Effect of starter unit availability on the spectrum of manumycin-type metabolites produced by Streptomyces nodosus ssp. asukaensis.

AIMS: Production of minor asukamycin congeners and its new derivatives by combination of targeted genetic manipulations with specific precursor feeding in the producer of asukamycin, Streptomyces nodosus ssp. asukaensis. METHODS AND RESULTS: Structural variations of manumycins lie only in the diverse initiation of the 'upper' polyketide chain. Inactivation of the gene involved in the biosynthesis of cyclohexanecarboxylic acid (CHC) turned off the production of asukamycin in the mutant strain and allowed an increased production of other manumycins with the branched end of the upper chain. The ratio of produced metabolites was further affected by specific precursor feeding. Precursor-directed biosynthesis of a new asukamycin analogue (asukamycin I, 28%) with linear initiation of the upper chain was achieved by feeding norleucine to the mutant strain. Another asukamycin analogue with the unbranched upper chain (asukamycin H, 14%) was formed by the CHC-deficient strain expressing a heterologous gene putatively involved in the formation of the n-butyryl-CoA starter unit of manumycin A. CONCLUSIONS: Combination of the described techniques proved to be an efficient tool for the biosynthesis of minor or novel manumycins. SIGNIFICANCE AND IMPACT OF THE STUDY: Production of two novel asukamycin derivatives, asukamycins H and I, was achieved. Variations appeared in the upper polyketide chain, the major determinant of enzyme-inhibitory features of manumycins, affecting their cancerostatic or anti-inflammatory features.

Glucosylglycerate is an osmotic solute and an extracellular metabolite produced by Streptomyces caelestis.

Streptomyces caelestis DSM 40084 produces two osmolytes, viz. 2-O-(alpha-D-glucopyranosyl)-zeta-glyceric acid (GG) and trehalose. Both compounds were isolated and identified by nuclear magnetic resonance spectroscopy and mass spectrometry. A very sensitive regulation of the cell osmolytes was demonstrated in exponentially growing cultures. The intracellular levels of GG and trehalose increased 2x in response to a step change of medium osmolarity caused by 0.3% NaCl. 1H NMR analysis of the cell extracts did not confirm the presence of additional osmolytes. GG is a S. caelestis metabolite commonly released from the cells; its concentration reached 3 g/L during the cultivation in a yeast extract--(NH4)2SO4-glycerol medium. This is the first report on the occurrence of the ionic osmolyte GG in the genus Streptomyces and on its free excretion to the medium.

Norleucine, a natural occurrence in a novel ergot alkaloid gamma-ergokryptinine.

A novel natural peptide ergot alkaloid gamma-ergokryptinine containing norleucine has been isolated from ergot sclerotia of the field-growing parasitic fungus Claviceps purpurea CCM 8059. Its structure was deduced from the NMR and mass spectral data. The final structural proof was provided by the crystal structure determination, which is the first X-ray structure of a natural Nle-containing secondary metabolite. The conformations of three ergopeptinines: gamma-ergokryptinine, ergoladinine, and alpha-ergokryptinine were compared.

11-Demethylcyclosporins exhibit a single conformation in methanol and dimethylsulfoxide.

NMR studies showed that 11-demethylcyclosporin A (cyclosporin E) and 11-demethylcyclosporin B exist as single species both in polar and nonpolar solvents. They adopt the same conformation that was found in the solid state.

Screening of basidiomycete fungi for the quinone-dependent sugar C-2/C-3 oxidoreductase, pyranose dehydrogenase, and properties of the enzyme from Macrolepiota rhacodes.

Mycelial cultures of 76 strains of lignocellulose-degrading basidiomycete fungi were screened for the activity of pyranose dehydrogenase, a novel sugar oxidoreductase recently detected in Agaricus bisporus. Of these fungi, 37 strains belonging to seven phylogenetically related genera of mostly litter-decomposing Agaricales were positive for the dehydrogenase, based on activity assays towards D-glucose with 1,4-benzoquinone or ferricenium ion as electron acceptors, and on TLC/HPLC analyses of the reaction products. Lack of activity with O(2) as the oxidant, specificity for C-3 of D-glucose, and active extracellular secretion of the enzyme were used as criteria to differentiate pyranose dehydrogenase from pyranose 2-oxidase (EC 1.1.3.10), known to be produced by numerous wood-rotting fungi. Extracellular pyranose dehydrogenase from Macrolepiota rhacodes was heavily glycosylated. The enzyme was characterized as a 78-kDa flavoprotein under denaturing conditions and a 76-kDa native protein using gel filtration. This enzyme had a maximum extracellular activity of 4.1 U ml(-1) in 39-day liquid cultures. It exhibited broad selectivity for sugar substrates and oxidized D-glucose (K(m)=1.82) exclusively at C-3 to 3-dehydro-D-glucose (D-ribo-hexos-3-ulose), in contrast to pyranose dehydrogenases from Agaricus species, which acted at both C-3 and C-2 of D-glucose. The N-terminal sequence, AVVYRHPDEL, showed significant similarity with that reported for A. bisporus.

