In the biosynthesis of lincosamide antibiotics lincomycin and celesticetin, the amino acid and amino sugar units are linked by an amide bond. The respective condensing enzyme lincosamide synthetase (LS) is expected to be an unusual system combining nonribosomal peptide synthetase (NRPS) components with so far unknown amino sugar related activities. The biosynthetic gene cluster of celesticetin was sequenced and compared to the lincomycin one revealing putative LS coding ORFs shared in both clusters. Based on a bioassay and production profiles of S. lincolnensis strains with individually deleted putative LS coding genes, the proteins LmbC, D, E, F and V were assigned to LS function. Moreover, the newly recognized N-terminal domain of LmbN (LmbN-CP) was also assigned to LS as a NRPS carrier protein (CP). Surprisingly, the homologous CP coding sequence in celesticetin cluster is part of ccbZ gene adjacent to ccbN, the counterpart of lmbN, suggesting the gene rearrangement, evident also from still active internal translation start in lmbN, and indicating the direction of lincosamide biosynthesis evolution. The in vitro test with LmbN-CP, LmbC and the newly identified S. lincolnensis phosphopantetheinyl transferase Slp, confirmed the cooperation of the previously characterized NRPS A-domain LmbC with a holo-LmbN-CP in activation of a 4-propyl-L-proline precursor of lincomycin. This result completed the functional characterization of LS subunits resembling NRPS initiation module. Two of the four remaining putative LS subunits, LmbE/CcbE and LmbV/CcbV, exhibit low but significant homology to enzymes from the metabolism of mycothiol, the NRPS-independent system processing the amino sugar and amino acid units. The functions of particular LS subunits as well as cooperation of both NRPS-based and NRPS-independent LS blocks are discussed. The described condensing enzyme represents a unique hybrid system with overall composition quite dissimilar to any other known enzyme system.

• MeSH
• cystein metabolismus   MeSH
• glykopeptidy metabolismus   MeSH
• inositol metabolismus   MeSH
• linkomycin biosyntéza   MeSH
• linkosamidy biosyntéza   MeSH
• peptidsynthasy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• celesticetin A MeSH Prohlížeč
• cystein MeSH
• glykopeptidy MeSH
• inositol MeSH
• linkomycin MeSH
• linkosamidy MeSH
• mycothiol MeSH Prohlížeč
• peptidsynthasy MeSH

A cosmid bearing an insert of 38 217 bp covering the gene cluster and its flanking regions of type strain Streptomyces lincolnensis ATCC 25466 was sequenced. Two relatively extensive sequence changes and several hundred point mutations were identified if compared with the previously published sequence of the lincomycin (Lin) industrial strain S. lincolnensis 78-11. Analysis of the cluster-flanking regions revealed its localization within the genome of the ATCC 25466 strain. The cluster-bearing cosmid was integrated into the chromosome of Lin non-producing strains S. coelicolor CH 999 and S. coelicolor M 145. The modified strains heterologously produced Lin but the level dropped to approximately 1-3% of the production in the ATCC 25466 strain.

• MeSH
• antibakteriální látky biosyntéza   chemie   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• biotechnologie MeSH
• bodová mutace MeSH
• genová knihovna MeSH
• kosmidy MeSH
• linkomycin biosyntéza   chemie   MeSH
• multigenová rodina * MeSH
• sekvenční analýza DNA * MeSH
• Streptomyces coelicolor genetika   metabolismus   MeSH
• Streptomyces genetika   růst a vývoj   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• bakteriální proteiny MeSH
• linkomycin 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
• antibakteriální látky biosyntéza   MeSH
• DNA bakterií genetika   MeSH
• hybridizace nukleových kyselin * metody   MeSH
• linkomycin analogy a deriváty   biosyntéza   MeSH
• linkosamidy MeSH
• mapování chromozomů * metody   MeSH
• multigenová rodina * MeSH
• operon genetika   MeSH
• regulace genové exprese u bakterií MeSH
• restrikční mapování MeSH
• sekvenční homologie nukleových kyselin MeSH
• Southernův blotting MeSH
• Streptomyces enzymologie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• celesticetin A MeSH Prohlížeč
• DNA bakterií MeSH
• linkomycin MeSH
• linkosamidy MeSH