Phylogenetic Relationships between different D-Xylose Biogroups in Wild-type Salmonella typhimurium strains and a suggested Evolutionary Pathway

Sixty-two wild-type Salmonella typhimurium strains were characterized for their D-xylose enzyme activities. Strains from the xylose strong biogroup synthesized high levels of D-xylose isomerase and D-xylulokinase and transported D-xylose. Strains from the xylose weak biogroup synthesized only low levels of D-xylulokinase and low, or no, levels of D-xylose isomerase and were deficient in the ability to transport D-xylose. These findings are discussed in the light of known phylogenetic relationships among the biotypes of Salm. typhimurium.     

The glucose/xylose facilitator Gxf1 from Candida intermedia expressed in a xylose-fermenting industrial strain of Saccharomyces cerevisiae increases xylose uptake in SSCF of wheat straw

Ethanolic fermentation of lignocellulose raw materials requires industrial xylose-fermenting strains capable of complete and efficient D-xylose consumption. A central question in xylose fermentation by Saccharomyces cerevisiae engineered for xylose fermentation is to improve the xylose uptake. In the current study, the glucose/xylose facilitator Gxf1 from Candida intermedia, was expressed in three different xylose-fermenting S. cerevisiae strains of industrial origin. The in vivo effect on aerobic xylose growth and the initial xylose uptake rate were assessed. The expression of Gxf1 resulted in enhanced aerobic xylose growth only for the TMB3400 based strain. It displayed more than a 2-fold higher affinity for D-xylose than the parental strain and approximately 2-fold higher initial specific growth rate at 4 g/L D-xylose. Enhanced xylose consumption was furthermore observed when the GXF1-strain was assessed in simultaneous saccharification and co-fermentation (SSCF) of pretreated wheat straw. However, the ethanol yield remained unchanged due to increased by-product formation. Metabolic flux analysis suggested that the expression of the Gxf1 transporter had shifted the control of xylose catabolism from transport to the NAD(+) dependent oxidation of xylitol to xylulose.     

Genetic analysis of D-xylose metabolism by endophytic yeast strains of Rhodotorula graminis and Rhodotorula mucilaginosa

Two novel endophytic yeast strains, WP1 and PTD3, isolated from within the stems of poplar (Populus) trees, were genetically characterized with respect to their xylose metabolism genes. These two strains, belonging to the species Rhodotorula graminis and R. mucilaginosa, respectively, utilize both hexose and pentose sugars, including the common plant pentose sugar, D-xylose. The xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) genes were cloned and characterized. The derived amino acid sequences of xylose reductase (XR) and xylose dehydrogenase (XDH) were 32%～41% homologous to those of Pichia stipitis and Candida. spp., two species known to utilize xylose. The derived XR and XDH sequences of WP1 and PTD3 had higher homology (73% and 69% identity) with each other. WP1 and PTD3 were grown in single sugar and mixed sugar media to analyze the XYL1 and XYL2 gene regulation mechanisms. Our results revealed that for both strains, the gene expression is induced by D-xylose, and that in PTD3 the expression was not repressed by glucose in the presence of xylose.     

Comparative study of xylose(glucose) isomerase synthesis in Arthrobacter strains

The substrate specificity of isomerases produced by six strains of Arthrobacter sp. was studied. The role of utilizable carbon sources in controlling enzyme biosynthesis was established. All of the strains studied were found to produce xylose isomerases efficiently, converting D-xylose into D-xylulose and D-glucose into D-fructose. All but A. ureafaciens B-6 strains showed low activity toward D-ribose, Arthrobacter sp. B-5 was slightly active toward L-arabinose, and A. ureafaciens B-6 and Arthrobacter sp. B-2239, toward L-rhamnose. In Arthrobacter sp. B-5, the synthesis of xylose/glucose isomerase was constitutive (i.e., it was not suppressed by readily metabolizable carbon sources). The synthesis of xylose/glucose isomerase induced by D-xylose in Arthrobacter sp. strains B-2239, B-2240, B-2241, and B-2242 and by D-xylose and xylitol in A. ureafaciens B-6 was suppressed by readily metabolizable carbon sources in a concentration-dependent manner. The data obtained suggest that D-xylose and/or its metabolites are involved in the regulation of xylose/glucose isomerase synthesis in the Arthrobacter sp. strains B-5, B-2239, B-2240, and B-2241.     

