Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism

The first enzyme in the pathway for l-arabinose catabolism in eukaryotic microorganisms is a reductase, reducing l-arabinose to l-arabitol. The enzymes catalyzing this reduction are in general nonspecific and would also reduce d-xylose to xylitol, the first step in eukaryotic d-xylose catabolism. It is not clear whether microorganisms use different enzymes depending on the carbon source. Here we show that Aspergillus niger makes use of two different enzymes. We identified, cloned, and characterized an l-arabinose reductase, larA, that is different from the d-xylose reductase, xyrA. The larA is up-regulated on l-arabinose, while the xyrA is up-regulated on d-xylose. There is however an initial up-regulation of larA also on d-xylose but that fades away after about 4 h. The deletion of the larA gene in A. niger results in a slow growth phenotype on l-arabinose, whereas the growth on d-xylose is unaffected. The l-arabinose reductase can convert l-arabinose and d-xylose to their corresponding sugar alcohols but has a higher affinity for l-arabinose. The K_m for l-arabinose is 54 ± 6 mm and for d-xylose 155 ± 15 mm.     

Metabolism of Pentose Sugars in the Hyperthermophilic Archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius

We have previously shown that the hyperthermophilic archaeon, Sulfolobus solfataricus, catabolizes d-glucose and d-galactose to pyruvate and glyceraldehyde via a non-phosphorylative version of the Entner-Doudoroff pathway. At each step, one enzyme is active with both C6 epimers, leading to a metabolically promiscuous pathway. On further investigation, the catalytic promiscuity of the first enzyme in this pathway, glucose dehydrogenase, has been shown to extend to the C5 sugars, d-xylose and l-arabinose. In the current paper we establish that this promiscuity for C6 and C5 metabolites is also exhibited by the third enzyme in the pathway, 2-keto-3-deoxygluconate aldolase, but that the second step requires a specific C5-dehydratase, the gluconate dehydratase being active only with C6 metabolites. The products of this pathway for the catabolism of d-xylose and l-arabinose are pyruvate and glycolaldehyde, pyruvate entering the citric acid cycle after oxidative decarboxylation to acetyl-coenzyme A. We have identified and characterized the enzymes, both native and recombinant, that catalyze the conversion of glycolaldehyde to glycolate and then to glyoxylate, which can enter the citric acid cycle via the action of malate synthase. Evidence is also presented that similar enzymes for this pentose sugar pathway are present in Sulfolobus acidocaldarius, and metabolic tracer studies in this archaeon demonstrate its in vivo operation in parallel with a route involving no aldol cleavage of the 2-keto-3-deoxy-pentanoates but direct conversion to the citric acid cycle C5-metabolite, 2-oxoglutarate.     

