Reviving GE23077: Investigating Biosynthetic Pathways and Antimicrobial Potential of a Promising RNAP Inhibitor

Discovered in the 1990s as a product of the metabolism of the so-called ‘rare’ filamentous actinomycete Actinomadura lepetitiana DSM 10909, GE23077 is a nonribosomally synthesised cyclic heptapeptide antibiotic that inhibits a wide range of bacterial RNA polymerases (RNAP) in vitro, while not affecting mammalian enzymes. After its discovery, the molecule was later overlooked, due to its limited in vivo antimicrobial activity, mainly exerted against Moraxella catarrhalis, Neisseria gonorrhoeae, and Mycobacterium smegmatis isolates. However, with antimicrobial resistance spreading, including towards the major approved RNAP inhibitors (fidaxomicin and rifamycin), the development of new drugs to fight bacterial infections is essential. In this context, GE23077 might prove promising, as its target includes functionally critical residues of the RNAP that are unique. Our project aims to better understand GE23077 production using multiple approaches as an essential step for its further development. High-quality genome sequence of A. lepetitiana was recently obtained using Illumina MiSeq and MinION, followed by the identification and annotation of a putative biosynthetic gene cluster (BGC) for GE23077 production. Following the development of new methods for the genetic manipulation of this recalcitrant actinomycete, BGC attribution was confirmed by the knock-out of key genes. Overexpression studies of putative regulatory genes are ongoing. In parallel, in vitro studies are being performed on purified A. lepetitiana RNAP to evaluate its susceptibility to GE23077. Elucidating the mechanisms behind the synthesis of this promising RNAP inhibitor may also open the way to combinatorial biochemistry approaches to modify the molecule and potentially improve its in vivo antimicrobial activity.