Fructose Utilization Research Articles

NMR bioreactor development for live in-situ microbial functional analysis

A live, in-situ metabolomics capability was developed for prokaryotic cultures under controlled growth conditions. Toward this goal, a radiofrequency-transparent bioreactor was developed and integrated with a commercial wide-bore nuclear magnetic resonance (NMR) imaging spectrometer and a commercial bioreactor controller. Water suppressed 1H NMR spectroscopy was used to monitor glucose and fructose utilization and byproduct excretion by Eubacterium aggregans (an anaerobic bacterial species relevant for biofuel production) under controlled batch and continuous culture conditions. The resulting metabolite profiles (short chain organic acids and ethanol) and trends are consistent with existing knowledge of its metabolism. However, our study also showed that E. aggregans produces lactate end product in significant concentrations—a result not previously reported. The advantages of live in-situ microbial metabolomics analysis and its complementariness with functional genomics/systems biology methods are discussed.

EFFECTS OF CHEMICAL AND PHYSICAL ENVIRONMENTS ON CELL CULTURE OF GYMNEMA SYLVESTRE

This study was carried out to investigate the effects of chemical (medium salt strength, sucrose, plant growth regulator) and physical (inoculum size, aeration volume) environments on cell culture of Gymnema sylvestre. Strength of MS medium (1/4 MS, 1/2 MS and 1 MS) did not significantly affect cell growth, while sucrose concentration affected cell growth, showing maximal cell growth at 3% sucrose. Sucrose concentration higher than 3% inhibited cell growth due to low water potential in the medium. Different kinds and concentrations of auxin were tested for cell culture of Gymnema sylvestre. Cell growth was greatest at 5.0 mg L -1 NAA, while significantly inhibited by IBA treatment. The optimal inoculum size was 60 g L -1 as evidenced by the highest growth rate. Increasing aeration volume from 0.05 vvm to 0.4 vvm during culture period was optimal for cell growth in bioreactors. Cell growth was investigated every 5 days during the entire culture period. Fresh weight increased until 20 days in culture and dry weight increased almost 10 times after 15 days in culture. Sucrose concentration in the medium steadily decreased at growth phase (from 10 day to 15 day), almost depleted after 15 days in culture, while the utilization of glucose and fructose reached 100% after 20 days of culture. The results suggested that 15 days of culture period were optimal for Gymnema sylvestre cell culture in bioreactor. Further experiments including analysis of secondary metabolites are in process.

Iron and carbon metabolism by a mineral-oxidizing Alicyclobacillus-like bacterium

A novel iron-oxidizing, moderately thermophilic, acidophilic bacterium (strain "GSM") was isolated from mineral spoil taken from a gold mine in Montana. Biomolecular analysis showed that it was most closely related to Alicyclobacillus tolerans, although the two bacteria differed in some key respects, including the absence (in strain GSM) of varpi-alicyclic fatty acids and in their chromosomal base compositions. Isolate GSM was able to grow in oxygen-free media using ferric iron as terminal electron acceptor confirming that it was a facultative anaerobe, a trait not previously described in Alicyclobacillus spp.. The acidophile used both organic and inorganic sources of energy and carbon, although growth and iron oxidation by isolate GSM was uncoupled in media that contained both fructose and ferrous iron. Fructose utilization suppressed iron oxidation, and oxidation of ferrous iron occurred only when fructose was depleted. In contrast, fructose catabolism was suppressed when bacteria were harvested while actively oxidizing iron, suggesting that both ferrous iron- and fructose-oxidation are inducible in this acidophile. Isolate GSM accelerated the oxidative dissolution of pyrite in liquid media either free of, or amended with, organic carbon, although redox potentials were significantly different in these media. The potential of this isolate for commercial mineral processing is discussed.

