Cloning, expression, and characterization of an arabitol dehydrogenase and coupled with NADH oxidase for effective production of L-xylulose.

A novel arabitol dehydrogenase (ArDH) gene was cloned from a bacterium named Aspergillus nidulans and expressed heterologously in Escherichia coli. The purified ArDH exhibited the maximal activity in pH 9.5 Tris-HCl buffer at 40 °C, showed Km and Vmax of 1.2 mg/mL and 9.1 U/mg, respectively. The ArDH was used to produce the L-xylulose and coupled with the NADH oxidase (Nox) for the regeneration of NAD+. In further optimization, a high conversion of 84.6% in 8 hours was achieved under the optimal conditions: 20 mM of xylitol, 100 µM NAD+ in pH 9.0 Tris-HCl buffer at 30 °C. The results indicated the coupling system with cofactor regeneration provides a promising approach for L-xylulose production from xylitol.

Interaction of (-)-Epigallocatechin Gallate and Deoxyosones Blocking the Subsequent Maillard Reaction and Improving the Yield of N-(1-Deoxy-d-xylulos-1-yl)alanine.

(-)-Epigallocatechin gallate (EGCG) had a significant effect on Maillard reaction intermediate formation in the xylose/alanine model system. A trapping effect of EGCG on the reactive deoxyosones was observed to change the reaction pathways. The rate constant of Amadori rearrangement product (ARP) conversion to deoxyosones was decreased with EGCG addition, indicating an inhibition of ARP degradation. Dehydration improved the ARP formation during the thermal reaction and synergistically improved the yield of ARP with the EGCG trapping effect on the deoxyosones. Additionally, EGCG decreased the activation energy for the conversion of xylose/alanine to ARP (from 77.8 to 62.8 kJ/mol) and in turn accelerated the ARP formation. The effect of EGCG was further facilitated at the optimal conditions of 90 °C, at pH 7.5, and a molar ratio of xylose to alanine of 2:1, which improved the yield of ARP (N-(1-deoxy-d-xylulos-1-yl)alanine) from 2 to 95%.

Modeling of Dolichol Mass Spectra Isotopic Envelopes as a Tool to Monitor Isoprenoid Biosynthesis.

The cooperation of the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways, operating in parallel in plants to generate isoprenoid precursors, has been studied extensively. Elucidation of the isoprenoid metabolic pathways is indispensable for the rational design of plant and microbial systems for the production of industrially valuable terpenoids. Here, we describe a new method, based on numerical modeling of mass spectra of metabolically labeled dolichols (Dols), designed to quantitatively follow the cooperation of MVA and MEP reprogrammed upon osmotic stress (sorbitol treatment) in Arabidopsis (Arabidopsis thaliana). The contribution of the MEP pathway increased significantly (reaching 100%) exclusively for the dominating Dols, while for long-chain Dols, the relative input of the MEP and MVA pathways remained unchanged, suggesting divergent sites of synthesis for dominating and long-chain Dols. The analysis of numerically modeled Dol mass spectra is a novel method to follow modulation of the concomitant activity of isoprenoid-generating pathways in plant cells; additionally, it suggests an exchange of isoprenoid intermediates between plastids and peroxisomes.

A dynamic flux balance model and bottleneck identification of glucose, xylose, xylulose co-fermentation in Saccharomyces cerevisiae.

A combination of batch fermentations and genome scale flux balance analysis were used to identify and quantify the rate limiting reactions in the xylulose transport and utilization pathway. Xylulose phosphorylation by xylulokinase was identified as limiting in wild type Saccharomyces cerevisiae, but transport became limiting when xylulokinase was upregulated. Further experiments showed xylulose transport through the HXT family of non-specific glucose transporters. A genome scale flux balance model was developed which included an improved variable sugar uptake constraint controlled by HXT expression. Model predictions closely matched experimental xylulose utilization rates suggesting the combination of transport and xylulokinase constraints is sufficient to explain xylulose utilization limitation in S. cerevisiae.

Synergistic effect of fermentable and non-fermentable carbon sources enhances TAG accumulation in oleaginous yeast Rhodosporidium kratochvilovae HIMPA1.

