Production of novel pladienolide analogues through native expression of a pathway-specific activator

Abstract

Aberrant splicing of pre-mRNA is implicated in many human genetic disorders. Small molecules that target the spliceosome are important leads as therapeutics and research tools, and one compound of significant interest is the polyketide natural product pladienolide B. Here, we describe the reactivation of quiescent pladienolide B production in the domesticated lab strain Streptomyces platensis AS6200 by overexpression of the pathway-specific activator PldR. The resulting dysregulation of the biosynthetic genes led to the accumulation and isolation of five additional intermediate or shunt metabolites of pladienolide B biosynthesis, including three previously unreported congeners. These compounds likely comprise the entire pladienolide biosynthetic pathway and demonstrate the link between polyketide tailoring reactions and bioactivity, particularly the importance of the 18,19-epoxide. Each congener demonstrated specific inhibitory activity against mammalian cell lines, with successive modifications leading to increased activity (IC₅₀: 8 mM to 5 μM).

Fig. 1. Chemical structures of natural product splicing modulators: (a) spliceostatin A from Burkholderia sp. No. 2663, herboxidiene from Streptomyces chromofuscus ATCC 49982, and pladienolide B (1) from Streptomyces platensis Mer-11107 and; (b) synthetic analogues of 1.

Fig. 2. Comparison of the pladienolide (pld) biosynthetic gene clusters of (a) S. platensis Mer-11107 and (b) S. platensis AS6200 and S. platensis MA5455 and (c) pladienolide B (1) biosynthetic pathway including polyketide synthesis and tailoring steps.

Fig. 3. Biosynthesis of pladienolide B (1) and congeners (2–6) produced by S. platensis AS6242 identified in this study. The polyketide product of PldAI-V is acetylated by PldC followed by hydroxylation/epoxidation by PldB/PldD to produce 1. Spontaneous intramolecular cyclisation of 1 yields 6.