Establishment of Oxidative d-Xylose Metabolism in Pseudomonas putida S12

The oxidative d-xylose catabolic pathway of Caulobacter crescentus, encoded by the xylXABCD operon, was expressed in the gram-negative bacterium Pseudomonas putida S12. This engineered transformant strain was able to grow on d-xylose as a sole carbon source with a biomass yield of 53% (based on g [dry weight] g d-xylose⁻¹) and a maximum growth rate of 0.21 h⁻¹. Remarkably, most of the genes of the xylXABCD operon appeared to be dispensable for growth on d-xylose. Only the xylD gene, encoding d-xylonate dehydratase, proved to be essential for establishing an oxidative d-xylose catabolic pathway in P. putida S12. The growth performance on d-xylose was, however, greatly improved by coexpression of xylXA, encoding 2-keto-3-deoxy-d-xylonate dehydratase and α-ketoglutaric semialdehyde dehydrogenase, respectively. The endogenous periplasmic glucose dehydrogenase (Gcd) of P. putida S12 was found to play a key role in efficient oxidative d-xylose utilization. Gcd activity not only contributes to d-xylose oxidation but also prevents the intracellular accumulation of toxic catabolic intermediates which delays or even eliminates growth on d-xylose.The requirement for renewable alternatives to replace oil-based chemicals and fuels necessitates development of novel technologies. Lignocellulose provides a promising alternative feedstock. However, since the pentose sugar fraction may account for up to 25% of lignocellulosic biomass (12), it is essential that this fraction is utilized efficiently to obtain cost-effective biochemical production. In a previous study, the solvent-tolerant bacterium Pseudomonas putida S12, known for its use as a platform host for the production of aromatic compounds (15, 16, 19, 22), was engineered to use d-xylose as a sole carbon source. This was achieved by introducing genes encoding the phosphorylative d-xylose metabolic pathway of Escherichia coli, followed by laboratory evolution (14). Prior to evolutionary improvement, extensive oxidation of d-xylose to d-xylonate occurred, resulting in a very low biomass-for-substrate yield as d-xylonate is a metabolic dead-end product in P. putida. The evolution approach resulted in elimination of the activity of periplasmic glucose dehydrogenase (Gcd), the enzyme responsible for d-xylose oxidation, which turned out to be a critical step in optimizing phosphorylative d-xylose utilization in P. putida S12.Instead of prevention of endogenous oxidation of d-xylose, this oxidation may be used to our advantage when it is combined with an oxidative d-xylose metabolic pathway, such as the pathways described for several Pseudomonas species, Caulobacter crescentus, and Haloarcula marismortui (7, 11, 18, 20). In these pathways, d-xylonate is dehydrated to 2-keto-3-deoxy-d-xylonate. This intermediate either can be cleaved into pyruvate and glycolaldehyde (7) or is further dehydrated to α-ketoglutaric semialdehyde (α-KGSA). In the final step of the latter pathway, α-KGSA is oxidized to the tricarboxylic acid (TCA) cycle intermediate α-ketoglutarate (18, 20).In addition to Gcd (PP1444), some of the enzymes required for oxidative d-xylose metabolism are expected to be endogenous in P. putida S12. Transport of d-xylonate into the cytoplasm likely occurs through the gluconate transporter (encoded by gntP [PP3417]). The enzyme catalyzing the final step of the pathway, α-KGSA dehydrogenase, is also likely to be present (presumably PP1256 and/or PP3602) because of the requirement for metabolism of 4-hydroxyproline (1), a compound that is efficiently utilized by P. putida S12. In view of these properties, the most obvious approach for constructing d-xylose-utilizing P. putida S12 is reconstruction of a complete oxidative d-xylose metabolic pathway by introducing the parts of such a pathway that complement the endogenous activities. Recently, the genetic information for one such oxidative d-xylose pathway has become available (18), enabling the approach used in the present study, i.e., expression of the oxidative d-xylose metabolic pathway of C. crescentus in P. putida S12 and investigation of the contribution of endogenous enzyme activities.     

Accumulation of d-Glucose from Pentoses by Metabolically Engineered Escherichia coli

Escherichia coli that is unable to metabolize d-glucose (with knockouts in ptsG, manZ, and glk) accumulates a small amount of d-glucose (yield of about 0.01 g/g) during growth on the pentoses d-xylose or l-arabinose as a sole carbon source. Additional knockouts in the zwf and pfkA genes, encoding, respectively, d-glucose-6-phosphate 1-dehydrogenase and 6-phosphofructokinase I (E. coli MEC143), increased accumulation to greater than 1 g/liter d-glucose and 100 mg/liter d-mannose from 5 g/liter d-xylose or l-arabinose. Knockouts of other genes associated with interconversions of d-glucose-phosphates demonstrate that d-glucose is formed primarily by the dephosphorylation of d-glucose-6-phosphate. Under controlled batch conditions with 20 g/liter d-xylose, MEC143 generated 4.4 g/liter d-glucose and 0.6 g/liter d-mannose. The results establish a direct link between pentoses and hexoses and provide a novel strategy to increase carbon backbone length from five to six carbons by directing flux through the pentose phosphate pathway.     

