Fermentation of D-xylose, xylitol, and D-xylulose by yeasts

Fifteen yeasts which can assimilate D-xylose were examined for the ability to convert this pentose to ethanol. In six of the seven genera investigated the conversion was enhanced when air had access to the medium. Therefore, the ability to convert D-xylose to ethanol under these conditions is probably common among yeasts. Growth under the same conditions on xylitol, a putative catabolite of D-xylose, led to only traces of ethanol. The effects of growth on another putative catabolite, D-xylose, were complex, but some of the strains which were among the better producers of ethanol from D-xylose produced less from D-xylulose.     

Complementation of the inability of Lactobacillus strains to utilize D-xylose with D-xylose catabolism-encoding genes of Lactobacillus pentosus.

The inability of two Lactobacillus strains to ferment D-xylose was complemented by the introduction of Lactobacillus pentosus genes encoding D-xylose isomerase, D-xylulose kinase, and a D-xylose catabolism regulatory protein. This result opens the possibility of using D-xylose fermentation as a food-grade selection marker for Lactobacillus spp.     

Complementation of the inability of Lactobacillus strains to utilize D-xylose with D-xylose catabolism-encoding genes of Lactobacillus pentosus.

The inability of two Lactobacillus strains to ferment D-xylose was complemented by the introduction of Lactobacillus pentosus genes encoding D-xylose isomerase, D-xylulose kinase, and a D-xylose catabolism regulatory protein. This result opens the possibility of using D-xylose fermentation as a food-grade selection marker for Lactobacillus spp.     

Sequential utilization of mixed monosaccharides by yeasts

Four yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilus, and Rhodotorula toruloides) were tested for their ability to grow and consume D-glucose, D-xylose, D-xylulose, and D-xylitol. Sequential utilization of substrates was observed when D-glucose as mixed with D-xylulose as the carbon source. Catabolite inhibition was tentatively concluded to be responsible for this regulatory mechanism. D-Glucose was also found to inhibit the utilization of D-xylose and D-xylitol in C. utilus and R. toruloides. D-Xylose, D-xylitol, and D-xylulose were consumed simultaneously by R. toruloides and C. utilus.     

Sequential utilization of mixed monosaccharides by yeasts.

Four yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilus, and Rhodotorula toruloides) were tested for their ability to grow and consume D-glucose, D-xylose, D-xylulose, and D-xylitol. Sequential utilization of substrates was observed when D-glucose as mixed with D-xylulose as the carbon source. Catabolite inhibition was tentatively concluded to be responsible for this regulatory mechanism. D-Glucose was also found to inhibit the utilization of D-xylose and D-xylitol in C. utilus and R. toruloides. D-Xylose, D-xylitol, and D-xylulose were consumed simultaneously by R. toruloides and C. utilus.     

Conversion of pentoses by yeasts

The utilization and conversion of D-xylose, D-xylulose, L-arabinose, and xylitol by yeast strains have been investigated with the following results: (1) The majority of yeasts tested utilize D-xylose and produce polyols, ethanol, and organic acids. The type and amount of products formed varies with the yeast strains used. The most commonly detected product is xylitol. (2)The majority of yeasts tested utilize D-xylulose aerobically and fermentatively to produce ethanol, xylitol, D-arabitol, and organic acids. The type and amount of products varies depending upon the yeast strains used. (3) Xylitol is a poor carbon and energy source for most yeasts tested. Some yeast strains produce small amounts of ethanol from xylitol. (4) Most yeast strains utilize L-arabinose, and L-arabitol is the common product. Small amounts of ethanol are also produced by some yeast strains. (5) Of the four substrates examined, D-xylulose was the perferred substrate, followed by D-xylose, L-arabinose, and xylitol. (6) Mutant yeast strains that exhibit different metabolic product patterns can be induced and isolated from Candida sp. Saccharomyces cerevisiae, and other yeasts. These mutant strains can be used for ethanol production from D-xylose as well as for the study of metabolic regulation of pentose utilization in yeasts.     

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.     

Growth of yeasts on D-xylulose 1

Nine of eleven yeasts of different species or genera grew in the presence of air on the intermediate of D-xylose catabolism, D-xylulose (D-threo-pentulose). Growth on this substrate was efficient as judged by the optical density in stationary phase being generally similar to that after growth on glucose. Yeasts which grew on D-xylose also did so on D-xylulose, but among those which grew are included several which utilise neither D-xylose nor xylitol: Saccharomyces cerevisiae, Saccharomyces carlsbergensis, and Schizosaccharomyces pombe. Since catabolism of a sugar generally requires an initial phosphorylation step, growth of these strains suggests that they contain an enzyme which can function as a D-xylulose kinase. The D-xylulose-5-phosphate formed thereby is considered to enter the pentose-phosphate pathway. Glucose-grown inocula of S. carlsbergensis and Schizosaccharomyces pombe, and of several other yeasts, began to grow logarithmically when placed on D-xylulose with no apparent delay, or one which was minimal, suggesting that the D-xylulose kinase was already present in such cells, or was rapidly induced. Petites of S. cerevisiae did not grow on D-xylulose indicating that, in this species, mitochondria are involved in its utilisation.     

