The lincomycin biosynthetic gene lmbX was deleted in Streptomyces lincolnensis ATCC 25466, and deletion of this gene led to abolition of lincomycin production. The results of complementation experiments proved the blockage in the biosynthesis of lincomycin precursor 4-propyl-L-proline. Feeding this mutant strain with precursor derivatives resulted in production of 4'-butyl-4'-depropyllincomycin and 4'-pentyl-4'-depropyllincomycin in high titers and without lincomycin contamination. Moreover, 4'-pentyl-4'-depropyllincomycin was found to be more active than lincomycin against clinical Staphylococcus isolates with genes determining low-level lincosamide resistance.

• MeSH
• antibakteriální látky farmakologie   chemie   metabolismus   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• lidé MeSH
• linkomycin analogy a deriváty   farmakologie   chemie   metabolismus   MeSH
• mikrobiální testy citlivosti MeSH
• molekulární struktura MeSH
• prolin analogy a deriváty   metabolismus   MeSH
• stafylokokové infekce mikrobiologie   MeSH
• Staphylococcus účinky léků   MeSH
• Streptomyces genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

The biosynthetic pathway of the clinically important antibiotic lincomycin is not known in details. The precise knowledge of the lincomycin biosynthesis is a prerequisite for generation of improved derivatives by means of combinatorial genetics. Methods allowing determination of the key intermediates are very important tools of the pathway investigation. Two new high-performance liquid chromatography methods with fluorescence detection for determination of lincomycin precursors in fermentation broth of Streptomyces lincolnensis and its lincomycin nonproducing mutants were developed. The first one enables simultaneous analysis of methylthiolincosamide (MTL) and N-demethyllincomycin (NDL), whereas the second one is suitable for 4-propyl-L-proline (PPL) assay. Both methods are based on the pre-column derivatization: MTL and NDL with 4-chloro-7-nitrobenzofurazan; PPL with o-phthaldialdehyde. The methods were validated with lower limit of quantification values of 2.50, 3.75, and 3.75 microg ml(-1) for MTL, NDL, and PPL, respectively. The inter- and intra-day accuracies and precisions were all within 12%. Stability of oxidized and derivatized analytes was investigated.

• MeSH
• amidy analýza   MeSH
• fermentace MeSH
• financování organizované MeSH
• fluorescence MeSH
• linkomycin analogy a deriváty   biosyntéza   MeSH
• molekulární struktura MeSH
• prolin analogy a deriváty   analýza   MeSH
• reprodukovatelnost výsledků MeSH
• Streptomyces metabolismus   MeSH
• sulfhydrylové sloučeniny analýza   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH

The first insight into celesticetin biosynthetic gene cluster of S. caelestis is presented. The genomic DNA of producing strain was digested, digoxigenin-labeled and hybridized with a set of probes designed according to S. lincolnensis gene sequences. Genes with high homology to the lincomycin biosynthetic genes coding for the predicted common parts of the pathway were identified in S. caelestis. Then, genomic DNA of S. caelestis treated by a multiple digestion was hybridized with five digoxigenin-labeled probes to construct a rough restriction map. Two consecutive islands formed by the genes with a putative function in biosynthesis of the shared saccharide moiety revealed an organization similar to the lincomycin biosynthetic gene cluster. The celesticetin cluster was mapped and essential information was obtained for subsequent steps, i.e. isolation and sequence analysis of the cluster.

• MeSH
• digoxigenin analogy a deriváty   MeSH
• DNA bakterií genetika   MeSH
• finanční podpora výzkumu jako téma MeSH
• hybridizace genetická genetika   imunologie   MeSH
• léková rezistence genetika   imunologie   MeSH
• linkomycin analogy a deriváty   analýza   chemie   MeSH
• mapování chromozomů metody   MeSH
• otevřené čtecí rámce genetika   MeSH
• polymerázová řetězová reakce metody   využití   MeSH
• regulace genové exprese u bakterií MeSH
• Southernův blotting metody   využití   MeSH
• Streptomyces coelicolor enzymologie   genetika   MeSH
• Streptomyces enzymologie   genetika   MeSH

• Klíčová slova
• CLINDAMYCIN,
• MeSH
• acne vulgaris farmakoterapie   MeSH
• léčivé přípravky kontraindikace   MeSH
• lékové interakce MeSH
• lidé MeSH
• linkomycin analogy a deriváty   MeSH
• nežádoucí účinky léčiv MeSH
• Check Tag
• lidé MeSH