D-xylose utilization by Saccharomyces cerevisiae

Although it is generally accepted that Saccharomyces cerevisiae is unable to assimilate D-xylose, four strains were found to utilize xylose aerobically at different efficiencies in the presence of a mixture of substrates. The degree of D-xylose utilization by S. cerevisiae ATCC 26602 depended upon the presence of other substrates or yeast extract. The greatest amount of xylose (up to 69% over 7 d) was utilized when sugar substrates such as D-ribose were co-metabolized. Much lower degrees of utilization occurred with co-metabolism of organic acids, polyols or ethanol. A mixture of D-glucose, D-ribose, D-raffinose, glycerol and D-xylose resulted in greater xylose utilization than the presence of a single substrate and xylose. The absence of growth on a co-substrate alone did not prevent the utilization of xylose in its presence. Xylose was co-metabolized with ribose under anaerobic conditions but at a much slower rate than under aerobic conditions. When [14C]xylose was utilized in the presence of ribose under anaerobic conditions, the radioactive label was detected mainly in xylitol and not in the small amounts of ethanol produced. Under aerobic conditions the radioactive label was distributed between xylitol (91.3 +/- 0.8%), CO2 (2.6 +/- 2.3%) and biomass (1.7 +/- 0.6%). No other metabolic products were detected. Whereas most xylose was dissimilated rather than assimilated by S. cerevisiae, the organism apparently possesses a pathway which completely oxidizes xylose in the presence of another substrate.     

Investigation of the synthesis of xylose/glucose isomerases in sixArthrobacter sp. strains

The substrate specificity of isomerases produced by six strains ofArthrobacter sp. was studied. The role of utilizable carbon sources in controlling enzyme biosynthesis was established. All of the strains studied were found to produce xylose isomerases efficiently, converting D-xylose into D-xylulose and D-glucose into D-fructose. All but A.ureafaciens B-6 strains showed low activity toward D-ribose,Arthrobacter sp. B-5 was slightly active toward L-arabinose, andA. ureafaciens B-6 andArthrobacter sp. B-2239, toward L-rhamnose. InArthrobacter sp. B-5, the synthesis of xylose/glucose isomerase was constitutive (i.e., it was not suppressed by readily metabolizable carbon sources. The synthesis of xylose/glucose isomerase induced by D-xylose inArthrobacter sp. strains B-2239, B-2240, B-2241, and B-2242 and by D-xylose and xylitol inA. ureafaciens B-6 was suppressed by readily metabolizable carbon sources in a concentration-dependent manner. The data obtained suggest that D-xylose and/or its metabolites are involved in the regulation of xylose/glucose isomerase synthesis in theArthrobacter sp. strains B-5, B-2239, B-2240, and B-2241.     

Isolation and characterization of the Salmonella typhimurium LT2 xylose regulon

Salmonella DNA was partially digested with EcoRI, and the digest was fractionated to obtain fragments larger than 8 kilobases (kb). These were ligated into EcoRI-cut pBR322, and the mixture was used to transform Salmonella Xyl- cells selecting for ampR xyl+ transformants. A 21- and a 27-kb plasmid were isolated, both of which contained the entire xylose regulon. The xylose regulon was localized to a 6.3-kb segment of a 13.5-kb EcoRI fragment. Subclones were constructed which contained either the genes for D-xylose isomerase and D-xylulokinase or the genes for the D-xylose transport and the D-xylose regulatory factors. The gene order determined by the subcloning experiments is consistent with that determined by genetic mapping. The spots corresponding to D-xylose isomerase and D-xylulokinase subunits were identified in two-dimensional gels of several xylose-induced strains. Each of them had a molecular weight of 45,000 and an isoelectric point of 6.2 +/- 0.1.     

Actinobacillus actinomycetem-comitans: Fermentative Capabilities of 140 Strains

Fermentative capabilities of 140 strains of Actinobacillus actinomycetem-comitans were studied. Findings correspond closely with those reported previously by Heinrich and Pulverer (12 strains), and by King and Tatum (33 strains). All strains ferment glucose, levulose, and maltose and reduce nitrate to nitrite. Reactions with glycogen and starch are exceedingly diverse. Eight different biotypes have been identified on the basis of their reactions with galactose, mannitol, and xylose.     