Oxidation of lincomycin by hydrogen peroxide restricts its potential biotransformation with haloperoxidases.

Lincomycin biotransformation was conducted by using Streptomyces venezuelae and Streptomyces phaeochromogenes cell-free extracts. Reaction products were isolated and identified by MS and NMR spectroscopy as lincomycin sulfoxide and lincomycin sulfone. Both compounds arise also by chemical oxidation with hydrogen peroxide; this reaction represents a new efficient way for the preparation of lincomycin sulfoxide and lincomycin sulfone and simultaneously excludes the biotransformation of lincomycin using haloperoxidases.

Chemoenzymatic preparation of silybin beta-glucuronides and their biological evaluation.

Chemoenzymatic glucuronidation of the optically pure silybin A (1) using ovine liver glucuronyl transferase afforded three beta-glucuronides of silybin, substituted at phenolic OH groups at the positions C-20 (2), C-7 (3), and C-5 (4) formed in the yields 27, 62.5, and 2.5%, respectively. Using these standards, it was shown that the main silybin conjugate in humans is its 20-beta-D-glucuronate (2), while the C-7 regioisomer (3) was formed in lower proportion. The rate of conjugation of (natural) silybin diastereomers 10S, 11S and 10R, 11R, and therefore also their metabolism in humans is rather different. The radical scavenging activity of 2 is considerably lower than that of its aglycone (1); however, the activity of 3 is higher than in the silybin. These findings corroborate the hypothesis that, at physiological pH, the exclusive target for one-electron oxidation of the silybin molecule is the o-methoxy-phenolic structure at C-19, C-20. This is first pharmacological study using optically pure silybin.

Double oxidation of D-xylose to D-glycero -pentos-2,3-diulose (2,3-diketo-D-xylose) by pyranose dehydrogenase from the mushroom Agaricus bisporus.

Pyranose dehydrogenase purified to homogeneity from the mycelia of the basidiomycete fungus Agaricus bisporus catalyzed the oxidation of D-xylose at C-2 to D-threo-pentos-2-ulose (2-keto-D-xylose) and successively at C-3 to D-glycero-pentos-2,3-diulose (2,3-diketo-D-xylose) using 1,4-benzoquinone as an electron acceptor. The sites of oxidation were deduced from the spectroscopic analysis (MS, NMR) of the N,N-diphenylhydrazone derivatives of the reaction products.

Destruxin Ed1 a cyclopeptide from the fungus Metarhizium anisopliae.

A new destruxin Ed1 has been isolated from the entomopathogenic fungus Metarhizium anisopliae. Its structure was deduced from the NMR and mass spectral data.

Rapid screening of destruxins by liquid chromatography/mass spectrometry.

A liquid chromatographic/mass spectrometric (LC/MS) method utilizing an atmospheric pressure chemical ionization (APCI) interface and in-source collisionally induced dissociation (CID) was developed for the rapid screening of the cyclic hexadepsipeptides destruxins, produced by the fungi Metarhizium anisopliae and Trichothecium roseum. The APCI mass spectra are fully consistent with the high-energy CID data but the sequence ions are abundant over the whole mass range. In addition to 22 known destruxins, two new representatives of this family were detected by LC/MS analysis: destruxin Ed1 and roseotoxin A were isolated and their structures were inferred from MS/MS and NMR data.

beta-Glucosylation of chitooligomers by galactosyltransferase.

Galactosyltransferase from bovine milk was found to be able to utilise UDP-Glc to transfer Glc onto GlcNAc and chitooligomers[-beta-GlcNAc-(1-->4)-]n, n = 2-4. beta-Glucosylated products were used in binding studies with NKR-P1A protein cloned from rat natural killer cells.

Biotransformation of diethenylbenzenes, V. Identification of urinary metabolites of 1,2-diethenylbenzene in the rat.

1. Biotransformation of 1,2-diethenylbenzene (1) in rat was studied. Five urinary metabolites were isolated by extraction of acid hydrolysed urine and identified by nmr and mass spectroscopy, namely, 1-(2-ethenylphenyl)ethane-1,2-diol (2) 2-ethenylmandelic acid (3), 2-ethenylphenylglyoxylic acid (4), 2-ethenylphenylacetylglycine (5) N-acetyl-S-[1-(2-ethenylphenyl)-2-hydroxyethyl]cysteine (6) and N-acetyl-S-[2-(2-ethenylphenyl)-2-hydroxy-ethyl]cysteine (7). 2. In addition, minor metabolites, namely, 2-ethenylbenzoic acid (8) and 2-ethenylphenyl-acetic acid (9) were identified by glc-mass spectral analysis of the hydrolysed urine extract treated subsequently with diazomethane, hydroxylamine and a trimethyl-silylating reagent. Several compounds, which could arise from biotransformation of both ethenyl groups in the molecule of 1, were detected but not identified unequivocally. 3. A glucuronide was detected by tlc analysis of urine as a blue spot after spraying with naphthoresorcinol. Compounds showing molecular fragments indicating the glucuronide moiety were also detected by glc-mass spectroscopy in non-hydrolysed urine samples. 4. The total thioether excretion amounted to 5.3 +/- 2.4, 5.1 +/- 3.4 and 5.0 +/- 1.9% of the dose at 500, 300 and 100 mg/kg, respectively (mean +/- SD; n = 5). 5. Like styrene and other diethenylbenzene isomers, 1,2-diethenylbenzene is metabolically activated to a reactive epoxide intermediate, 2-ethenylphenyloxirane (10), which is further converted to the urinary metabolites mentioned above. The main detoxification pathways are hydrolysis to the glycol 2 followed by several oxidation steps, and conjugation with glutathione. The latter reaction is both regioselective and stereoselective. 6. The ratio of mercapturic acids 6:7 was 83:17. Each regioisomer consists of two diastereomers which show distinct resonance signals in the 13C-nmr. The diastereomer ratio was 82:28 and 79:21 for 6 and 7 respectively.

