ANALYSIS OF CARBON SOURCE AND PH-DEPENDENT TRANSCRIPTIONAL REGULATION OF HUMICOLA GRISEA VAR. THERMOIDEA LIGNOCELLULOLYTIC SYSTEM

Thiago Machado Mello de Sousa (UnB); Lorena da Silveira Derengowiski (UnB); Ildinete Silva Pereira (UnB); Marcio José Poças Fonseca (UnB)

Environmental pH is an important signal for fungi physiology, intervening at the transcriptional regulation of several gene products. In filamentous fungi and yeasts, the PacC zinc-finger transcription factor regulates gene expression in response to alkaline external pH. The production of enzymes involved in plant cell wall breakdown is regulated mainly at the transcriptional level. Nonetheless, the involvement of the pH-related regulatory pathway in the lignocellulolytic enzymes expression has not been extensively studied. We have demonstrated that the thermophilic deuteromycete Humicola grisea var. thermoidea is a potent cellulases producer, presenting a considerable potential for agricultural wastes bioconvertion processes. Results of our group support the existence of a pH regulatory pathway for H. grisea var. thermoidea transcriptional regulation. In this work, we have performed by quantitative real time RT-PCR a time course trascriptional analysis of several H. grisea genes. Eight lignocellulolytic genes (cbh1.1, cbh1.2, egl1, egl2, egl3, egl4, bgl4 and xyn1) and two transcription factors (pacC and creA) were analyzed in the presence of simple (glucose) or complex (sugarcane bagasse) carbon sources and in acid or alkaline medium conditions. The qRT-PCR analysis reveled an early and strong induction of transcription of almost all lignocellulolitc genes, in a synergistic way, when the mycelia were grown with the complex carbon source and in alkali conditions (pH 8.0). The only exception was egl4, that was acid induced. An opposite pattern of expression of the two trascription factors was observed. While pacC was induced in alkaline conditions and strongly repressed in presence of glucose, creA was induced by glucose and repressed in alkaline conditions. By eletrophoretic mobility shift assays (EMSAs) with upstream regulatory sequences of pacC, we showed that exists an in vitro interaction between the proteins PacC and CreA with pacC upstream regulatory sequence, where the both factor compete by the same binding site. Taken together, this data corroborates our previous evidences supporting the existence of a pH regulatory pathway for H. grisea transcriptional regulation by PacC. Moreover, PacC is probably transcriptionaly regulated by itself and may suffer influence of the carbon repression mechanism mediated by CreA.