Application of Saccharomyces cerevisiae and Pichia stipitis karyoductants to the production of ethanol from xylose

Karyoductants of Saccharomyces cerevisiae V30 and Pichia stipitis CCY 39501 with the ability to ferment D-xylose to ethanol were isolated. The ability of these isolates to assimilate different sugars, ethanol tolerance and ethanol production from D-xylose was investigated. Karyoductants didn't grow on starch, lactose and cellobiose, like S. cerevisiae, but showed good growth on xylose and L-arabinose, like P. stipitis. All isolates fermented xylose to ethanol slower than P. stipitis and with lower yields, 0.09 - 0.16 g/g. They secreted also about 3.4 - 7.1 g/dm3 of xylitol to the culture medium (P. stipitis only 0.06 g/dm3). The karyoductants showed an average tolerance to ethanol when compared with the parent strains and fermented glucose in the presence of 6% alcohol whereas parent strain S. cerevisiae and P. stipitis showed exogenic ethanol tolerance of 9% and 3%, respectively.     

Isolation and identification of xylitol-producing yeasts from agricultural residues

Selected yeast strains isolated from corn silage and viticulture residues were screened for their capacities to convert D-xylose into xylitol A conventional TLC was adapted for easy determination of xylose and xylitol in the culture supernatant solutions. This technique is suitable for the first steps of a screening program to select xylitol-producing yeasts from natural environments. Candida tropicalis ASM III (NRRL Y-27290), isolated from corn silage, appears to be a promising strain for xylitol production with a high yield (0.88 g xylitol per g of xylose consumed).     

NADP(+)-dependent D-xylose dehydrogenase from pig liver. Purification and properties.

An NADP(+)-dependent D-xylose dehydrogenase from pig liver cytosol was purified about 2000-fold to apparent homogeneity with a yield of 15% and specific activity of 6 units/mg of protein. An Mr value of 62,000 was obtained by gel filtration. PAGE in the presence of SDS gave an Mr value of 32,000, suggesting that the native enzyme is a dimer of similar or identical subunits. D-Xylose, D-ribose, L-arabinose, 2-deoxy-D-glucose, D-glucose and D-mannose were substrates in the presence of NADP+ but the specificity constant (ratio kcat./Km(app.)) is, by far, much higher for D-xylose than for the other sugars. The enzyme is specific for NADP+; NAD+ is not reduced in the presence of D-xylose or other sugars. Initial-velocity studies for the forward direction with xylose or NADP+ concentrations varied at fixed concentrations of the nucleotide or the sugar respectively revealed a pattern of parallel lines in double-reciprocal plots. Km values for D-xylose and NADP+ were 8.8 mM and 0.99 mM respectively. Dead-end inhibition studies to confirm a ping-pong mechanism showed that NAD+ acted as an uncompetitive inhibitor versus NADP+ (Ki 5.8 mM) and as a competitive inhibitor versus xylose. D-Lyxose was a competitive inhibitor versus xylose and uncompetitive versus NADP+. These results fit better to a sequential compulsory ordered mechanism with NADP+ as the first substrate, but a ping-pong mechanism with xylose as the first substrate has not been ruled out. The presence of D-xylose dehydrogenase suggests that in mammalian liver D-xylose is utilized by a pathway other than the pentose phosphate pathway.     

Biosynthetic features and properties of xylose isomerases from Arthrobacter nicotianae, Escherichia coli, and Erwinia carotovota subsp. atroseptica

The characteristics of xylose isomerase biosynthesis in the bacteria Arthrobacter nicotianae BIM B-5, Erwinia carotovota subsp atroseptica jn42xylA, and Escherichia coli HB101xylA have been studied. The bacteria formed the enzyme constitutively. Out of the carbon sources studied, D-glucose and D-xylose were most favorable for the biosynthesis of xylose isomerase in E. carotovota subsp atroseptica, but the least appropriate in terms of the enzyme production efficiency in E. coli. Minimum and maximum levels of xylose isomerase formation in A. nicotianae were noted, respectively, during D-xylose and sucrose utilization. An addition to the nutrient medium of 0.1-1.5% D-glucose (together with D-xylose) did not affect the enzyme synthesis in A. nicotianae, but suppressed it in Erwinia carotovota subsp atroseptica (by 7% at the highest concentration) and Escherichia coli (by 63 and 75% at concentrations of 0.1 and 1.0%, respectively). The enzyme proteins produced by the bacteria exhibited the same substrate specificity and electrophoretic mobility (PAGE) as xylose isomerase A. nicotianae, although insignificant differences in the major physicochemical properties were noted.     