Demonstration of D-xylose reductase and D-xylitol dehydrogenase in Pachysolen tannophilus

Summary The yeast, Pachysolen tannophilus, can utilize the pentose D-xylose with accumulation of significant quantities of ethanol. Cell extracts of the organism contain NADPH-linked D-xylose reductase (aldose reductase EC 1.1.1.21) and NAD-dependent D-xylitol dehydrogenase (D-xylulose reductase EC 1.1.1.9). D-Xylose was required for induction of both the D-xylitol dehydrogenase and the D-xylose reductase. Neither enzyme was found in glucose grown cell-free extracts.     

Role of invariant water molecules and water-mediated ionic interactions in D-xylose isomerase from Streptomyces rubiginosus

The enzyme, D-xylose isomerase (D-xylose keto-isomerase; EC 5.3.1.5) is a soluble enzyme that catalyzes the conversion of the aldo-sugar D-xylose to the keto-sugar D-xylulose. A total of 27 subunits of D-xylose isomerase from Streptomyces rubiginosus were analyzed in order to identify the invariant water molecules and their water-mediated ionic interactions. A total of 70 water molecules were found to be invariant. The structural and/or functional roles of these water molecules have been discussed. These invariant water molecules and their ionic interactions may be involved in maintaining the structural stability of the enzyme D-xylose isomerase. Fifty-eight of the 70 invariant water molecules (83%) have at least one interaction with the main chain polar atom.     

Uptake and catabolism of D-xylose in Salmonella typhimurium LT2.

Salmonella typhimurium LT2 grows on D-xylose as sole carbon source with a generation time of 105 to 110 min. The following activities are induced at the indicated time after the addition of the inducer, D-xylose: D-xylulokinase (5 min), D-xylose isomerase (7 to 8 min), and D-xylose transport (10 min). All other pentoses and pentitols tested failed to induce isomerase or kinase. Synthesis of D-xylose isomerase was subject to catabolite repression, which was reversed by the addition of cyclic adenosine monophosphate. Most of the radioactive counts from D-[14C]xylose were initially accumulated in the cell in the form of D-xylose or D-xylulose. D-Xylose uptake in a mutant which was deficient in D-xylose isomerase was equal to that of the wild type. The apparent Km for D-xylose uptake was 0.41 mM. Some L-arabinose was accumulated in D-xylose-induced cells, and some D-xylose was accumulated in L-arabinose-induced cells. D-Xylitol and L-arabinose competed against C-xylose uptake, but D-arabinose, D-lyxose, and L-lyxose did not. Osmotic shock reduced the uptake of D-xylose by about 50%; by equilibrium dialysis, a D-xylose-binding protein was detected in the supernatant fluid after spheroplasts were formed from D-xylose-induced cells.     

Regulation of D-xylose utilization by hexoses in pentose-fermenting yeasts

The aldopentose D-xylose is one of the most abundant sugars in plant biomass and its efficient microbial utilization is of fundamental importance in the overall bioconversion of lignocellulosic materials into liquid fuels and chemicals. The discovery of pentose-fermenting yeasts in the early 1980's led to world wide interest because of the perceived potential for improved D-xylose fermentation to enhance the prospect of biomass conversions. However, the utilization of D-xylose by pentose-fermenting yeasts can be adversely affected by the hexoses, mainly D-glucose and D-mannose, which are usually present in high amounts in lignocellulosic hydrolysates. Research in the past several years has uncovered some of the regulatory effects of D-glucose on D-xylose utilization. However, much remains unknown about the mechanisms responsible for these effects. This review summarizes the current state of knowledge on the induction, repression and inactivation of D-xylose utilization in pentose-fermenting yeasts.     

Amplification of D-xylose and D-glucose isomerase activities in Escherichia coli by gene cloning

A recombinant plasmid, designated pUC1002, was constructed by ligation of a HindIII restriction endonuclease fragment of Escherichia coli chromosomal DNA to vector plasmid pMB9. Strains carrying this plasmid were selected by transformation of an E. coli strain bearing the xyl-7 mutation to a xylose-positive (Xyl+) phenotype. Strains containing pUC1002 produced coordinately elevated levels of D-xylose isomerase and D-xylulose kinase. Under appropriate conditions, the isomerase also efficiently catalyzed the conversion of D-glucose to D-fructose.     