Crystal structure of a zinc-dependent D-serine dehydratase from chicken kidney

d-Serine is a physiological co-agonist of the N-methyl-d-aspartate receptor. It regulates excitatory neurotransmission, which is important for higher brain functions in vertebrates. In mammalian brains, d-amino acid oxidase degrades d-serine. However, we have found recently that in chicken brains the oxidase is not expressed and instead a d-serine dehydratase degrades d-serine. The primary structure of the enzyme shows significant similarities to those of metal-activated d-threonine aldolases, which are fold-type III pyridoxal 5′-phosphate (PLP)-dependent enzymes, suggesting that it is a novel class of d-serine dehydratase. In the present study, we characterized the chicken enzyme biochemically and also by x-ray crystallography. The enzyme activity on d-serine decreased 20-fold by EDTA treatment and recovered nearly completely by the addition of Zn²⁺. None of the reaction products that would be expected from side reactions of the PLP-d-serine Schiff base were detected during the >6000 catalytic cycles of dehydration, indicating high reaction specificity. We have determined the first crystal structure of the d-serine dehydratase at 1.9 Å resolution. In the active site pocket, a zinc ion that coordinates His³⁴⁷ and Cys³⁴⁹ is located near the PLP-Lys⁴⁵ Schiff base. A theoretical model of the enzyme-d-serine complex suggested that the hydroxyl group of d-serine directly coordinates the zinc ion, and that the ϵ-NH₂ group of Lys⁴⁵ is a short distance from the substrate Cα atom. The α-proton abstraction from d-serine by Lys⁴⁵ and the elimination of the hydroxyl group seem to occur with the assistance of the zinc ion, resulting in the strict reaction specificity.     

Metabolic Studies on Intermediates in the myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen: III. Polysaccharidic Origin of Labeled Glucose

myo-Inositol-linked glucogenesis in germinated lily (Lilium longiflorum Thunb., cv. Ace) pollen was investigated by studying the effects of added l-arabinose or d-xylose on metabolism of myo-[2-³H]inositol and by determining the distribution of radioisotope in pentosyl and hexosyl residues of polysaccharides from pollen labeled with myo-[2-¹⁴C]inositol, myo-[2-³H]inositol, l-[5-¹⁴C]arabinose, and d-[5R,5S-³H]xylose.     

Identification and Characterization of d-Hydroxyproline Dehydrogenase and Δ1-Pyrroline-4-hydroxy-2-carboxylate Deaminase Involved in Novel l-Hydroxyproline Metabolism of Bacteria: METABOLIC CONVERGENT EVOLUTION*

l-Hydroxyproline (4-hydroxyproline) mainly exists in collagen, and most bacteria cannot metabolize this hydroxyamino acid. Pseudomonas putida and Pseudomonas aeruginosa convert l-hydroxyproline to α-ketoglutarate via four hypothetical enzymatic steps different from known mammalian pathways, but the molecular background is rather unclear. Here, we identified and characterized for the first time two novel enzymes, d-hydroxyproline dehydrogenase and Δ¹-pyrroline-4-hydroxy-2-carboxylate (Pyr4H2C) deaminase, involved in this hypothetical pathway. These genes were clustered together with genes encoding other catalytic enzymes on the bacterial genomes. d-Hydroxyproline dehydrogenases from P. putida and P. aeruginosa were completely different from known bacterial proline dehydrogenases and showed similar high specificity for substrate (d-hydroxyproline) and some artificial electron acceptor(s). On the other hand, the former is a homomeric enzyme only containing FAD as a prosthetic group, whereas the latter is a novel heterododecameric structure consisting of three different subunits (α₄β₄γ₄), and two FADs, FMN, and [2Fe-2S] iron-sulfur cluster were contained in αβγ of the heterotrimeric unit. These results suggested that the l-hydroxyproline pathway clearly evolved convergently in P. putida and P. aeruginosa. Pyr4H2C deaminase is a unique member of the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein family, and its activity was competitively inhibited by pyruvate, a common substrate for other dihydrodipicolinate synthase/N-acetylneuraminate lyase proteins. Furthermore, disruption of Pyr4H2C deaminase genes led to loss of growth on l-hydroxyproline (as well as d-hydroxyproline) but not l- and d-proline, indicating that this pathway is related only to l-hydroxyproline degradation, which is not linked to proline metabolism.     

