Structural characterization of extracellular polysaccharides produced by the marine fungus Epicoccum nigrum JJY-40 and their antioxidant activities

Two extracellular polysaccharides, ENP1 and ENP2, were isolated from the fermentation liquid of the marine fungus Epicoccum nigrum JJY-40 by anion-exchange chromatography and gel-filtration chromatography, and their structures were investigated using chemical and spectroscopic methods including methylation analysis and NMR spectroscopy. The results demonstrated that ENP1 was composed of mannose, glucose, and galactose in the molar ratio of 5.0:2.1:1.0, and the main chain of the polysaccharide consisted of (1?→?2)-linked mannose, (1?→?3)-linked mannose, terminal mannose, (1?→?6)-linked glucose, (1?→?4)-linked glucose, and (1?→?4)-linked galactose. ENP2 was composed of mannose, galactose, glucose, and glucuronic acid in a molar ratio of 12.4:11.2:8.3:1.0, and its glycosidic linkage patterns included terminal mannose, (1?→?6)-linked glucose, (1?→?4)-linked galactose, and (1?→?3)-linked mannose. The two polysaccharides had a partially branched structure with branch point located at C-3 position of (1?→?6)-linked glucose residue. The molecular weights of ENP1 and ENP2 were 19.2 kDa and 32.7 kDa, respectively. Antioxidant properties of the two polysaccharides were evaluated with hydroxyl, superoxide, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities and lipid peroxidation inhibition in vitro, and results showed that ENP2 and ENP1 had good antioxidant activities, especially ENP2. ENP2 could be effective as a potential antioxidant.     

Expression of an inducible enzyme II fructose and activation of a cryptic enzyme II glucose in glucose-grown cells of spontaneous mutants of Streptococcus salivarius lacking the low-molecular-mass form of IIIman, a component of the phosphoenolpyruvate:mannose phosphotransferase system.

We have reported previously that the phosphoenolpyruvate:mannose phosphotransferase system (mannose PTS) of Streptococcus salivarius, consisting of an Enzyme II mannose (EIIman) and two forms of Enzyme III mannose (IIIman) with Mr values of 38,900 and 35,200, respectively, concomitantly transports and phosphorylates mannose, as well as glucose and fructose. In this paper, we report the presence, in S. salivarius, of alternative specific fructose and glucose PTSs encoded by inducible and cryptic genes, respectively. Protein phosphorylation experiments conducted with [32P]phosphoenolpyruvate have allowed us to identify by SDS-PAGE and autoradiography the EII fructose (EIIfru) (Mr 57,500) and the EII glucose (EIIglc) (Mr 58,700). No proteins corresponding to IIIfru or IIIglc could be detected. EIIfru phosphorylated fructose on the C-1 position rather than, as with the constitutive mannose PTS, on the C-6 position. Growth on fructose resulted in the induction of EIIfru as well as an increase of 1-phosphofructokinase activity. Nevertheless, the genes encoding these proteins were independently regulated. Studies carried out with spontaneous mutants lacking the low-molecular-mass form of IIIman (mutants A37, G29 and B31) showed that EIIfru was expressed in glucose-grown cells of strains G29 and B31, but not in strain A37, whereas the cryptic gene encoding EIIglc was activated in all three mutant strains. The results obtained with the mutants suggest that the three spontaneous mutants were not all mutated on the gene encoding IIIman although all of them lacked IIIman.     

Further identification of the nature and linkage of the carbohydrate in hemoglobin A1c.

We have found that Hb A_1c contains neutral sugars which are only partially hydrolyzed from the N-termini of β chains. In both normal and diabetic Hb A_1c, 0.2–0.3 equivalents of hexose were released, composed primarily of glucose and mannose in a 3:1 ratio. When Hb A_1c was reduced with ³HNa BH₄ and then treated with periodic acid, most of the radioactivity was recovered as ³H-formic acid with much lesser amounts of ³H-formaldehyde. From these results, we propose that in the red cell, glucose binds to the α-amino position of hemoglobin β-chains (valine) in an aldimine (Schiff base) linkage. This aldimine can then partially rearrange in a reversible manner to form a ketoamine linkage which is stable to acid hydrolysis. This Amadori-type rearrangement accounts for the formation of mannose, the C-2 epimer of glucose, as well as the inability to demonstrate ³HNa BH₄ reduction at the C-1 position.     