Ergot alkaloid glycosides with immunomodulatory activities.

New glycosides derived from ergot alkaloids elymoclavine and DH-lysergol were synthesized by chemoenzymatic methods. beta-Glucosides were obtained either by chemical method or by transglycosylation (glycosidase from Aspergillus oryzae), lactosides were prepared by further extension of carbohydrate chain using beta-1,4-galactosyltransferase (bovine milk) and alpha-5-N-acetylneuraminyl-(2-->6)-beta-D-galactopyranosyl-(l-->4)-2- acetamido-2-deoxy-beta-D-glucopyranosyl-(1-->O)-elymoclavine was prepared using alpha-2,6-sialyltransferase (rat liver). Immunomodulatory activity of elymoclavine and 9,10-dihydrolysergol and their glycosylated derivatives on natural killer (NK) cell-mediated cytotoxicity of human resting and activated human peripheral blood mononuclear cells (PBMC) was investigated. Addition of ergot alkaloid glycosides to the mixtures of effector and target cells potentiated the PBMC cytotoxicity against both NK-sensitive and -resistant target cells. The glycoconjugates of elymoclavine enhanced cytotoxicity of PBMC against NK-resistant target cells. The glycoconjugates of DH-lysergol potentiated NK cytotoxicity of PBMC against NK-sensitive target cells.

Direct resolution of optically active isomers on chiral packings containing ergoline skeletons. 5. Enantioseparation of amino Acid derivatives.

A new procedure for ergot alkaloid-based chiral stationary phase preparation is described. Synthesis is based on bonding the allyl derivative of terguride to mercaptopropylsilanized silica gel. The packing exhibits higher content of chiral selector, stability, reproducibility, and enantioselectivity toward amino acids compared to that previously studied. The chromatographic behavior of amino acids with different side chains and substituent groups is investigated in order to obtain a deeper insight into the enantiodiscriminative mechanism as well as to determine the limitations and strengths of terguride as a chiral selector for this class of compounds. A variety of factors, including mobile phase parameters such as pH, ionic strength, content and nature of organic modifier, and temperature, are examined.

An unusual side chain C-C cleavage at the MeBmt amino acid in cyclosporin A.

The mixture of products obtained by alkaline treatment of cyclosporin A was analyzed by HPLC-continuous-flow-FAB/MS. The changes involve the atypical amino acid (4R)-4-((E)-2-butenyl)-4,N-dimethyl-L-threonine (MeBmt) without affecting the cyclic structure. The main degradation pathway is dehydration producing all four possible anhydro-MeBmt containing cyclosporins. A new cyclosporin, [Sar(1)]CS, resulting from the side chain cleavage of MeBmt has been isolated and characterized.

Cyclosporins from Tolypocladium terricola.

New natural cyclosporins were isolated from the mycelium of surface cultivated fungus Tolypocladium terricola. The chemical structures of [Leu4] CS and [MeLeu1] CS = cyclosporin-J, were deduced from the NMR and mass spectral data. Biological activity of new cyclosporins is reported based on the proliferative mitogen stimulation test.

BF3-catalysed methanolysis combined with fast atom bombardment tandem mass spectrometry as a new tool for the preparation and analysis of linear secocyclosporins.

BF3-catalysed methanolysis is presented for cyclic peptide cleavage using cyclosporins as model compounds. The reaction conditions for BF3-catalysed methanolysis of cyclosporins were optimized to favour the formation of linear undecapeptides. The resulting secocyclosporins were analysed by fast atom bombardment tandem mass spectrometry. Only one dominant and two side mechanisms of the ring opening are operating so that the complete sequence determination of all prepared oligopeptides was achieved.

Beauverolides L and La from Beauveria tenella and Paecilomyces fumosoroseus.

New beauverolides L and La were isolated and identified from the entomopathogenic fungi, Beauveria tenella and Paecilomyces fumosoroseus. Their structures, cyclo-[3-hydroxy-4-methyldecanoyl-L-phenylalanyl-L-alanyl-D-leucyl ], and cyclo-[3-hydroxy-4-methyldecanoyl-L-phenylalanyl-L-alanyl-D-allo-i soleucyl] were deduced from HPLC and GC-mass spectrometric analyses of their hydrolysates and NMR and mass spectral data.