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

Abstract

Polyene macrolides, such as nystatin A1, amphotericin B, and NPP A1, belong to a large family of valuable antifungal polyketide compounds that are typically produced by soil actinomycetes. Previously, NPP B1, a novel NPP A1 derivative harboring a heptaene core structure, was generated 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 in Pseudonocardia autotrophica. This derivative showed superior antifungal activity to NPP A1. In this study, another novel derivative called NPP B2 was developed, which lacks a hydroxyl group at the C10 position by site-specific gene disruption of the P450 hydroxylase NppL. To stimulate the extremely low expression of the NPP B2 biosynthetic pathway genes, the 32-kb NPP-specific regulatory gene cluster was overexpressed via site-specific chromosomal integration. The extra copy of the six NPP-specific regulatory genes led to a significant increase in the NPP B2 yield from 0.19 to 7.67 mg/L, which is the highest level of NPP B2 production ever achieved by the P. autotrophica strain. Subsequent in vitro antifungal activity and toxicity studies indicated that NPP B2 exhibited similar antifungal activity but significantly lower hemolytic toxicity than NPP B1. These results suggest that an NPP biosynthetic pathway refactoring and overexpression of its pathway-specific regulatory genes is an efficient approach to stimulating the production of an extremely low-level metabolite, such as NPP B2 in a pathway-engineered rare actinomycete strain.

Update of the list of QPS‐recommended microbiological agents intentionally added to food or feed as notified to EFSA 16: suitability of taxonomic units notified to EFSA until March 2022

“…P. autotrophica has been reported to produce an antifungal compound belonging to the tetraene‐family, nystatin‐like Pseudonocardia polyene A1 (NPP A1) (Lee et al., 2012 ; Han et al., 2019 ; Park et al., 2020 ). It is also used for the production of biosurfactants (Kuznetsov et al., 2020 ), and as a biocatalyst for the commercial production of a cytochrome P‐450 hydroxylase of vitamin D3 (Fujii et al., 2009 ).…”

New Glycosylated Polyene Macrolides: Refining the Ore from Genome Mining

“…autotrophica KCTC9441 has been genetically engineered extensively. This work has delivered four disaccharide-containing analogues of NPP A1: (1) a 10-deoxy tetraene, NPP A2 [86]; (2) a heptaene with a double bond between C28 and C29, NPP B1 [87]; (3) a 10-deoxy heptaene, NPP B2 [88]; and (4) NPP A3, a tetraene in which mannose replaces the GlcNAc sugar [89]. The 10-deoxy tetraene and the heptaene NPP B1 had increased antifungal activity.…”

“…autotrophica KCTC9441 has been genetically engineered extensively. This work has delivered four disaccharide-containing analogues of NPP A1: (1) a 10-deoxy tetraene, NPP A2 [86]; (2) a heptaene with a double bond between C28 and C29, NPP B1 [87]; (3) a 10-deoxy heptaene, NPP B2 [88]; and (4) NPP A3, a tetraene in which mannose replaces Yields of the potentially improved NPP B1 and NPP B2 analogues were increased by strain improvement of the producer organisms. This was achieved by nitrosoguanidine mutagenesis and screening for increased production, and by eliminating a 128 kb plasmid carrying a BGC for a competing polyketide.…”

“…In addition, an integrating plasmid was used to introduce an extra copy of the 32 kb region containing positive regulatory genes. These measures increased the yields to over 31 mg per liter of production culture for NPP B1 [90] and 7 mg per liter for NPP B2 [88].…”

Evaluation of Physically and/or Chemically Modified Chitosan Hydrogels for Proficient Release of Insoluble Nystatin in Simulated Fluids

“…Nystatin is known for a wide range of antifungal activity, being intended for the prophylaxis and treatment of candidiasis located on the skin and mucous membranes [ 8 ]. Derived from Streptomyces noursei actinobacterium, the polyene macrolide possesses a macrolactone ring structure with a polyketide core [ 9 ]. Acting like an ionophore, nystatin is highly selective towards fungi, interacting with the membranes’ ergosterol and forming pores that allow K+ ions to pass through, resulting in the death of fungal cells [ 10 ].…”