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.
Highlights
More than 45% of the bioactive compounds discovered from microbial secondary metabolites are derived from actinomycetes (Bu et al, 2019)
Based on the previous results that characterization of the P450 hydroxylases, which were a region-specific hydroxylation such as AmphL, NysL, and NppL (NPP) (Byrne et al, 2003; Volokhan et al, 2006; Kim et al, 2016), the NPP B1 production strain was engineered by inactivation of the nppL gene, which was involved in NPP C10 region-specific hydroxylation (Figures 2A,B; Supplementary Figure S1)
The in vitro toxicity was 4.5-fold lower than the minimum hemolysis concentration (MHC) value of NPP B1 (17.72 μg/ml). These results suggest that the absence of the hydroxyl moiety at the C10 position of NPP B2 could play an important role in controlling both antifungal acidity and hemolytic toxicity
Summary
More than 45% of the bioactive compounds discovered from microbial secondary metabolites are derived from actinomycetes (Bu et al, 2019). Among them are polyene macrolide antibiotics, such as nystatin, Pseudonocardia NPP B2 Regulatory Genes Overexpression amphotericin, candicidin, and pimaricin, which are potent antifungal compounds that are comprised typically of a polyketide core macrolactone ring with about 20–40 carbon atoms, including 3–8 conjugated double bonds (Figure 1; Caffrey et al, 2016). The discovery of C35deOAmB revealed that the antifungal activity was critical for binding to ergosterol, not for ion-channel formation (Gray et al, 2012), which generated toxicity-reduced amphotericin B derivatives (Cioffi et al, 2015; Muraglia et al, 2019). The engineering of polyene PKS to change the number of conjugated double bonds as well as post-PKS modifications for manipulation of the sugar and hydroxyl moieties is considered an important strategy for the generation of pharmacokinetically improved novel polyene derivatives (Bruheim et al, 2004; Gray et al, 2012; Caffrey et al, 2016; Walmsley et al, 2017; Muraglia et al, 2019)
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