Metabolic control analysis of Aspergillus niger L-arabinose catabolism

A mathematical model of the L-arabinose/D-xylose catabolic pathway of Aspergillus niger was constructed based on the kinetic properties of the enzymes. For this purpose L-arabinose reductase, L-arabitol dehydrogenase and D-xylose reductase were purified using dye-affinity chromatography, and their kinetic properties were characterized. For the other enzymes of the pathway the kinetic data were available from the literature. The metabolic model was used to analyze flux and metabolite concentration control of the L-arabinose catabolic pathway. The model demonstrated that flux control does not reside at the enzyme following the intermediate with the highest concentration, L-arabitol, but is distributed over the first three steps in the pathway, preceding and following L-arabitol. Flux control appeared to be strongly dependent on the intracellular L-arabinose concentration. At 5 mM intracellular L-arabinose, a level that resulted in realistic intermediate concentrations in the model, flux control coefficients for L-arabinose reductase, L-arabitol dehydrogenase and L-xylulose reductase were 0.68, 0.17 and 0.14, respectively. The analysis can be used as a guide to identify targets for metabolic engineering aiming at either flux or metabolite level optimization of the L-arabinose catabolic pathway of A. niger. Faster L-arabinose utilization may enhance utilization of readily available organic waste containing hemicelluloses to be converted into industrially interesting metabolites or valuable enzymes or proteins.     

Production of ethanol from L-arabinose by Saccharomyces cerevisiae containing a fungal L-arabinose pathway

The fungal pathway for L-arabinose catabolism converts L-arabinose to D-xylulose 5-phosphate in five steps. The intermediates are, in this order: L-arabinitol, L-xylulose, xylitol and D-xylulose. Only some of the genes for the corresponding enzymes were known. We have recently identified the two missing genes for L-arabinitol 4-dehydrogenase and L-xylulose reductase and shown that overexpression of all the genes of the pathway in Saccharomyces cerevisiae enables growth on L-arabinose. Under anaerobic conditions ethanol is produced from L-arabinose, but at a very low rate. The reasons for the low rate of L-arabinose fermentation are discussed.     

L-arabinose metabolism in Azospirillum brasiliense

An oxidative pathway by which L-arabinose is converted to alpha-ketoglutarate in crude extracts of Azospirillum brasiliense is demonstrated. Specific activities of enzymes involved in the pathway were determined, and several pathway intermediates were identified.     

Characterization of an Aspergillus nidulans L-arabitol dehydrogenase mutant

Abstract The degradation pathway for L-arabinose, which consists of a sequence of alternating reduction and oxidation reactions prior to ultimate phosphorylation, was studied in Aspergillus nidulans wild-type as well as in an L-arabinose non-utilizing mutant. The inability of the mutant to use L-arabinose was caused by the absence of L-arabitol dehydrogenase activity. The effect of the mutation on polyol accumulation patterns was studied upon growth on various carbon sources. The presence of L-arabinose resulted in intracellular accumulation of arabitol in this mutant. Moreover, the mutant secreted arabitol under these conditions and, in contrast to the wild-type, featured enhanced expression of enzymes involved in L-arabinose catabolism as well as of extracellular glycosyl hydrolases involved in degradation of the plant cell wall polysaccharide L-arabinan.     

alpha-Ketoglutarate dehydrogenase mutant of Rhizobium meliloti.

A mutant of Rhizobium meliloti selected as unable to grow on L-arabinose also failed to grow on acetate or pyruvate. It grew, but slower than the parental strain, on many other carbon sources. Assay showed it to lack alpha-ketoglutarate dehydrogenase (kgd) activity, and revertants of normal growth phenotype contained the activity again. Other enzymes of the tricarboxylic acid cycle and of the glyoxylate cycle were present in both mutant and parent strains. Enzymes of pyruvate metabolism were also assayed. L-Arabinose degradation in R. meliloti was found to differ from the known pathway in R. japonicum, since the former strain lacked 2-keto-o-deoxy-L-arabonate aldolase but contained alpha-ketoglutarate semialdehyde dehydrogenase; thus, it is likely that R. meliloti has the L-arabinose pathway leading to alpha-ketoglutarate rather than the one to glycolaldehyde and pyruvate. This finding accounts for the L-arabinose negativity of the mutant. Resting cells of the mutant were able to metabolize the three substrates which did not allow growth.     

