Properties and engineering of a mutant STA promoter of Saccharomyces diastaticus

A new allelic variant of the STA2 gene of S. diastaticus, designated as STA2K, was cloned and characterized (1; accompanying paper). An application-oriented analysis of the promoter region of STA2K is described, with an emphasis on its peculiar structural feature: A 1.1-kb natural deletion located 189 nucleotides upstream of the translation start codon. The strength of the STA2K promoter was found comparable to that of known strong constitutive yeast promoters (ADH1, GAPDH). Regulated glucoamylase expression was demonstrated by chimeric promoters, which were constructed by placing the STA2K promoter under the control of either the PHO5 or CYC1 upstream regulatory sequences. On high-copy-number vectors, induction of the UASPHO5-STA2K chimeric promoter by phosphate depletion resulted in a destructive overexpression of the secreted glucoamylase, which completely halted cell growth, and promoted cell decay. In contrast, UASCYC1 was shown to mediate a fine-tuned regulation both by glucose concentration and, indirectly, by starch, the substrate for the glucoamylase to produce glucose.