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

The induction of xylose reductase and xylitol dehydrogenase activities on mixed sugars was investigated in the yeasts Pachysolen tannophilus and Pichia stipitis. Enzyme activities induced on d-xylose served as the controls. In both yeasts, d-glucose, d-mannose, and 2-deoxyglucose inhibited enzyme induction by d-xylose to various degrees. Cellobiose, l-arabinose, and d-galactose were not inhibitory. In liquid batch culture, P. tannophilus utilized d-glucose and d-mannose rapidly and preferentially over d-xylose, while d-galactose consumption was poor and lagged behind that of the pentose sugar. In P. stipitis, all three hexoses were used preferentially over d-xylose. The results showed that the repressibility of xylose reductase and xylitol dehydrogenase may limit the potential of yeast fermentation of pentose sugars in hydrolysates of lignocellulosic substrates.     

Physiological Properties of a Mutant of Pachysolen tannophilus Deficient in NADPH-Dependent d-Xylose Reductase

A d-xylose reductase mutant of Pachysolen tannophilus was isolated on the basis of its poor growth on d-xylose but normal growth on xylitol and d-glucose. Fractionation of cell extracts indicated that the mutant was deficient in d-xylose reductase activity that used NADPH exclusively as a cofactor, but not in activity that used both NADH and NADPH. Mutant cultures grown on d-xylose as the sole carbon source exhibited some properties that would be desired in improved strains. Growth rate, growth yield, and d-xylose consumption rate of the mutant were less sensitive than those of the wild type to changes in aeration rate. d-Xylose was utilized more efficiently in that less of a by-product, xylitol, was produced. In addition, under low aeration conditions, more ethanol was produced. A disadvantage was a relatively slow rate of d-xylose utilization.     

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

Induced mutants, selected for their defective growth on d-xylose while retaining the ability to grow normally on d-glucose, were studied in Pachysolen tannophilus, a yeast capable of converting d-xylose to ethanol. Fourteen of the mutations were found to occur at nine distinct loci, and data indicated that many more loci remain to be detected. Most of the mutations were pleiotropic in character, and the expression of some of them was much affected by nutritional conditions and by genetic background. Mutations at several loci resulted in poor growth on at least one compound that was either an intermediate of the tricarboxylic acid cycle, succinate or alpha-ketoglutarate, or on compounds metabolizable via this cycle, ethanol or glycerol. An initial biochemical characterization of the mutants was undertaken. Analysis for xylose reductase, xylitol dehydrogenase, and xylulose kinase activity showed that one or more of these activities was affected in 12 of 13 mutants. However, drastic reduction in activity of a single enzyme was confined to that of xylitol dehydrogenase by mutations at three different loci and to that of d-xylose reductase by mutation at another locus. Growth of these latter four mutants was normal on all carbon sources tested that were not five-carbon sugars.     

Concurrent Production and Consumption of Ethanol by Cultures of Pachysolen tannophilus Growing on d-Xylose

Growing cultures of Pachysolen tannophilus concurrently consumed and produced ethanol in the presence of substantial concentrations of d-xylose. Ethanol was also assimilated in the presence of other sugars, the amount depending on the sugar. Less ethanol assimilation occurred with d-glucose than with d-xylose. The rate of ethanol consumption decreased as the concentration of glucose was increased, but some consumption still occurred when 2% glucose was present. The rate increased with the amount of oxygen available to the culture when d-xylose or ethanol was the carbon source. In most instances, estimates of consumption were based on the extent of incorporation of C from [1-C]ethanol into trichloroacetic acid-insoluble material. The results are pertinent to the use of P. tannophilus for the production of ethanol from d-xylose.     

