CYTOPLASMIC EXPRESSION OF THE ASPERGILLUS GLUCOAMYLASE GENE INTEGRATED INTO A LINEAR PLASMID IN SACCHAROMYCES-CEREVISIAE

The glaA gene encoding glucoamylase I (GAI) of Aspergillus awamori var . kawachi was successfully expressed in cytoplasm of Saccharomyces cer evisiae, using two cytoplasmic linear plasmids, pGKL1 and pGKL2. In or der to integrate the glaA gene, two pGKL1-derived plasmids, designated pKTF951 and pKTF952, were constructed. The glaA gene was placed downs tream of the ORF2 promoter (UCS2) between ORF1 and ORF3 of pGKL1 to ob tain pKTF951. pKTF952 contained an additional ORF5 promoter (UCS5) fro m pGKL2, connected downstream of the UCS2 in pKTF951. pKTF951 and pKTF 952 were respectively transformed to S. cerevisiae carrying the origin al pGKL1 and pGKL2. Southern analysis revealed that in vivo homologous recombination took between the indigenous pGKL1 and the recombinant p KTF951 or pKTF952 within the common ORF1 and ORF3 regions, which resul ted in the replacement of ORF2 by the glaA gene In pGKL1. Transformant s carrying the resultant linear recombinant plasmids, pNS951 or pNS952 , produced GAI. S. cerevisiae (pNS952) secreted 2.6-fold more GAI than S. cerevisiae (pNS951).