Identification of the Biosynthetic Pathway for the Antibiotic Bicyclomycin

Abstract

Diketopiperazines (DKPs) make up a large group of natural products with diverse structures and biological activities. Bicyclomycin is a broad-spectrum DKP antibiotic with unique structure and function: it contains a highly oxidized bicyclic [4.2.2] ring and is the only known selective inhibitor of the bacterial transcription termination factor, Rho. Here, we identify the biosynthetic gene cluster for bicyclomycin containing six iron-dependent oxidases. We demonstrate that the DKP core is made by a tRNA-dependent cyclodipeptide synthase, and hydroxylations on two unactivated sp carbons are performed by two mononuclear iron, α-ketoglutarate-dependent hydroxylases. Using bioinformatics, we also identify a homologous gene cluster prevalent in a human pathogen Pseudomonas aeruginosa. We detect bicyclomycin by overexpressing this gene cluster and establish P. aeruginosa as a new producer of bicyclomycin. Our work uncovers the biosynthetic pathway for bicyclomycin and sheds light on the intriguing oxidation chemistry that converts a simple DKP into a powerful antibiotic.

A Comprehensive Overview of the Cyclodipeptide Synthase Family Enriched with the Characterization of 32 New Enzymes

“…We extended our analysis to all CDPSs biochemically characterized to date. This set now consists of 98 active CDPSs (Gondry et al, 2009 ; Seguin et al, 2011 ; Giessen et al, 2013a , b ; Alqahtani et al, 2015 ; Jacques et al, 2015 ; James et al, 2016 ; Brockmeyer and Li, 2017 ; Patteson et al, 2017 ). The products and P1 and P2 motifs of all the enzymes previously characterized are shown in blue in Supplemental Data Set 4 .…”

“…The number of characterized biosynthetic pathways for DKPs is relatively low relative to their known diversity (Belin et al, 2012 ; Giessen and Marahiel, 2015 ). Ten pathways that involve NRPSs have been decoded (Belin et al, 2012 ; Giessen and Marahiel, 2015 ), whereas only six have been shown to depend on CDPSs: albonoursin (Lautru et al, 2002 ), pulcherrimin (Cryle et al, 2010 ), mycocyclosin (Belin et al, 2009 ), methylated ditryptophan (Giessen et al, 2013a ), nocazines (Giessen et al, 2013b ), and, most recently bicyclomycin (Meng et al, 2017 ; Patteson et al, 2017 ). In this context, mapping the diversity of cyclodipeptides attainable by CDPSs allows the identification of new gene clusters responsible for the synthesis of DKPs with high pharmacological potential, as illustrated by the recent identification of the biosynthetic pathway of bicyclomycin, which involves a CDPS predicted to belong to the cLI/cLL-synthesizing group (Patteson et al, 2017 ).…”

“…Cyclodipeptide synthases (CDPSs) are a novel family of enzymes that use aminoacyl-tRNAs (aa-tRNAs) as substrates for the biosynthesis of various cyclodipeptides (Gondry et al, 2009 ; Moutiez et al, 2017 ), precursors of diketopiperazines (DKPs), a large class of natural products with noteworthy biological activities (Borthwick, 2012 ). Since the first description of a CDPS enzyme in 2002 (Lautru et al, 2002 ), 66 members have been characterized for their cyclodipeptide-synthesizing activities (Gondry et al, 2009 ; Seguin et al, 2011 ; Giessen et al, 2013a , b ; Alqahtani et al, 2015 ; Jacques et al, 2015 ; James et al, 2016 ; Brockmeyer and Li, 2017 ; Patteson et al, 2017 ). Most CDPSs are promiscuous enzymes, synthesizing one main cyclodipeptide and one or several minor cyclodipeptides.…”

Genome mining of cyclodipeptide synthases unravels unusual tRNA-dependent diketopiperazine-terpene biosynthetic machinery

“…As a matter of fact, CDPSs generally occur associated with tailoring enzymes 15 . Nonetheless only four CDPS-dependent pathways have been fully understood so far, including albonoursin 7 , 16 , nocazines 17 , 18 , pulcherriminic acid 19 , and bicyclomycin 20 , 21 . Compared to the canonic NRPS pathways, CDPS pathways are still underexplored.…”

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

“…287 One of the more impressive series of transformations reported to occur during bacterial DKP synthesis is found in the biosynthesis of bicyclomycin. 293,294 In this pathway, the initial cyclo(L-Leu-L-Ile) DKP undergoes six oxidation events (catalysed by ve a-ketoglutarate/Fe 2+ -dependant dioxygenases (BcmB/C/E/F/G) and one cytochrome P450 (BcmD)) to afford the nal, bicyclic structure of bicyclomycin. Within this pathway, the BcmD has been identied as carrying out the penultimate oxidation reaction, which affords hydroxylation of the C6 position of the DKP ring (Fig.…”