Enhanced production of anticoagulant hirudin in recombinant Saccharomyces cerevisiae by chromosomal delta-integration

Recombinant Saccharomyces cerevisiae strains were developed to overproduce an anticoagulant hirudin. The delta -sequences of the yeast retrotransposon Ty1 and URA3 were used as target sites for a hirudin expression cassette. High copy-number transformants were successfully selected using a dominant selection antibiotic, G418. The copy numbers of the hirudin expression cass ette integrated into F-sequences of the yeast chromosome ranged from five t o ten copies per cell. Production of hirudin in the delta -integrated recom binant S. cerevisiae system increased over two-fold compared with the YEp-b ased episomal hirudin expression system. A linear relationship between the copy number of the hirudin expression cassette and hirudin expression level was observed up to 10 copies. The hirudin expression cassettes integrated into the yeast chromosome were stably maintained in non-selective culture c onditions. (C) 2001 Elsevier Science B.V. All rights reserved.