Metathramycin, a new bioactive aureolic acid discovered by heterologous expression of a metagenome derived biosynthetic pathway

Luke J Stevenson,David F Ackerley,Jeremy G Owen,Robert A Keyzers,Liwei Liu,Abigail V Sharrock,Joe Bracegirdle

doi:10.1039/d0cb00228c

Luke J Stevenson, David F Ackerley + Show 5 more

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https://doi.org/10.1039/d0cb00228c

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Abstract

Bacterial natural products have been a rich source of bioactive compounds for drug development, and advances in DNA sequencing, informatics and molecular biology have opened new avenues for their discovery. Here, we describe the isolation of an aureolic acid biosynthetic gene cluster from a metagenome library derived from a New Zealand soil sample. Heterologous expression of this pathway in Streptomyces albus resulted in the production and isolation of two new aureolic acid compounds, one of which (metathramycin, 6) possesses potent bioactivity against a human colon carcinoma cell line (HCT-116, IC50 = 14.6 nM). As metathramycin was a minor constituent of the fermentation extract, its discovery relied on a combination of approaches including bioactivity guided fractionation, MS/MS characterisation and pathway engineering. This study not only demonstrates the presence of previously uncharacterised aureolic acids in the environment, but also the value of an integrated natural product discovery approach which may be generally applicable to low abundance bioactive metabolites.

Highlights

Aureolic acids are a family of glycosylated aromatic polyketides that have potent bioactivities against human cancer cells and Gram-positive bacteria.[1,2] All naturally occurring aureolic acids have been discovered from soil or marine actinomycete bacteria, and the biosynthetic gene clusters (BGCs) for two of these have been described: mithramycin (Fig. 1, 1) from Streptomyces argillaceus,[1,3] and chromomycin A3 (Fig. 1, 2) produced by Streptomyces griseus subsp. griseus.[4]
The resulting library of 410 million cosmid clones, each containing 30–40 kb of metagenome DNA, was arrayed over four 96 well plates. These arrayed wells were screened by PCR with degenerate primers targeting bacterial ketosynthase alpha (KSa) genes,[23] using previously established serial dilution plating protocols.[10]
Following assembly with SPAdes,[24] contigs were matched to individual cosmid insert end sequences by Sanger sequencing, and the assembled metagenome insert sequences analysed with antiSMASH v4.25 One of the recovered cosmids had 68% gene cluster similarity by clusterBLAST to the aureolic acid pathway producing mithramycin and 58% to chromomycin A3

Summary

Introduction

Aureolic acids are a family of glycosylated aromatic polyketides that have potent bioactivities against human cancer cells and Gram-positive bacteria.[1,2] All naturally occurring aureolic acids have been discovered from soil or marine actinomycete bacteria, and the biosynthetic gene clusters (BGCs) for two of these have been described: mithramycin (Fig. 1, 1) from Streptomyces argillaceus,[1,3] and chromomycin A3 (Fig. 1, 2) produced by Streptomyces griseus subsp. griseus.[4]. The first is the significant sugar decoration of the polyketide core (premithramycinone, Fig. 1, 3), which can be modified by deletion or complementation with different sugar biosynthesis pathways and/or glycosyltransferase enzymes Mtm/ CmmGI-GIV.[7,8] The second is the aliphatic tail which is generated by oxidative cleavage of the fourth ring in the tetracyclic precursor (premithramycin B, Fig. 1, 4) by Mtm/CmmOIV, and subsequent sidechain reduction by Mtm/CmmW. Gene knockout of the sidechain reductase has been demonstrated to produce a set of analogue compounds with distinct toxicity profiles.[2,4]

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