A new synthetic biology system for investigating the biosynthesis of antibiotics and other secondary metabolites in streptomycetes.

We have created a novel synthetic biology expression system allowing easy refactoring of biosynthetic gene clusters (BGCs) as monocistronic transcriptional units. The system is based on a set of plasmids containing a strong kasOp* promoter, RBS and terminators. It allows the cloning of biosynthetic genes into transcriptional units kasOp*-gene(s)-terminator flanked by several rare restriction cloning sites that can be sequentially combined into the artificial BGC in three compatible Streptomyces integration vectors. They allow a simultaneous integration of these BGCs at three different attB sites in the Streptomyces chromosome. The system was validated with biosynthetic genes from two known BGCs for aromatic polyketides landomycin and mithramycin.

A Structural Analysis of the Angucycline-Like Antibiotic Auricin from Streptomyces lavendulae Subsp. Lavendulae CCM 3239 Revealed Its High Similarity to Griseusins.

We previously identified the aur1 gene cluster in Streptomyces lavendulae subsp. lavendulae CCM 3239 (formerly Streptomyces aureofaciens CCM 3239), which is responsible for the production of the angucycline-like antibiotic auricin (1). Preliminary characterization of 1 revealed that it possesses an aminodeoxyhexose d-forosamine and is active against Gram-positive bacteria. Here we determined the structure of 1, finding that it possesses intriguing structural features, which distinguish it from other known angucyclines. In addition to d-forosamine, compound 1 also contains a unique, highly oxygenated aglycone similar to those of spiroketal pyranonaphthoquinones griseusins. Like several other griseusins, 1 also undergoes methanolysis and displays modest cytotoxicity against several human tumor cell lines. Moreover, the central core of the aur1 cluster is highly similar to the partial gris gene cluster responsible for the biosynthesis of griseusin A and B in both the nature of the encoded proteins and the gene organization.

Magnetization of active inclusion bodies: comparison with centrifugation in repetitive biotransformations.

BACKGROUND: Physiological aggregation of a recombinant enzyme into enzymatically active inclusion bodies could be an excellent strategy to obtain immobilized enzymes for industrial biotransformation processes. However, it is not convenient to recycle "gelatinous masses" of protein inclusion bodies from one reaction cycle to another, as high centrifugation forces are needed in large volumes. The magnetization of inclusion bodies is a smart solution for large-scale applications, enabling an easier separation process using a magnetic field. RESULTS: Magnetically modified inclusion bodies of UDP-glucose pyrophosphorylase were recycled 50 times, in comparison, inclusion bodies of the same enzyme were inactivated during ten reaction cycles if they were recycled by centrifugation. Inclusion bodies of sialic acid aldolase also showed good performance and operational stability after the magnetization procedure. CONCLUSIONS: It is demonstrated here that inclusion bodies can be easily magnetically modified by magnetic iron oxide particles prepared by microwave-assisted synthesis from ferrous sulphate. The magnetic particles stabilize the repetitive use of the inclusion bodies .

Exopolysaccharides of Lactobacillus reuteri: Their influence on adherence of E. coli to epithelial cells and inflammatory response.

The aim of the study was to characterize exopolysaccharides (EPS) originated from Lactobacillus reuteri strain DSM 17938 (EPS-DSM17938) and L. reuteri strain L26 Biocenol™ (EPS-L26) and evaluate their influence on adherence of enterotoxigenic Escherichia coli (ETEC) to IPEC-1 cells and proinflammatory gene expression. Both EPS were d-glucan polysaccharides with higher molecular weight (Mw), but differing in spatial conformation and elicited variable cytokine profile. EPS-DSM17938, relatively linear polysaccharide with (1→4) and (1→6) glycosidic linkages, increased IL-1ß gene expression (0.1mg/mL; P<0.05), while EPS-L26, more branched polysaccharide with (1→3) and (1→6) glycosidic linkages, exerted slight but statistically significant up-regulation of NF-κB, TNF-α and IL-6 mRNA (P<0.05). The most significant finding is that preincubation of IPEC-1 cells with both EPS followed by ETEC infection inhibit ETEC adhesion on IPEC-1 cells (P<0.01) and ETEC-induced gene expression of proinflammatory cytokine IL-1ß and IL-6 (P<0.01).

Enzymatic oxidation and separation of various saccharides with immobilized glucose oxidase.

