Niddamycin

A previously unknown chemical structure, 6-desmethyl-6-ethylerythromycin A (6-ethylErA), was produced through directed genetic manipulation of the erythromycin (Er)-producing organism Saccharopolyspora erythraea. In an attempt to replace the methyl side chain at the C-6 position of the Er polyketide backbone with an ethyl moiety, the methylmalonate-specific acyltransferase (AT) domain of the Er polyketide synthase was replaced with an ethylmalonate-specific AT domain from the polyketide synthase involved in the synthesis of the 16-member macrolide niddamycin. The genetically altered strain was found to produce ErA, however, and not the ethyl-substituted derivative. When the strain was provided with precursors of ethylmalonate, a small quantity of a macrolide with the mass of 6-ethylErA was produced in addition to ErA. Because substrate for the heterologous AT seemed to be limiting, crotonyl-CoA reductase, a primary metabolic enzyme involved in butyryl-CoA production in streptomycetes, was expressed in the strain. The primary macrolide produced by the reengineered strain was 6-ethylErA.

The genes encoding the polyketide synthase (PKS) portion of the niddamycin biosynthetic pathway were isolated from a library of Streptomyces caelestis NRRL-2821 chromosomal DNA. Analysis of 40 kb of DNA revealed the presence of five large open reading frames (ORFs) encoding the seven modular sets of enzymatic activities required for the synthesis of a 16-membered lactone ring. The enzymatic motifs identified within each module were consistent with those predicted from the structure of niddamycin. Disruption of the second ORF of the PKS coding region eliminated niddamycin production, demonstrating that the cloned genes are involved in the biosynthesis of this compound.

A new actinomycete strain designated US24 producing antibacterial activities against Gram-positive and Gram-negative bacteria was isolated from Tunisian soil. Culture characteristic studies strongly suggested that the US24 strain belonged to the genus Streptomyces. Analysis of the nucleotide sequence of the 16S rRNA gene of the Streptomyces sp. US24 strain showed high similarity (98%) with the 16S rRNA gene of Streptomyces caelestis which produces two antibiotics, niddamycin and celesticetin. Study of the influence of different nutritional compounds on antibiotic biosynthesis showed that the highest antibacterial activities were obtained when starch at 1% (w/v) was used as sole carbon source in the presence of traces of mineral oligoelements. Application to the supernatant culture of the Streptomyces sp. US24 strain of various separation steps led to isolation of two pure active molecules having a retention time of 34 and 37.26 min, respectively. P(34 min) possessed antibacterial activity against Gram-positive and Gram-negative bacteria, whereas P(37.26 min) inhibited only Gram-positive bacteria. Partial characterization of the P(34 min) molecule using spectroscopic studies showed that this active molecule is different from the two antibiotics produced by the S. caelestis strain.