Structural studies of water-soluble glycoproteins from Cannabis sativa L

Two carbohydrate-protein fractions, isolated from Cannabis sativa L. by extraction with water and chromatography on DEAE-cellulose, contained arabinose, galactose, glucose, mannose, galacturonic acid, 2-acetamido-2-deoxyglucose, and 2-acetamido-2-deoxygalactose. The structure of the carbohydrate moieties was investigated by methylation analysis and Smith degradation. A high percentage of end-groups indicates a large degree of branching, glucose and galactose being the main branch-points, linked at C-3 and C-6. The hexoses are also present as unbranched residues in the chain, largely as (1→3)- and (1→4)-linked units and as end-groups. Arabinofuranosyl units constitute the main part of the non-reducing end-groups, and are also present as part of the chain. The polysaccharide chains are probably linked to protein through the hydroxyl group of hydroxyproline.     

Biosynthesis of Novel Exopolymers by Aureobasidium pullulans

Aureobasidium pullulans ATCC 42023 was cultured under aerobic conditions with glucose, mannose, and glucose analogs as energy sources. The exopolymer extracts produced under these conditions were composed of glucose and mannose. The molar ratio of glucose to mannose in the exopolymer extract and the molecular weight of the exopolymer varied depending on the energy source and culture time. The glucose content of exopolymer extracts formed with glucose and mannose as the carbon sources was between 91 and 87%. The molecular weight decreased from 3.5 × 10⁶ to 2.12 × 10⁶ to 0.85 × 10⁶ to 0.77 × 10⁶ with culture time. As the culture time increased, the glucose content of the exopolymer extract formed with glucosamine decreased from 55 ± 3 to 29 ± 2 mol%, and the molecular weight increased from 2.73 × 10⁶ to 4.86 × 10⁶. There was no evidence that glucosamine was directly incorporated into exopolymers. The molar ratios of glucose to mannose in exopolymer extracts ranged from 87 ± 3:13 ± 3 to 28 ± 2:72 ± 2 and were affected by the energy source added. On the basis of the results of an enzyme hydrolysis analysis of the exopolymer extracts and the compositional changes observed, mannose (a repeating unit) was substituted for glucose, which gave rise to a new family of exopolymer analogs.     

Synthesis and evaluation of affinity adsorbents for glycoproteins: an artificial lectin

A combination of rational design based on mimicking natural protein–carbohydrate interactions and solid-phase combinatorial chemistry has led to the identification of an affinity ligand which displays selectivity for the mannose moiety of glycoproteins. The ligand, denoted 18/18 and comprising a triazine scaffold bis-substituted with 5-aminoindan, has been synthesised in solution, characterised by TLC, ¹H-NMR and MS. When immobilised to amine-derivatised agarose at concentrations >24 μmol/g moist weight gel, ligand 18/18 selectively binds glucose oxidase. The adsorbed enzyme was quantitatively eluted with 0.5 M α- -methyl-mannoside and to a lesser extent with the equivalent glucoside. An investigation of the comparative retention times of saccharidic solutes showed that significant retardation was observed for α- -mannose, mannobiose and mannan, with little or no evidence for selective retention of other saccharides, with the exception of α- -fucose. Interestingly, α- -fucose and α- -mannose share an identical configuration of the hydroxyl groups on C-2, C-3 and C-4. Analysis of Scatchard plots from partition equilibrium studies on the interaction of glucose oxidase and the p-nitrophenyl-glycosides of -mannose, -glucose, -fucose and -galactose with immobilised 18/18 establish that the affinity constants (K_AX) for the enzyme, the glycosides of mannose, glucose and fucose, and the p-nitrophenyl-galactoside are 4.3×10⁵ M⁻¹, 1.9×10⁴ M⁻¹ and 1.2×10⁴ M⁻¹ respectively. ¹H-NMR studies on the interaction of α- -methyl-mannoside with ligand 18/18 in solution confirm the involvement of the hydroxyl group in the C-2 position. Molecular modelling suggests the formation of four hydrogen bonds between the hydroxyl groups at positions C-2, C-3 and C-4 of α- -methyl-mannoside and the bridging and ring nitrogen atoms of the triazine scaffold, with aromatic stacking of a second ligand against the carbohydrate face. The greater specificity of ligand 18/18 for mannose and glucose than for galactose parallels that exhibited by concanavalin A.     

