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

Respiratory and fermentative pathways coexist 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 cerevisiae URA1 (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. Introduction N.-Q: Shi · T. W. Jeffries (* ) Department of Bacteriology, University of Wisconsin, Madison,1550 Linden Drive, Madison, Wisconsin 53706. USA Present address: USDA, Forest Service, Forest Products Laboratory, One Gifford Pinchot Drive, Madison, WI 53705. USA e-mail:[email protected] Tel.: + 1 608-231-9453 Fax: + 1 608-231-9262 Most yeast and fungi are Crabtree-negative, which is to say that they lack the ability to grow anaerobically, and must rely on respirofermentative metabolism to support cell growth (Siso et al. 1996). Molecular oxygen acts as a terminal electron acceptor of respiratory metabolism. and it is required for biosynthesis of membrane sterols in yeasts (Gancedo and Serrano 1989). However. rare species such as Saccharomyces cerevisiae can grow anaerobically using energy solely generated from fermentation when essential lipids are present (Andreasen and Stier 1953). Nagy et al. (1992) showed that S. cerevisiae possesses a unique form of dihydroorotate dehydrogenase (EC 1.3.3.1) that confers the ability to grow anaerobically. Dihydroorotate dehydrogenase. encoded by ScURA1 (Roy 1992), catalyses a single redox reaction converting dihydroorotate to orotate in the pyrimidine biosynthesis pathway. Dihgydroorotate dehydrogenases of higher eukaryotes are functional components of the respiratory chains. using oxygen as the ultimate (Vorisek et al. 1993). but not necessarily the only, electron acceptor (Hines et al. 1986). In S. cererisiae, the enzyme is found in the cytosol where its activity is also coupled to the reaction that reduces fumarate to succinate (Nagy et al. 1992). S. cererisiae is incapable of using xylose because it lacks two key xylose-metabolizing enzymes, xylose reductase (PsXYL1) and xylitol dehydrogenase (PsXYL2). However, this yeast can ferment xylulose (Yu et al. 1995) because of the presence of a xylulokinase (ScXUK) (Ho and Chang 1989). Recently. XYL1 and XYL2 genes from the xylose-fermenting yeast, Pichia stipitis, have been used to impart xylose fermentation to S. cerevisiae (Kotter and Ciriacy 1993; Tantirungkij et al. 1994; Meinander et al. 1996) but the resulting strains have failed to ferment xylose effectively. Toon et al. (1997) improved the Saccharomyces xylose fermentation by generating a fusion strain that overexpresses PsXYL1.