Abstract
In this study, we report the rapid characterisation of a novel microbial natural product resulting from the rational derepression of a silent gene cluster. A conserved set of five regulatory genes was used as a query to search genomic databases and identify atypical biosynthetic gene clusters (BGCs). A 20-kb BGC from the genetically intractable Streptomyces sclerotialus bacterial strain was captured using yeast-based homologous recombination and introduced into validated heterologous hosts. CRISPR/Cas9-mediated genome editing was then employed to rationally inactivate the key transcriptional repressor and trigger production of an unprecedented class of hybrid natural products exemplified by (2-(benzoyloxy)acetyl)-l-proline, named scleric acid. Subsequent rounds of CRISPR/Cas9-mediated gene deletions afforded a selection of biosynthetic gene mutant strains which led to a plausible biosynthetic pathway for scleric acid assembly. Synthetic standards of scleric acid and a key biosynthetic intermediate were also prepared to confirm the chemical structures we proposed. The assembly of scleric acid involves two unique condensation reactions catalysed by a single NRPS module and an ATP-grasp enzyme that link a proline and a benzoyl residue to each end of a rare hydroxyethyl-ACP intermediate, respectively. Scleric acid was shown to exhibit moderate inhibition activity against Mycobacterium tuberculosis, as well as inhibition of the cancer-associated metabolic enzyme nicotinamide N-methyltransferase (NNMT).
Highlights
Actinomycete bacteria have been the foremost producers of antibiotics since the mid-1940s
In S. coelicolor A3(2), these ve genes are responsible for regulation of methylenomycin biosynthesis: mmyR and mmfR both code for TetR-like transcriptional repressors; mmfLHP are responsible for the biosynthesis of signalling molecules, known as methylenomycin furans (MMFs) that trigger production of the methylenomycin antibiotics.[9]
Specialised metabolites from actinomycete bacteria are one of the most valuable sources of novel antibiotics, as well as of other useful bioactive compounds employed in various elds, from human medicine to crop protection
Summary
Actinomycete bacteria have been the foremost producers of antibiotics since the mid-1940s. Highthroughput DNA sequencing technologies and novel bioinformatics tools have highlighted an immense number of uncharacterised biosynthetic gene clusters (BGCs) predicted to direct the assembly of bioactive natural products.[1] The presence of BGCs has been revealed in actinomycete genomes and in those of human commensal and pathogenic bacteria (i.e. Staphylococcus lugdunensis, Burkholderia cepacia complex), as well as in the genomes of unculturable bacteria and in metagenomic libraries.[2,3,4,5,6,7]. Despite the conspicuous number of specialised metabolites isolated from actinomycetes, only a small fraction of the natural products ‘encrypted’ at the DNA level has been exploited to date.
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