Novel strategy for enhancing TAG accumulation by simultaneous utilization of fermentable and non-fermentable carbon sources as substrate for cultivation of oleaginous yeast Rhodosporidium kratochvilovae HIMPA1 were undertaken in this investigation. The yeast strain showed direct correlation between the size of lipid bodies, visualized by BODIPY stain (493-515 nm) and TAG accumulation when examined on individual fermenting and non-fermenting carbon sources and their mixtures. Maximum TAG accumulation (µm) in glucose (2.38 ± 0.52), fructose (4.03 ± 0.38), sucrose (4.24 ± 0.45), glycerol (4.35 ± 0.54), xylulose (3.94 ± 0.12), and arabinose (2.98 ± 0.43) were observed. Synergistic effect of the above carbon sources (fermentable and non-fermentable) in equimolar concentration revealed maximum lipid droplet size of 5.35 ± 0.76 µm and cell size of 6.89 ± 0.97 µm. Total lipid content observed in mixed carbon sources was 9.26 g/l compared to glucose (6.2g/l). FAME profile revealed enhanced longer chain (C14:0-C24:0) fatty acids in mix carbon sources.

Production of rare sugars from common sugars in subcritical aqueous ethanol.

A new isomerization reaction was developed to synthesize rare ketoses. D-tagatose, D-xylulose, and D-ribulose were obtained in the maximum yields of 24%, 38%, and 40%, respectively, from the corresponding aldoses, D-galactose, D-xylose, and D-ribose, by treating the aldoses with 80% (v/v) subcritical aqueous ethanol at 180°C. The maximum productivity of D-tagatose was ca. 80 g/(Lh). Increasing the concentration of ethanol significantly increased the isomerization of D-galactose. Variation in the reaction temperature did not significantly affect the production of D-tagatose from D-galactose. Subcritical aqueous ethanol converted both 2,3-threo and 2,3-erythro aldoses to the corresponding C-2 ketoses in high yields. Thus, the treatment of common aldoses in subcritical aqueous ethanol can be regarded as a new method to synthesize the corresponding rare sugars.

Alteration of the flexible loop in 1-deoxy-D-xylulose-5-phosphate reductoisomerase boosts enthalpy-driven inhibition by fosmidomycin.

1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), which catalyzes the first committed step in the 2-C-methyl-d-erythritol 4-phosphate pathway of isoprenoid biosynthesis used by Mycobacterium tuberculosis and other infectious microorganisms, is absent in humans and therefore an attractive drug target. Fosmidomycin is a nanomolar inhibitor of DXR, but despite great efforts, few analogues with comparable potency have been developed. DXR contains a strictly conserved residue, Trp203, within a flexible loop that closes over and interacts with the bound inhibitor. We report that while mutation to Ala or Gly abolishes activity, mutation to Phe and Tyr only modestly impacts kcat and Km. Moreover, pre-steady-state kinetics and primary deuterium kinetic isotope effects indicate that while turnover is largely limited by product release for the wild-type enzyme, chemistry is significantly more rate-limiting for W203F and W203Y. Surprisingly, these mutants are more sensitive to inhibition by fosmidomycin, resulting in Km/Ki ratios up to 19-fold higher than that of wild-type DXR. In agreement, isothermal titration calorimetry revealed that fosmidomycin binds up to 11-fold more tightly to these mutants. Most strikingly, mutation strongly tips the entropy-enthalpy balance of total binding energy from 50% to 75% and 91% enthalpy in W203F and W203Y, respectively. X-ray crystal structures suggest that these enthalpy differences may be linked to differences in hydrogen bond interactions involving a water network connecting fosmidomycin's phosphonate group to the protein. These results confirm the importance of the flexible loop, in particular Trp203, in ligand binding and suggest that improved inhibitor affinity may be obtained against the wild-type protein by introducing interactions with this loop and/or the surrounding structured water network.

pH-rate profiles of L-arabinitol 4-dehydrogenase from Hypocrea jecorina and its application in L-xylulose production.

l-Arabinitol 4-dehydrogenase (LAD) from Hypocrea jecorina (HjLAD) was cloned and overexpressed in Escherichia coli BL21 (DE3). The kinetics of l-arabinitol oxidation by NAD(+), catalyzed by HjLAD, was studied within the pH range of 7.0-9.5 at 25°C. The turnover number (kcat) and the catalytic efficiency (kcat/Km) were 4200min(-1) and 290mM(-1)min(-1), respectively. HjLAD showed the highest turnover number and catalytic efficiency among all previously characterized LADs. In further application of HjLAD, rare l-sugar l-xylulose was produced by the enzymatic oxidation of arabinitol to give a yield of approximately 86%.

