Properties and engineering of a mutantSTA promoter ofSaccharomyces diastaticus

A new allelic variant of theSTA2 gene ofS. diastaticus, designated asSTA2 ^K, was cloned and characterized (1; accompanying paper). An application-oriented analysis of the promoter region ofSTA2 ^K 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 theSTA2 ^K 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 theSTA2 ^K promoter under the control of either thePH05 orCYC1 upstream regulatory sequences. On high-copy-number vectors, induction of the UASpho5-STA2^K 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.