Novel xylose dehydrogenase in the halophilic archaeon Haloarcula marismortui

During growth of the halophilic archaeon Haloarcula marismortui on D-xylose, a specific D-xylose dehydrogenase was induced. The enzyme was purified to homogeneity. It constitutes a homotetramer of about 175 kDa and catalyzed the oxidation of xylose with both NADP+ and NAD+ as cosubstrates with 10-fold higher affinity for NADP+. In addition to D-xylose, D-ribose was oxidized at similar kinetic constants, whereas D-glucose was used with about 70-fold lower catalytic efficiency (kcat/Km). With the N-terminal amino acid sequence of the subunit, an open reading frame (ORF)-coding for a 39.9-kDA protein-was identified in the partially sequenced genome of H. marismortui. The function of the ORF as the gene designated xdh and coding for xylose dehydrogenase was proven by its functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione, and substrates. Xylose dehydrogenase showed the highest sequence similarity to glucose-fructose oxidoreductase from Zymomonas mobilis and other putative bacterial and archaeal oxidoreductases. Activities of xylose isomerase and xylulose kinase, the initial reactions of xylose catabolism of most bacteria, could not be detected in xylose-grown cells of H. marismortui, and the genes that encode them, xylA and xylB, were not found in the genome of H. marismortui. Thus, we propose that this first characterized archaeal xylose dehydrogenase catalyzes the initial step in xylose degradation by H. marismortui.     

Transient-state and steady-state kinetic studies of the mechanism of NADH-dependent aldehyde reduction catalyzed by xylose reductase from the yeast Candida tenuis

Microbial xylose reductase, a representative aldo-keto reductase of primary sugar metabolism, catalyzes the NAD(P)H-dependent reduction of D-xylose with a turnover number approximately 100 times that of human aldose reductase for the same reaction. To determine the mechanistic basis for that physiologically relevant difference and pinpoint features that are unique to the microbial enzyme among other aldo/keto reductases, we carried out stopped-flow studies with wild-type xylose reductase from the yeast Candida tenuis. Analysis of transient kinetic data for binding of NAD(+) and NADH, and reduction of D-xylose and oxidation of xylitol at pH 7.0 and 25 degrees C provided estimates of rate constants for the following mechanism: E + NADH right arrow over left arrow E.NADH right arrow over left arrow E.NADH + D-xylose right arrow over left arrow E.NADH.D-xylose right arrow over left arrow E.NAD(+).xylitol right arrow over left arrow E.NAD(+) right arrow over left arrow E.NAD(+) right arrow over left arrow E + NAD(+). The net rate constant of dissociation of NAD(+) is approximately 90% rate limiting for k(cat) of D-xylose reduction. It is controlled by the conformational change which precedes nucleotide release and whose rate constant of 40 s(-)(1) is 200 times that of completely rate-limiting E.NADP(+) --> E.NADP(+) step in aldehyde reduction catalyzed by human aldose reductase [Grimshaw, C. E., et al. (1995) Biochemistry 34, 14356-14365]. Hydride transfer from NADH occurs with a rate constant of approximately 170 s(-1). In reverse reaction, the E.NADH --> E.NADH step takes place with a rate constant of 15 s(-1), and the rate constant of ternary-complex interconversion (3.8 s(-1)) largely determines xylitol turnover (0.9 s(-1)). The bound-state equilibrium constant for C. tenuis xylose reductase is estimated to be approximately 45 (=170/3.8), thus greatly favoring aldehyde reduction. Formation of productive complexes, E.NAD(+) and E.NADH, leads to a 7- and 9-fold decrease of dissociation constants of initial binary complexes, respectively, demonstrating that 12-fold differential binding of NADH (K(i) = 16 microM) vs NAD(+) (K(i) = 195 microM) chiefly reflects difference in stabilities of E.NADH and E.NAD(+). Primary deuterium isotope effects on k(cat) and k(cat)/K(xylose) were, respectively, 1.55 +/- 0.09 and 2.09 +/- 0.31 in H(2)O, and 1.26 +/- 0.06 and 1.58 +/- 0.17 in D(2)O. No deuterium solvent isotope effect on k(cat)/K(xylose) was observed. When deuteration of coenzyme selectively slowed the hydride transfer step, (D)()2(O)(k(cat)/K(xylose)) was inverse (0.89 +/- 0.14). The isotope effect data suggest a chemical mechanism of carbonyl reduction by xylose reductase in which transfer of hydride ion is a partially rate-limiting step and precedes the proton-transfer step.     