C-2 Epimer Formation of Tetrose,Pentose and Hexose Sugars by Xylose Isomerase

We describe novel tetrose isomerizations and C-2 epimerizations by the industrially important d -xylose ketol-isomerase (E.C.5.3.1.5) with both the d - and l -forms of the sugars. We further show that in addition to isomerization to d -fructose, d -glucose is slowly C-2 epimerized to d -mannose. The formation rate of the C-2 epimer was 0.03 mg mg &#109 1 min &#109 1 from d -glucose, 0.56 mg mg &#109 1 min &#109 1 from d -arabinose and 3.0 mg mg &#109 1 min &#109 1 from d -erythrose. The equilibria of the reaction products as a function of temperature were measured for threose/erythrulose/erythrose, arabinose/ribulose/ ribose and glucose/fructose/mannose.     

Metabolism of position-labelled glucose in anoxic methanogenic paddy soil and lake sediment

Abstract Turnover times of radioactive glucose were shorter in paddy soil (4–16 min) than in Lake Constance sediment (18–62 min). In the paddy soil, 65–75% of the radioactive glucose was converted to soluble metabolites. In the sediment, only about 25% of the radioactive glucose was converted to soluble metabolites, the rest to particulate material. In anoxic paddy soil, the degradation pattern of position-labelled glucose was largely consistent with glucose degradation via the Embden-Meyerhof-Parnas (EMP) pathway followed by methanogenic acetate cleavage: CO₂ mainly originated from C-3,4, whereas CH₄ mainly originated from C-1 and C-6 of glucose. Acetate-carbon originated from C-1, C-2 and C-6 rather than from C-3,4 of glucose. In both paddy soil and Lake Constance sediment acetate and CO₂ were the most important early metabolites of radioactive glucose. Other early products included propionate, ethanol/butyrate, succinate, and lactate, but accounted each for less than 1–8% of the glucose utilized. The labelling of propionate by [3,4-¹⁴C]glucose suggests that it was mainly produced from glucose or lactate rather than from ethanol. Isopropanol and caproate were also detectable in paddy soil, but were not produced from radioactive glucose. Chloroform inhibited methanogenesis, inhibited the further degradation of radioactive acetate and resulted in the accumulation of H₂, however, did not inhibit glucose degradation. Since acetate was the main soluble fermentation product of glucose and was produced at a relatively high molar acetate: CO₂ ratio (2.5:1), homoacetogenesis appeared to be the most important glucose fermentation pathway.     

C-2 Epimer Formation of Tetrose, Pentose and Hexose Sugars by Xylose Isomerase

We describe novel tetrose isomerizations and C-2 epimerizations by the industrially important d -xylose ketol-isomerase (E.C.5.3.1.5) with both the d - and l -forms of the sugars. We further show that in addition to isomerization to d -fructose, d -glucose is slowly C-2 epimerized to d -mannose. The formation rate of the C-2 epimer was 0.03 mg mg -1 min -1 from d -glucose, 0.56 mg mg -1 min -1 from d -arabinose and 3.0 mg mg -1 min -1 from d -erythrose. The equilibria of the reaction products as a function of temperature were measured for threose/erythrulose/erythrose, arabinose/ribulose/ ribose and glucose/fructose/mannose.     