Identification and characterization of d-arabinose reductase and d-arabinose transporters from Pichia stipitis

d-xylose and l-arabinose are the major constituents of plant lignocelluloses, and the related fungal metabolic pathways have been extensively examined. Although Pichia stipitis CBS 6054 grows using d-arabinose as the sole carbon source, the hypothetical pathway has not yet been clarified at the molecular level. We herein purified NAD(P)H-dependent d-arabinose reductase from cells grown on d-arabinose, and found that the enzyme was identical to the known d-xylose reductase (XR). The enzyme activity of XR with d-arabinose was previously reported to be only 1% that with d-xylose. The k_cat/K_m value with d-arabinose (1.27 min^?1 mM^?1), which was determined using the recombinant enzyme, was 13.6- and 10.5-fold lower than those with l-arabinose and d-xylose, respectively. Among the 34 putative sugar transporters from P. stipitis, only seven genes exhibited uptake ability not only for d-arabinose, but also for d-glucose and other pentose sugars including d-xylose and l-arabinose in Saccharomyces cerevisiae.     

Uptake of monosaccharides by guinea-pig cerebral-cortex slices

By the use of 1mm-iodoacetate to inhibit glycolysis in guinea-pig cerebral tissue slices, the kinetics of the uptake of monosaccharides on transfer of tissue from 0° to 37° were studied. d-Ribose, d-galactose, d-mannose, l-sorbose, and d-fructose showed diffusion kinetics, whereas 2-deoxy-d-glucose, d-glucose, d-arabinose and d-xylose showed saturation kinetics.     

Identification of Glycosyltransferase 8 Family Members as Xylosyltransferases Acting on O-Glucosylated Notch Epidermal Growth Factor Repeats

The epidermal growth factor repeats of the Notch receptor are extensively glycosylated with three different O-glycans. O-Fucosylation and elongation by the glycosyltransferase Fringe have been well studied and shown to be essential for proper Notch signaling. In contrast, biosynthesis of O-glucose and O-N-acetylglucosamine is less well understood. Recently, the isolation of the Drosophila mutant rumi has shown that absence of O-glucose impairs Notch function. O-Glucose is further extended by two contiguous α1,3-linked xylose residues. We have identified two enzymes of the human glycosyltransferase 8 family, now named GXYLT1 and GXYLT2 (glucoside xylosyltransferase), as UDP-d-xylose:β-d-glucoside α1,3-d-xylosyltransferases adding the first xylose. The enzymes are specific for β-glucose-terminating acceptors and UDP-xylose as donor substrate. Generation of the α1,3-linkage was confirmed by nuclear magnetic resonance. Activity on a natural acceptor could be shown by in vitro xylosylation of a Notch fragment expressed in a UDP-xylose-deficient cell line and in vivo by co-expression of the enzymes and the Notch fragment in insect cells followed by mass spectrometric analysis of peptide fragments.     

Substrate specificity of the Bacillus licheniformis lyxose isomerase YdaE and its application in in vitro catalysis for bioproduction of lyxose and glucose by two-step isomerization

Enzymatic processes are useful for industrially important sugar production, and in vitro two-step isomerization has proven to be an efficient process in utilizing readily available sugar sources. A hypothetical uncharacterized protein encoded by ydaE of Bacillus licheniformis was found to have broad substrate specificities and has shown high catalytic efficiency on d-lyxose, suggesting that the enzyme is d-lyxose isomerase. Escherichia coli BL21 expressing the recombinant protein, of 19.5 kDa, showed higher activity at 40 to 45°C and pH 7.5 to 8.0 in the presence of 1.0 mM Mn²⁺. The apparent K_m values for d-lyxose and d-mannose were 30.4 ± 0.7 mM and 26 ± 0.8 mM, respectively. The catalytic efficiency (k_cat/K_m) for lyxose (3.2 ± 0.1 mM⁻¹ s⁻¹) was higher than that for d-mannose (1.6 mM⁻¹ s⁻¹). The purified protein was applied to the bioproduction of d-lyxose and d-glucose from d-xylose and d-mannose, respectively, along with the thermostable xylose isomerase of Thermus thermophilus HB08. From an initial concentration of 10 mM d-lyxose and d-mannose, 3.7 mM and 3.8 mM d-lyxose and d-glucose, respectively, were produced by two-step isomerization. This two-step isomerization is an easy method for in vitro catalysis and can be applied to industrial production.     

