Transcriptional control of ADH genes in the xylose-fermenting yeast Pichia stipitis

Abstract

We studied the expression of the genes encoding group I alcohol dehydrogenases (PsADH1 and PsADH2) in the xylose-fermenting yeast Pichia stipitis CBS 6054. The cells expressed PsADH1 approximately 10 times higher under oxygen-limited conditions than under fully aerobic conditions when cultivated on xylose. Transcripts of PsADH2 were not detectable under either aeration condition. We used a PsADH1::lacZ fusion to monitor PsADH1 expression and found that expression increased as oxygen decreased. The level of PsADH1 transcript was repressed about 10-fold in cells grown in the presence of heme under oxygen-limited conditions. Concomitantly with the induction of PsADH1, PsCYC1 expression was repressed. These results indicate that oxygen availability regulates PsADH1 expression and that regulation may be mediated by heme. The regulation of PsADH2 expression was also examined in other genetic backgrounds. Disruption of PsADH1 dramatically increased PsADH2 expression on nonfermentable carbon sources under fully aerobic conditions, indicating that the expression of PsADH2 is subject to feedback regulation under these conditions.

FIG. 1

Zymogram analysis of ADH isozymes from P. stipitis CBS 6054 grown on 8% xylose under fully aerobic conditions (0 h) and after shifting to oxygen-limited conditions (12 to 72 h).

FIG. 2

Induction of PsADH1 mRNA following a shift to oxygen-limited conditions. CBS 6054 cells were grown in either xylose (A) or glucose (B) under fully aerobic conditions and shifted to oxygen-limited conditions. Aliquots of cells were harvested at the indicated times, and total RNA was prepared for Northern analysis. Blots were probed with PsADH1. The positions of rRNA and PsADH1 mRNA are indicated. ADH activity (C) was also measured on samples prepared at the same time, as indicated. ■, xylose; □, glucose.

FIG. 3

Kinetics of induction of PsADH1 mRNA level upon shift to oxygen-limited conditions. Cells were grown on xylose under aerobic conditions (repressed state) until they reached an OD₆₀₀ of 1.0, collected by centrifugation, and shifted to oxygen-limited conditions by suspension in the same medium. Aliquots of cells were taken at the indicated times, and total RNA was prepared for Northern analysis. Blots were probed with PsADH1.

FIG. 4

Effect of heme addition on PsADH1 and PsCYC1 expression in anaerobic cells. Cells were grown aerobically on xylose until they reached an OD₆₀₀ of 1.0, collected by centrifugation, and shifted to oxygen-limited conditions by suspension in the same medium. After the cells had been shifted to oxygen limitation, heme (50 μg/ml) or protoporphyrin IX (p.p. IX [50 μg/ml]) was added as indicated, and growth was continued under oxygen-limited conditions for 4 h. The RNA blot was hybridized with PsADH1- or PsCYC1-specific oligonucleotides as probes.

FIG. 5

Effect of PsADH1 disruption on expression of the PsADH2 gene during batch growth on nonfermentable carbon sources under fully aerobic conditions. Wild-type (WT) cells (lanes 1 and 2) and cells from PSU218 (lanes 3 and 4) carrying two disrupted Psadh1 alleles were grown in either glycerol (Gly [lanes 1 and 3]) or ethanol (E [lanes 2 and 4]) medium under aerobic conditions. Total RNA was extracted from these cultures for Northern analysis with PsADH1- or PsADH2-specific oligonucleotides as probes.