Ethanol production from d-galactose and glycerol by Pachysolen tannophilus

Pachysolen tannophilus has recently been shown to be able to convert d-xylose, a pentose, to ethanol. Previously, d-xylose had been considered to be nonfermentable by yeasts. The present study shows that the organism can be used to obtain ethanol from other carbohydrates previously considered as nonfermentable, either by P. tannophilus in particular, d-galactose, or by yeasts in general, glycerol. Such identification for d-galactose allows P. tannophilus to be considered for fermentation of four of the five major plant monosaccharides: d-glucose, d-mannose, d-galactose and d-xylose. The ability to ferment glycerol is of potential use, in part, for the conversion of glycerol derived from algae into ethanol.     

Production of Ethanol from d-Xylose by Using d-Xylose Isomerase and Yeasts

d-Xylulose, an intermediate of d-xylose catabolism, was observed to be fermentable to ethanol and carbon dioxide in a yield of greater than 80% by yeasts (including industrial bakers' yeast) under fermentative conditions. This conversion appears to be carried out by many yeasts known for d-glucose fermentation. In some yeasts, xylitol, in addition to ethanol, was produced from d-xylulose. Fermenting yeasts are also able to produce ethanol from d-xylose when d-xylose isomerizing enzyme is present. The results indicate that ethanol could be produced from d-xylose in a yield of greater than 80% by a two-step process. First, d-xylose is converted to d-xylulose by xylose isomerase. d-Xylulose is then fermented to ethanol by yeasts.     

Isolation of a nucleotide activated disaccharide pentapeptide precursor from Methanobacterium thermoautotrophicum

The yeasts Pachysolen tannophilus and Pichia stipitis differed in their ability to utilize D-xylose in the presence of D-fructose. When P. tannophilus was grown aerobically in fructose-xylose mixture, the ketohexose was utilized preferentially over the pentose. However, in P. stipitis cultures, the converse was observed. The effect was associated with the ability of D-fructose to repress the induction of xylose reductase and xylitol dehydrogenase activities in P. tannophilus but not in P. stipitis. Both yeasts grew on D-fructose and fermented it to ethanol when it was supplied as the sole carbon source. The results suggest that there may exist some fundamental difference in the regulation of D-fructose metabolism between P. tannophilus and P. stipitis.     

Oxygen requirements for d-xylose fermentation to ethanol and polyols by Pachysolen tannophilus

The oxygen requirements for ethanol production from d-xylose (10 or 20 g l^?1) by Pachysolen tannophilus have been determined by controlling the availability of oxygen to shake flasks. Under anaerobic conditions no ethanol was produced whereas under aerobic conditions mainly biomass was formed. Semi-anaerobic conditions resulted in maximum ethanol production. By varying the stirring speed of a fermenter and supplying air to the liquid surface at various rates, the oxygen transfer rate (OTR) was controlled under semi-anaerobic conditions. By increasing the OTR from 0.05 to 16.04 mmol l^?1 h^?1, the ethanol yield coefficient decreased from 0.28 to 0.18 while the cell yield coefficient increased from 0.14 to 0.22. The accumulation of polyols decreased from 0.88 to 0.56 g l^?1 with increasing OTR. At OTRs between 0.09 and 1.18 mmol l^?1 h^?1, specific ethanol productivity attained a maximum value of 0.07 h^?1 and decreased with either increasing or decreasing OTR. The results indicate that the OTR must be carefully controlled for efficient ethanol production from d-xylose by P. tannophilus.     

Ethanol production from pentoses and sugar-cane bagasse hemicellulose hydrolysate by Mucor and Fusarium species

The fermentation of carbohydrates and hemicellulose hydrolysate by Mucor and Fusarium species has been investigated, with the following results. Both Mucor and Fusarium species are able to ferment various sugars and alditols, including d-glucose, pentoses and xylitol, to ethanol. Mucor is able to ferment sugar-cane bagasse hemicellulose hydrolysate to ethanol. Fusarium F5 is not able to ferment sugar-cane bagasse hemicellulose hydrolysate to ethanol. During fermentation of hemicellulose hydrolysates, d-glucose was utilized first, followed by d-xylose and l-arabinose. Small amounts of xylitol were produced by Mucor from d-xylose through oxidoreduction reactions, presumably mediated by the enzyme aldose reductase¹ (alditol: NADP⁺ 1-oxidoreductase, EC 1.1.1.21). For pentose fermentation, d-xylose was the preferred substrate. Only small amounts of ethanol were produced from l-arabinose and d-arabitol. No ethanol was produced from l-xylose, d-arabinose or l-arabitol.     