Structural investigations of the extracellular polysaccharide elaborated by s19, a Xanthomonas-type bacterium

The extracellular, acidic heteropolysaccharide from Xanthomonas S19 consists of D-glucuronic acid, D-glucose, D-galactose, and D-mannose residues in the approximate molar ratios of 1.6:3:1:1, plus acetyl groups liked to C-2 and/or C-3 of a large proportion of the glucose residues. Methylation studies showed that the glucose is present as non-reducing end-group also as 1,2- and 1,4-linked units, the galactose residues are solely 1,3-linked, a major proportion of the mannose residues are 1,2,4-linked and the rest 1,2-linked. A high proportion of the glucuronic acid units are 1,4-linked. Periodate oxidation confirmed the presence of these linkages. The disaccharides D-Glc-(1→4)-D-Glc,D-Glc-(1→2)-D-Man, D-Glc-(1→3)-D-Gal, D-Gal-(1→2)-D-Glc, D-GlcA-(1→4)-D-GlcA, and β-D-GlcA-(1→4)-D-Man were isolated from a partial hydrolysate of the polysaccharide, and characterised. The similarities and differences between this polysaccharide and those from other Xanthomonas species are discussed.     

The activity of the pentose phosphate pathway in isolated liver cells

Isolated liver cells have been used to assess the relative contribution of the pentose phosphate pathway to glucose metabolism. The incorporation of carbon from specifically labelled glucose into ¹⁴CO₂ by isolated cells gave values (μg.atoms/g.cells/hr) of: C-1, 7.9; C-6, 1.3; C-U, 3.4. The corresponding figures for liver slices were: C-1, 2.3; C-6, 1.6; C-U, 3.0. The most striking difference was the 3.5-fold increase in the oxidation of C-1 of glucose. Isolated cells retain more than 50% of ATP and have a content of intermediates of the glycolytic pathway closely similar to freeze-clamped liver. The relative importance of the pentose phosphate pathway in isolated liver cells, approximately 16% of glucose catabolised, is consistent with the enzyme profile of liver and the reductive synthetic reactions of the tissue.     

Glucose Catabolism in Rhizobium japonicum

Glucose catabolism in Rhizobium japonicum ATCC 10324 was investigated by the radiorespirometric method and by assaying for key enzymes of the major energy-yielding pathways. Specifically labeled glucose gave the following results for resting cells, with values expressed as per cent (14)CO(2) evolution: C-1=59%, C-2=51%, C-3=45%, C-4=59%, and C-6=43%. These values indicate that glucose was degraded by the Entner-Doudoroff pathway alone. Cells which grew in glucose-yeast extract-salts medium gave essentially the same pattern except for retardation of the C-6 carbon. The rates were: C-1=54%, C-2=42%, C-3=51%, C-4=59%, and C-6=32%. Hexokinase, glucose-6-phosphate dehydrogenase, transketolase, and an enzyme system which produces pyruvate from 6-phosphogluconate were found to be present in these cells. No 6-phosphogluconate dehydrogenase was detected. Oxidation of specifically labeled pyruvate gave the following (14)CO(2) evolution pattern: C-1=78%, C-2=48%, and C-3=37%; the pattern from acetate was C-1=73%; and C-2=56%. Oxidation of glutamate showed the preferential rate of (14)CO(2) evolution to be C-1 > C-2=C-5 > C-3, 4, whereas a higher yield of (14)CO(2) was obtained from the C-1 and C-4 carbons of succinate than from the C-2 and C-3 carbons. These data are consistent with the operation of the Entner-Doudoroff pathway and tricarboxylic acid cycle as the catabolic pathways of glucose oxidation in R. japonicum.     

[14C]Glucose Metabolism in Sympathetic Ganglia of Chicken Embryos and in Primary Cultures of Neurons and of Other Cells from These Ganglia

