The ENA1 Na+-ATPase Gene Is Regulated by the SPS Sensing Pathway and the Stp1/Stp2 Transcription Factors.

The Saccharomyces cerevisiae ENA1 gene, encoding a Na+-ATPase, responds transcriptionally to the alkalinization of the medium by means of a network of signals that involves the Rim101, the Snf1 and PKA kinases, and the calcineurin/Crz1 pathways. We show here that the ENA1 promoter also contains a consensus sequence, located at nt -553/-544, for the Stp1/2 transcription factors, the downstream components of the amino acid sensing SPS pathway. Mutation of this sequence or deletion of either STP1 or STP2 decreases the activity of a reporter containing this region in response to alkalinization as well as to changes in the amino acid composition in the medium. Expression driven from the entire ENA1 promoter was affected with similar potency by the deletion of PTR3, SSY5, or simultaneous deletion of STP1 and STP2 when cells were exposed to alkaline pH or moderate salt stress. However, it was not altered by the deletion of SSY1, encoding the amino acid sensor. In fact, functional mapping of the ENA1 promoter reveals a region spanning from nt -742 to -577 that enhances transcription, specifically in the absence of Ssy1. We also found that the basal and alkaline pH-induced expression from the HXT2, TRX2, and, particularly, SIT1 promoters was notably decreased in an stp1 stp2 deletion mutant, whereas the PHO84 and PHO89 gene reporters were unaffected. Our findings add a further layer of complexity to the regulation of ENA1 and suggest that the SPS pathway might participate in the regulation of a subset of alkali-inducible genes.

Proteomic analysis reflects an environmental alkalinization-coupled pH-dependent mechanism of regulating lignocellulases in Trichoderma guizhouense NJAU4742.

BACKGROUND: Filamentous fungi have the ability to efficiently decompose plant biomass, and thus are widely used in the biofuel and bioprocess industries. In process, ambient pH has been reported to strongly affect the performance of the applied functional filamentous fungi. In this study, Trichoderma guizhouense NJAU4742 was investigated under the fermentation of rice straw at different initial pH values for a detailed study. RESULTS: The results showed that NJAU4742 strain could tolerate ambient pH values ranging from 3.0 to 9.0, but had significantly higher growth speed and extracellular enzyme activities under acidic conditions. At low ambient pH (< 4), NJAU4742 strain achieved rapid degradation of rice straw by elevating the ambient pH to an optimal range through environmental alkalinization. Further proteomic analysis identified a total of 1139 intracellular and extracellular proteins during the solid-state fermentation processes, including the quantified 190 carbohydrate-active enzymes (CAZymes) responsible for rice straw degradation, such as 19 cellulases, 47 hemicellulases and 11 chitinases. Meanwhile, the analysis results clearly showed that the secreted lignocellulases had a synergistic trend in distribution according to the ambient pH, and thus led to a pH-dependent classification of lignocellulases in T. guizhouense NJAU4742. CONCLUSIONS: Most functional lignocellulases were found to be differently regulated by the ambient pH in T. guizhouense NJAU4742, which had the ability of speeding up biomass degradation by elevating the ambient pH through environmental alkalinization. These findings contribute to the theoretical basis for the biodegradation of plant biomass by filamentous fungi in the biofuel and bioprocess industries.