Decreased xylitol formation during xylose fermentation in Saccharomyces cerevisiae due to overexpression of water-forming NADH oxidase

The recombinant xylose-fermenting Saccharomyces cerevisiae strain harboring xylose reductase (XR) and xylitol dehydrogenase (XDH) from Scheffersomyces stipitis requires NADPH and NAD(+), creates cofactor imbalance, and causes xylitol accumulation during growth on d-xylose. To solve this problem, noxE, encoding a water-forming NADH oxidase from Lactococcus lactis driven by the PGK1 promoter, was introduced into the xylose-utilizing yeast strain KAM-3X. A cofactor microcycle was set up between the utilization of NAD(+) by XDH and the formation of NAD(+) by water-forming NADH oxidase. Overexpression of noxE significantly decreased xylitol formation and increased final ethanol production during xylose fermentation. Under xylose fermentation conditions with an initial d-xylose concentration of 50 g/liter, the xylitol yields for of KAM-3X(pPGK1-noxE) and control strain KAM-3X were 0.058 g/g xylose and 0.191 g/g, respectively, which showed a 69.63% decrease owing to noxE overexpression; the ethanol yields were 0.294 g/g for KAM-3X(pPGK1-noxE) and 0.211 g/g for the control strain KAM-3X, which indicated a 39.33% increase due to noxE overexpression. At the same time, the glycerol yield also was reduced by 53.85% on account of the decrease in the NADH pool caused by overexpression of noxE.     

d-Xylulose Fermentation to Ethanol by Saccharomyces cerevisiae

We used commercial bakers' yeast (Saccharomyces cerevisiae) to study the conversion of d-xylulose to ethanol in the presence of d-xylose. The rate of ethanol production increased with an increase in yeast cell density. The optimal temperature for d-xylulose fermentation was 35 degrees C, and the optimal pH range was 4 to 6. The fermentation of d-xylulose by yeast resulted in the production of ethanol as the major product; small amounts of xylitol and glycerol were also produced. The production of xylitol was influenced by pH as well as temperature. High pH values and low temperatures enhanced xylitol production. The rate of d-xylulose fermentation decreased when the production of ethanol yielded concentrations of 4% or more. The slow conversion rate of d-xylulose to ethanol was increased by increasing the yeast cell density. The overall production of ethanol from d-xylulose by yeast cells under optimal conditions was 90% of the theoretical yield.     

Influence of oxygen on ethanol and xylitol production by xylose fermenting yeasts

The behaviour of Pichia stipitis, Pachysolen tannophilus, Candida shehatae and Candida parapsilosis was investigated to select the most suitable yeast to convert xylose either to ethanol or to xylitol, with little or no formation of by-products. The aeration rate was used as a variable parameter. P. stipitis and C. parapsilosis were the most effective producers or ethanol and xylitol, respectively, both reaching productivities at very low levels of oxygenation. With P. stipitis, better ethanol productivity was attained under microaerobic conditions (K_La = 4·8 h⁻¹) while with C. parapsilosis high yields and rates of xylitol production were detected at K_La values of about 16·3 h⁻¹. P. tannophilus and C. shehatae showed lower performances under all conditions used while changes in oxygenation modified the ratio of ethanol to xylitol produced by these yeasts, suggesting that they are more dependent on the oxygen power input than P. stipitis and C. parapsilosis. The influence of oxygen transfer rates on ethanol and xylitol formation with the best producers is discussed.     