A metal-mediated hydride shift mechanism for xylose isomerase based on the 1.6 A Streptomyces rubiginosus structures with xylitol and D-xylose

The crystal structure of recombinant Streptomyces rubiginosus D-xylose isomerase (D-xylose keto-isomerase, EC 5.3.1.5) solved by the multiple isomorphous replacement technique has been refined to R = 0.16 at 1.64 A resolution. As observed in an earlier study at 4.0 A (Carrell et al., J. Biol. Chem. 259: 3230-3236, 1984), xylose isomerase is a tetramer composed of four identical subunits. The monomer consists of an eight-stranded parallel beta-barrel surrounded by eight helices with an extended C-terminal tail that provides extensive contacts with a neighboring monomer. The active site pocket is defined by an opening in the barrel whose entrance is lined with hydrophobic residues while the bottom of the pocket consists mainly of glutamate, aspartate, and histidine residues coordinated to two manganese ions. The structures of the enzyme in the presence of MnCl2, the inhibitor xylitol, and the substrate D-xylose in the presence and absence of MnCl2 have also been refined to R = 0.14 at 1.60 A, R = 0.15 at 1.71 A, R = 0.15 at 1.60 A, and R = 0.14 at 1.60 A, respectively. Both the ring oxygen of the cyclic alpha-D-xylose and its C1 hydroxyl are within hydrogen bonding distance of NE2 of His-54 in the structure crystallized in the presence of D-xylose. Both the inhibitor, xylitol, and the extended form of the substrate, D-xylose, bind such that the C2 and C4 OH groups interact with one of the two divalent cations found in the active site and the C1 OH with the other cation. The remainder of the OH groups hydrogen bond with neighboring amino acid side chains. A detailed mechanism for D-xylose isomerase is proposed. Upon binding of cyclic alpha-D-xylose to xylose isomerase, His-54 acts as the catalytic base in a ring opening reaction. The ring opening step is followed by binding of D-xylose, involving two divalent cations, in an extended conformation. The isomerization of D-xylose to D-xylulose involves a metal-mediated 1,2-hydride shift. The final step in the mechanism is a ring closure to produce alpha-D-xylulose. The ring closing is the reverse of the ring opening step. This mechanism accounts for the majority of xylose isomerase's biochemical properties, including (1) the lack of solvent exchange between the 2-position of D-xylose and the 1-pro-R position of D-xylulose, (2) the chemical modification of histidine and lysine, (3) the pH vs. activity profile, and (4) the requirement for two divalent cations in the mechanism.     

Amplification of D-xylose and D-glucose isomerase activities in Escherichia coli by gene cloning.

A recombinant plasmid, designated pUC1002, was constructed by ligation of a HindIII restriction endonuclease fragment of Escherichia coli chromosomal DNA to vector plasmid pMB9. Strains carrying this plasmid were selected by transformation of an E. coli strain bearing the xyl-7 mutation to a xylose-positive (Xyl+) phenotype. Strains containing pUC1002 produced coordinately elevated levels of D-xylose isomerase and D-xylulose kinase. Under appropriate conditions, the isomerase also efficiently catalyzed the conversion of D-glucose to D-fructose.     

Specific features of fermentation of D-xylose and D-glucose by xylose-assimilating yeasts

The ability to assimilate D-glucose and D-xylose was studied in 21 yeast species of the following genera: Candida, Kluyveromyces, Pachysolen, Pichia, and Torulopsis. All the cultures fermented D-glucose with the formation of ethanol. During the assimilation of D-xylose, ethanol was produced by P. stipitis and C. shehatae, whereas xylitol was produced by C. didensiae, C. intermediae, C. parapsilosis, C. silvanorum, C. tropicalis, K. fragilis, K. marxianus, P. guillermondii, and T. molishiama. The yeast P. tannophilus produced comparable amounts of both alcohols. The possible use of xylose-assimilating yeasts for the production of xylitol and ethanol is discussed.     

Quantitative production of xylitol from D-xylose by a high-xylitol producing yeast mutant Candida tropicalis HXP2

Summary Xylitol was produced as a metabolic by-product by a number of yeasts when grown on medium containing D-xylose as carbon and energy sources. Among the yeast strains tested, a mutant strain of Candida tropicalis (HXP2) was found to produce xylitol from D-xylose with a high yield (>90%). Ethanol was also produced by HXP2 when D-glucose, D-fructose, or sucrose were used as substrates. The high-xylitol-producing yeast mutant is a good organism for the production of xylitol from biomass that contains D-xylose.     

Fermentation of D-xylose by free and immobilized Saccharomyces cerevisiae

With D-xylose (50 g l ) as sole carbon substrate, aerobic cultures of S. cerevisiae consumed significant amounts of sugar (26.4 g l ), producing 4.0 g xylitol l but no ethanol. In the presence of a mixture of glucose (35 g l ) and xylose (15 g l ), yeasts consumed 1.6 g xylose l that was converted nearly stoichiometrically to xylitol. Anaerobic conditions lessened xylose consumption and its conversion into xylitol. Traces of ethanol (0.4 g l ) were produced when xylose was the only carbon source, however. Agar-entrapped yeasts behaved as anaerobically-grown cultures but with higher specific rates of xylose consumption and xylitol production.     

Ethanol productions from D-xylose and cellobiose by Kluyveromyces cellobiovorus

Yeasts capable of fermenting both D-xylose and cellobiose to ethanol were screened. Of 213 species of yeasts surveyed, Kluyveromyces cellobiovorus sp. nov., a new species belonging to genus of Kluyveromyces, was selected as the sole strain. This strain accumulated 32, 22, and 19 g/L of ethanol from 8% glucose, D-xylose, and cellobiose, respectively. It was also shown that this strain produced ethanol from the enzymatic bagasse hydrolysate containing hexoses and pentoses more efficiently than Saccharomyces cerevisiae.