Xylitol dehydrogenase mutants ofPachysolen tannophilusand the role of xylitol in d-xylose catabolism
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Cited by 5 publications
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~~ ~~Increasing chromosome number of Pachysolen tannophilus above the haploid level increased the yield of ethanol from D-XylOSe. There was a large increment on going from the haplophase to the diplophase, and the highest yields were obtained with either a triploid or a probable tetraploid, depending on the concentration of D-xylose. In addition, the rate of ethanol production from Dxylose and D-glucose increased, with the increment being larger on D-xylose. On D-galactose, the amount of ethanol produced within a given time also increased. The level of a by-product from D-XylOSe, xylitol, decreased on going from a haploid to higher ploidy, but there was no discernible trend for two other by-products, acetic acid and arabinitol. Increasing ploidy increased growth rate on D-galactose, but not appreciably on D-xylose. The altered properties on D-XylOSe of the strains tested were not due to increased levels of either D-xylOSe reductase or xylitol dehydrogenase, nor probably of alcohol dehydrogenase. I N T R O D U C T I O NPachysolen tannophilus is one of the better producers of ethanol from D-XylOSe . It also functions well in some mixtures of pentoses and hexoses (Neirinck et al., 1982). However, several of its properties require improvement before it can be used for industrial purposes. The rate at which alcohol is produced from D-xylose was found to be about a quarter of that from D-glucose, while the rate of growth on D-XylOSe was found to vary with conditions from a quarter to a tenth of that of Saccharomyces cerevisiae on D-glucose. Although yields can be high with low concentrations of D-xylose, -79% of theoretical on 2% (w/v) , they decrease with higher sugar concentrations. In addition, several by-products can be produced at the expense of ethanol: xylitol, arabinitol and acetic acid (Neirinck et al., 1982).Industrial strains of yeast have been reported to be polyploid or aneuploid (Burrows, 1979), suggesting that an increased number of chromosomes is advantageous. A systematic increase with increasing ploidy in the rate of ethanol formation by S. cerevisiae from D-glucose has been demonstrated (Scheda, 1963). Recently, a genetic system for P. tannophilus was described which permits tetrad analysis and the construction of diploids (James & Zahab, 1982) and polyploids (James & Zahab, 1983). The present paper describes several aspects of ethanol production and by-product formation from several carbon sources in a ploidy series of P. tannophilus. METHODSMedia. The medium was 2 or 4% (w/v) sugar in 0.67% (w/v) yeast nitrogen base (YNB), complete or without amino acids (Difco), as specified. Auxotrophic requirements were added at a level of 40 pg ml-' . Sterilization was by filtration. The temperature for all experiments was 30 "C.Growth rate. Growth was followed by optical density measurements at 600 nm using a Coleman Model 275 spectrophotometer. Fifty millilitres of medium in a 500 ml Erlenmeyer flask with a side arm was inoculated to an OD,,, of 0.04-0.07. The flask was stoppered with a porous foam plastic pl...
~~ ~~Increasing chromosome number of Pachysolen tannophilus above the haploid level increased the yield of ethanol from D-XylOSe. There was a large increment on going from the haplophase to the diplophase, and the highest yields were obtained with either a triploid or a probable tetraploid, depending on the concentration of D-xylose. In addition, the rate of ethanol production from Dxylose and D-glucose increased, with the increment being larger on D-xylose. On D-galactose, the amount of ethanol produced within a given time also increased. The level of a by-product from D-XylOSe, xylitol, decreased on going from a haploid to higher ploidy, but there was no discernible trend for two other by-products, acetic acid and arabinitol. Increasing ploidy increased growth rate on D-galactose, but not appreciably on D-xylose. The altered properties on D-XylOSe of the strains tested were not due to increased levels of either D-xylOSe reductase or xylitol dehydrogenase, nor probably of alcohol dehydrogenase. I N T R O D U C T I O NPachysolen tannophilus is one of the better producers of ethanol from D-XylOSe . It also functions well in some mixtures of pentoses and hexoses (Neirinck et al., 1982). However, several of its properties require improvement before it can be used for industrial purposes. The rate at which alcohol is produced from D-xylose was found to be about a quarter of that from D-glucose, while the rate of growth on D-XylOSe was found to vary with conditions from a quarter to a tenth of that of Saccharomyces cerevisiae on D-glucose. Although yields can be high with low concentrations of D-xylose, -79% of theoretical on 2% (w/v) , they decrease with higher sugar concentrations. In addition, several by-products can be produced at the expense of ethanol: xylitol, arabinitol and acetic acid (Neirinck et al., 1982).Industrial strains of yeast have been reported to be polyploid or aneuploid (Burrows, 1979), suggesting that an increased number of chromosomes is advantageous. A systematic increase with increasing ploidy in the rate of ethanol formation by S. cerevisiae from D-glucose has been demonstrated (Scheda, 1963). Recently, a genetic system for P. tannophilus was described which permits tetrad analysis and the construction of diploids (James & Zahab, 1982) and polyploids (James & Zahab, 1983). The present paper describes several aspects of ethanol production and by-product formation from several carbon sources in a ploidy series of P. tannophilus. METHODSMedia. The medium was 2 or 4% (w/v) sugar in 0.67% (w/v) yeast nitrogen base (YNB), complete or without amino acids (Difco), as specified. Auxotrophic requirements were added at a level of 40 pg ml-' . Sterilization was by filtration. The temperature for all experiments was 30 "C.Growth rate. Growth was followed by optical density measurements at 600 nm using a Coleman Model 275 spectrophotometer. Fifty millilitres of medium in a 500 ml Erlenmeyer flask with a side arm was inoculated to an OD,,, of 0.04-0.07. The flask was stoppered with a porous foam plastic pl...