Microaerophilic metabolism of Pachysolen tannophilus at different pH values

Microaerophilic production of xylitol by Pachysolen tannophilus from detoxified hemicellulose hydrolyzate was optimal between pH values 6.0 to 7.5 when about 90% of xylose was utilized for xylitol production, the rest being fermented to ethanol. At pH values of 3.0 and 12.0, respiration became important, consuming up to 30% of available xylose. A graphic procedure suggests that histamine and cysteine are at the active site of xylose reductase in this yeast.     

Alcoholic Fermentation of d-Xylose by Yeasts

Type strains of 200 species of yeasts able to ferment glucose and grow on xylose were screened for fermentation of d-xylose. In most of the strains tested, ethanol production was negligible. Nineteen were found to produce between 0.1 and 1.0 g of ethanol per liter. Strains of the following species produce more than 1 g of ethanol per liter in the fermentation test with 2% xylose: Brettanomyces naardenensis, Candida shehatae, Candida tenuis, Pachysolen tannophilus, Pichia segobiensis, and Pichia stipitis. Subsequent screening of these yeasts for their capacity to ferment d-cellobiose revealed that only Candida tenuis CBS 4435 was a good fermenter of both xylose and cellobiose under the test conditions used.     

Induction of NADPH-linked D-xylose reductase and NAD-linked xylitol dehydrogenase activities in Pachysolen tannophilus by D-xylose, L-arabinose, or D-galactose

Considerable interest in the D-xylose catabolic pathway of Pachysolen tannophilus has arisen from the discovery that this yeast is capable of fermenting D-xylose to ethanol. In this organism D-xylose appears to be catabolized through xylitol to D-xylulose. NADPH-linked D-xylose reductase is primarily responsible for the conversion of D-xylose to xylitol, while NAD-linked xylitol dehydrogenase is primarily responsible for the subsequent conversion of xylitol to D-xylulose. Both enzyme activities are readily detectable in cell-free extracts of P. tannophilus grown in medium containing D-xylose, L-arabinose, or D-galactose and appear to be inducible since extracts prepared from cells growth in media containing other carbon sources have only negligible activities, if any. Like D-xylose, L-arabinose and D-galactose were found to serve as substrates for NADPH-linked reactions in extracts of cells grown in medium containing D-xylose, L-arabinose, or D-galactose. These L-arabinose and D-galactose NADPH-linked activities also appear to be inducible, since only minor activity with L-arabinose and no activity with D-galactose is detected in extracts of cells grown in D-glucose medium. The NADPH-linked activities obtained with these three sugars may result from the actions of distinctly different enzymes or from a single aldose reductase acting on different substrates. High-performance liquid chromatography and gas-liquid chromatography of in vitro D-xylose, L-arabinose, and D-galactose NADPH-linked reactions confirmed xylitol, L-arabitol, and galactitol as the respective conversion products of these sugars. Unlike xylitol, however, neither L-arabitol nor galactitol would support comparable NAD-linked reaction(s) in cellfree extracts of induced P. tannophilus. Thus, the metabolic pathway of D-xylose diverges from those of L-arabinose or D-galactose following formation of the pentitol.     

Xylitol production by a methanol yeast,Candida boidinii (Kloeckera sp.) No. 2201

A methanol yeast, Candida boidinii no. 2201, could produce xylitol and also d-xylulose during cultivation on d-xylose medium. These fermentative products were identified by high performance liquid chromatography. A large amount of xylitol was obtained from a d-xylose medium containing ammonium acetate and yeast extract at the initial pH of 7.0. Maximum productivities of xylitol and enzymes concerned with the production were observed after 4–5 d of cultivation. A d-xylose (100 g/l) medium supplemented with 2% (v/v) methanol gave higher amounts of xylitol (48.5 g/l) and d-xylulose (3.3 g/l). Enzyme activities for d-xylose reduction, d-xylulose reduction, d-xylose isomerization, and d-xylulose phosphorylation, which could be involved in the xylitol production, were measured in cell-free extracts during cultivation and a possible pathway of xylitol production was discussed.     

