Cremimycin is a 19-membered macrolactam glycoside antibiotic based on three distinctive substructures: 1) a β-amino fatty acid starter moiety, 2) a bicyclic macrolactam ring, and 3) a cymarose unit. To elucidate the biosynthetic machineries responsible for these three structures, the cremimycin biosynthetic gene cluster was identified. The cmi gene cluster consists of 33 open reading frames encoding eight polyketide synthases, six deoxysugar biosynthetic enzymes, and a characteristic group of five β-amino-acid-transfer enzymes. Involvement of the gene cluster in cremimycin production was confirmed by a gene knockout experiment. Further, a feeding experiment demonstrated that 3-aminononanoate is a direct precursor of cremimycin. Two characteristic enzymes of the cremimycin-type biosynthesis were functionally characterized in vitro. The results showed that a putative thioesterase homologue, CmiS1, catalyzes the Michael addition of glycine to the β-position of a non-2-enoic acid thioester, followed by hydrolysis of the thioester to give N-carboxymethyl-3-aminononanoate. Subsequently, the resultant amino acid was oxidized by a putative FAD-dependent glycine oxidase homologue, CmiS2, to produce 3-aminononanoate and glyoxylate. This represents a unique amino transfer mechanism for β-amino acid biosynthesis.
Quinolidomicin A 1 is the largest macrolide compound from terrestrial sources, consisting of a 60membered ring, and its biosynthetic gene cluster was revealed to be over 200 kb. The gene cluster for quinolidomicin was cloned and heterologously expressed. Confirmation of the product led to a structural revision, in which the hydroxy group in the chromophore moiety of the reported structure was replaced by an amine group.
Telomestatin, a strong telomerase inhibitor with G-quadruplex stabilizing activity, is a potential therapeutic agent for treating cancers. Difficulties in isolating telomestatin from microbial cultures and in chemical synthesis are bottlenecks impeding the wider use. Therefore, improvement in telomestatin production and structural diversification are required for further utilization and application. Here, we discovered the gene cluster responsible for telomestatin biosynthesis, and achieved production of telomestatin by heterologous expression of this cluster in the engineered Streptomyces avermitilis SUKA strain. Utilization of an optimal promoter was essential for successful production. Gene disruption studies revealed that the tlsB, tlsC, and tlsO–T genes play key roles in telomestatin biosynthesis. Moreover, exchanging TlsC core peptide sequences resulted in the production of novel telomestatin derivatives. This study sheds light on the expansion of chemical diversity of natural peptide products for drug development.
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