Gene cloning and characterization of L-ribulose 3-epimerase from Mesorhizobium loti and its application to rare sugar production.

A gene encoding L-ribulose 3-epimerase (L-RE) from Mesorhizobium loti, an important enzyme for rare sugar production by the Izumoring strategy, was cloned and overexpressed. The enzyme showed highest activity toward L-ribulose (230 U/mg) among keto-pentoses and keto-hexoses. This is the first report on a ketose 3-epimerase showing highest activity toward keto-pentose. The optimum enzyme reaction conditions for L-RE were determined to be sodium phosphate buffer (pH 8.0) at 60 °C. The enzyme showed of higher maximum reaction a rate (416 U/mg) and catalytic efficiency (43 M(-1) min(-1)) for L-ribulose than other known ketose 3-epimerases. It was able to produce L-xylulose efficiently from ribitol in two-step reactions. In the end, 7.2 g of L-xylulose was obtained from 20 g of ribitol via L-ribulose at a yield of 36%.

Insights into the isomerization of xylose to xylulose and lyxose by a Lewis acid catalyst.

We present electronic structure calculations on the isomerization and epimerization of xylose to xylulose and lyxose, respectively, by a zeolite Sn-BEA model at the MP2 and B3LYP theory levels. Benchmarking calculations at the CCSD(T) theory level are also presented. We show that lyxose is formed from a stable intermediate in the xylose-xylulose isomerization pathway. In agreement with experimental observations, we predict that xylulose is thermodynamically and kinetically favoured over lyxose. We find that the slowest step for both reactions involves hydrogen transfer from the C2 to the C1 carbon of the carbohydrate molecule and we characterize it using natural population analysis. We conclude that the hydrogen transfer does not take place as a hydride ion but rather as concerted neutral hydrogen-electron transfer that involves different centres for the hydrogen and electron transfer.

Cloning and characterization of a novel NAD+ -dependent xylitol dehydrogenase from Gluconobacter oxydans CGMCC 1. 637.

OBJECTIVE: To clone the xylitol dehydrogenase gene from Gluconobacter oxydans CGMCC 1.637, to characterize enigmatic properties of xylitol dehydrogenase and to investigate the induction abilities of various carbon sources on the oxidative activity of xylitol dehydrogenase and the effect of various carbon sources on the bioconversion of d-xylulose to xylitol in G. oxydans CGMCC 1.637. METHODS: Touch-down polymerase chain reaction (PCR) was applied to clone the xylitol dehydrogenase gene from chromosomal DNA of G. oxydans CGMCC 1.637. RESULTS: The 798-bp open reading frame of xylitol dehydrogenase encoded a protein of 265 amino acids, with the molecular mass of 27.95 kDa. Sequence analysis of the putative protein revealed it to be a member of short-chain dehydrogenase/reductase family. Xylitol dehydrogenase showed oxidative activity with xylitol and sorbitol and no activity with other polyols, such as d-arabitol. K(m) and V(max) with xylitol was 78.97 mmol/L and 40.17 U/mg, respectively. The highest oxidative activity of xylitol dehydrogenase for xylitol was only 23.27 U/mg under optimum conditions (pH 10.0, 35 degrees C). However, the activity of its reverse reaction, d-xylulose reduction, reached 255.55 U/mg under optimum conditions (pH 6.0, 30 degrees C), 10-times higher than that of xylitol oxidation. Oxidative activity of xylitol dehydrogenase was induced when G. oxydans CGMCC 1.637 was cultivated on d-sorbitol. D-arabitol, which supported a high cell growth, inhibited the oxidative activity of xylitol dehydrogenase and the bioconversion ability of G. oxydans CGMCC 1.637. CONCLUSIONS: The obtained gene from G. oxydans CGMCC 1.637 was a novel gene encoding xylitol dehydrogenase. Oxidative activity of xylitol dehydrogenase in G. oxydans CGMCC 1.637 and the bioconversion ability of G. oxydans CGMCC 1.637 after grown on d-arabitol were inhibited, which provided a valuable clue for further study to increase xylitol yield from d-arabitol.

Synthesis of furfural from xylose, xylan, and biomass using AlCl3·6H2O in biphasic media via xylose isomerization to xylulose.