Dissecting differential binding of fructose and phosphate as leaving group/nucleophile of glucosyl transfer catalyzed by sucrose phosphorylase

Site-directed mutagenesis was used to examine the specificity of Leuconostoc mesenteroides sucrose phosphorylase for utilization of fructose and phosphate as leaving group/nucleophile of the reaction. The largest catalytic defect in Arg 137 → Ala (≈60-fold) and Tyr 340 → Ala (≈2500-fold) concerned phosphate dependant half-reactions whereas that in Asp 338 → Asn (≈7000-fold) derived from disruption of steps where fructose departs or attacks. The relative efficiencies for enzyme glucosylation by sucrose compared with α- d-glucose-1-phosphate and enzyme deglucosylation by phosphate compared with fructose were 5.5 and 6.2 for wild-type, 19 and 2.0 for Arg 137 → Ala, 950 and 0.17 for Tyr 340 → Ala, and 0.05 and 180 for Asp 338 → Asn, respectively. Asp 338 and Tyr 340 have a key role in differential binding of fructose and phosphate, respectively.

Flour carbohydrate catabolism and metabolite production by sourdough lactic acid bacteria

The aim of this study was to assess the mode of carbohydrate catabolism by lactic acid bacteria isolated from traditional sourdoughs, as well as to study their effect on the metabolites produced. For this purpose, single cultures of the heterofermentative lactic acid bacteria Lactobacillus sanfranciscensis, Lactobacillus brevis, Weissella cibaria, and the homofermentative Lactobacillus paralimentarius and Pediococcus pentosaceus were grown in liquid media containing glucose, fructose, maltose and sucrose, either as a single carbon source or in combination with glucose. Carbon catabolism and the production of metabolites were determined by HPLC analysis. W. cibaria could ferment all carbon sources, L. sanfranciscensis, L. paralimentarius and P. pentosaceus could not ferment sucrose, while L. brevis could only ferment maltose. The presence of glucose did not influence the utilization of fructose and maltose by L. sanfranciscensis, while it repressed the fermentation of fructose, maltose and sucrose by W. cibaria, and fructose and maltose by L. paralimentarius and P. pentosaceus. Moreover, L. sanfranciscensis and L. brevis could obtain extra ATP through the reduction of fructose to mannitol, which favored the production of acetic acid against ethanol. The utilization of fructose as an electron acceptor has a decisive effect on the prevailing of L. sanfranciscensis and L. brevis in spontaneously fermented sourdough and in the scarce appearance of the other lactic acid bacteria studied.

Molecular Basis of Fructose Utilization by the Wine YeastSaccharomyces cerevisiae: a MutatedHXT3Allele Enhances Fructose Fermentation

Fructose utilization by wine yeasts is critically important for the maintenance of a high fermentation rate at the end of alcoholic fermentation. A Saccharomyces cerevisiae wine yeast able to ferment grape must sugars to dryness was found to have a high fructose utilization capacity. We investigated the molecular basis of this enhanced fructose utilization capacity by studying the properties of several hexose transporter (HXT) genes. We found that this wine yeast harbored a mutated HXT3 allele. A functional analysis of this mutated allele was performed by examining expression in an hxt1-7Delta strain. Expression of the mutated allele alone was found to be sufficient for producing an increase in fructose utilization during fermentation similar to that observed in the commercial wine yeast. This work provides the first demonstration that the pattern of fructose utilization during wine fermentation can be altered by expression of a mutated hexose transporter in a wine yeast. We also found that the glycolytic flux could be increased by overexpression of the mutant transporter gene, with no effect on fructose utilization. Our data demonstrate that the Hxt3 hexose transporter plays a key role in determining the glucose/fructose utilization ratio during fermentation.

A route for fructose utilization by Escherichia coli involving the fucose regulon

Fructose can be taken up by Escherichia coli via a variety of membrane-spanning proteins that recognize sugars with the 3,4,5-d-arabino-hexose configuration. Here, we describe a mutant that is devoid of those proteins but takes up fructose via the FucP carrier normally used for the transport of alpha-L-fucose; this implies that the fructose is taken up in the alpha- or beta-fructopyranose form. For growth, the assimilated fructose is sequentially phosphorylated by ATP and (i) manno(fructo)kinase, to form fructose 6-phosphate, and (ii) phosphofructokinase, to form fructose 1,6-bisphosphate, which is a member of central routes of glycolysis and gluconeogenesis. The mutation that confers on the organism the ability to take up fructose via the fucose regulon was located as a deletion of the fucA gene with consequent induction of the proton-linked fucose transporter, FucP.

Sugar metabolism and pathogenicity of Spiroplasma citri.