Glucose oxidase from Aspergillus niger, the specific enzyme for beta-D-glucose oxidation, can also oxidize other related saccharides at very slow or negligible rates. The present study aimed to compare the kinetics of D-glucose oxidation using immobilized glucose oxidase on bead cellulose for the oxidation of related saccharides using the same biocatalyst. The significant differences were observed between the reaction rates for D-glucose and other saccharides examined. As a result, k (cat)/K (M) ratio for D-glucose was determined to be 42 times higher than D-mannose, 61.6 times higher than D-galactose, 279 times higher than D-xylose, and 254 times higher than for D-fructose and D-cellobiose. On the basis of these differences, the ability of immobilized glucose oxidase to remove D-glucose from D-cellobiose, D-glucose from D-xylose, and D-xylose from D-lyxose was examined. Immobilized catalase on Eupergit and mixed with immobilized glucose oxidase on bead cellulose or co-immobilized with glucose oxidase on bead cellulose was used for elimination of hydrogen peroxide from the reaction mixture. The accelerated elimination of D-glucose and D-xylose in the presence of co-immobilized catalase was observed. The co-immobilized glucose oxidase and catalase were able to decrease D-glucose or D-xylose content to 0-0.005% of their initial concentrations, while a minimum decrease of low oxidized saccharides D-xylose, D-cellobiose, and D-lyxose, respectively, was observed.

Enzymatic synthesis of sialylation substrates powered by a novel polyphosphate kinase (PPK3).

Active inclusion bodies of polyphosphate kinase 3 and cytidine 5'-monophosphate kinase were combined with whole cells that co-express sialic acid aldolase and CMP-sialic acid synthetase. The biocatalytic mixture was used for the synthesis of CMP-sialic acid, which was then converted to 3'-sialyllactose by whole cells.

Off-line FIA monitoring of D-sorbitol consumption during L-sorbose production using a sorbitol biosensor.

A ferricyanide mediated amperometric biosensor system implementing D-sorbitol dehydrogenase together with diaphorase for sensitive detection of D-sorbitol was used. The biosensor system was successfully integrated into an off-line FIA system with a throughput of detection of 10 h(-1). The device exhibited limit of detection of 20 microM with an average relative standard deviation of analysis of samples of 2.2%. The signal of the biosensor was linear up to 1.1 mM for D-sorbitol with sensitivity of (72 +/- 2) nA mM(-1), while a dynamic range was much wider up to 18 mM. The sorbitol biosensor gave reliable results even in the presence of a high molar excess of L-sorbose, a product of the biotransformation process, as judged from an excellent agreement with HPLC and GC.

Production of oligosaccharides and cellobionic acid by Fibrobacter succinogenes S85 growing on sugars, cellulose and wheat straw.

Extracellular culture fluid of Fibrobacter succinogenes S85 grown on glucose, cellobiose, cellulose or wheat straw was analysed by 2D-NMR spectroscopy. Cellodextrins did not accumulate in the culture medium of cells grown on cellulose or straw. Maltodextrins and maltodextrin-1P were identified in the culture medium of glucose, cellobiose and cellulose grown cells. New glucose derivatives were identified in the culture fluid under all the substrate conditions. In particular, a compound identified as cellobionic acid accumulated at high levels in the medium of F. succinogenes S85 cultures. The production of cellobionic acid (and cellobionolactone also identified) was very surprising in an anaerobic bacterium. The results suggest metabolic shifts when cells were growing on solid substrate cellulose or straw compared to soluble sugars.

Lectinomics II. A highway to biomedical/clinical diagnostics.

The review assesses current status and attempts to forecast trends in the development of lectin biorecognition technology. The progressive trend is characterized scientometrically and reflects the current transient situation, when standard low-throughput lectin-based techniques are being replaced by a novel microarray-based techniques offering high-throughput of detection. The technology is still in its infancy (validation phase), but already shows promise as an efficient tool to decipher the enormous complexity of the glycocode that influences physiological status of the cell. Further enhancement in robustness and flexibility of lectin microarrays is predicted by using recombinant and artificial lectins that will render production of lectin microarrays cost-effective and more affordable. Mass spectrometry is expected to play an important role to characterize the binding profile of new lectins. Differences in glycan recognition by lectins and anti-carbohydrate antibodies are given on a molecular basis, and strong and weak points of both biorecognition molecules in diagnosis are briefly discussed.

