Analysis of the hypoxia‐induced ADH2 promoter of the respiratory yeast Pichia stipitis reveals a new mechanism for sensing of oxygen limitation in yeast

Abstract

We introduced a reporter gene system into Pichia stipitis using the gene for the artificial green fluorescent protein (GFP), variant yEGFP. This system was used to analyse hypoxia-dependent PsADH2 regulation. Reporter gene activity was only found under oxygen limitation on a fermentable carbon source. The promoter was not induced by oxygen limitation in the Crabtree-positive yeast Saccharomyces cerevisiae. Promoter deletions revealed that a region of 15 bp contained the essential site for hypoxic induction. This motif was different from the known hypoxia response elements of S. cerevisiae but showed some similarity to the mammalian HIF-1 binding site. Electrophoretic mobility shift assays demonstrated specific protein binding to this region under oxygen limitation. Similar to the S. cerevisiae heme sensor system, the promoter was induced by Co 2+ . Cyanide was not able to mimic the effect of oxygen limitation. The activation mechanism of PsADH2 also, in this respect, has similarities to the mammalian HIF-1 system, which is inducible by Co 2+ but not by cyanide. Thus, the very first promoter analysis in P. stipitis revealed a hitherto unknown mechanism of oxygen sensing in yeast.

Effect of cell immobilization and pH on Scheffersomyces stipitis growth and fermentation capacity in rich and inhibitory media

“…Biomass variation suggests that sugars, organic acids and even ethanol were used for growth and cell division. These results were probably arising from the presence of fully aerobic conditions in the bioreactor, since S. stipitis favors growth under excess of oxygen and ethanol production is maximized when microaerophilic conditions are present [14,15]. Figure 3B shows the changes in S. stipitis cultivation profile caused by the immobilization.…”

“…stipitis was described as having the highest native capacity for D-xylose fermentation of any known microbe [1,13]. This yeast induces fermentative activity in response to oxygen limitation and reaches the optimal activity with microaerophilic conditions (1% to 15% O 2 ) [14,15]. One of the major drawbacks of S. stipitis is the low tolerance towards inhibitors and high ethanol concentrations [16].…”

Saccharomyces cerevisiae Engineered for Xylose Metabolism Exhibits a Respiratory Response

“…Metabolic regulation by glucose has been studied in S. cerevisiae for many years (16). The regulatory and physiological properties of xylose metabolism have been extensively studied only in the xylose-fermenting yeast Pichia stipitis (8,43), which has served as the source of genes for engineering xylose metabolism in S. cerevisiae.…”

Bioethanol a Microbial Biofuel Metabolite; New Insights of Yeasts Metabolic Engineering

“…Xylose and glucose are not equal fermentations for many reasons, but increasing the capacity of P. stipitis for rapid xylose fermentation can greatly develop its usefulness in commercial applications. One of fermentative regulatory machinery is that S. cerevisiae regulates fermentation process by sensing the existence of glucose, but P. stipitis promotes fermentative activity in response to oxygen limitation [114][115][116][117]. Universal expression array analysis has shown special response patterns for rahamnose, cellobiose, arabinose, xylose and other lignocellulosic substrates.…”

“…This suggests that P. stipitis XDH competes with ADH for reductant, apparently NADH. In native xylose-fermenting yeast P. stipitis, the primary ADH is heavily expressed with decreasing the availability of O 2 [116,188]. Moreover, P. stipitis produces xylitol and arabitol, when the xylulokinase is deleted [172].…”