Triggering the expression of a silent gene cluster from genetically intractable bacteria results in scleric acid discovery

Herein we report a strategy for the rapid and rational characterisation of novel microbial natural products from silent gene clusters. 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. Scleric acid and a key biosynthetic intermediate were also synthesised and used as authentic standards. The assembly of scleric acid involves two unique enzymatic condensation reactions that respectively link a proline and a benzoyl residue to each end of a rare hydroxyethyl-ACP intermediate. Scleric acid was then shown to exhibit moderate activity against Mycobacterium tuberculosis, as well as modest inhibition of the cancer-associated metabolic enzyme Nicotinamide N-methyltransferase (NNMT).