A NEW PROMOTER-PROBE VECTOR FOR SACCHAROMYCES-CEREVISIAE USING FUNGALGLUCOAMYLASE CDNA AS THE REPORTER GENE

A system is described for the selection of DNA sequences showing promo ter activity in the yeast Saccharomyces cerevisiae using a heterologou s reporter enzyme which is efficiently secreted by the yeast host. A m ulticopy shuttle plasmid of the YEp-type was constructed so as to carr y multiple unique cloning sites at the 5' end of the Aspergillus awamo ri glucoamylase cDNA. Glucoamylase can only be expressed upon insertio n at one of these unique cloning sites of a DNA fragment from any sour ce, provided it is endowed with promoter function in S. cerevisiae. As the glucoamylase signal-peptide is functional in S. cerevisiae, the e nzyme is efficiently secreted by the yeast transformants. This phenoty pe can be very easily detected on plate assays and accurately quantifi ed by spectrophotometric analysis of the culture supernatant. Since S. cerevisiae naturally lacks amylolytic activity, any wild-type strain can be used as a host in this system. To evaluate the system, a DNA po ol of random fusions was created by ligating sau 3A digested S. cerevi siae genomic DNA to the BglII-linearized vector. The resulting hybrid plasmids were transformed into S. cerevisiae and several transformants secreting glucoamylase to varying degrees were obtained.