Spiroplasma citri is a plant-pathogenic mollicute, phylogenetically related to Gram-positive bacteria. Spiroplasma cells are restricted to the phloem sieve elements and are transmitted by leafhopper vectors. Recent research has allowed depicting a unique scenario in S. citri pathogenicity, where sugar metabolism plays a major role. In vitro S. citri uses fructose, glucose, and trehalose, which are imported through the Phosphoenolpyruvate phosphoTransferase System (PTS). When both fructose and glucose are present, fructose is used preferentially. The fructose-PTS permease is made of one single polypeptide IIABCFru, whereas the glucose permease is split into two polypeptides IICBGlc and IIAGlc encoded by two separated genes ptsG and crr. The glucose IICBGlc and trehalose IIBCTre permeases function with a single IIAGlc domain, enabling the spiroplasma to rapidly adapt from glucose to trehalose and vice versa. Upregulation of expression of the PTS permease genes by the relevant sugar also illustrates the adaptive capacity of the spiroplasma. S. citri mutants unable to import glucose and trehalose are still highly pathogenic. In contrast, mutants unable to import fructose induce very mild and delayed symptoms, indicating that fructose and glucose play distinct roles in spiroplasmal pathogenicity. While fructose is used as a substrate by the spiroplasma to grow, glucose accumulates in source leaves of the host plant, leading to physiological disorders and down-regulation of photosynthesis genes. Fructose utilization by the spiroplasmas is postulated to deprive companion cells of fructose, thereby impairing sucrose loading in the sieve elements. Alternatively, preferential use of fructose is hypothesized to increase invertase activity, leading to glucose accumulation, and inhibition of photosynthesis. Both model mechanisms may contribute to S. citri pathogenicity.

Characterization of genes involved in fructose utilization by Lactobacillus fermentum

The genes encoding phosphoglucose isomerase (fruI) and fructokinase (fruK) of Lactobacillus fermentum NRRL-B-1932 were sequenced. They constituted an operon, which is involved in fructose metabolism of this strain by channeling intracellular fructose into the phosphoketolase pathway. A third open reading frame, unkR, upstream of the operon was identified as homologous to genes of LacI/GalR family repressors. The UnkR repressor's role in transcriptional control of the fruIK operon could, however, not be established by electrophoretic mobility shift assay (EMSA) analysis. Sequence analysis revealed two putative catabolite responsive elements (cre) in the promoter region of fruIK suggesting that the fruIK operon is under negative regulatory control by carbon catabolite repression. Expression and enzyme activity data were compatible with the assumption that the fruIK operon is repressed by glucose. No sugar specific phosphoenolpyruvate sugar transferase system activity for the transport of fructose, glucose, sucrose or mannose could be detected in L. fermentum NRRL-B-1932 cells, which suggest that fructose is taken up by a permease system.

Bacterial Synthesis of PHA Block Copolymers

Polyhydroxyalkanoates (PHA) containing block copolymers were synthesized in Cupriavidus necator using periodic substrate addition. Poly(3-hydroxybutyrate) (PHB) segments were formed during fructose utilization. Pulse feeds of pentanoic acid resulted in the synthesis of 3-hydroxyvalerate monomers, forming poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) random copolymer. PHA synthesis was controlled using analysis of oxygen uptake and carbon evolution rates from the bioreactor off-gas. A combination of characterization techniques applied to the polymer batches strongly suggests the presence of block copolymers: (i) Thermodynamically stable polymer samples obtained by fractionation and analyzed by differential scanning calorimetry (DSC) and nuclear magnetic resonance spectroscopy (NMR) indicate that some fractions, representing approximately 30% of the total polymer sample, exhibit melting characteristics and nearest-neighbor statistics indicative of block copolymers, (ii) preliminary rheology experiments indicate additional mesophase transitions only found in block copolymer materials, (iii) dynamic mechanical analysis shows extension of the rubbery plateaus in block copolymer samples, and (iv) uniaxial extension tests result in differences in mechanical properties (modulus and elongation at failure) expected of similarly prepared block copolymer and single polymer type materials.

