Efficient production of recombinant DNA proteins in Saccharomyces cerevisiae by controlled high-cell-density fermentation.
High levels of expression of heterologous proteins (from 5 to 15% of total cell proteins) in the budding yeast Saccharomyces cerevisiae have been obtained previously by the use of the inducible strong hybrid promoter UASGAL/CYC1, in batch as well in continuous cultures. However, in order to maximize the yield of heterologous proteins, a computer controlled fed-batch fermentation is essential. For this reason we have developed a fed-batch system based on a semiconductor gas detector that measures ethanol in the outflow gases. The optimal conditions are described for very high production (up to 1550 mg/liter), with both high productivity (up to 100-120 mg/liter/h) and high yield (up to 15 mg of protein/g of dry biomass), of heterologous protein driven by the UASGAL/CYC1 promoter in a completely computer controlled fed-batch fermentation of budding yeast. However, high production was dependent upon the addition of a large amount of galactose. The process was improved by developing a new, more easily inducible, vector system obtained by subcloning the GAL4 gene.
Alberghina
L
L
Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Italy.
Porro
D
D
Martegani
E
E
Ranzi
B M
BM
D001709:Biotechnology / Q000379:methods; D003001:Cloning, Molecular; D000431:Ethanol / Q000378:metabolism; D005285:Fermentation; D005656:Fungal Proteins / Q000096:biosynthesis*; D005690:Galactose / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
D005656:Fungal Proteins; D011994:Recombinant Proteins; D000431:Ethanol; D005690:Galactose