Abstract
BackgroundPolyene antibiotics are important as antifungal medicines albeit with serious side effects such as nephrotoxicity. Reedsmycin (RDM) A (1), produced by marine-derived Streptomyces youssoufiensis OUC6819, is a non-glycosylated polyene macrolide antibiotic with antifungal activity comparable to that of clinically used nystatin. To elucidate its biosynthetic machinery, herein, the rdm biosynthetic gene cluster was cloned and characterized.ResultsThe rdm cluster is located within a 104 kb DNA region harboring 21 open reading frames (ORFs), among which 15 ORFs were designated as rdm genes. The assembly line for RDM A is proposed on the basis of module and domain analysis of the polyketide synthetases (PKSs) RdmGHIJ, which catalyze 16 rounds of decarboxylative condensation using malonyl-CoA as the starter unit (loading module), two methylmalonyl-CoA (module 1 and 2), and fourteen malonyl-CoA (module 3–16) as extender units successively. However, the predicted substrate specificity of AT0 in the loading module is methylmalonyl-CoA instead of malonyl-CoA. Interestingly, the rdm cluster contains a five-gene regulation system RdmACDEF, which is different from other reported polyene gene clusters. In vivo experiments demonstrated the XRE family regulator RdmA and the PAS/LuxR family regulator RdmF function in negative and positive manner, respectively. Notably, inactivation of rdmA and overexpression of rdmF led to increased production of RDM A by ~ 2.0-fold and ~ 2.5-fold, reaching yields of 155.3 ± 1.89 and 184.8 ± 9.93 mg/L, respectively.ConclusionsBiosynthesis of RDM A is accomplished on a linear assembly line catalyzed by Rdm PKSs harboring a unique AT0 under the control of a complex regulatory system. These findings enable generation of new biologically active RDM derivatives at high yield and with improved properties by engineered biosynthesis.
Highlights
Polyene antibiotics are important as antifungal medicines albeit with serious side effects such as nephrotoxicity
By inactivation of the corresponding P450 gene in each cluster, the exocyclic carboxyl group was replaced with a methyl group in amphotericin B [13], nystatin [14], candicidin [15], and rimocidin [16], leading to decreased haemolytic activity but not antifungal activity
The sequence gaps within the polyketide synthetase (PKS) genes were filled by chromosomal walking using the pWLI512 or pWLI513 as template
Summary
Polyene antibiotics are important as antifungal medicines albeit with serious side effects such as nephrotoxicity. Reedsmycin (RDM) A (1), produced by marine-derived Streptomyces youssoufiensis OUC6819, is a nonglycosylated polyene macrolide antibiotic with antifungal activity comparable to that of clinically used nystatin. The emergence of rare or unidentified species of drug-resistant fungal pathogens and the clinical need for non-toxic antifungal drugs demand us to develop new polyene derivatives and formulations, given the apparent toxicity and serious side effects of existing polyene macrolides [2, 3]. By inactivation of the corresponding P450 gene in each cluster, the exocyclic carboxyl group was replaced with a methyl group in amphotericin B [13], nystatin [14], candicidin [15], and rimocidin [16], leading to decreased haemolytic activity but not antifungal activity. Albeit many potentially valuable polyene analogues have been developed, none of them has advanced into clinical medicine so far [11]
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