Selective Utilization of Fructose to Glucose by <i>Candida magnoliae</i>, an Erythritol Producer

Candida magnoliae isolated from honeycomb is an industrially important yeast with high erythritol-producing ability. Erythritol has been used as functional sugar substitute for various foods. In order to analyze the physiological properties of C. magnoliae, a study on sugar utilization pattern was carried out. The fermentation kinetics of glucose and fructose revealed that C. magnoliae has the discrepancy in glucose and fructose utilization when it produces erythritol. In contrast to most yeasts, C. magnoliae showed preference for fructose to glucose as a carbon source, deserving the designation of fructophilic yeast. Such a peculiar pattern of sugar utilization in C. magnoliae seems to be related to the evolutionary environment.

Effects of Two Energy-Restricted Diets Containing Different Fruit Amounts on Body Weight Loss and Macronutrient Oxidation

The consumption of specific foods in energy-restricted diets may affect the weight loss process. The purpose of this research was to evaluate whether obese women following two hypocaloric diets with distinct fruit content differ in weight loss and metabolic responses. Fifteen obese women were included, who were randomly assigned to follow a low or a high-fruit energy-restricted diet for 8 weeks. The main outcome variables were weight and fat losses. Metabolic measurements concerning macronutrient oxidation were also assessed by using (13)C labelled fructose and indirect calorimetry. The induced weight loss was similar for both diets (6.9 +/- 2% vs. 6.6 +/- 2%, p = 0.785). Both experimental diets similarly improved the lipid plasma profile in the participants, but the cholesterol fall was higher in obese subjects receiving the diet containing more fruit. No statistical differences in lipids carbohydrates and (13)C labelled fructose utilisation were observed, but protein oxidation was differently affected by the experimental diets. The compensatory effects of the associated fibre/fructose intake may explain the lack of a specific effect of the fruit amount on hypocaloric diets designed to weight loss, although the increased fibre content from enriched fruit diets may be involved in the favourable effects on cholesterol plasma levels.

Fructose Utilization inLactococcus lactisas a Model for Low-GC Gram-Positive Bacteria: Its Regulator, Signal, and DNA-Binding Site

In addition to its role as carbon and energy source, fructose metabolism was reported to affect other cellular processes, such as biofilm formation by streptococci and bacterial pathogenicity in plants. Fructose genes encoding a 1-phosphofructokinase and a phosphotransferase system (PTS) fructose-specific enzyme IIABC component reside commonly in a gene cluster with a DeoR family regulator in various gram-positive bacteria. We present a comprehensive study of fructose metabolism in Lactococcus lactis, including a systematic study of fru mutants, global messenger analysis, and a molecular characterization of its regulation. The fru operon is regulated at the transcriptional level by both FruR and CcpA and at the metabolic level by inducer exclusion. The FruR effector is fructose-1-phosphate (F1P), as shown by combined analysis of transcription and measurements of the intracellular F1P pools in mutants either unable to produce this metabolite or accumulating it. The regulation of the fru operon by FruR requires four adjacent 10-bp direct repeats. The well-conserved organization of the fru promoter region in various low-GC gram-positive bacteria, including CRE boxes as well as the newly defined FruR motif, suggests that the regulation scheme defined in L. lactis could be applied to these bacteria. Transcriptome profiling of fruR and fruC mutants revealed that the effect of F1P and FruR regulation is limited to the fru operon in L. lactis. This result is enforced by the fact that no other targets for FruR were found in the available low-GC gram-positive bacteria genomes, suggesting that additional phenotypical effects due to fructose metabolism do not rely directly on FruR control, but rather on metabolism.

The Effects Of Humic Substances On Pinus Callus Are Reversed By 2,4-Dichlorophenoxyacetic Acid

