A highly sensitive in vivo footprinting technique for condition-dependent identification of cis elements.

Knowing which regions of a gene are targeted by transcription factors during induction or repression is essential for understanding the mechanisms responsible for regulation. Therefore, we re-designed the traditional in vivo footprinting method to obtain a highly sensitive technique, which allows identification of the cis elements involved in condition-dependent gene regulation. Data obtained through DMS methylation, HCl DNA cleavage and optimized ligation-mediated PCR using fluorescent labelling followed by capillary gel electrophoresis are analysed by ivFAST. In this work we have developed this command line-based program, which is designed to ensure automated and fast data processing and visualization. The new method facilitates a quantitative, high-throughput approach because it enables the comparison of any number of in vivo footprinting results from different conditions (e.g. inducing, repressing, de-repressing) to one another by employing an internal standard. For validation of the method the well-studied upstream regulatory region of the Trichoderma reesei xyn1 (endoxylanase 1) gene was used. Applying the new method we could identify the motives involved in condition-dependent regulation of the cbh2 (cellobiohydrolase 2) and xyn2 (endoxylanase 2) genes.

In Vitro Characterization of a Nuclear Receptor-like Domain of the Xylanase Regulator 1 from Trichoderma reesei.

Engineering transcription factors is an interesting research target gaining increasing attention, such as in the case of industrially used organisms. With respect to sustainability, biomass-degrading saprophytic fungi, such as Trichoderma reesei, are promising industrial work horses because they exhibit a high secretory capacity of native and heterologously expressed enzymes and compounds. A single-point mutation in the main transactivator of xylanase and cellulase expressions in T. reesei Xyr1 led to a strongly deregulated and enhanced xylanase expression. Circular dichroism spectroscopy revealed a change in secondary structure caused by this mutation. According to electrophoretic mobility shift assays and determination of the equilibrium-binding constants, the DNA-binding affinity of the mutated Xyr1 was considerably reduced compared to the wild-type Xyr1. Both techniques were also used to investigate the allosteric response to carbohydrates (D-glucose-6-phosphate, D-xylose, and sophorose) signalling the repression or induction of Xyr1 target genes. The mutated Xyr1 no longer exhibited a conformational change in response to these carbohydrates, indicating that the observed deregulation is not a simple matter of a change in DNA-binding of the transactivator. Altogether, we postulate that the part of Xyr1 where the mutation is located functions as a nuclear receptor-like domain that mediates carbohydrate signals and modulates the Xyr1 transactivating activity.

Synthesis of an antiviral drug precursor from chitin using a saprophyte as a whole-cell catalyst.

BACKGROUND: Recent incidents, such as the SARS and influenza epidemics, have highlighted the need for readily available antiviral drugs. One important precursor currently used for the production of Relenza, an antiviral product from GlaxoSmithKline, is N-acetylneuraminic acid (NeuNAc). This substance has a considerably high market price despite efforts to develop cost-reducing (biotechnological) production processes. Hypocrea jecorina (Trichoderma reesei) is a saprophyte noted for its abundant secretion of hydrolytic enzymes and its potential to degrade chitin to its monomer N-acetylglucosamine (GlcNAc). Chitin is considered the second most abundant biomass available on earth and therefore an attractive raw material. RESULTS: In this study, we introduced two enzymes from bacterial origin into Hypocrea, which convert GlcNAc into NeuNAc via N-acetylmannosamine. This enabled the fungus to produce NeuNAc from the cheap starting material chitin in liquid culture. Furthermore, we expressed the two recombinant enzymes as GST-fusion proteins and developed an enzyme assay for monitoring their enzymatic functionality. Finally, we demonstrated that Hypocrea does not metabolize NeuNAc and that no NeuNAc-uptake by the fungus occurs, which are important prerequisites for a potential production strategy. CONCLUSIONS: This study is a proof of concept for the possibility to engineer in a filamentous fungus a bacterial enzyme cascade, which is fully functional. Furthermore, it provides the basis for the development of a process for NeuNAc production as well as a general prospective design for production processes that use saprophytes as whole-cell catalysts.

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.