| The macrotetrolides are a family of cyclic polyether polyketide antibiotics produced by Streptomyces griseus DSM40695. These structurally unique natural products are composed of four units of (±)-nonactic (NA) and or (±)-homonactic acids (HNA), stereospecifically linked via ester bonds in an alternating (+)-(−)-(+)-(−) pattern of enantiomeric forms. Previous feeding experiments with various isotopically labeled precursors raised the suggestion that each enantiomer is produced by a pair of enantiospecific polyketide pathways. Thus, macrotetrolide biosynthesis can be viewed as the perfect model system in which to understand how polyketide synthases (PKS's) control the stereochemistry of their products.; The 25-kb macrotetrolide biosynthetic gene cluster was isolated from a S. griseus genomic library using the previously isolated resistance gene (nonR) as probe. Sequence analysis revealed an atypical PKS lacking an acyl carrier protein (ACP) gene, and targeted gene inactivation and heterologous expression of the cluster in S. lividans 1326 unambiguously confirmed that the cloned DNA contains all genes necessary to support macrotetrolide biosynthesis.; Functions of individual genes putatively involved in biosynthesis on the basis of sequence homology were investigated by generation of S. griseus DSM40695 mutants via targeted gene replacement and in vivo complementation. Four ketosynthases (KS's) ( nonQPKJ), four ketoreductases (KR's) (nonONME), an enoyl CoA-hydratase (ECH) (nonXS), and a CoA-transferase (CoAT) (nonT) were inactivated in S. griseus or the S. lividans 1326 harboring the macrotetrolide cluster, and production restored by in trans expression of the gene in the mutant, demonstrating an essential biosynthetic role. Similar experiments showed that KS gene (nonU) and nonC (no homologue) influenced macrotetrolide production but were not essential, presumably via utilization of precursors or by regulatory role (activator), respectively.; The role of the essential biosynthetic genes in the enantiospecific pathways was investigated by fermenting the non-producing mutants in the presence of (±)-NA. Results suggested that nonXS and nonON encode for pairs of enantiospecific enzymes, nonT, nonQP, for enzymes in an achiral pathway, and nonKJ for proteins responsible for the tetramerization activity. Additionally, the potential for precursor-directed biosynthesis was demonstrated by the apparent production of several macrotetrolide analogs by feeding of a non-natural analog, (±)-n-propyl-nonactic acid. |