The reversal of humic matter-induced inhibition of callus growth and metabolism by 2,4-dichlorophenoxyacetic acid (2,4-D) was studied in Pinus laricio. Two forest humic fractions (relative molecular mass (Mr) > 3500), derived from soil under Fagus sylvatica (Fs) and Abies alba (Aa) plantation, were used. Pinus laricio callus was grown for a subculture period (4 weeks) on Basal Murashige and Skoog (MS) medium plus forest humic matters (Fs or Aa), at a concentration of 1 mg C/l, and then was transferred, for an additional four weeks, to a MS medium culture without humic matter, but with different hormones: indole-3-acetic acid (IAA, 2 mg/l) or 2,4-dichlorophenoxyacetic acid (2,4-D, 0.5 mg/l) and/or 6-benzylaminopurine (BAP, 0.25 mg/l). Growth of calluse, glucose, fructose, and sucrose contents, and activities of soluble and bound invertases, glucokinase, phosphoglucose isomerase, aldolase, and pyruvate kinase were monitored. The results show a negative effect of humic fractions on callus growth, due to decreased utilization of glucose and fructose, and decreased activities of glycolytic enzymes. The effects are reversible. Substitution of humic fractions with 2,4-D+BAP or 2,4-D is followed by an increase of glycolytic enzyme activities and, consequently, by the utilization of glucose and fructose that induces a restart of growth. In contrast, the inhibitory effects of humic fractions persist when they are substituted with BAP alone, indicating that only the auxin 2,4-D is capable of reversing the negative effects. A possible competitive action on the auxin-binding site between 2,4-D and the chemical structures in the forest humic fractions is suggested.

Fructose utilization in Lactococcus lactis as a model for low-GC gram-positive bacteria

Fructose utilization in Lactococcus lactis as a model for low-GC gram-positive bacteria

Sugar Import and Phytopathogenicity ofSpiroplasma citri: Glucose and Fructose Play Distinct Roles

We have shown previously that the glucose PTS (phosphotransferase system) permease enzyme II of Spiroplasma citri is split into two distinct polypeptides, which are encoded by two separate genes, crr and ptsG. A S. citri mutant was obtained by disruption of ptsG through homologous recombination and was proved unable to import glucose. The ptsG mutant (GII3-glc1) was transmitted to periwinkle (Catharanthus roseus) plants through injection to the leaf-hopper vector. In contrast to the previously characterized fructose operon mutant GMT 553, which was found virtually nonpathogenic, the ptsG mutant GII3-glc1 induced severe symptoms similar to those induced by the wild-type strain GII-3. These results, indicating that fructose and glucose utilization were not equally involved in pathogenicity, were consistent with biochemical data showing that, in the presence of both sugars, S. citri used fructose preferentially. Proton nuclear magnetic resonance analyses of carbohydrates in plant extracts revealed the accumulation of soluble sugars, particularly glucose, in plants infected by S. citri GII-3 or GII3-glc1 but not in those infected by GMT 553. From these data, a hypothetical model was proposed to establish the relationship between fructose utilization by the spiroplasmas present in the phloem sieve tubes and glucose accumulation in the leaves of S. citri infected plants.

In vitro sugar uptake by grapefruit (Citrus paradisi) juice-sac cells.

To further our understanding of the mechanisms of sugar uptake and accumulation into grapefruit (Citrus paradisi Macf. cv. Marsh seedless), the patterns of uptake and utilisation of sucrose, glucose and fructose by Citrus juice cells was investigated. Analyses were conducted on sliced juice sacs that were incubated in radioactive [14C]-sugar solutions with unlabelled sugars, in the presence or absence of metabolic inhibitors. Both hexoses demonstrated an initial uptake peak in December and a second uptake peak in February-March. From March through April the rates of sucrose uptake increased to levels comparable to those of glucose and fructose. Sucrose and its moieties fructose and glucose entered the juice cells of Citrus juice fruit by an insaturable, and mostly by an independent, process. However, NaN3 and carbonylcyanide m-chlorophenylhydrazone (CCCP) produced slight inhibition of these processes. Cells took up hexoses at a greater rate than sucrose, with accumulation reaching a plateau by 4-8 h, and then continuing unabated, in the case of glucose, for 42 h. Uptake of all three sugars increased linearly in the range of sugar concentrations tested, which extended from 0.01 to 320 mm, denoting an insaturable system for sugar uptake. 14CO2 evolution was relatively low in all the experiments, the lowest evolution being recorded when the uptake of [14C]-sucrose was studied, while the highest 14CO2 evolution was recorded when the uptake of [14C]-glucose was studied. The data demonstrate a preferential utilisation of glucose over fructose and sucrose. In all the experiments, the two metabolic inhibitors significantly inhibited the decarboxylation of the three sugars.

