ANAEROBIC GROWTH AND IMPROVED FERMENTATION OF PICHIA-STIPITIS BEARINGA URA1 GENE FROM SACCHAROMYCES-CEREVISIAE

Respiratory and fermentative pathways coexist to support growth and pr oduct formation in Pichia stipitis. This yeast grows rapidly without e thanol production under fully aerobic conditions, and it ferments gluc ose or xylose under oxygen-limited conditions, but it stops growing wi thin one generation under anaerobic conditions. Expression of Saccharo myces cerevisiae URA1 (ScURA1) in P. stipitis enabled rapid anaerobic growth in minimal defined medium containing glucose when essential lip ids were present. ScURA1 encodes a dihydroorotate dehydrogenase that u ses fumarate as an alternative electron acceptor to confer anaerobic g rowth. Initial P. stipitis transformants grew and produced 32 g/l etha nol from 78 g/l glucose. Cells produced even more ethanol faster follo wing two anaerobic serial subcultures. Control strains without ScURA1 were incapable of growing anaerobically and showed only limited fermen tation. P. stipitis cells bearing ScURA1 were viable in anaerobic xylo se medium for long periods, and supplemental glucose allowed cell grow th, but xylose alone could not support anaerobic growth even after ser ial anaerobic subculture on glucose. These data imply that P. stipitis can grow anaerobically using metabolic energy generated through ferme ntation but that it exhibits fundamental differences in cofactor selec tion and electron transport with glucose and xylose metabolism. This i s the first report of genetic engineering to enable anaerobic growth o f a eukaryote.