Metabolism of [1-14C]glucose and [6-14C]glucose was measured in sympathetic ganglia excised from chicken embryos 12-16 days old and in primary cultures of neurons or nonneurons prepared from these ganglia. Some metabolic rates tended to change with the tissue/medium ratio, so this variable had to be controlled. Less C-6 than C-1 od glucose was put out in CO2 by all three types of preparations, indicating operation of the hexosemonophosphate shunt. The C-6/C-1 ratio was greater for the neuronal cultures and for intact ganglia than for the nonneuronal cultures. The C-6/C-1 ratio for the neurons increased with the amount of tissue added to a given volume of incubation medium, in agreement with previous experiments on embryonic dorsal root ganglia (Larrabee, 1978). Per unit of protein, the output of C-1 of glucose in CO2 was higher in both the neuronal and the nonneural cultures than in intact ganglia, whereas that of C-6 was higher in the neuronal cultures and lower in the nonneuronal ones than in the ganglia. The rates of release in lactate of C-1 and C-6 of glucose were 3-5 times higher from both types of cultures than from intact ganglia. The average rates of incorporation of C-1 and C-6 of glucose into tissue constituents were lower in the cultures than in intact ganglia, significantly so for incorporation of C-6 in the nonneuronal cultures.     

Alternative pathways of glucose transport in Prevotella bryantii B(1)4(1)

Prevotella bryantii B(1)4 grew faster on glucose than mannose (0.70 versus 0.45 h(-1)), but these sugars were used simultaneously rather than diauxically. 2-deoxy-glucose (2DG) decreased the growth rate of cells that were provided with either glucose or mannose, but 2DG did not completely prevent growth. Cells grown on glucose or mannose transported both (14)C-glucose and (14)C-mannose, but cells grown on glucose had over three-fold higher rates of (14)C-glucose transport than cells grown on mannose. The (14)C-mannose transport rates of glucose- and mannose-grown cells were similar. Woolf-Augustinsson-Hofstee plots were not linear, and it appeared that the glucose/mannose/2DG carrier acted as a facilitated diffusion system at high substrate concentrations. When cultures were grown on nitrogen-deficient (excess sugar) medium, isolates had three-fold lower (14)C-glucose transport, but the (14)C-mannose transport did not change significantly. (14)C-glucose and (14)C-mannose transport rates could be inhibited by 2DG and either mannose or glucose, respectively. The (14)C-glucose transport of mannose-grown cells was inhibited more strongly by mannose and 2DG than those grown on glucose. Cells grown on glucose or mannose had similar ATP-dependent glucokinase activity, and 2DG was a competitive inhibitor (K(i)=0.75 mM). Thin layer chromatography indicated that cell extracts also had ATP-dependent mannose phosphorylation, but only a small amount of phosphorylated 2DG was detected. Glucose, mannose or 2DG were not phosphorylated in the presence of PEP. Based on these results, it appeared that P. bryantii B(1)4 had: (1) two mechanisms of glucose transport, a constitutive glucose/mannose/2DG carrier and an alternative glucose carrier that was regulated by glucose availability, (2) an ATP-dependent glucokinase that was competitively inhibited by 2DG but was unable to phosphorylate 2DG at a rapid rate, and (3) virtually no PEP-dependent glucose, mannose or 2DG phosphorylation activities.     

Sequential assembly and polymerization of the polyprenol-linked pentasaccharide repeating unit of the xanthan polysaccharide in Xanthomonas campestris.

Lipid-linked intermediates are involved in the synthesis of the exopolysaccharide xanthan produced by the bacterium Xanthomonas campestris (L. Ielpi, R. O. Couso, and M. A. Dankert, FEBS Lett. 130:253-256, 1981). In this study, the stepwise assembly of the repeating pentasaccharide unit of xanthan is described. EDTA-treated X. campestris cells were used as both enzyme preparation and lipid-P acceptor, and UDP-Glc, GDP-Man, and UDP-glucuronic acid were used as sugar donors. A linear pentasaccharide unit is assembled on a polyprenol-P lipid carrier by the sequential addition of glucose-1-P, glucose, mannose, glucuronic acid, and mannose. The in vitro synthesis of pentasaccharide-P-P-polyprenol was also accompanied by the incorporation of radioactivity into a polymeric product, which was characterized as xanthan, on the basis of gel filtration and permethylation studies. Results from two-stage reactions showed that essentially pentasaccharide-P-P-polyprenol is polymerized. In addition, the direction of chain elongation has been studied by in vivo experiments. The polymerization of lipid-linked repeat units occurs by the successive transfer of the growing chain to a new pentasaccharide-P-P-polyprenol. The reaction involves C-1 of glucose at the reducing end of the polyprenol-linked growing chain and C-4 of glucose at the nonreducing position of the newly formed polyprenol-linked pentasaccharide, generating a branched polymer with a trisaccharide side chain.     