The missing link in the fungal L-arabinose catabolic pathway,identification of the L-xylulose reductase gene

The fungal L-arabinose pathway consists of five enzymes, aldose reductase, L-arabinitol 4-dehydrogenase, L-xylulose reductase, xylitol dehydrogenase, and xylulokinase. All the genes encoding the enzymes of this pathway are known except for that of L-xylulose reductase (EC 1.1.1.10). We identified a gene encoding this enzyme from the filamentous fungus Trichoderma reesei (Hypocrea jecorina). The gene was named lxr1. It was overexpressed in the yeast Saccharomyces cerevisiae, and the enzyme activity was confirmed in a yeast cell extract. Overexpression of all enzymes of the L-arabinose pathway in S. cerevisiae led to growth of S. cerevisiae on L-arabinose; i.e., we could show that the pathway is active in a heterologous host. The lxr1 gene encoded a protein with 266 amino acids and a calculated molecular mass of 28 428 Da. The LXRI protein is an NADPH-specific reductase. It has activity with L-xylulose, D-xylulose, D-fructose, and L-sorbose. The highest affinity is toward L-xylulose (K(m) = 16 mM). In the reverse direction, we found activity with xylitol, D-arabinitol, D-mannitol, and D-sorbitol. It requires a bivalent cation for activity. It belongs to the protein family of short chain dehydrogenases. The enzyme is catalytically similar and homologous in sequence to a D-mannitol:NADP 2-dehydrogenase (EC 1.1.1.138).     

Characterization of the mmsAB-araD1 (gguABC) genes of Agrobacterium tumefaciens

The chvE-gguABC operon plays a critical role in both virulence and sugar utilization through the activities of the periplasmic ChvE protein, which binds to a variety of sugars. The roles of the GguA, GguB, and GguC are not known. While GguA and GguB are homologous to bacterial ABC transporters, earlier genetic analysis indicated that they were not necessary for utilization of sugars as the sole carbon source. To further examine this issue, in-frame deletions were constructed separately for each of the three genes. Our growth analysis clearly indicated that GguA and GguB play a role in sugar utilization and strongly suggests that GguAB constitute an ABC transporter with a wide range of substrates, including L-arabinose, D-fucose, D-galactose, D-glucose, and D-xylose. Site-directed mutagenesis showed that a Walker A motif was vital to the function of GguA. We therefore propose renaming gguAB as mmsAB, for multiple monosaccharide transport. A gguC deletion affected growth only on L-arabinose medium, suggesting that gguC encodes an enzyme specific to L-arabinose metabolism, and this gene was renamed araD1. Results from bioinformatics and experimental analyses indicate that Agrobacterium tumefaciens uses a pathway involving nonphosphorylated intermediates to catabolize L-arabinose via an L-arabinose dehydrogenase, AraA(At), encoded at the Atu1113 locus.     

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.     

L-Arabinose transport and catabolism in yeast

Two yeasts, Candida arabinofermentans PYCC 5603(T) and Pichia guilliermondii PYCC 3012, which show rapid growth on L-arabinose and very high rates of L-arabinose uptake on screening, were selected for characterization of L-arabinose transport and the first steps of intracellular L-arabinose metabolism. The kinetics of L-arabinose uptake revealed at least two transport systems with distinct substrate affinities, specificities, functional mechanisms and regulatory properties. The L-arabinose catabolic pathway proposed for filamentous fungi also seems to operate in the yeasts studied. The kinetic parameters of the initial L-arabinose-metabolizing enzymes were determined. Reductases were found to be mostly NADPH-dependent, whereas NAD was the preferred cofactor of dehydrogenases. The differences found between the two yeasts agree with the higher efficiency of L-arabinose metabolism in C. arabinofermentans. This is the first full account of the initial steps of L-arabinose catabolism in yeast including the biochemical characterization of a specific L-arabinose transporter.     

