Abstract
Streptomyces are well-known producers of a range of different secondary metabolites, including antibiotics and other bioactive compounds. Recently, it has been demonstrated that “silent” biosynthetic gene clusters (BGCs) can be activated by heterologously expressing transcriptional regulators from other BGCs. Here, we have activated a silent BGC in Streptomyces sp. CA-256286 by overexpression of a set of SARP family transcriptional regulators. The structure of the produced compound was elucidated by NMR and found to be an N-acetyl cysteine adduct of the pyranonaphtoquinone polyketide 3′-O-α-d-forosaminyl-(+)-griseusin A. Employing a combination of multi-omics and metabolic engineering techniques, we identified the responsible BGC. These methods include genome mining, proteomics and transcriptomics analyses, in combination with CRISPR induced gene inactivations and expression of the BGC in a heterologous host strain. This work demonstrates an easy-to-implement workflow of how silent BGCs can be activated, followed by the identification and characterization of the produced compound, the responsible BGC, and hints of its biosynthetic pathway.
Highlights
Bacteria of the genus Streptomyces are well-known producers of bioactive compounds with anti-bacterial activity. Most of these compounds are produced by large enzyme complexes, such as the polyketide synthases (PKSs) [1] and the non-ribosomal peptide synthetases (NRPSs) [2,3]
One class of transcriptional regulators, that are frequently used for similar purposes, is the Streptomyces antibiotic regulatory protein (SARP) family regulators
It has previously been shown that overexpressing a SARP family regulator can activate production from silent biosynthetic gene clusters (BGCs) in Streptomyces
Summary
Bacteria of the genus Streptomyces are well-known producers of bioactive compounds with anti-bacterial activity. The four plasmids encode: cluster specific regulators (CSRs) containing the four genes aur1P-pntR-strR-fkbN; Streptomyces antibiotic regulatory proteins (SARPs) containing the five genes actIIORF4-griR-aur1PR3-papR2-redD; gamma butyrolactone synthases (GBLs) containing the two genes scbA-afsA; and global regulators containing the five genes afsR-adpA-crp-absB-dasR. Overexpression of these different classes of transcriptional regulators are hypothesized to activate different silent BGCs. The objective of this study was to employ the four plasmids to activate production of novel compounds in a collection of actinomycete strains isolated from soil samples. A combination of multi-omics methods, gene inactivations, and heterologous expression was used to identify and characterize the compound and the corresponding BGC
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