The bifidogenic effect of Taraxacum officinale root

The infusion of dandelion root ( Taraxacum officinale) stimulated in vitro the growth of 14 strains of bifidobacteria. The utilization of oligofructans, glucose, fructose and total saccharides was determined by enzymatic and phenol-sulfuric methods. Dandelion oligofructans were important source of carbon and energy for bifidobacteria tested.

Fructose transport and metabolism in adipose tissue of Zucker rats: diminished GLUT5 activity during obesity and insulin resistance.

Fructose is a major dietary sugar, which is elevated in the serum of diabetic humans, and is associated with metabolic syndromes important in the pathogenesis of diabetic complications. The facilitative fructose transporter, GLUT5, is expressed in insulin-sensitive tissues (skeletal muscle and adipocytes) of humans and rodents, where it mediates the uptake of substantial quantities of dietary fructose, but little is known about its regulation. We found that GLUT5 abundance and activity were compromised severely during obesity and insulin resistance in Zucker rat adipocytes. Adipocytes from young obese (fa/fa), highly insulin-responsive Zucker rats contained considerably more plasma membrane GLUT5 than those from their lean counterparts (1.8-fold per microgram membrane protein), and consequently exhibited higher fructose transport (fivefold) and metabolism (threefold) rates. Lactate production was the preferred route for fructose metabolism in these cells. As the rats aged and become more obese and insulin-resistant, adipocyte GLUT5 surface density (12-fold) and fructose transport (10-fold) and utilisation rates (threefold) fell markedly. The GLUT5 loss was more dramatic in adipocytes from obese animals, which developed a more marked insulin resistance than lean counterparts. The decline of GLUT5 levels in adipocytes from older, obese animals was not a generalised effect, and was not observed in kidney, nor was this expression pattern shared by the alpha1 subunit of the Na+/K+ ATPase. Our findings suggest that plasma membrane GLUT5 levels and thus fructose utilisation rates in adipocytes are dependent upon cellular insulin sensitivity, inferring a possible role for GLUT5 in the elevated circulating fructose observed during diabetes, and associated pathological complications.

Glucose and trehalose PTS permeases of Spiroplasma citri probably share a single IIA domain, enabling the spiroplasma to adapt quickly to carbohydrate changes in its environment.

Spiroplasma citri is a plant-pathogenic mollicute phylogenetically related to Gram-positive bacteria. Spiroplasma cells are restricted to the phloem sieve tubes and are transmitted from plant to plant by the leafhopper vector Circulifer haematoceps. In the plant sieve tubes, S. citri grows on glucose and fructose, whereas in the leafhopper haemolymph the spiroplasma must grow on trehalose, the major sugar in insects. Previous studies in this laboratory have shown that fructose utilization was a key factor of spiroplasmal pathogenicity. To further study the implication of sugar metabolism in the interactions of S. citri with its plant host and its leafhopper vector, genes encoding permease enzymes II (EII(Glc) and EII(Tre)) of the S. citri phosphoenolpyruvate : glucose and phosphoenolpyruvate : trehalose phosphotransferase systems (PTS) were characterized. Mapping studies revealed that the EII(Glc) complex was split into two distinct polypeptides, IIA(Glc) and IICB(Glc), encoded by two separate genes, crr and ptsG, respectively. As expected, S. citri polypeptides IIA(Glc) and IICB(Glc) were more phylogenetically related to their counterparts from Gram-positive than to those from Gram-negative bacteria. The trehalose operon consisted of three genes treR, treP and treA, encoding a transcriptional regulator, the PTS permease (EII(Tre)) and the amylase, respectively. However, in contrast to the fructose-PTS permease, which is encoded as a single polypeptide (IIABC(Fru)) containing the three domains A, B and C, the trehalose-PTS permease (IIBC(Tre)) lacks its own IIA domain. No trehalose-specific IIA could be identified in the spiroplasmal genome, suggesting that the IIBC(Tre) permease probably functions with the IIA(Glc) domain. In agreement with this statement, yeast two-hybrid system experiments revealed that the IIA(Glc) domain interacted not only with IIB(Glc) but also with the IIB(Tre) domain. The results are discussed with respect to the ability of the spiroplasma to adapt from the phloem sap of the host plant to the haemolymph and salivary gland cells of the insect vector.