Activity of pentose phosphate pathway enzymes in alkane-oxidizing yeast cells]

The activity of the key enzymes of the pentose phosphate pathway (glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, transketolase) was determined in cell-free homogenates of Candida lipolytica 695 and Candida tropicalis 303 growing on different carbon sources. The activity of these enzymes remained almost the same in the course of growth of both cultures. The activity of the enzymes differed only slightly in the cells metabolizing hexadecane and glucose. The activity of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the cell-free homogenates of C. tropicalis 303 was twice as high as in the cells of C. lipolytica 695. The activity of transketolase was the same in both cultures. The main role of the pentose phosphate pathway is presumed to consist not in catabolism of the carbon source, but in biosynthesis of pentoses and other important intermediates.     

Thiobacillus acidophilus sp. nov.; isolation and some physiological characteristics.

After a brief exposition to glucose, Thiobacillus acidophilus was isolated from a culture of iron-grown T. ferrooxidans. Physicochemical analysis of its DNA showed a G+C content of 62.9-63.2%. The new isolate grows best at 25-30 degrees C and at pH 3.0. Growth is possible between pH 1.5 and 6.0. Thiobacillus acidophilus is apparently strictly aerobic. Ammonium salts are the only suitable source of nitrogen. The bacterium is a facultative autotroph. In addition to elemental sulfur, it obtains energy from organic compounds such as D-glucose, D-galactose, D-fructose, D-mannitol, D-xylose, D-ribose, D-arabinose, L-arabinose, sucrose, sodium citrate, malic acid,dl-aspartic acid, and dl-glutamic acid. Thiobacillus acidophilus possesses the key enzymes of the tricarboxylic acid (TCA) cycle including NAD-and NADP-linked isocitric dehydrogenase and alpha-ketoglutarate dehydrogenase, and the key enzymes of the hexose monophosphate pathway (glucose-6-phosphate and 6-phosphogluconate dehydrogenase, and fructose 1,6-diphosphate aldolase). NADH oxidase has been found in particulate fraction of extracts. Rhodanese and thiosulfate oxidase have also been detected.     

Interaction of aspartate and aspartate-derived antimetabolites with the enzymes of the threonine biosynthetic pathway of Escherichia coli

The five enzymes responsible for the conversion of L-aspartate to L-threonine in Escherichia coli were purified to homogeneity and subsequently reconstituted in vitro in ratios approximating those found in vivo. 31P NMR was used to conveniently monitor the rates of consumption of the substrates ATP and NADPH, the accumulation of the intermediates beta-aspartyl phosphate and homoserine phosphate, and the formation of the products ADP, NADP+, and Pi in a single experiment. By this method, the flux of aspartic acid through the enzymes of the pathway was monitored in the absence and in the presence of several alternative substrates and inhibitors. Several known antimetabolites were found to be alternative substrates that ultimately became inhibitors of pathway flux. L-threo-3-Hydroxyaspartic acid was converted to 3-hydroxyhomoserine phosphate by the first four enzymes of the pathway. The antimetabolite L-threo-3-hydroxyhomoserine was found to bind to and inhibit aspartokinase-homoserine dehydrogenase I in a cooperative fashion (I 0.5 = 3 mM, nH = 2.5), similar to the action of the allosteric end product inhibitor L-threonine (I 0.5 = 0.36 mM, nH = 2.4). In the presence of the remaining enzymes of the pathway, however, L-threo-3-hydroxyhomoserine was phosphorylated to the apparent ultimate antimetabolite L-threo-3-hydroxyhomoserine phosphate that was a potent inhibitor of threonine synthase and consequently of L-threonine biosynthesis. When aspartic acid alone was examined as a substrate of the enzymes of the pathway, no accumulation of the beta-aspartyl phosphate and homoserine phosphate intermediates was observed. However, in the presence of either 5 mM L-threo-3-hydroxyhomoserine or 5 mM L-threo-3-hydroxyhomoserine phosphate, homoserine phosphate was found to accumulate. In contrast to the homoserine phosphate and 3-hydroxyhomoserine phosphate intermediates, both of which were very stable, the acylphosphate intermediates beta-aspartyl phosphate and beta-3-hydroxyaspartyl phosphate were highly susceptible to hydrolysis, with first-order rate constants of 4.6 X 10(-3) min-1 and 4.5 X 10(-2) min-1 (pH 7.8, 25 degrees C), respectively.     

