Study of bicyclomycin biosynthesis in Streptomyces cinnamoneus by genetic and biochemical approaches

Jerzy Witwinowski,Antonio Ruzzini,Muriel Gondry,Jean-Luc Pernodet,Laëtitia Caraty,Matthieu Coupet,Emmanuel Favry,Isabelle Correia,Sylvie Lautru,Corinne Saulnier,Pascal Belin,Jérôme Seguin,Olivier Lequin,Emmanuelle Darbon,Mireille Moutiez

doi:10.1038/s41598-019-56747-7

Abstract

The 2,5-Diketopiperazines (DKPs) constitute a large family of natural products with important biological activities. Bicyclomycin is a clinically-relevant DKP antibiotic that is the first and only member in a class known to target the bacterial transcription termination factor Rho. It derives from cyclo-(l-isoleucyl-l-leucyl) and has an unusual and highly oxidized bicyclic structure that is formed by an ether bridge between the hydroxylated terminal carbon atom of the isoleucine lateral chain and the alpha carbon of the leucine in the diketopiperazine ring. Here, we paired in vivo and in vitro studies to complete the characterization of the bicyclomycin biosynthetic gene cluster. The construction of in-frame deletion mutants in the biosynthetic gene cluster allowed for the accumulation and identification of biosynthetic intermediates. The identity of the intermediates, which were reproduced in vitro using purified enzymes, allowed us to characterize the pathway and corroborate previous reports. Finally, we show that the putative antibiotic transporter was dispensable for the producing strain.

Highlights

The 2,5-Diketopiperazines (DKPs) constitute a large family of natural products with important biological activities
Having generated and analysed a draft genome sequence of the bicyclomycin producer S. cinnamoneus, we had identified as candidates to direct bicyclomycin biosynthesis the same genes as those identified by other groups studying this topic[11,12,13]
These genes constitute a cluster composed of a cyclodipeptide synthases (CDPSs) gene, five genes encoding 2-oxoglutarate/ iron-dependent dioxygenases (2OG/Fe dioxygenases), one gene encoding a cytochrome P450 monooxygenase, and one gene encoding a transporter belonging to the Major Facilitator Superfamily (MFS) (Fig. 1b)

Summary

Introduction

The 2,5-Diketopiperazines (DKPs) constitute a large family of natural products with important biological activities. Our work provides in vivo data that corroborate the results obtained in vitro on the biosynthetic pathway of bicyclomycin, establish the stereochemistry of several biosynthetic intermediates and demonstrate the dispensable nature of the transporter associated with the biosynthetic gene cluster in S. cinnamoneus. Deletion of the whole bcm cluster from S. cinnamoneus (Δbcm::aphII mutant, Supplementary methods) abolished bicyclomycin production (Fig. 2c).

Results

Conclusion