Effect of hydrogen acceptors on D-xylose fermentation by anaerobic culture of immobilized Pachysolen tannophilus cells

The effect of hydrogen acceptors on the kinetic parameters of D-xylose fermentation under anaerobic conditions was studied in a transient culture of immobilized Pachysolen tannophilus cells. Addition of oxygen to a steady-state culture resulted in a rapid increase (up to fivefold) in the rates of ethanol production and D-xylose uptake, but the rate of xylitol production was unaffected. Furthermore, the molar ethanol yield increased from 0.97 to 1.43 in the presence of oxygen. The moles of ethanol produced per moles of oxygen utilized were considerably greater than would be predicted from the stoichiometry of D-xylose fermentation, which suggests that the organism required oxygen for other functions in addition to its role as a hydrogen acceptor in D-xylose metabolism. When the artificial hydrogen acceptors acetone, acetaldehyde, and acetoin were added to the culture, the rate of ethanol production increased while the xylitol production rate decreased but the rate of xylose uptake was unaffected. The molar ethanol yields increased from 1.03 to 1.63, 1.43, and 1.24 upon addition of acetaldehyde, acetone, and acetoin, respectively, at the expense of the molar xylitol yields. The hydrogen acceptors sodium acetate, methylene blue, benzyl viologen, phenazine methosulfate, indigo carmine, and tetrazolium chloride had no effect on ethanol production.     

Affinity Purifications of Aldose Reductase and Xylitol Dehydrogenase from the Xylose-Fermenting Yeast Pachysolen tannophilus

Although xylose is a major product of hydrolysis of lignocellulosic materials, few yeasts are able to convert it to ethanol. In Pachysolen tannophilus, one of the few xylose-fermenting yeasts found, aldose reductase and xylitol dehydrogenase were found to be key enzymes in the metabolic pathway for xylose fermentation. This paper presents a method for the rapid and simultaneous purification of both aldose reductase and xylitol dehydrogenase from P. tannophilus. Preliminary studies indicate that this method may be easily adapted to purify similar enzymes from other xylose-fermenting yeasts.     

Mutants of Pachysolen tannophilus showing enhanced rates of growth and ethanol formation from d-xylose

Mutants of Pachysolen tannophilus NRRL Y-2460 have been sought that show enhanced rates of d-xylose fermentation. Mutagenesis followed by enrichment in urea-xylitol broth generally resulted in a lower frequency of good ethanol producers than enrichment in nitrate-xylitol broth. Under aerobic conditions, the best xylose-fermenting strains (which were obtained from nitrate-xylitol broth) produced ethanol from xylose twice as fast and in 32% better yield than the parent strain. Under anaerobic conditions, these strains produced ethanol from xylose 50% faster than (but in the same yield as) the parent strain. These findings show that enrichment in nitrate-xylitol broth is a promising method for obtaining mutants of Pachysolen having enhanced fermentation rates.     

Xylitol dehydrogenase from Pachysolen tannophilus

Abstract NAD-xylitol-dehydrogenase (EC 1.1.1.9) from Pachysolen tannophilus was investigated in relation to xylitol byproduction during xylose fermentation by this yeast. For this purpose the enzyme was partially purified by a combination of affinity chromatography and fast liquid protein chromatography. The enzyme catalyzes an equilibrium reaction which at physiological pH values favours the accumulation of xylitol. The kinetics of the enzyme were shown to be Michaelis-Menten type with respect to both reaction directions. The activity of the enzyme was shown to be under the influence of the 'catabolic reduction charge' (NADH/NAD + NADH) and ATP. The apparent equilibrium constant of the enzyme may explain the considerable byproduction of xylitol during xylose fermentation by P. tannophilus .