Evidence for a pentose phosphate pathway in Helicobacter pylori

Abstract Evidence for the presence of enzymes of the pentose phosphate pathway in Helicobacter pylori was obtained using ³¹P nuclear magnetic resonance spectroscopy. Activities of enzymes which are part of the oxidative and non-oxidative phases of the pathway were observed directly in incubations of bacterial lysates with pathway intermediates. Generation of NADPH and 6-phosphogluconate from NADP⁺ and glucose 6-phosphate indicated the presence of glucose 6-phosphate dehydrogenase and 6-phosphogluconolactonase. Reduction of NADP⁺ with production of ribulose 5-phosphate from 6-phosphogluconate revealed 6-phosphogluconate dehydrogenase activity. Phosphopentose isomerase and transketolase activities were observed in incubations containing ribulose 5-phosphate and xylulose 5-phosphate, respectively. The formation of erythrose 4-phosphate from xylulose 5-phosphate and ribose 5-phosphate suggested the presence of transaldolase. The activities of this enzyme and triosephosphate isomerase were observed directly in incubations of bacterial lysates with dihydroxyacetone phosphate and sedoheptulose 7-phosphate. Glucose-6-phosphate isomerase activity was measured in incubations with fructos 6-phosphate. The presence of these enzymes in H. pylori suggested the existence of a pentose phosphate pathway in the bacterium, possibly as a mechanism to provide NADPH for reductive biosynthesis and ribose 5-phosphate for synthesis of nucleic acids.     

Genetic analysis of a novel pathway for D-xylose metabolism in Caulobacter crescentus

Genetic data suggest that the oligotrophic freshwater bacterium Caulobacter crescentus metabolizes D-xylose through a pathway yielding alpha-ketoglutarate, comparable to the recently described L-arabinose degradation pathway of Azospirillum brasilense. Enzymes of the C. crescentus pathway, including an NAD(+)-dependent xylose dehydrogenase, are encoded in the xylose-inducible xylXABCD operon (CC0823-CC0819).     

Aerobic benzoyl-coenzyme A (CoA) catabolic pathway in Azoarcus evansii: conversion of ring cleavage product by 3,4-dehydroadipyl-CoA semialdehyde dehydrogenase