Structural investigation of a fucoidan containing a fucose-free core from the brown seaweed, Hizikia fusiforme

A fucoidan, obtained from the hot-water extract of the brown seaweed, Hizikia fusiforme, was separated into five fractions by DEAE Sepharose CL-6B and Sepharose CL-6B column chromatography. All five fractions contained predominantly fucose, mannose and galactose and also contained sulfate groups and uronic acid. The fucoidans had MWs from 25 to 950 kDa. The structure of fraction F32 was investigated by desulfation, carboxyl-group reduction, partial hydrolysis, methylation analysis and NMR spectroscopy. The results showed that the sugar composition of F32 was mainly fucose, galactose, mannose, xylose and glucuronic acid; sulfate was 21.8%, and the MW was 92.7 kDa. The core of F32 was mainly composed of alternating units of -->2)-alpha-D-Man(1--> and -->4)-beta-D-GlcA(1-->, with a minor portion of -->4)-beta-D-Gal(1--> units. The branch points were at C-3 of -->2)-Man-(1-->, C-2 of -->4)-Gal-(1--> and C-2 of -->6)-Gal-(1-->. About two-thirds of the fucose units were at the nonreducing ends, and the remainder were (1-->4)-, (1-->3)- and (1-->2)-linked. About two-thirds of xylose units were at the nonreducing ends, and the remainder were (1-->4)-linked. Most of the mannose units were (1-->2)-linked, and two-thirds of them had a branch at C-3. Galactose was mainly (1-->6)-linked. The absolute configurations of the sugar residues were alpha-D-Manp, alpha-L-Fucp, alpha-D-Xylp, beta-D-Galp and beta-D-GlcpA. Sulfate groups in F32 were at C-6 of -->2,3)-Man-(1-->, C-4 and C-6 of -->2)-Man-(1-->, C-3 of -->6)-Gal-(1-->, C-2, C-3 or C-4 of fucose, while some fucose had two sulfate groups. There were no sulfate groups in either the GlcA or xylose residues.     

Analysis of the biosynthetic process of cellulose and curdlan using C-labeled glucoses

In order to elucidate the biosynthetic process of cellulose and curdlan, ¹³C-labeled polysaccharides were biosynthesized by Acetobacter xylinum (IFO 13693) and Agrobacterium sp. (ATCC 31749), from culture media containing -(1-¹³C)glucose, -(2-¹³C)glucose, -(4-¹³C)glucose, or -(6-¹³C)glucose as the carbon source, and their structures were determined by ¹³C NMR spectroscopy. The labeling was mainly found in the original position, indicating direct polymerization of introduced glucoses. In addition, the transfer of labeling from C-2 to C-1, C-3 and C-5, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 was found in celluloses. In curdlan, the transfer of labeling from C-1 to C-3, from C-2 to C-1 and C-3, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 and C-3 was observed. From analysis of this labeling, the biosynthetic process of cellulose and curdlan was explained as involving six routes. The percentages of each route via which cellulose or curdlan is biosynthesized were estimated for upper (C-1 to C-3) and lower portions (C-4 to C-6) of glucosidic units in the polysaccharides. It is noted that very few polysaccharides are formed via the Embden-Meyerhof pathway. The lower half (C-4 to C-6) structure of introduced glucoses is well preserved in the polysaccharides.     

