Triinu Visnapuu

Levansucrases from Pseudomonas syringae pv. tomato and P. chlororaphis subsp. aurantiaca: substrate specificity, polymerizing properties and usage of different acceptors for fructosylation.

Levansucrases of Pseudomonas syringae pv. tomato DC3000 (Lsc3) and Pseudomonas chlororaphis subsp. aurantiaca (also Pseudomonas aurantiaca) (LscA) have 73% identity of protein sequences, similar substrate specificity and kinetic properties. Both enzymes produce levan and fructooligosaccharides (FOS) of varied length from sucrose, raffinose and sugar beet molasses. A novel high-throughput chip-based nanoelectrospray mass spectrometric method was applied to screen alternative fructosyl acceptors for levansucrases. Lsc3 and LscA could both transfructosylate D-xylose, D-fucose, L- and D-arabinose, D-ribose, D-sorbitol, xylitol, xylobiose, D-mannitol, D-galacturonic acid and methyl-α-D-glucopyranoside and heterooligofructans with degree of polymerization up to 5 were detected. The ability of D-sorbitol, xylobiose, D-galacturonic acid, D-mannitol, xylitol and methyl-α-D-glucopyranoside to serve as fructosyl acceptors for levansucrases is shown for the first time. Expectedly, site-directed mutagenesis of His321 in Lsc3 to Arg, Lys, Leu and Ser resulted in proteins with decreased catalytic activity, affinity for sucrose and polymerizing ability. Random mutagenesis yielded a Lsc3 mutant Thr302Pro with reduced synthesis of levan and long-chain FOS. Thr302 is located in conserved DQTERP region of levansucrases adjacent to predicted acid-base catalyst Glu303. Thr302 and His321 are predicted to belong to +1 subsite of the substrate binding region of Lsc3.

Fully automated chip-based negative mode nanoelectrospray mass spectrometry of fructooligosaccharides produced by heterologously expressed levansucrase from Pseudomonas syringae pv. tomato DC3000.

Pseudomonas syringae pathovars possess multiple levansucrases with still unclear specific roles for bacteria. We have cloned and expressed three levansucrase genes, lsc1, lsc2 and lsc3, from P. syringae DC3000 in Escherichia coli. Levansucrases synthesize a high molecular weight fructan polymer, levan, from sucrose and in the case of some levansucrases, fructooligosaccharides (FOS) with potential prebiotic effects are also produced. The ability of purified Lsc3 protein of DC3000 to synthesize FOS was tested using prolonged incubation time and varied concentrations of sugar substrates. Thin-layer chromatography (TLC) analysis of reaction products disclosed formation of FOS from both sucrose and raffinose, revealing a new catalytic property for P. syringae levansucrases. In order to analyze Lsc3-produced FOS in underivatized form, we optimized a novel method recently introduced in carbohydrate research, based on fully automated chip-based nanoelectrospray ionization (nanoESI) high-capacity ion trap mass spectrometry (HCT-MS). Uding chip-based nanoESI MS in negative ion mode, FOS, with degrees of polymerization up to five, were detected in reaction mixtures of Lsc3 with sucrose and raffinose. For confirmation, further structural analysis by tandem mass spectrometry (MS/MS) employing collision-induced dissociation at low energies was performed. To validate the method, commercial inulin-derived FOS preparations Orafti P95 and Orafti Synergy1, which are currently used as prebiotics, were used as controls. By chip-based nanoESI HCT-MS, similar FOS distribution was observed in these reference mixtures. Thereby, the obtained data allowed us to postulate that FOS produced by the Lsc3 protein of P. syringae DC3000 may be prebiotic as well.

Degradation of Fructans and Production of Propionic Acid by Bacteroides thetaiotaomicron are Enhanced by the Shortage of Amino Acids.

Bacteroides thetaiotaomicron is commonly found in the human colon and stabilizes its ecosystem by catabolism of various polysaccharides. A model of cross-talk between the metabolism of amino acids and fructans in B. thetaiotaomicron was proposed. The growth of B. thetaiotaomicron DSM 2079 in two defined media containing mineral salts and vitamins, and supplemented with either 20 or 2 amino acids, was studied in an isothermal microcalorimeter. The polyfructans inulin (from chicory) and levan (synthesized using levansucrase from Pseudomonas syringae), two fructooligosaccharide preparations with different composition, sucrose and fructose were tested as substrates. The calorimetric power-time curves were substrate specific and typically multiauxic. A surplus of amino acids reduced the consumption of longer oligosaccharides (degree of polymerization > 3). Bacterial growth was not detected either in the carbohydrate free medium containing amino acids or in the medium with inulin as a sole carbohydrate. In amino acid-restricted medium, fermentation leading to acetic acid formation was dominant at the beginning of growth (up to 24 h), followed by increased lactic acid production, and mainly propionic and succinic acids were produced at the end of fermentation. In the medium supplemented with 20 amino acids, the highest production of d-lactate (82 ± 33 mmol/gDW) occurred in parallel with extensive consumption (up to 17 mmol/gDW) of amino acids, especially Ser, Thr, and Asp. The production of Ala and Glu was observed at growth on all substrates, and the production was enhanced under amino acid deficiency. The study revealed the influence of amino acids on fructan metabolism in B. thetaiotaomicron and showed that defined growth media are invaluable in elucidating quantitative metabolic profiles of the bacteria. Levan was shown to act as an easily degradable substrate for B. thetaiotaomicron. The effect of levan on balancing or modifying colon microbiota will be studied in further experiments.