Negative matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry fragmentation of synthetic analogs of the O-specific polysaccharide of Vibrio cholerae O:1 in the presence of anionic dopants.

Oligosaccharides (tri- to hexamers) that represent terminal epitopes of O-antigens of Vibrio cholerae O:1, serotypes Ogawa and Inaba, have been studied by negative matrix-assisted laser desorption/ionization time of flight/time-of-flight mass spectrometry (MALDI ToF/ToF MS). The [M - H(+)](-) ions are formed after expulsion of a proton from molecules studied under condition of MALDI MS analysis in the negative mode. Several ammonium salts (chloride, nitrate, hydrogencarbonate and hydrogensulfate) were used as additives to increase the formation of negative ions from saccharides. The most efficient was the addition of ammonium hydrogencarbonate, which increased the amount of [M - H(+)](-) ions more than six times. Between three fragmentation pathways, the new conjugated transfer of electrons within the second downstream unit of oligosaccharides was discovered. Production of these ions, which has not been observed in any other kinds of measurement, distinguishes substances belonging to Ogawa and Inaba serotypes. The negative MALDI ToF/ToF mass spectra are simpler and, at the same time, more informative, as compared with positive and negative electrospray ionization ion trap as well as with positive MALDI ToF/ToF analysis.

Positive-ion fragmentation in matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry of synthetic analogs of the O-specific polysaccharide of Vibrio cholerae O:1.

Saccharides (mono- to hexamers) that mimic the terminal epitopes of O-antigens of Vibrio cholerae O:1, serotypes Ogawa and Inaba have been studied by matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI ToF/ToF) mass spectrometry (MS). Cationized adducts are the characteristic ions formed through the capture of sodium or potassium cations under MALDI MS conditions. Three characteristic pathways dominate in the fragmentation of model substances under MALDI ToF/ToF collision-induced dissociation MS/MS conditions of measurement. The first is the elimination which shortens the length of the oligosaccharide. In this way, conversion of the Ogawa to Inaba fragments takes place under the conditions of measurement. In the second, the conjugated transfer of electrons in the upstream unit of oligosaccharides takes place. The third route brings about the elimination of one alpha-hydroxy-gamma-butyrolactone molecule from the 4-(3-deoxy-L-glycero-tetronamido) group. The MALDI ToF/ToF MS/MS provided sufficient information about molecular mass, the number of saccharide residues, the structure of saccharides, the C(4)- amide of 3-deoxy-L-glycero-tetronic acid (DGT) of the compounds investigated, and allows to distinguish between Ogawa and Inaba serotypes.

Flow calorimetry--a useful tool for determination of immobilized cis-epoxysuccinate hydrolase activity from Nocardia tartaricans.

Bacterial cells Nocardia tartaricans with cis-epoxysuccinate hydrolase activity were entrapped in hardened calcium pectate gel by a commercial high performance encapsulator. This enzyme (in a single step reaction with no formation of side products) was used to hydrolyze disodium cis-epoxysuccinate to a pure enantiomer--disodium L-(+)-tartrate. Activities of this enzyme were determined using flow calorimetry. The validity of this method was corroborated by HPLC and isotachophoresis. The immobilized biocatalyst has activity (75.8 U/mgdry) able to convert disodium cis-epoxysuccinate to disodium tartrate at 94% yield in 5.5h. Immobilization of N. tartaricans in hardened calcium pectate gel beads had a positive effect on the activity of cis-epoxysuccinate hydrolase, storage stability, yield, and time of bioconversion.

Electron ionization mass spectrometric study of monomeric models of O-polysaccharides of Vibrio cholerae O:1, serotypes Ogawa and Inaba.

Fragmentation mechanisms of electron ionization (EI) mass spectrometry of the title compounds have been elucidated by high-resolution (HR) mass spectrometric measurements of the elemental composition and measurements of the metastable transitions (B(2)/E, CID). The experimental results were interpreted with the help of Mass Frontier 3.0 software, which aided the elucidation of fragmentation mechanisms and helped to deduce structures of the ions formed. Characteristic under the conditions of EI-MS measurement was the production of protonated adducts. Three distinct pathways observed include the formation of oxonium type ions, the conjugated transfer of electrons in the pyranose ring, and cleavage of the acylamide side chains. By applying the results obtained, the molecular mass, as well as the structures of both the saccharide and acylamide side chain involved in related substances, can be determined.