ABSTRACT
Colabomycin E is a new member of the manumycin-type metabolites produced by the strain Streptomyces aureus SOK1/5-04 and identified by genetic screening from a library of streptomycete strains. The structures of colabomycin E and accompanying congeners were resolved. The entire biosynthetic gene cluster was cloned and expressed in Streptomyces lividans. Bioinformatic analysis and mutagenic studies identified components of the biosynthetic pathway that are involved in the formation of both polyketide chains. Recombinant polyketide synthases (PKSs) assembled from the components of colabomycin E and asukamycin biosynthetic routes catalyzing the biosynthesis of "lower" carbon chains were constructed and expressed in S. aureus SOK1/5-04 ΔcolC11-14 deletion mutant. Analysis of the metabolites produced by recombinant strains provided evidence that in both biosynthetic pathways the length of the lower carbon chain is controlled by an unusual chain-length factor supporting biosynthesis either of a triketide in asukamycin or of a tetraketide in colabomycin E. Biological activity assays indicated that colabomycin E significantly inhibited IL-1ß release from THP-1 cells and might thus potentially act as an anti-inflammatory agent.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/metabolism , Polyunsaturated Alkamides/chemistry , Polyunsaturated Alkamides/metabolism , Streptomyces/metabolism , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line , Dose-Response Relationship, Drug , Humans , Interleukin-1beta/metabolism , Molecular Structure , Polyunsaturated Alkamides/pharmacology , Streptomyces/chemistry , Structure-Activity RelationshipABSTRACT
A new simple ultra-high-performance liquid chromatography method with diode array detection (UHPLC-DAD) was developed for chemical fingerprinting analysis of extracellular metabolites in fermentation broth of Geosmithia spp. The SPE method employing Oasis MCX strong cation-exchange mixed-mode polymeric sorbent was chosen for extraction of the metabolites. The analyses were performed on an Acquity UPLC BEH C18 column (100 × 2.1 mm i.d.; particle size, 1.7 µm; Waters) using a gradient elution program with an aqueous solution of trifluoroacetic acid and acetonitrile as the mobile phase. The applicability of the method was proved by analysis of 38 strains produced by different species and isolated from different sources (hosts). The results revealed the correlation of obtained UHPLC-DAD fingerprints with taxonomical identity.
Subject(s)
Chromatography, High Pressure Liquid/methods , Hypocreales/chemistry , Solid Phase Extraction/methods , Chromatography, High Pressure Liquid/economics , Fermentation , Hypocreales/metabolism , Reproducibility of Results , Solid Phase Extraction/economicsABSTRACT
Antibiotics contained in animal manure can contaminate soil, groundwater and eventually surface and drinking water. To reduce the usage of antibiotics in livestock industry the EU banned their application as growth promoters in 2006. Even though the antibiotics are still used for this purpose and therefore it is necessary to control their applications. An Ultra Performance Liquid Chromatography method (UPLC) with UV detection for determination of tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), and doxycycline (DOX) including their epimers in the liquid hog manure was developed. The antibiotics were extracted with ethyl acetate and separated on UPLC BEH Shield RP18 column. The validated method was selective for all analytes and system suitability was assessed. Calibration curves ranged from 7.8 to 250.0mugmL(-1) with determination coefficient of 0.9999. The method limits of quantification ranged from 0.9 to 1.6mgkg(-1). Recoveries were 52.4+/-3.8%, 72.4+/-5.0%, 83.8+/-5.7% and 95.9+/-4.7% for TC, OTC, CT, and DOX, respectively. The method was used for the determination of TC, OTC, CT, and DOX in liquid hog manure samples.
