ArpA-like regulatory control and discovery of new biosynthetic genes driving scleric acid biosynthesis.

Abstract

Scleric acid is a natural product that was discovered by heterologous expression of the silent scl gene cluster from Streptomyces sclerotialus in Streptomyces albidoflavus and rational de-repression of the biosynthetic genes through deletion of the ArpA-like regulator SclM4. It is a weak inhibitor of the cancer-associated enzyme nicotinamide N-methyltransferase (NNMT). In this work we aimed to improve understanding of scleric acid biosynthesis and of gene regulation of the scl gene cluster. We performed CRISPR/Cas9-mediated deletion of the biosynthetic gene sclG, which confirmed the essential role of the corresponding ATP-grasp family enzyme in the biosynthesis of scleric acid. We also carried out whole-genome Nanopore long-read sequencing of S. sclerotialus to investigate the borders of the scl gene cluster, which led us to discover two additional genes, sclC and sclK, proposed to be involved in biosynthetic precursor supply. Transcriptomics analyses were performed to investigate the effect of the deletion of the ArpA-like gene sclM4 on the expression of the scl biosynthetic genes, shedding light on the role of the corresponding transcriptional repressor. The present work improves understanding of scleric acid biosynthesis and provides new insights into the ArpA-like mediated regulation of biosynthetic gene expression in Streptomyces bacteria.