SHAM-sensitive alternative respiration in the xylose-metabolizing yeast Pichia stipitis

SHAM-sensitive (STO) alternative respiration is present in the xylose-metabolizing, Crabtree-negative yeast, Pichia stipitis, but its pathway components and physiological roles during xylose metabolism are poorly understood. We cloned PsSTO1, which encodes the SHAM-sensitive terminal oxidase (PsSto1p), by genome walking from wild-type CBS 6054 and subsequently deleted PsSTO1 by targeted gene disruption. The resulting sto1-delta deletion mutant, FPL-Shi31, did not contain other isoforms of Sto protein that were detectable by Western blot analysis using an alternative oxidase monoclonal antibody raised against the Sto protein from Sauromatum guttatum. Levels of cytochromes b, c, c(1) and a.a(3) did not change in the sto1-delta mutant, which indicated that deleting PsSto1p did not alter the cytochrome pool. Interestingly, the sto1-delta deletion mutant stopped growing earlier than the parent and produced 20% more ethanol from xylose. Heterologous expression of PsSTO1 in Saccharomyces cerevisiae increased its total oxygen consumption rate and imparted cyanide-resistant oxygen uptake but did not enable growth on ethanol, indicating that PsSto1p is not coupled to ATP synthesis. We present evidence that the mitochondrial NADH dehydrogenase complex (Complex I) was present in wild-type CBS 6054 but was bypassed in the cells during xylose metabolism. Unexpectedly, deleting PsSto1p led to the use of Complex I in the mutant cells when xylose was the carbon source. We propose that the non-proton-translocating NAD(P)H dehydrogenases are linked to PsSto1p in xylose-metabolizing cells and that this non-ATP-generating route serves a regulatory function in the complex redox network of P. stipitis.