Levansucrases of a Pseudomonas syringae pathovar as catalysts for the synthesis of potentially prebiotic oligo- and polysaccharides.

Gut microbiota influences more physiological and developmental processes of humans and animals than earlier expected. Therefore, the possibility to shape the composition and activity of this bacterial population by prebiotics becomes especially important. Inulin, a β-2,1 linked fructan polymer, from plants and fructooligosaccharides (FOS) derived from it are recognized and already widely used as prebiotics while β-2,6 linked fructans have received much less attention from scientific community. In this mini-review, we will address β-2,6 linked fructans: levan and levan-type FOS as novel potential prebiotics and summarize the literature data on levansucrases of Pseudomonas bacteria which are producing these fructans. The major attention is drawn to stable and highly efficient levansucrases of Pseudomonas syringae pv. tomato, among which the Lsc3 protein has been most thoroughly studied using biochemical methods as well as extensive mutagenesis of the protein.

Levansucrases of Pseudomonas bacteria: novel approaches for protein expression, assay of enzymes, fructooligosaccharides and heterooligofructans

Levansucrases are bacterial extracellular enzymes that act on sucrose producing b-2,6-linked fructans of varied chain length. We summarize here our up-to-date results on (i) novel methods for the production of heterologous levansucrase proteins in Escherichia coli, (ii) biochemical characterization of the levansucrases encoded in the genome of a tomato and Arabidopsis pathogen Pseudomonas syringae pv. tomato DC3000, (iii) innovative chipbased mass spectrometric analysis of oligosaccharidic reaction products, (iv) isolation and characterization of the first mutants and structure-function analysis of a levansucrase of P. syringae pv. tomato. Data on the levansucrase LscA of P. chlororaphis subsp. aurantiaca will be presented for comparison. As most important features of the levansucrases from Pseudomonas bacteria we emphasize their ability to synthesize potentially prebiotic fructooligosaccharides from sucrose or raffinose and to transfructosylate a variety of alternative acceptor sugars with production of heterooligofructans.

Levan Enhances Associated Growth of Bacteroides, Escherichia, Streptococcus and Faecalibacterium in Fecal Microbiota.

The role of dietary fiber in supporting healthy gut microbiota and overall well-being of the host has been revealed in several studies. Here, we show the effect of a bacterial polyfructan levan on the growth dynamics and metabolism of fecal microbiota in vitro by using isothermal microcalorimetry. Eleven fecal samples from healthy donors were incubated in phosphate-buffered defined medium with or without levan supplementation and varying presence of amino acids. The generation of heat, changes in pH and microbiota composition, concentrations of produced and consumed metabolites during the growth were determined. The composition of fecal microbiota and profile of metabolites changed in response to substrate (levan and amino acids) availability. The main products of levan metabolism were acetic, lactic, butyric, propionic and succinic acids and carbon dioxide. Associated growth of levan-degrading (e.g. Bacteroides) and butyric acid-producing (e.g. Faecalibacterium) taxa was observed in levan-supplemented media. The study shows that the capacity of levan and possibly also other dietary fibers/prebiotics to modulate the composition and function of colon microbiota can be predicted by using isothermal microcalorimetry of fecal samples linked to metabolite and consortia analyses.

Mutational analysis of conserved regions harboring catalytic triad residues of the levansucrase protein encoded by the lsc-3 gene (lsc3) of Pseudomonas syringae pv. tomato DC3000

Levansucrase encoded by the lsc‐3 (lsc3) gene at genomic locus PSPTOA0032 of Pseudomonas syringae pv. tomato DC3000 was mutationally analyzed. Altogether, 18 single‐amino‐acid mutants of 13 positions of Lsc3 were studied for catalytic properties, including production of fructooligosaccharides (FOS). Asp62, Asp219, and Glu303 were proved as members of the catalytic triad. Respective alanine replacement mutants were practically inactive with their kcat values reduced up to ∼130,000 times. Additionally, the requirements of Trp61, Gln301, and Arg304, located in conserved sequence blocks around the catalytic triad positions for the catalysis were shown. The catalytic significance of the position equivalent to Arg304 was shown for levansucrases for the first time. Replacement of Gln301 specifically affected the polymerizing ability of Lsc3. The Gln301Ala mutant was largely hydrolytic and produced 31 times less FOS than the wild type. Despite high conservation grades, Leu66, Pro220, Asp225, and His306 tolerated replacement well. Quantification of produced FOS showed a high biotechnological potential of Lsc3. Using 1 mg of Lsc3 protein, 15.4 g of FOS with a degree of polymerization from 3 to 7 can be synthesized in a 20 H reaction with 1,200 mM sucrose. Our expression system allowed us to produce up to 30 mg of Lsc3 protein from 1 L of induced culture of recombinant Escherichia coli.