Inositol Metabolism in Plants. V. Conversion of Myo-inositol to Uronic Acid and Pentose Units of Acidic Polysaccharides in Root-tips of Zea mays

The metabolism of myo-inositol-2-¹⁴C, d-glucuronate-1-¹⁴C, d-glucuronate-6-¹⁴C, and l-methionine-methyl-¹⁴C to cell wall polysaccharides was investigated in excised root-tips of 3 day old Zea mays seedlings. From myo-inositol, about one-half of incorporated label was recovered in ethanol insoluble residues. Of this label, about 90% was solubilized by treatment, first with a preparation of pectinase-EDTA, then with dilute hydrochloric acid. The only labeled constituents in these hydrolyzates were d-galacturonic acid, d-glucuronic acid, 4-O-methyl-d-glucuronic acid, d-xylose, and l-arabinose, or larger oligosaccharide fragments containing these units. Medium external to excised root-tips grown under sterile conditions in myo-inositol-2-¹⁴C contained labeled polysaccharide.     

The metabolism of galactarate, d-glucarate and various pentoses by species of Pseudomonas

1. When NAD⁺ was present, cell extracts of Pseudomonas (A) grown with d-glucarate or galactarate converted 1mol. of either substrate into 1mol. each of 2-oxoglutarate and carbon dioxide; 70–80% of the gas originated from C-1 of the hexarate. 2. The enzyme system that liberated carbon dioxide from galactarate was inactive in air and was stabilized by galactarate or Fe²⁺ ions; the system that acted on d-glucarate was more stable and was stimulated by Mg²⁺ ions. 3. When NAD⁺ was not added, 2-oxoglutarate semialdehyde accumulated from either substrate. This compound was isolated as its bis-2,4-dinitrophenylhydrazone, and several properties of the derivative were compared with those of the chemically synthesized material. Methods were developed for the determination of 2-oxoglutarate semialdehyde. 4. Synthetic 2-oxoglutarate semialdehyde was converted into 2-oxoglutarate by an enzyme that required NAD⁺; the reaction rate with NADP⁺ was about one-sixth of that with NAD⁺. 5. For extracts of Pseudomonas (A) grown with d-glucarate or galactarate, or for those of Pseudomonas fragi grown with l-arabinose or d-xylose, specific activities of 2-oxoglutarate semialdehyde–NAD oxidoreductase were much higher than for extracts of the organisms grown with (+)-tartrate and d-glucose respectively. 6. Extracts of Pseudomonas fragi grown with l-arabinose or d-xylose converted l-arabonate or d-xylonate into 2-oxoglutarate when NAD⁺ was added to reaction mixtures and into 2-oxoglutarate semialdehyde when NAD⁺ was omitted.     

Biosynthesis of ethylene glycol in Escherichia coli

Ethylene glycol (EG) is an important platform chemical with steadily expanding global demand. Its commercial production is currently limited to fossil resources; no biosynthesis route has been delineated. Herein, a biosynthesis route for EG production from d-xylose is reported. This route consists of four steps: d-xylose?→?d-xylonate?→?2-dehydro-3-deoxy-d-pentonate?→?glycoaldehyde?→?EG. Respective enzymes, d-xylose dehydrogenase, d-xylonate dehydratase, 2-dehydro-3-deoxy-d-pentonate aldolase, and glycoaldehyde reductase, were assembled. The route was implemented in a metabolically engineered Escherichia coli, in which the d-xylose?→?d-xylulose reaction was prevented by disrupting the d-xylose isomerase gene. The most efficient construct produced 11.7 g?L^?1 of EG from 40.0 g?L^?1 of d-xylose. Glycolate is a carbon-competing by-product during EG production in E. coli; blockage of glycoaldehyde?→?glycolate reaction was also performed by disrupting the gene encoding aldehyde dehydrogenase, but from this approach, EG productivity was not improved but rather led to d-xylonate accumulation. To channel more carbon flux towards EG than the glycolate pathway, further systematic metabolic engineering and fermentation optimization studies are still required to improve EG productivity.     

