Abstract
Polyene macrolides such as nystatin A1 and amphotericin B belong to a large family of very valuable antifungal polyketide compounds typically produced by soil actinomycetes. Recently, nystatin-like Pseudonocardia polyene (NPP) A1 has been identified as a unique disaccharide-containing tetraene antifungal macrolide produced by Pseudonocardia autotrophica. Despite its significantly increased water solubility and decreased hemolytic activity, its antifungal activity remains limited compared with that of nystatin A1. In this study, we developed NPP B1, a novel NPP A1 derivative harboring a heptaene core structure, by introducing two amino acid substitutions in the putative NADPH-binding motif of the enoyl reductase domain in module 5 of the NPP A1 polyketide synthase NppC. The low level NPP B1 production yield was successfully improved by eliminating the native plasmid encoding a polyketide biosynthetic gene cluster present in P. autotrophica. In vitro and in vivo antifungal activity and toxicity studies indicated that NPP B1 exhibited comparable antifungal activity against Candida albicans and was less toxic than the most potent heptaene antifungal, amphotericin B. Moreover, NPP B1 showed improved pharmacokinetic parameters compared to those of amphotericin B, suggesting that NPP B1 could be a promising candidate for development into a pharmacokinetically improved and less-toxic polyene antifungal antibiotic.
Highlights
Polyenes typically comprise a polyketide core macrolactone ring with 20–40 carbon atoms including 3–8 conjugated double bonds
During biosynthesis of the nystatin-like Pseudonocardia polyene (NPP) A1 polyketide backbone, the enoyl reductase (ER) domain in module 5 (ER5) of NppC is responsible for the nicotinamide adenine dinucleotide phosphate (NADPH)-mediated reduction of the C28-C29 unsaturated bond
The production yield of NPP B1 (0.34 mg/L, 5-day flask cultivation) was extremely poor compared with that of the wild-type (8.91 mg/L, 5-day flask cultivation) (Fig. 1), implying that the in-frame deletion of the ER5 domain might affect the three-dimensional (3D) structure of NPP polyketide synthase, rendering it less efficient in subsequent biosynthetic steps. This low titer problem was overcome by generating an alternative NPP B1 producing strain by substituting only two amino acids in the NADPH-binding motif in the ER5 domain of NppC
Summary
Polyenes typically comprise a polyketide core macrolactone ring with 20–40 carbon atoms including 3–8 conjugated double bonds. The antifungal drugs primarily used to treat systemic fungal infections are polyene macrolides such as the tetraene-containing nystatin A1 and heptaene-containing amphotericin B1,2. In contrast to the heptaene-containing amphotericin B, the macrolide core structures of both NPP A1 and nystatin A1 are tetraenes, which have a saturated C28-C-29 bond in the polyene region. We successfully developed the heptaene form of NPP A1 named NPP B1 by manipulating the specific polyketide enoyl reductase (ER) domain in the NPP A1 biosynthetic pathway gene, followed by substantial efforts to enhance the NPP B1 production yield. We expect this study to be the basis for the development of a novel less-toxic disaccharide heptaene macrolide of P. autotrophica, which has comparable antifungal efficacy to that of the most potent heptaene antifungal antibiotic, amphotericin B
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