~ ~ ~~A hexokinase (ATP : D-hexose 6-phosphotransferase; EC 2.7.1.. 1) associated with catabolite repression was isolated and purified from the yeast Pachysolen tannophilus. The enzyme phosphorylated D-fructose at a rate 1.5 times greater than that for D-glUCOSe. The K , values for D-glucose and D-fructose were 0.36 and 2.28 mM, respectively. Neither xylose reductase nor xylitol dehydrogenase were subject to catabolite repression in mutants defective in this enzyme.Two of the enzymes associated with xylose catabolism, aldose (xylose) reductase and xylitol dehydrogenase, have been purified from Pachysolen tannophilus and examined (Ditzelmuller et al., 1984a(Ditzelmuller et al., , b, 1985 Verduyn et al., 1985;Bolen et al., 1986;Morimoto et al., 1986 Morimoto et al., , 1987. However, little is known about other enzymes in this yeast. We have recently demonstrated the existence of three hexose-phosphorylating enzymes in P . tannophilus; hexokinases A and B and a glucokinase specific for D-glucose (Wedlock et al., 1989; accompanying paper). In this paper, we report the isolation and partial purification of a hexokinase (ATP : D-hexose 6-phosphotransferase; EC 2.7.1 , I), designated A, from a mutant which is defective in the glucokinase. The abcPg1ce of the glucokinase, which co-elutes with hexokinase A in the wild-type strain of P . tannophilus, aids the isolation of the hexokinase without contamination by the glucokinase. The hexokinase PI1 enzyme of Saccharomyces cerevisiae has been implicated in catabolite repression (Kopetzki & Entian, 1985;Hong & Botstein, 1986) as has a hexokinase isolated from Schwanniomyces occidentalis (McCann et al., 1987). Xylose utilization by P. tannophilus is subject to hexose-sugar catabolite repression (Slininger et al., 1987; Bicho et al., 1988). The effect of the P . tannophilus hexokinase A on the activities of xylose reductase and xylitol dehydrogenase and on D-xylose utilization is described here. METHODSYeast strains and growth conditions. The Pachysolen tannophilus strains used in enzyme analyses and sugar utilization experiments were P444-3 the wild-type strain, P5 10-5A defective in a glucokinase enzyme (glul), P509-3C defective in hexokinase A (hxk2) and P509-1B defective in both hexokinase A and the glucokinase (hxk2 glul). Strain P510-5A was also used in the purification of hexokinase A.Cells were grown in 1 litre flasks containing 250 ml of YEP-glucose [ 1 %, w/v, yeast extract (Difco), 2%, w/v, peptone (Difco) and 2%, w/v, ~-glucose]. The culture was incubated for 42 h at 30 "C and 180 r.p.m. on a gyratory shaker. Cells grown for 48 h in YEP-glucose were used as the inoculum. For the measurement of xylose reductase and xylitol dehydrogenase activities, cells were grown for 16 h with either Dglucose, Dxylose, or D-glucose and Dxylose as carbon source.Growth and sugar utilization were followed in YNB [0.67% yeast nitrogen base (Difco), 2%, w/v, sugar] liquid media. A 48 h YNB-xylose culture (100 ml) was washed with deionized water and resuspended in water to an optical...
Mutants of the yeast Pachysolen tannophilus, exhibiting decreased ability to utilize D-glucose as the sole carbon source, were obtained by selecting for resistance to 2-deoxyglucose. Enzyme studies confirmed that these strains are defective in the hexose-phosphorylating enzymes and are unable to phosphorylate D-glucose to D-glucose 6-phosphate. The results confirmed the presence of two hexokinases, A and B, with ratios of D-glUCOSe to D-fructose phosphorylation activity of 1 *3/ 1.0 and 3.0/ 1.0, respectively, and a D-glucose-specific glucokinase. The behaviour of a hexose-negative strain, able to ferment D-XylOSe in the presence of D-glucose, is described.
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