Furfural was prepared in high yields (75 %) from the reaction of xylose in a water-tetrahydrofuran biphasic medium containing AlCl(3)·6H2O and NaCl under microwave heating at 140 °C. The reaction profile revealed the formation of xylulose as an intermediate en route to the dehydration product (furfural). The reaction under these conditions reached completion in 45 min. The aqueous phase containing AlCl(3)·6H(2)O and NaCl could be recycled multiple times (>5) without any loss of activity or selectivity for furfural. Extension of this biphasic reaction system to include xylan as the starting material afforded furfural in 64 % yield. The use of corn stover, pinewood, switchgrass, and poplar gave furfural in 55, 38, 56, and 64 % yield, respectively, at 160 °C. Even though AlCl(3)·6H(2)O did not affect the conversion of crystalline cellulose, moderate yields of the by-product 5-hydroxymethylfurfural (HMF) were noted. The highest HMF yield of 42 % was obtained from pinewood. The coproduction of HMF and furfural from biomass was attributed to the weakening of the cellulose network in the biomass, as a result of hemicellulose hydrolysis. The multifunctional capacity of AlCl(3)·6H(2)O (hemicellulose hydrolysis, xylose isomerization, and xylulose dehydration) in combination with its ease of recyclability make it an attractive candidate/catalyst for the selective synthesis of furfural from various biomass feedstocks.

Glycerol conversion to D-xylulose by a two-stage microbial reaction using Candida parapsilosis and Gluconobacter oxydans.

A yeast strain, 25N-2B, that produces D-arabitol from glycerol, was identified as Candida parapsilosis based on phylogenetic, morphological, physiological, and biochemical analyses. It produced 32.2 g/L D-arabitol from 170 g/L glycerol in a jar fermentor. The D-arabitol in the reaction mixture was then completely converted to D-xylulose using Gluconobacter oxydans NBRC3293. The product was isolated from the reaction mixture and confirmed to be D-xylulose by (1)H and (13)C-NMR and optical rotation.

Hydrogen location in stages of an enzyme-catalyzed reaction: time-of-flight neutron structure of D-xylose isomerase with bound D-xylulose.

The time-of-flight neutron Laue technique has been used to determine the location of hydrogen atoms in the enzyme d-xylose isomerase (XI). The neutron structure of crystalline XI with bound product, d-xylulose, shows, unexpectedly, that O5 of d-xylulose is not protonated but is hydrogen-bonded to doubly protonated His54. Also, Lys289, which is neutral in native XI, is protonated (positively charged), while the catalytic water in native XI has become activated to a hydroxyl anion which is in the proximity of C1 and C2, the molecular site of isomerization of xylose. These findings impact our understanding of the reaction mechanism.

Biosynthesis of isoprenoids via the non-mevalonate pathway.

The mevalonate pathway for the biosynthesis of the universal terpenoid precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), is known in considerable detail. Only recently, the existence of a second mevalonate-independent pathway for the biosynthesis of IPP and DMAPP was detected in plants and certain eubacteria. Experiments with 13C and/or 2H-labelled precursors were crucial in the elucidation of this novel route. The pathway is essential in plants, many eubacteria and apicomplexan parasites, but not in archaea and animals. The genes, enzymes and intermediates of this pathway were rapidly unravelled over the past few years. Detailed knowledge about the mechanisms of this novel route may benefit the development of novel antibiotics, antimalarials and herbicides.

A novel NADH-linked l-xylulose reductase in the l-arabinose catabolic pathway of yeast.

An NADH-dependent l-xylulose reductase and the corresponding gene were identified from the yeast Ambrosiozyma monospora. The enzyme is part of the yeast pathway for l-arabinose catabolism. A fungal pathway for l-arabinose utilization has been described previously for molds. In this pathway l-arabinose is sequentially converted to l-arabinitol, l-xylulose, xylitol, and d-xylulose and enters the pentose phosphate pathway as d-xylulose 5-phosphate. In molds the reductions are NADPH-linked, and the oxidations are NAD(+)-linked. Here we show that in A. monospora the pathway is similar, i.e. it has the same two reduction and two oxidation reactions, but the reduction by l-xylulose reductase is not performed by a strictly NADPH-dependent enzyme as in molds but by a strictly NADH-dependent enzyme. The ALX1 gene encoding the NADH-dependent l-xylulose reductase is strongly expressed during growth on l-arabinose as shown by Northern analysis. The gene was functionally overexpressed in Saccharomyces cerevisiae and the purified His-tagged protein characterized. The reversible enzyme converts l-xylulose to xylitol. It also converts d-ribulose to d-arabinitol but has no activity with l-arabinitol or adonitol, i.e. it is specific for sugar alcohols where, in a Fischer projection, the hydroxyl group of the C-2 is in the l-configuration and the hydroxyl group of C-3 is in the d-configuration. It also has no activity with C-6 sugars or sugar alcohols. The K(m) values for l-xylulose and d-ribulose are 9.6 and 4.7 mm, respectively. To our knowledge this is the first report of an NADH-linked l-xylulose reductase.