Comparative study of Agrobacterium biotypes 1, 2 and 3 by electrophoresis and serological methods

A comparative study by electrophoresis and serology of strains representing the three Agrobacterium biotypes was carried out. Thirteen Spanish isolates and strains from international collections were included. Ten antisera were prepared by using strains from the three biotypes and different types of antigens. The strains were studied by immunodiffusion, indirect immunofluorescence and indirect ELISA. Serological relationship among all the strains was observed, although serological heterogeneity within each of the biotypes occurred. Biotype 3 appears as serologically related to biotypes 1 and 2, having an intermediate position. This observation is in agreement with their biochemical characteristics. Electrophoretic analysis of the three biotypes showed that there was high variability. Three main bands appeared in the six strains studied. One specific band occurred in the biotype 1 strains and another in the biotype 3 strains.     

Controlled transient changes reveal differences in metabolite production in two<Emphasis Type="Italic"> Candida</Emphasis> yeasts

Physiological responses during growth on xylose and the xylose-degrading pathway of Candida tropicalis and Candida guilliermondii yeasts were investigated. The responses to a linearly decreasing oxygen transfer rate and a simultaneously increasing dilution rate were compared. C. guilliermondii produced acetate but no ethanol, and C. tropicalis ethanol but no acetate under oxygen limitation. Both strains produced glycerol. The D-xylose reductase of C. guilliermondii is exclusively NADPH-dependent. and acetate production regenerated NADPH. The xylose'reductase of C. tropicalis has a dual dependency for both NADH and NADPH. It regenerated NAD by producing ethanol. Both strains regenerated NAD by producing glycerol. The effect of intracellular NADH accumulation to xylose uptake and metabolite production was studied by using formate as a cosubstrate. Formate feeding in C. tropicalis triggered the accumulation of glycerol, ethanol and xylitol. Consequently, the specific xylose consumption increased 28% during formate feeding, from 477 to 609 C-mmol/C-mol cell dry-weight (CDW)/h. In C. guilliermondii cultures. formate feeding resulted only in glycerol accumulation. The specific xylose consumption increased 6%, from 301 to 319 C-mmol/C-mol CDW/h, until glycerol started to accumulate.     

Endogenous xylose pathway in Saccharomyces cerevisiae

The baker's yeast Saccharomyces cerevisiae is generally classified as a non-xylose-utilizing organism. We found that S. cerevisiae can grow on D-xylose when only the endogenous genes GRE3 (YHR104w), coding for a nonspecific aldose reductase, and XYL2 (YLR070c, ScXYL2), coding for a xylitol dehydrogenase (XDH), are overexpressed under endogenous promoters. In nontransformed S. cerevisiae strains, XDH activity was significantly higher in the presence of xylose, but xylose reductase (XR) activity was not affected by the choice of carbon source. The expression of SOR1, encoding a sorbitol dehydrogenase, was elevated in the presence of xylose as were the genes encoding transketolase and transaldolase. An S. cerevisiae strain carrying the XR and XDH enzymes from the xylose-utilizing yeast Pichia stipitis grew more quickly and accumulated less xylitol than did the strain overexpressing the endogenous enzymes. Overexpression of the GRE3 and ScXYL2 genes in the S. cerevisiae CEN.PK2 strain resulted in a growth rate of 0.01 g of cell dry mass liter(-1) h(-1) and a xylitol yield of 55% when xylose was the main carbon source.     

Transcriptional analysis of catabolite repression in Clostridium acetobutylicum growing on mixtures of D-glucose and D-xylose

Clostridium acetobutylicum is a strict anaerobic organism that is used for biotechnological butanol fermentation. It ferments various hexoses and pentoses to solvents but prefers glucose presumably using a catabolite repression mechanism. Accordingly during growth on a mixture of D-glucose and D-xylose a typical diauxic growth pattern was observed. We used DNA microarrays and real-time RT-PCR to study gene expression during growth on D-glucose, D-xylose mixtures on a defined minimal medium together with monitoring substrate consumption and product formation. We identified two putative operons involved in D-xylose degradation. The first operon (CAC1344-CAC1349) includes a transporter, a xylulose-kinase, a transaldolase, a transketolase, an aldose-1-epimerase and a putative xylose isomerase that has been annotated as an arabinose isomerase. This operon is induced by D-xylose but was catabolite repressed by D-glucose. A second operon (CAC2610-CAC2612) consists of a xylulose-kinase, a hypothetical protein and a gene that has been annotated as a L-fucose isomerase that might in fact code for a xylose isomerase. This operon was induced by D-xylose but was not subject to catabolite repression. In accordance with these results we identified a CRE site in the catabolite repressed operon but not in the operon that was not subject to catabolite repression.     

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.