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Martins-Santana, L.

Publications and source records attributed to Martins-Santana, L..

2 recordsLinked to original sources

CRZ1 regulator and calcium cooperatively modulate holocellulases gene expression in Trichoderma reesei QM6a

BackgroundTrichoderma reesei is the main filamentous fungus used in industry to produce cellulases. Over the last decades, there have been a strong increase in the understanding of the regulatory network controlling cellulase-coding gene expression in response to a number of inducers and environmental signals. In this sense, the role of calcium and the Calcineurin-responsive protein (CRZ1) has been investigated in industrially relevant strains of T. reesei RUT-C30, but this system has not been investigated in wild-type reference strain of this fungus.\n\nResultsHere, we investigated the role of CRZ1 and Ca2+ signaling in the fungus T. reesei QM6a. For this, we first searched for potential CRZ1 binding sites in promoter regions of key genes coding holocellulases, as well as transcriptional regulators and sugar and calcium transporters. Using a nearly constructed T. reesei {Delta}crzl strain, we demonstrated that most of the genes expected to be regulated by CRZ1 were affected in the mutant strain induced with sugarcane bagasse (SCB) and cellulose. In particular, our data demonstrate that Ca2+ acts synergistically with CRZ1 to modulate gene expression, but also exerts CRZ1-independent regulatory role in gene expression in T. reesei, suggesting the existence of additional Ca2+ sensing mechanisms in this fungus.\n\nConclusionsThis work presents new evidence on the regulatory role of CRZ1 and Ca2+ sensing in the regulation of cellulolytic enzymes in T. reesei, evidencing significant and previously unknown function of this Ca2+ sensing system in the control key transcriptional regulators (XYR1 and CRE1) and on the expression of genes related to sugar and Ca2+ transport. Taken together, the data obtained here provide new evidence on the regulatory network of T. reesei related to plant biomass deconstruction.

microbiology↗

Reverse engineering of an aspirin-responsive regulator in bacteria

Bacterial transcriptional factors (TFs) and their target promoters are key devices for engineering of complex circuits in many biotechnological applications. Yet, there is a dearth of well characterized inducer-responsive TFs that could be used in the context of an animal or human host. In this work we have deciphered the inducer recognition mechanism of two AraC/XylS regulators from Pseudomonas putida (BenR and XylS) for creating a novel expression system responsive to acetyl salicylate (i.e. Aspirin). Using protein homology modeling and molecular docking with the cognate inducer benzoate and a suite of chemical analogues, we identified the conserved binding pocket of these two proteins. Using site directed mutagenesis, we identified a single amino acid position required for efficient inducer recognition and transcriptional activation. While modification of this position in BenR abolishes protein activity, its modification in XylS increases the response to several aromatic compounds, including acetyl salicylic acid to levels close to those achieved by the canonical inducer. Moreover, by constructing chimeric proteins with swapped N-terminal domains, we created novel regulators with mixed promoter and inducer recognition profiles. As a result, a collection of engineered TFs was generated with enhanced response to a well characterized and largely innocuous molecule with a potential for eliciting heterologous expression of bacterial genes in animal carriers.

synthetic biology↗