Mutants of Pachysolen tannophilus with Improved Production of Ethanol from d -Xylose

Abstract

The conversion of D-xylose to ethanol by the yeast Pachysolen tannophilus is relatively inefficient in batch culture. The inefficiency has been attributed in part to concurrent utilization of ethanol in the presence of appreciable concentrations of D-xylose and to the formation of xylitol and other by-products. To increase the concentration of ethanol accumulated in batch cultures, UV-induced mutants of P. tannophilus were selected on the basis of diminished growth on ethanol. Eleven independent mutant loci that conferred the ethanoldefective phenotype were identified. Three led to a greater yield and volumetric rate of production of ethanol than the wild type. (ne also produced less xylitol and was characterized by a deficiency in activity for malate dehydrogenase.

Genetic and Biochemical Characterization of Mutations Affecting the Ability of the Yeast Pachysolen tannophilus To Metabolize d -Xylose

“…Portions of 1 ml were transferred into 100 ml of a medium containing 20 g of glycerol (locus 5 strain in 20 g of a-ketoglutarate per liter) and 10 g of Difco yeast extract per liter and incubated for 16 h. D-Xylose was then added to a final concentration of 100 mM, and the cells were incubated at 30°C for an additional period of 5 h. In instances specified, induction was also carried out for 20 h. Cells were harvested (optical density, 0.6 to 0.8; Coleman model 35 spectrophotometer; wavelength, 600 nm; 14-mm-inside-diameter cuvette) and washed with 0.5 M phosphate buffer (pH 7.5). Cell extracts were obtained by sonication (8). The final protein concentration was 4 to 6 mg/ml.…”

“…It is noteworthy that the present suggestion that metabolism in P. tannophilus leading to growth on D-xylose, or its catabolities xylitol and D-xylulose, is linked to oxidative metabolism is in accord with previous observations. A TCA cycle mutant metabolizes D-xylose differently than does the wild type in that the mutant produces more ethanol (8). Also, sustained growth on D-xylose requires oxygen (5,10,11,14,16,18,20).…”

Induction of Xylose Reductase and Xylitol Dehydrogenase Activities in Pachysolen tannophilus and Pichia stipitis on Mixed Sugars

“…A loopful of cells from a potato glucose agar slant was transferred to 20 ml of medium containing 0.67% (wt/vol) yeast nitrogen base (YNB; Difco) without amino acids and 2% (wt/vol) glycerol. The culture was kept at 30°C in a loosely capped 125-ml Erlenmeyer flask which was agitated at 200 rpm in a New Brunswick model G24 bench-top Gyrotory shaker for 48 h (14).…”

“…Both methods provided adequate cell breakage, as ascertained by microscopic examination. Cell extracts were then obtained by centrifugation (14).…”

Additive Effects of Alcohols, Their Acidic By-Products, and Temperature on the Yeast Pachysolen tannophilus

“…The measurements used 200 mM Tris buffer (pH 9.0)-30 mM semicarbazide hydrochloride-1 mM NAD-20 to 40 ,ug of protein ml-'. The reaction was initiated by adding alcohol to a final concentration of 25 mM (14). Blanks for both enzyme and substrates (ethanol, 1-propanol, 1-butanol, and benzyl alcohol) were performed, and the endogenous activity was subtracted.…”