycete Trichoderma reesei is used for the production of plant cell wall-degrading enzymes in industrial scale. The interplay of the transactivator Xyr1 and the repressor Cre1 mainly regulates the expression of these enzymes. During induc-ing conditions, such as in the presence of sophorose, the transcription of the two major cellulase-encoding genes, cbh1 and cbh2, is activated as well as the expression of xyr1. In the presence of D-glucose carbon catabolite repression mediated by Cre1 takes place and the expression of Xyr1 and the plant cell wall-degrading enzymes is down-regulated. In this study we compare the chromatin status of xyr1, cbh1, and cbh2 promoters in the wild-type strain and the Cre1-deficient strain Rut-C30. Chromatin rearrangement occurs in the xyr1 promoter during induction on sophorose. Chromatin opening and protein-DNA interactions in the xyr1 promoter were detected especially in a region located 0.9 kb upstream the translation start co-don, which bears several putative Cre1-binding sites and a CCAAT-box. Moreover, the xyr1 promoter is overall more acces-sible in a cre1-truncated background, no matter which carbon source is present. This makes the xyr1 regulatory sequence a good target for promoter engineering aiming at the enhancement of cellulase production.

Xpp1 regulates the expression of xylanases, but not of cellulases in Trichoderma reesei.

BACKGROUND: The ascomycete Trichoderma reesei is industrially used for the production of cellulases. During the production process xylanases are co-secreted, which uses energy and nutrients. Cellulases and xylanases share the same main regulators, which makes a knowledge-based strain design difficult. However, previously a cis-element in the promoter of the main xylanase-encoding gene was identified as binding site for a putative repressor. Subsequently, three candidate repressors were identified in a pull-down approach. The expression of the most promising candidate, Xpp1 (Xylanase promoter-binding protein 1), was reported to be up-regulated on the repressing carbon source d-glucose and to bind the cis-element in vitro. RESULTS: In this study, Xpp1 was deleted and over-expressed in T. reesei. An in vivo DNA-footprint assay indicated that Xpp1 binds a palindromic sequence in the xyn2 promoter. Comparison of the deletion, the over-expression, and the parent strain demonstrated that Xpp1 regulates gene expression of xylanolytic enzymes at later cultivation stages. Xpp1 expression was found to be up-regulated, additionally to d-glucose, by high d-xylose availability. These findings together with the observed xyn2 transcript levels during growth on xylan suggest that Xpp1 is the mediator of a feedback mechanism. Notably, Xpp1 has neither influence on the d-xylose metabolism nor on the expression of cellulases. CONCLUSIONS: Xpp1 as regulator acting on the expression of xylanases, but not cellulases, is a highly promising candidate for knowledge-based strain design to improve the cellulases-to-xylanases ratio during industrial cellulase production.

The impact of chromatin remodelling on cellulase expression in Trichoderma reesei.

BACKGROUND: Trichoderma reesei is used for industry-scale production of plant cell wall-degrading enzymes, in particular cellulases, but also xylanases. The expression of the encoding genes was so far primarily investigated on the level of transcriptional regulation by regulatory proteins. Otherwise, the impact of chromatin remodelling on gene expression received hardly any attention. In this study we aimed to learn if the chromatin status changes in context to the applied conditions (repressing/inducing), and if the presence or absence of the essential transactivator, the Xylanase regulator 1 (Xyr1), influences the chromatin packaging. RESULTS: Comparing the results of chromatin accessibility real-time PCR analyses and gene expression studies of the two prominent cellulase-encoding genes, cbh1 and cbh2, we found that the chromatin opens during sophorose-mediated induction compared to D-glucose-conferred repression. In the strain bearing a xyr1 deletion the sophorose mediated induction of gene expression is lost and the chromatin opening is strongly reduced. In all conditions the chromatin got denser when Xyr1 is absent. In the case of the xylanase-encoding genes, xyn1 and xyn2, the result was similar concerning the condition-specific response of the chromatin compaction. However, the difference in chromatin status provoked by the absence of Xyr1 is less pronounced. A more detailed investigation of the DNA accessibility in the cbh1 promoter showed that the deletion of xyr1 changed the in vivo footprinting pattern. In particular, we detected increased hypersensitivity on Xyr1-sites and stronger protection of Cre1-sites. Looking for the players directly causing the observed chromatin remodelling, a whole transcriptome shotgun sequencing revealed that 15 genes encoding putative chromatin remodelers are differentially expressed in response to the applied condition and two amongst them are differentially expressed in the absence of Xyr1. CONCLUSIONS: The regulation of xylanase and cellulase expression in T. reesei is not only restricted to the action of transcription factors but is clearly related to changes in the chromatin packaging. Both the applied condition and the presence of Xyr1 influence chromatin status.

A truncated form of the Carbon catabolite repressor 1 increases cellulase production in Trichoderma reesei.

BACKGROUND: Rut-C30 is a cellulase-hyperproducing Trichoderma reesei strain and, consequently, became the ancestor of most industry strains used in the production of plant cell wall-degrading enzymes, in particular cellulases. Due to three rounds of undirected mutagenesis its genetic background differs from the wild-type QM6a in many ways, of which two are the lack of a 83 kb large sequence in scaffold 15 and the partial lack of the gene encoding the Carbon catabolite repressor 1 (CREI). However, it is still unclear, what exactly enhances cellulase production in Rut-C30. RESULTS: The investigation of the expression of two genes encoding cellulases (cbh1 and cbh2) and the gene encoding their main transactivator (xyr1) revealed that the presence of the truncated form of CREI (CREI-96) contributes more to the Rut-C30 phenotype than a general loss of CREI-mediated carbon catabolite repression (cre1 deletion strain) or the deletion of 29 genes encoded in the scaffold 15 (83 kb deletion strain). We found that the remaining cre1 in Rut-C30 (cre1-96) is transcribed into mRNA, that its putative gene product (Cre1-96) is still able to bind DNA, and that the CREI-binding sites in the upstream regulatory regions of the chosen CREI-target genes are still protected in Rut-C30. As it was previously reported that CREI acts on the nucleosome positioning, we also analyzed chromatin accessibility of the core promoters of CREI-target genes and found them open even on D-glucose in the presence of CREI-96. CONCLUSIONS: The lack of the full version of CREI in Rut-C30 corresponds with a partial release from carbon catabolite repression but is not completely explained by the lack of CREI. In contrast, the truncated CREI-96 of Rut-C30 exerts a positive regulatory influence on the expression of target genes. Mechanistically this might be explained at least partially by a CREI-96-mediated opening of chromatin.