A study of the phosphate linkages in phosphomannan in cell walls of Saccharomyces cerevisiae

1. The phosphomannan of Saccharomyces cerevisiae was released by Pronase digestion of cell walls and isolated by chromatography on DEAE-cellulose or by precipitation with borate-Cetavlon solutions. Mannose and phosphorus were present in the molar ratio 18:1 and the phosphate groups were in the diester form. 2. Hydrolysis with acid gave mannose 6-phosphate. Under mild acid conditions (autohydrolysis) the phosphate groups were converted into the monoester form, mannose was released and the molecular size of the phosphomannan was substantially decreased. 3. Hydrolysis with alkali also gave a monoester phosphate and a similar decrease in molecular weight. Under mild alkaline conditions the serine and threonine content of the phosphomannan was decreased by about 80%. The phosphate content was not altered. 4. Treatment with 40% (v/v) HF removed 70% of the phosphorus from the phosphomannan with no detectable decrease in molecular weight. 5. Periodate oxidation gave an oxophosphomannan from which 80% of the phosphorus was eliminated under mild alkaline conditions. 6. The properties of the phosphomannan are consistent with a structure in which the phosphate groups are located on the outside of the molecule and link C-1 of a terminal mannose unit with C-6 of another mannose unit, which is in turn attached to the polysaccharide backbone of the molecule. 7. The implications of this structure are discussed in relation to flocculation.     

Identification of mannose uptake and catabolism genes in Corynebacterium glutamicum and genetic engineering for simultaneous utilization of mannose and glucose

Here, focus is on Corynebacterium glutamicum mannose metabolic genes with the aim to improve this industrially important microorganism’s ability to ferment mannose present in mixed sugar substrates. cgR_0857 encodes C. glutamicum’s protein with 36% amino acid sequence identity to mannose 6-phosphate isomerase encoded by manA of Escherichia coli. Its deletion mutant did not grow on mannose and exhibited noticeably reduced growth on glucose as sole carbon sources. In effect, C. glutamicum manA is not only essential for growth on mannose but also important in glucose metabolism. A double deletion mutant of genes encoding glucose and fructose permeases (ptsG and ptsF, respectively) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was not able to grow on mannose unlike the respective single deletion mutants with mannose utilization ability. A mutant deficient in ptsH, a general PTS gene, did not utilize mannose. These indicate that the glucose-PTS and fructose-PTS are responsible for mannose uptake in C. glutamicum. When cultured with a glucose and mannose mixture, mannose utilization of manA-overexpressing strain CRM1 was significantly higher than that of its wild-type counterpart, but with a strong preference for glucose. ptsF-overexpressing strain CRM2 co-utilized mannose and glucose, but at a total sugar consumption rate much lower than that of the wild-type strain and CRM1. Strain CRM3 overexpressing both manA and ptsF efficiently co-utilized mannose and glucose. Under oxygen-deprived conditions, high volumetric productivity of organic acids concomitant with the simultaneous consumption of the mixed sugars was achieved by the densely packed growth-arrested CRM3 cells.     

Sugar recognition by the glucose and mannose permeases of Escherichia coli. Steady-state kinetics and inhibition studies

The glucose (EII(Glc)) and mannose (EII(Man)) permeases of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of Escherichia coli belong to structurally different families of PTS transporters. The sugar recognition mechanism of the two transporters is compared using as inhibitors and pseudosubstrates all possible monodeoxy analogues, monodeoxyfluoro analogues, and epimers of D-glucose. The analogues were tested as phosphoryl acceptors in vitro and as uptake inhibitors with intact cells. Both EII have a high K(m) of phosphorylation for glucose modified at C-4 and C-6, and these analogues also are weak inhibitors of uptake. Conversely, modifications at C-1 (and also at C-2 with EII(Man)) were well tolerated. OH-3 is proposed to interact with hydrogen bond donors on EII(Glc) and EII(Man), since only substitution by fluorine was tolerated. Glucose-6-aldehydes, which exist as gem-diols in aqueous solution, are potent and highly selective inhibitors of "nonvectorial" phosphorylation by EII(Glc) (K(I) 3-250 microM). These aldehydes are comparatively weak inhibitors of transport by EII(Glc) and of phosphorylation and transport by EII(Man). Both transporters display biphasic kinetics (with glucose and some analogues) but simple Michaelis-Menten kinetics with 3-fluoroglucose (and other analogues). Kinetic simulations of the phosphorylation activities measured with different substrates and inhibitors indicate that two independent activities are present at the cytoplasmic side of the transporter. A working model that accounts for the kinetic data is presented.