Maltase protein of Ogataea (Hansenula) polymorpha is a counterpart to the resurrected ancestor protein ancMALS of yeast maltases and isomaltases.

Saccharomyces cerevisiae maltases use maltose, maltulose, turanose and maltotriose as substrates, isomaltases use isomaltose, α‐methylglucoside and palatinose and both use sucrose. These enzymes are hypothesized to have evolved from a promiscuous α‐glucosidase ancMALS through duplication and mutation of the genes. We studied substrate specificity of the maltase protein MAL1 from an earlier diverged yeast, Ogataea polymorpha (Op), in the light of this hypothesis. MAL1 has extended substrate specificity and its properties are strikingly similar to those of resurrected ancMALS. Moreover, amino acids considered to determine selective substrate binding are highly conserved between Op MAL1 and ancMALS. Op MAL1 represents an α‐glucosidase in which both maltase and isomaltase activities are well optimized in a single enzyme. Substitution of Thr200 (corresponds to Val216 in S. cerevisiae isomaltase IMA1) with Val in MAL1 drastically reduced the hydrolysis of maltose‐like substrates (α‐1,4‐glucosides), confirming the requirement of Thr at the respective position for this function. Differential scanning fluorimetry (DSF) of the catalytically inactive mutant Asp199Ala of MAL1 in the presence of its substrates and selected monosaccharides suggested that the substrate‐binding pocket of MAL1 has three subsites (–1, +1 and +2) and that binding is strongest at the –1 subsite. The DSF assay results were in good accordance with affinity (Km) and inhibition (Ki) data of the enzyme for tested substrates, indicating the power of the method to predict substrate binding. Deletion of either the maltase (MAL1) or α‐glucoside permease (MAL2) gene in Op abolished the growth of yeast on MAL1 substrates, confirming the requirement of both proteins for usage of these sugars.

High-throughput assay of levansucrase variants in search of feasible catalysts for the synthesis of fructooligosaccharides and levan.

Bacterial levansucrases polymerize fructose residues of sucrose to β-2,6 linked fructans—fructooligosaccharides (FOS) and levan. While β-2,1-linked FOS are widely recognized as prebiotics, the health-related effects of β-2,6 linked FOS are scarcely studied as they are not commercially available. Levansucrase Lsc3 (Lsc-3) of Pseudomonas syringae pv. tomato has very high catalytic activity and stability making it a promising biotechnological catalyst for FOS and levan synthesis. In this study we evaluate feasibility of several high-throughput methods for screening and preliminary characterization of levansucrases using 36 Lsc3 mutants as a test panel. Heterologously expressed and purified His-tagged levansucrase variants were studied for: (1) sucrose-splitting activity; (2) FOS production; (3) ability and kinetics of levan synthesis; (4) thermostability in a Thermofluor assay. Importantly, we show that sucrose-splitting activity as well as the ability to produce FOS can both be evaluated using permeabilized levansucrase-expressing E. coli transformants as catalysts. For the first time we demonstrate the key importance of Trp109, His113, Glu146 and Glu236 for the catalysis of Lsc3. Cost-effective and high-throughput methods presented here are applicable not only in the levansucrase assay, but have a potential to be adapted for high-throughput (automated) study of other enzymes.

A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal Bacteroides thetaiotaomicron Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass

Bacteroides thetaiotaomicron, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a β-2,6-linked polyfructan levan produced by Zymomonas mobilis degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by B. thetaiotaomicron, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the levansucrase Lsc3 of Pseudomonas syringae pv. tomato, its mutant Asp300Asn, levansucrases of Zymomonas mobilis, Erwinia herbicola, Halomonas smyrnensis as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (<60 kDa) levans, including timothy grass levan and levan synthesized from sucrose by the Lsc3Asp300Asn, were degraded most rapidly whilst levan produced by Lsc3 from raffinose least rapidly. BT1760 catalyzed finely at human body temperature (37°C) and in moderately acidic environment (pH 5–6) that is typical for the gut lumen. According to differential scanning fluorimetry, the Tm of the endo-levanase was 51.5°C. All tested levans were sufficiently stable in acidic conditions (pH 2.0) simulating the gastric environment. Therefore, levans of both bacterial and plant origin may serve as a prebiotic fiber for B. thetaiotaomicron and contribute to short-chain fatty acids synthesis by gut microbiota. In the genome of Bacteroides xylanisolvens of human origin a putative levan degradation locus was disclosed.