Benzoate, a strategic intermediate in aerobic aromatic metabolism, is metabolized in various bacteria via an unorthodox pathway. The intermediates of this pathway are coenzyme A (CoA) thioesters throughout, and ring cleavage is nonoxygenolytic. The fate of the ring cleavage product 3,4-dehydroadipyl-CoA semialdehyde was studied in the beta-proteobacterium Azoarcus evansii. Cell extracts contained a benzoate-induced, NADP(+)-specific aldehyde dehydrogenase, which oxidized this intermediate. A postulated putative long-chain aldehyde dehydrogenase gene, which might encode this new enzyme, is located on a cluster of genes encoding enzymes and a transport system required for aerobic benzoate oxidation. The gene was expressed in Escherichia coli, and the maltose-binding protein-tagged enzyme was purified and studied. It is a homodimer composed of 54 kDa (without tag) subunits and was confirmed to be the desired 3,4-dehydroadipyl-CoA semialdehyde dehydrogenase. The reaction product was identified by nuclear magnetic resonance spectroscopy as the corresponding acid 3,4-dehydroadipyl-CoA. Hence, the intermediates of aerobic benzoyl-CoA catabolic pathway recognized so far are benzoyl-CoA; 2,3-dihydro-2,3-dihydroxybenzoyl-CoA; 3,4-dehydroadipyl-CoA semialdehyde plus formate; and 3,4-dehydroadipyl-CoA. The further metabolism is thought to lead to 3-oxoadipyl-CoA, the intermediate at which the conventional and the unorthodox pathways merge.     

Functional expression and extension of staphylococcal staphyloxanthin biosynthetic pathway in Escherichia coli

The biosynthetic pathway for staphyloxanthin, a C(30) carotenoid biosynthesized by Staphylococcus aureus, has previously been proposed to consist of five enzymes (CrtO, CrtP, CrtQ, CrtM, and CrtN). Here, we report a missing sixth enzyme, 4,4'-diaponeurosporen-aldehyde dehydrogenase (AldH), in the staphyloxanthin biosynthetic pathway and describe the functional expression of the complete staphyloxanthin biosynthetic pathway in Escherichia coli. When we expressed the five known pathway enzymes through artificial synthetic operons and the wild-type operon (crtOPQMN) in E. coli, carotenoid aldehyde intermediates such as 4,4'-diaponeurosporen-4-al accumulated without being converted into staphyloxanthin or other intermediates. We identified an aldH gene located 670 kilobase pairs from the known staphyloxanthin gene cluster in the S. aureus genome and an aldH gene in the non-staphyloxanthin-producing Staphylococcus carnosus genome. These two putative enzymes catalyzed the missing oxidation reaction to convert 4,4'-diaponeurosporen-4-al into 4,4'-diaponeurosporenoic acid in E. coli. Deletion of the aldH gene in S. aureus abolished staphyloxanthin biosynthesis and caused accumulation of 4,4'-diaponeurosporen-4-al, confirming the role of AldH in staphyloxanthin biosynthesis. When the complete staphyloxanthin biosynthetic pathway was expressed using an artificial synthetic operon in E. coli, staphyloxanthin-like compounds, which contained altered fatty acid acyl chains, and novel carotenoid compounds were produced, indicating functional expression and coordination of the six staphyloxanthin pathway enzymes.     

Regulation of growth of Acinetobacter calcoaceticus NCIB8250 on L-mandelate in batch culture.

Batch culture of Acinetobacter calcoaceticus in L-mandelate- or phenylglyoxylate-salts medium showed an unusual non-exponential pattern unless the inoculum had been grown on benzyl alcohol. There were transient accumulations of benzaldehyde and benzyl alcohol caused by the limitation of L-mandelate oxidation by low activities of benzaldehyde dehydrogenase and the diversion of reducing power to the formation of benzyl alcohol. In vivo enzymic activities were estimated from patterns of substrate utilization in batch cultures containing pairs of substrates. When bacteria previously grown in L-mandelate-salts medium were inoculated into media containing L-mandelate and a second carbon source, metabolism of L-mandelate was arithmetical in the presence of benzoate, catechol or succinate, but accelerated on exhaustion of the second substrate. This indicated repression of the enzymes involved in L-mandelate oxidation. Inoculation of bacteria grown in benzoate-salts medium into medium containing L-mandelate and benzoate gave diauxie with initial utilization of benzoate. Similar experiments showed that benzoate oxidation was not repressed by catechol and only partially repressed by succinate. Measurement of L-mandelate dehydrogenase, phenylglyoxylate carboxy-lyase and benzaldehyde dehydrogenase I in bacterial extracts showed no evidence for feedback inhibition by intermediates of the pathway. The rates of L-mandelate and benzoate utilization by bacterial suspensions were inhibited by succinate and catechol but not by other intermediates of the pathway.     

