Cloning of the URA1 gene of Saccharomyces cerevisiae

A 5.7-kilobase segment of Saccharomyces cerevisiae deoxyribonucleic acid which complements both the yeast ura1 and Escherichia coli pyrD mutations in dihydroorotate dehydrogenase has been cloned in plasmid YRp7.     

Structure and expression of the PHO80 gene of Saccharomyces cerevisiae.

In yeast, the repression of acid phosphatase under high phosphate growth conditions requires the trans-acting factor PHO80. We have determined the DNA sequence of the PHO80 gene and found that it encodes a protein of 293 amino acids. The expression of the PHO80 gene, as measured by Northern analysis and level of a PHO80-LacZ fusion protein is independent of the level of phosphate in the growth medium. Disruption of the PHO80 gene is a non-lethal event and causes a derepressed phenotype, with acid phosphatase levels which are 3-4 fold higher than the level found in derepressed wild type cells. Furthermore, over-expression of the PHO80 gene causes a reduction in the level of acid phosphatase produced under derepressed growth conditions. Finally, we have cloned, localized and sequenced a temperature-sensitive allele of PHO80 and found the phenotype to be due to T to C transition causing a substitution of a Ser for a Leu at amino acid 163 in the protein product.     

Anaerobic growth and improved fermentation of Pichia stipitis bearing a URA1 gene from Saccharomyces cerevisiae

Respiratory and fermentative pathways co-exist to support growth and product formation in Pichia stipitis. This yeast grows rapidly without ethanol production under fully aerobic conditions, and it ferments glucose or xylose under oxygen-limited conditions, but it stops growing within one generation under anaerobic conditions. Expression of Saccharomyces cerevisiaeURA1 (ScURA1) in P. stipitis enabled rapid anaerobic growth in minimal defined medium containing glucose when essential lipids were present. ScURA1 encodes a dihydroorotate dehydrogenase that uses fumarate as an alternative electron acceptor to confer anaerobic growth. Initial P. stipitis transformants grew and produced 32 g/l ethanol from 78 g/l glucose. Cells produced even more ethanol faster following two anaerobic serial subcultures. Control strains without ScURA1 were incapable of growing anaerobically and showed only limited fermentation. P. stipitis cells bearing ScURA1 were viable in anaerobic xylose medium for long periods, and supplemental glucose allowed cell growth, but xylose alone could not support anaerobic growth even after serial anaerobic subculture on glucose. These data imply that P. stipitis can grow anaerobically using metabolic energy generated through fermentation but that it exhibits fundamental differences in cofactor selection and electron transport with glucose and xylose metabolism. This is the first report of genetic engineering to enable anaerobic growth of a eukaryote. Received: 6 January 1998 / Received revision: 9 April 1998 / Accepted: 19 April 1998