A new separation and quantification method using ultra-performance liquid chromatography (UPLC) with UV detection was developed for detection of lincomycin traces in fermentation broth of different Streptomyces spp. A similar high-performance liquid chromatography (HPLC) protocol was simultaneously developed for comparison purposes. Both methods were validated and showed a linear range of detector response for quantification of lincomycin in concentration from 3.125 to 1000.0 microgml(-1) with correlation coefficient 0.999 and recoveries ranging from 81.5 to 89.85% with precision < or =5%. Compared with the HPLC, the UPLC method offered high sample throughput and about 10 times lower consumption of solvents. The developed assays were used for determination of lincomycin production in genetically manipulated production strain Streptomyces lincolnensis and for determination of lincomycin production after heterologous expression of lincomycin biosynthetic gene cluster in non-producing strain Streptomyces coelicolor.
- MeSH
- chromatografie kapalinová metody MeSH
- fermentace MeSH
- financování organizované MeSH
- geneticky modifikované organismy metabolismus MeSH
- linkomycin analýza MeSH
- senzitivita a specificita MeSH
- spektrofotometrie ultrafialová metody MeSH
- Streptomyces genetika metabolismus 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
