Transcriptional regulation of ARO9 and ARO10 genes for the catabolism of aromatic amino acids in Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae can utilize aromatic amino acids as the sole nitrogen source. The final products of aromatic amino acid catabolism are aromatic alcohols, including tryptophol, phenylethanol, and tyrosol. Production of aromatic alcohols from aromatic amino acids is known to proceed via the Ehrlich pathway. It consists of three main steps, transamination of an amino acid to an α-keto acid, decarboxylation of an α-keto acid to an aldehyde, and reduction of an aldehyde to an alcohol. ARO9 and ARO10 genes encode transaminase and decarboxylase, respectively, in the Ehrlich pathway. In this thesis, the activation mechanisms of ARO9 and ARO10 genes via Aro80, a pathway-specific transcriptional activator, and GATA factors, global nitrogen regulators, were elucidated. Firstly, inducer-dependent activation mechanism of Aro80 was investigated. It was found that Aro80 constitutively localized in the nucleus irrespective of the availability of inducers such as aromatic amino acids, methionine, or aromatic alcohols. In addition, it was demonstrated that Aro80 is constitutively bound to its target promoters and is activated by inducers at the level of transactivation. Although Aro80 was also shown to bind to its own promoter, ARO80 expression was not induced by tryptophan. Secondly, direct involvement of GATA factors in ARO9, ARO10 and ARO80 was demonstrated. It was shown that Aro80 is absolutely required for Gat1 binding to the ARO9, ARO10, and ARO80 promoters upon TORC1 inhibition by rapamycin treatment. Gln3 binding to these promoters showed a partial requirement for Aro80. Rapamycin-dependent Gat1 and Gln3 binding to the Aro80 target promoters was not affected by tryptophan availability, suggesting that transactivation activity of Aro80 is not necessary for the recruitment of GATA factors. Rapamycin-dependent induction of Aro80 target genes also required PP2A phosphatase complex, but not Sit4 phosphatase, acting downstream of TORC1 Thirdly, it was shown that the transcription of ARO9 and ARO10 is also induced by heat shock in an Aro80-dependent manner. However, heat shock-related signaling pathways including PKA, PKC, and HOG pathways were not involved in the heat shock activation of Aro80. It was elucidated that heat-induced increase in aromatic amino acid influx can lead to the inducer-dependent activation of Aro80 upon heat shock. Known aromatic amino acid permeases play an insignificant role in the heat-induced expression of ARO9 and ARO10, suggesting that an increase in plasma membrane fluidity might be responsible for the influx of aromatic amino acids during heat shock stress. Fourthly, a series of novel inducible promoters were generated based on the ARO9 promoter. The promoters with various promoter strengths could be generated by modulating the number of Aro80 binding site combined with either ARO9 or ARO80 core promoter. Expression levels from these promoters could be further regulated by tryptophan concentration, suggesting potential application of these inducible promoters for fine tuning of gene expression levels in metabolic engineering.