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

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).


Strains and Culture Conditions
A list of strains used in this study can be found in Table S1. Streptomyces spp. were maintained at 30 °C on SF+M (soy flour 20.0 g L -1 , mannitol 20.0 g L -1 ) containing 20 % agar. E. coli were maintained at 37 °C on LB (NaCl 10.0 g L -1 , tryptone 10.0 g L -1 , yeast extract 5.0 g L -1 ) containing 15% agar.
Glycerol stocks were generated from a 1:1 mixture of liquid culture and 40% glycerol and stored at -80 °C. Streptomyces spore stocks were generated using a modified version of the protocol published by Kieser et al. 1 . Spores were harvested in sterile water (5 mL) and filtered through sterile cotton wool.

Extraction and Sequencing of Genomic DNA
High molecular weight genomic DNA was extracted according to the salting out procedure. Wet mycelium (0.5 mL) from 30-hour-old SV2 culture was washed with 10% sucrose (10 mL) before S5 resuspension in SET buffer (5 mL; 75 mM NaCl, 25 mM EDTA, 20 mM Tris HCl pH 8.0) to which lysozyme (200 µL; 50 mg mL -1 ) and ribonuclease A (15 µL; 10 mg mL -1 ) were added. The cells were incubated overnight at 37 °C; fresh lysozyme (300 µL) was added after ca. 17 h followed by an additional 2 h incubation. The subsequent steps were performed according to Kieser et al. 1 .
Genomic DNA of Streptomyces platensis AS6200 and Streptomyces platensis MA5455 were sequenced using Pacific Biosciences (PacBio) RSII SMRT technology (commissioned to The Genome Analysis Centre (TGAC) Norwich, UK) 2 and assembled via the HGAP2.0 pipeline. Detection of specialised metabolic BGCs and annotation of gDNA was performed in antiSMASH v4.0 3 including ClusterBlast and whole-genome PFAM analysis 4 . Gene homologues were identified from translated protein sequences using BLASTp 5 . The BGC sequence was deposited into the GenBank database under the accession: MN974405. Whole genome alignments were performed using LASTZ 6 . Genomes were submitted to autoMLST 7 for taxonomic classification. The resulting alignment (95,515 bp) was used to generate a phylogenetic tree using FastTree 2 8 .

Molecular Cloning and Transformation
The positive regulator pldR was amplified from Streptomyces platensis AS6200 genomic DNA via polymerase chain reaction (PCR) using the primers pldR_F (ttaattaaggaggacacatatgcatctcttcgggcgggac) and pldR_R (ccaagctcagctaattaagcttcatgccgtcggggcag) and Q5 high-fidelity DNA polymerase in 25 -50 µL aliquots according to the manufacturer's protocol supplemented with 3% DMSO. For screening purposes, PCR was performed using GoTaq Green Master Mix (Promega) in 10 µL reactions. Thermal cycling was carried out according to manufacturer's protocol (initial denaturation at 98 °C for 30 s followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 60 °C for 30 s and extension at 72 °C for 1 min 30 s, followed by a final extension at 72 °C for 2 min). The plasmid pGP9 was digested with NdeI and HindIII (New England Biolabs) according to manufacturer's protocols. PCR and digestion products were purified using Wizard SV gel and PCR clean up system (Promega) according to manufacturer's protocols. Molecular cloning was performed via Gibson Assembly 9 in 10 µL reactions using Gibson

Screening for Pladienolide Production
The exconjugants S. platensis AS6200 pGP9+pldR and S. platensis AS6200 pGP9 were screened on  which is one oxygen atom less than both 2 and 3. The 1 H and 13 C NMR data for 4 (Table S10) (Table S10) confirmed 4 to be the 6-deoxy analogue of 3. H-18 to C-21 were indicative of an intramolecular 5-exo-tet cyclisation from 21-OH to C-18 to form a tetrahydrofuran ring system. Detailed analysis of the 2D NMR data (Table S12) confirmed 6 to be the cyclised analogue of 1.

Assays for Biological Activity
Purified metabolites (1 -6) were dissolved in DMSO to provide stock solutions (1,000 µg/mL or 40 µg/mL). Bioassays were carried out in 96-well microtitre plates containing two-fold serial dilutions to yield ranges of 10 to 0.005 µg mL -1 or 0.4 to 0.0002 µg mL -1 , in 1% DMSO. Wells containing no test compound as a reference for no inhibition, and uninoculated wells were used as a reference for complete inhibition.