Structurally different and functionally diverse natural compounds - antitumour agents pyrrolo[1,4]benzodiazepines, bacterial hormone hormaomycin, and lincosamide antibiotic lincomycin - share a common building unit, 4-alkyl-L-proline derivative (APD). APDs arise from L-tyrosine through a special biosynthetic pathway. Its generally accepted scheme, however, did not comply with current state of knowledge. Based on gene inactivation experiments and in vitro functional tests with recombinant enzymes, we designed a new APD biosynthetic scheme for the model of lincomycin biosynthesis. In the new scheme at least one characteristic in each of five final biosynthetic steps has been changed: the order of reactions, assignment of enzymes and/or reaction mechanisms. First, we demonstrate that LmbW methylates a different substrate than previously assumed. Second, we propose a unique reaction mechanism for the next step, in which a putative γ-glutamyltransferase LmbA indirectly cleaves off the oxalyl residue by transient attachment of glutamate to LmbW product. This unprecedented mechanism would represent the first example of the C-C bond cleavage catalyzed by a γ-glutamyltransferase, i.e., an enzyme that appears unsuitable for such activity. Finally, the inactivation experiments show that LmbX is an isomerase indicating that it transforms its substrate into a compound suitable for reduction by LmbY, thereby facilitating its subsequent complete conversion to APD 4-propyl-L-proline. Elucidation of the APD biosynthesis has long time resisted mainly due to the apparent absence of relevant C-C bond cleaving enzymatic activity. Our proposal aims to unblock this situation not only for lincomycin biosynthesis, but generally for all above mentioned groups of bioactive natural products with biotechnological potential.

• Keywords
• 4-propyl-L-proline, antibiotics, anticancer drug, hormaomycin, lincomycin, natural product biosynthesis, pyrrolobenzodiazepine, secondary metabolism,
• Publication type
• Journal Article MeSH

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
• Anti-Bacterial Agents chemistry   metabolism   pharmacology   MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Humans MeSH
• Lincomycin analogs & derivatives   chemistry   metabolism   pharmacology   MeSH
• Microbial Sensitivity Tests MeSH
• Molecular Structure MeSH
• Proline analogs & derivatives   metabolism   MeSH
• Staphylococcal Infections microbiology   MeSH
• Staphylococcus drug effects   MeSH
• Streptomyces genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Bacterial Proteins MeSH
• Lincomycin MeSH
• Proline 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
• Anti-Bacterial Agents biosynthesis   chemistry   MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Biotechnology MeSH
• Point Mutation MeSH
• Gene Library MeSH
• Cosmids MeSH
• Lincomycin biosynthesis   chemistry   MeSH
• Multigene Family * MeSH
• Sequence Analysis, DNA * MeSH
• Streptomyces coelicolor genetics   metabolism   MeSH
• Streptomyces genetics   growth & development   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Bacterial Proteins MeSH
• Lincomycin MeSH