Efficient Bioethanol Production by a Recombinant Flocculent Saccharomyces cerevisiae Strain with a Genome-Integrated NADP+-Dependent Xylitol Dehydrogenase Gene

The recombinant industrial Saccharomyces cerevisiae strain MA-R5 was engineered to express NADP⁺-dependent xylitol dehydrogenase using the flocculent yeast strain IR-2, which has high xylulose-fermenting ability, and both xylose consumption and ethanol production remarkably increased. Furthermore, the MA-R5 strain produced the highest ethanol yield (0.48 g/g) from nonsulfuric acid hydrolysate of wood chips.Successful fermentation of lignocellulosic biomass to ethanol is dependent on efficient utilization of d-xylose, which is the second most common fermentable sugar in the hydrolysate. Although the well-known fermentative yeast Saccharomyces cerevisiae is one of the most effective ethanol-producing organisms for hexose sugars, it is not able to ferment d-xylose. However, S. cerevisiae can metabolize an isomerization product of d-xylose, d-xylulose, which is phosphorylated to d-xylulose 5-phosphate, channeled through the pentose phosphate pathway and glycolysis.S. cerevisiae transformed with the XYL1 and XYL2 genes encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis (referred to as PsXR and PsXDH, respectively) acquires the ability to ferment d-xylose to ethanol (2, 5, 6, 9, 10, 12, 22). Furthermore, overexpression of the XKS1 gene encoding xylulokinase (XK) from S. cerevisiae (ScXK) has been shown to aid d-xylose utilization (4, 7, 11, 16, 23), with xylitol still a major by-product. Kuyper et al. (14) also demonstrated the successful fermentation of d-xylose to ethanol using recombinant S. cerevisiae strains expressing fungal xylose isomerase. However, these approaches are insufficient for industrial bioprocesses, mainly due to the low rate of d-xylose fermentation.Xylitol excretion has been ascribed mainly to the difference in coenzyme specificities between PsXR (with NADPH) and PsXDH (with NAD⁺), which creates an intracellular redox imbalance (1). Therefore, modifying the coenzyme specificity of XR and/or XDH by protein engineering is an attractive approach for achieving efficient fermentation of ethanol from d-xylose using recombinant S. cerevisiae. Watanabe et al. (24) previously succeeded in generating several PsXDH mutants (e.g., quadruple ARSdR mutant) with a complete reversal of coenzyme specificity toward NADP⁺ by multiple site-directed mutagenesis on amino acids from the coenzyme-binding domain. The ARSdR mutant (D207A/I208R/F209S/N211R) has more that 4,500-fold-higher catalytic efficiency (k_cat/K_m) with NADP⁺ than the wild-type PsXDH. In addition, we recently found that several laboratory recombinant S. cerevisiae strains, in which the ARSdR mutant, along with PsXR and ScXK, is expressed through a strong constitutive promoter, increased ethanol production from d-xylose at the expense of xylitol excretion (17, 18), probably by maintaining the intracellular redox balance. However, commercialization of lignocellulosic hydrolysate fermentation requires industrial strains that are more robust than laboratory strains (5, 19, 21).A potential host for developing d-xylose-fermenting strains requires an active and efficient pentose phosphate pathway linking the d-xylose-to-d-xylulose pathway to glycolysis. In the case of S. cerevisiae, strains have different d-xylulose fermentation abilities (3, 25), indicating inherent differences in the capacities of these strains to ferment pentose sugars. Furthermore, anaerobic d-xylulose fermentation was investigated to identify genetic backgrounds potentially beneficial to anaerobic d-xylose fermentation in strains not exhibiting product formation related to the redox imbalance generated by the first two steps of the eukaryotic d-xylose metabolism (3), although the physiological purpose of the different d-xylulose fermentation abilities of S. cerevisiae is not yet clear. Therefore, we selected an efficient industrial d-xylulose-fermenting S. cerevisiae strain as a host for constructing a recombinant strain through chromosomal integration of the NADP⁺-dependent XDH gene and the XR and endogenous XK genes. Using this recombinant strain, we characterized the enzyme activity and ability to ferment both d-xylose and lignocellulosic hydrolysate.