Rapid stimulation of free glucuronate formation by non-glucuronidable xenobiotics in isolated rat hepatocytes.

Vitamin C synthesis in rat liver is enhanced by several xenobiotics, including aminopyrine and chloretone. The effect of these agents has been linked to induction of enzymes potentially involved in the formation of glucuronate, a precursor of vitamin C. Using isolated rat hepatocytes as a model, we show that a series of agents (aminopyrine, antipyrine, chloretone, clotrimazole, metyrapone, proadifen, and barbital) induced in a few minutes an up to 15-fold increase in the formation of glucuronate, which was best observed in the presence of sorbinil, an inhibitor of glucuronate reductase. They also caused an approximately 2-fold decrease in the concentration of UDP-glucuronate but little if any change in the concentration of UDP-glucose. Depletion of UDP-glucuronate with resorcinol or d-galactosamine markedly decreased the formation of glucuronate both in the presence and in the absence of aminopyrine, confirming the precursor-product relationship between UDP-glucuronate and free glucuronate. Most of the agents did not induce the formation of detectable amounts of glucuronides, indicating that the formation of glucuronate is not due to a glucuronidation-deglucuronidation cycle. With the exception of barbital (which inhibits glucuronate reductase), all of the above mentioned agents also caused an increase in the concentration of ascorbic acid. They had little effect on glutathione concentration, and their effect on glucuronate and vitamin C formation was not mimicked by glutathione-depleting agents such as diamide and buthionine sulfoximine. It is concluded that the stimulation of vitamin C synthesis exerted by some xenobiotics is mediated through a rapid increase in the conversion of UDP-glucuronate to glucuronate, which does not apparently involve a glucuronidation-deglucuronidation cycle.

Catalytic asymmetric synthesis of a 1-deoxy-1,1-difluoro-D-xylulose.

[reaction: see text] A new route, of potential strategic importance, to a difluorosugar analogue has been developed. Key steps included a Stille coupling and a highly regio- and enantioselective dihydroxylation of a highly substituted diene. Protecting groups were chosen to enhance the reactivity of the disubstituted allylic fragment in the AD reaction and allow deprotection under orthogonal conditions.

Feeding of [5,5-2H(2)]-1-desoxy-D-xylulose and [4,4,6,6,6-2H(5)]-mevalolactone to a geosmin-producing Streptomyces sp. and Fossombronia pusilla.

The biosynthesis of the trisnor sesquiterpenoid geosmin (4,8a-dimethyl-octahydro-naphthalen-4a-ol) (1) was investigated by feeding labeled [5,5-2H(2)]-1-desoxy-D-xylulose (11), [4,4,6,6,6-(2)H(5)]-mevalolactone (7) and [2,2-2H(2)]-mevalolactone (9) to Streptomyces sp. JP95 and the liverwort Fossombronia pusilla. The micro-organism produced geosmin via the 1-desoxy-D-xylulose pathway, whereas the liverwort exclusively utilized mevalolactone for terpenoid biosynthesis. Analysis of the labeling pattern in the resulting isotopomers of geosmin (1) by mass spectroscopy (EI/MS) revealed that geosmin is synthesized in both organisms by cyclization of farnesyl diphosphate to a germacradiene-type intermediate 4. Further transformations en route to geosmin (1) involve an oxidative dealkylation of an i-propyl substituent, 1,2-reduction of a resulting conjugated diene, and bicyclization of a germacatriene intermediate 13. The transformations largely resemble the biosynthesis of dehydrogeosmin (2) in cactus flowers but differ with respect to the regioselectivity of the side chain dealkylation and 1,2-reduction