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 cassette integrated into delta-sequences of the yeast chromosome ranged from five to ten copies per cell. Production of hirudin in the delta-integrated recombinant S. cerevisiae system increased over two-fold compared with the YEp-based 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 conditions.
Kim
M D
MD
Department of Food Science and Technology and School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, South Korea.
Rhee
S K
SK
Seo
J H
JH
D000991:Antithrombins / Q000096:biosynthesis / Q000235:genetics*; D015825:Chromosomes, Fungal / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D018628:Gene Dosage; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000235:genetics; D006629:Hirudins / Q000096:biosynthesis / Q000235:genetics*; D016254:Mutagenesis, Insertional; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D018626:Retroelements / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D014170:Transformation, Genetic
D000991:Antithrombins; D006629:Hirudins; D011994:Recombinant Proteins; D018626:Retroelements