Characterization of enzymes involved in the central metabolism of Gluconobacter oxydans

Gluconobacter oxydans is an industrially important bacterium that lacks a complete Embden–Meyerhof pathway (glycolysis). The organism instead uses the pentose phosphate pathway to oxidize sugars and their phosphorylated intermediates. However, the lack of glycolysis limits the amount of NADH as electron donor for electron transport phosphorylation. It has been suggested that the pentose phosphate pathway contributes to NADH production. Six enzymes predicted to play central roles in intracellular glucose and gluconate flux were heterologously overproduced in Escherichia coli and characterized to investigate the intracellular flow of glucose and gluconates into the pentose phosphate pathway and to explore the contribution of the pentose phosphate pathway to NADH generation. The key pentose phosphate enzymes glucose 6-phosphate dehydrogenase (Gox0145) and 6-phosphogluconate dehydrogenase (Gox1705) had dual cofactor specificities but were physiologically NADP- and NAD-dependent, respectively. Putative glucose dehydrogenase (Gox2015) was NADP-dependent and exhibited a preference for mannose over glucose, whereas a 2-ketogluconate reductase (Gox0417) displayed dual cofactor specificity for NAD(P)H. Furthermore, a putative gluconokinase and a putative glucokinase were identified. The gluconokinase displayed high activities with gluconate and is thought to shuttle intracellular gluconate into the pentose phosphate pathway. A model for the trafficking of glucose and gluconates into the pentose phosphate pathway and its role in NADH generation is presented. The role of NADPH in chemiosmotic energy conservation is also discussed.     

Identification of nicotine biotransformation intermediates by Agrobacterium tumefaciens strain S33 suggests a novel nicotine degradation pathway

Nicotine, a major alkaloid in tobacco plants and the main toxic chemical in tobacco wastes, can be transformed by bacteria into hydroxylated-pyridine intermediates, which are important precursors for the chemical synthesis of valuable drugs and insecticides. Such biotransformation could be a useful approach to utilize tobacco and its wastes. In this study, we explored nicotine degradation by a recently isolated Agrobacterium tumefaciens S33 by identifying the intermediates during its growth on nicotine and during transformation of nicotine with its resting cells. Five hydroxylated-pyridine intermediates were detected through multiple approaches, including GC-HR-MS, HPLC, and ESI-Q-TOF MS analyses. Surprisingly, these identified intermediates suggest that strain S33 employs a novel pathway that is different from the two characterized pathways described in Arthrobacter and Pseudomonas. Based on these findings, we propose that strain S33 is able to transform nicotine to 6-hydroxy-pseudooxynicotine first via the pyridine pathway through 6-hydroxy-L: -nicotine and 6-hydroxy-N-methylmyosmine, and then, it turns to the pyrrolidine pathway with the formation of 6-hydroxy-3-succinoylpyridine and 2,5-dihydroxypyridine. The activities of the key enzymes, nicotine dehydrogenase, 6-hydroxy-L: -nicotine oxidase, and 6-hydroxy-3-succinoylpyridine hydroxylase, were demonstrated in the cell extract of strain S33 and by partially enriched enzymes. Moreover, the cell extract could transform 6-hydroxy-pseudooxynicotine into 6-hydroxy-3-succinoylpyridine by coupling with 6-hydroxy-L: -nicotine oxidation reaction by 6-hydroxy-L: -nicotine oxidase. These results indicated that strain S33 can transform nicotine into renewable hydroxylated-pyridine intermediates by the special pathway, in which at least three intermediates, 6-hydroxy-L: -nicotine, 6-hydroxy-3-succinoylpyridine, and 2,5-dihydroxypyridine, have potential to be further chemically modified into useful compounds.