Disruption of the cytochromec gene in xylose-utilizing yeastPichia stipitis leads to higher ethanol production

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Abstract
The xylose‐utilizing yeast, Pichia stipitis, has a complex respiratory system that contains cytochrome and non‐cytochrome alternative electron transport chains in its mitochondria. To gain primary insights into the alternative respiratory pathway, a cytochrome c gene (PsCYC1, Accession No. AF030426) was cloned from wild‐type P. stipitis CBS 6054 by cross‐hybridization to CYC1 from Saccharomyces cerevisiae. The 333 bp open reading frame of PsCYC1 showed 74% and 69% identity to ScCYC1 and ScCYC7, respectively, at the DNA level. Disruption of PsCYC1 resulted in a mutant that uses the salicylhydroxamic acid (SHAM)‐sensitive respiratory pathway for aerobic energy production. Cytochrome spectra revealed that cytochromes c and a·a3 both disappeared in the cyc1‐Δ mutant, so no electron flow through the cytochrome c oxidase was possible. The cyc1‐Δ mutant showed 50% lower growth rates than the parent when grown on fermentable sugars. The cyc1‐Δ mutant was also found to be unable to grow on glycerol. Interestingly, the mutant produced 0·46 g/g ethanol from 8% xylose, which was 21% higher in yield than the parental strain (0·38 g/g). These results suggested that the alternative pathway might play an important role in supporting xylose conversion to ethanol under oxygen‐limiting conditions. Copyright © 1999 John Wiley & Sons, Ltd.