Nystatin-like Pseudonocardia polyene B1, a novel disaccharide-containing antifungal heptaene antibiotic

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.

Actinomycete-Derived Polyketides as a Source of Antibiotics and Lead Structures for the Development of New Antimicrobial Drugs

“…Compared to nystatin A1, NPP A1 displayed a 300-fold increase in water solubility, 10-fold reduced hemolytic activity, but also~50% lower antifungal activity against Candida albicans. The issue of lowered bioactivity was later solved through the manipulation of the ER domain in module five (ER5) of the NPP biosynthetic cluster [312]. Deleting this gene disables the reduction at the C28-C29 unsaturated bond in the aglycone of NPP A1 hence, generating a heptaene instead of the original tetraene.…”

“…However, the production yields of NPP B1 in the pathway-engineered strain (P. autotrophica ER5 mutant) were extremely low. In an attempt to solve this issue, the P. autotrophica ER5 mutant strain was subjected to N-methyl-N -nitro-N-nitrosoguanidine (NTG) iterative random mutagenesis [312]. The resulting mutants were screened in zone-of-inhibition agar plug assays in which the mutant strain 3R-42 produced the largest inhibition zone.…”

Stimulated Biosynthesis of an C10-Deoxy Heptaene NPP B2 via Regulatory Genes Overexpression in Pseudonocardia autotrophica

“…Previously, several strategies, including overexpression of the pathway-specific regulatory gene, deletion of the global antibiotic downregulator, in situ screening of random mutants, co-culture system, and cultivation with xenobiotics, all failed to improve NPP B1 production (Kim et al, 2018). Recently, the entire cluster containing all six NPP pathway-specific genes in a pSBAC system followed by its re-integration into the P. autotrophica chromosome led to a significant increase in the NPP B1 titer (Han et al, 2019).…”

“…An in vitro antifungal assay confirmed that the NPP B1, a heptaene version of NPP A1, showed much higher antifungal activity than the tetraene NPP A1 (Kim et al, 2018). Therefore, we tried to generate pharmacokinetically improved heptaene NPP B1 derivative through the engineering of post-PKS modification.…”

“…NPP A2 lacks a C10 hydroxyl group that was generated by the inactivation of the P450 hydroxylase-encoding nppL gene in P. autotrophica (Kim et al, 2016). NPP B1 was generated by changing its core macrolactone structure from tetraene to heptaene through site-specific, 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 (Figure 1; Kim et al, 2018). Interestingly, both NPP A2 and NPP B1 exhibited higher antifungal activities than those of NPP A1.…”