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

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).

Fig. 1. Overview of the approach used in this study to characterise scleric acid, a novel natural product from a cryptic and silent gene cluster.

Fig. 2. Genetic organisation of the scl gene cluster from S. sclerotialus NRRL ISP-5269 characterised in this study and schematic representation of mutants generated. (a) Gene cluster (19 782 bp) sequenced from S. sclerotialus NRRL ISP-5269. (b) Configuration of the scl gene cluster after CRISPR/Cas9-mediated targeted gene deletion within the heterologous host S. albus. The 20-bp out of frame deletion of sclM4 generated a truncated gene, represented here as a rectangular shape for the derived gene sequence. (c) Configuration of the scl cluster within S. albus double mutants; deletion of genes sclN, sclQ1-4 and sclA were generated starting from strain S. albus/scl ΔsclM4, and are represented here as dotted lines.

Fig. 3. Identification and characterisation of scleric acid. (a) UHPLC-HRMS detection of metabolites produced in S. albus/pCAP03 (grey trace), S. albus/scl (black), S. albus/scl ΔsclM4 (orange), S. albus/scl ΔsclM4 ΔsclQ1-4 (blue), S. albus/scl ΔsclM4 ΔsclN (green) and S. albus/scl ΔsclM4 ΔsclA (red). Extracted ion chromatograms in positive mode for m/z = 278.1020 are shown, highlighting accumulation of a new metabolite (scleric acid) at retention time 16.4 minutes in S. albus/scl ΔsclM4 (orange trace). (b) UV chromatogram of scleric acid. (c) High-resolution mass spectrum in positive mode of scleric acid. (d) Selected correlations observed in the COSY (bold lines) and HMBC (arrows) NMR spectra of scleric acid. (e) Chemical structure of scleric acid that can adopt two main rotamer conformations.

Fig. 4. Proposed biosynthetic pathway to scleric acid.