Synthesis and secretion of Providencia rettgeri and Escherichia coli heterodimeric penicillin amidases in Saccharomyces cerevisiae

The Providencia rettgeri and Escherichia coli pac genes encoding heterodime ric penicillin G amidases (PAC) were successfully expressed in Saccharomyce s cerevisiae. Furthermore, these recombinant enzymes are secreted from the yeast cell into the medium which is in contrast to bacterial hosts, where t he enzymes are retained in the periplasm. Contrary to the P. rettgeri PAC-e ncoding gene, the E. coli pac is poorly expressed in yeast. The highest yie ld of P. rettgeri PAC was obtained with a multi-copy plasmid, resulting in of 1500 units per liter. This yield is higher by an order of magnitude than that obtained in the best recombinant bacterial expression system. The rec ombinant P. rettgeri enzyme is only partially and selectively O-glycosylate d. Only every sixth or seventh alpha-subunit is glycosylated, while the bet a-subunit is not glycosylated at all. N-Glycosylation has not been detected . (C) 1999 Elsevier Science B.V. All rights reserved.