Discovery and Biosynthesis of the Antibiotic Bicyclomycin in Distantly Related Bacterial Classes

Abstract

Bicyclomycin (BCM) is a clinically promising antibiotic that is biosynthesized by Streptomyces cinnamoneus DSM 41675. BCM is structurally characterized by a core cyclo(l-Ile-l-Leu) 2,5-diketopiperazine (DKP) that is extensively oxidized. Here, we identify the BCM biosynthetic gene cluster, which shows that the core of BCM is biosynthesized by a cyclodipeptide synthase, and the oxidative modifications are introduced by five 2-oxoglutarate-dependent dioxygenases and one cytochrome P450 monooxygenase. The discovery of the gene cluster enabled the identification of BCM pathways encoded by the genomes of hundreds of Pseudomonas aeruginosa isolates distributed globally, and heterologous expression of the pathway from P. aeruginosa SCV20265 demonstrated that the product is chemically identical to BCM produced by S. cinnamoneus. Overall, putative BCM gene clusters have been found in at least seven genera spanning Actinobacteria and Proteobacteria (Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria). This represents a rare example of horizontal gene transfer of an intact biosynthetic gene cluster across such distantly related bacteria, and we show that these gene clusters are almost always associated with mobile genetic elements.IMPORTANCE Bicyclomycin is the only natural product antibiotic that selectively inhibits the transcription termination factor Rho. This mechanism of action, combined with its proven biological safety and its activity against clinically relevant Gram-negative bacterial pathogens, makes it a very promising antibiotic candidate. Here, we report the identification of the bicyclomycin biosynthetic gene cluster in the known bicyclomycin-producing organism Streptomyces cinnamoneus, which will enable the engineered production of new bicyclomycin derivatives. The identification of this gene cluster also led to the discovery of hundreds of bicyclomycin pathways encoded in highly diverse bacteria, including in the opportunistic pathogen Pseudomonas aeruginosa. This wide distribution of a complex biosynthetic pathway is very unusual and provides an insight into how a pathway for an antibiotic can be transferred between diverse bacteria.

The hidden enzymology of bacterial natural product biosynthesis

“…NRPSs, for example, are traditionally considered to be nature's go-to machinery for catalysing amide bond formation independently of the ribosome during NP biosynthesis, but in recent years, many pathways have been characterized that employ alternative strategies for assembling amino and carboxylic acid substrates 182,183 . Examples include a transglutaminase homologue that catalyses N-acyl-β-peptide link formation during andrimid (36) biosynthesis in various γ-proteobacterial species 184 , the use of ATP-grasp enzymes such as that involved in the assembly of the β-lactone belactosin (37) 185 and cyclodipeptide synthases that utilize activated aminoacyl-tRNAs to biosynthesize diketopiperazine NPs, such as bicyclomycin (38) [186][187][188] . Indolmycin (39) is a potent antibacterial compound produced by various bacteria that is derived from the condensation of two nonproteinogenic amino acids, indolmycenic acid and D-4,5-dehydroarginine.…”

Continuous bioactivity-dependent evolution of an antibiotic biosynthetic pathway

“…Antibiotic BGCs are found in nearly all sequenced bacteria, where their genes, proteins, and chemical products continuously undergo natural selection, evolving their fitness-enhancing bioactivities 1 . This bioactivity-dependent selection pressure can favor the retention and adaptation of BGCs acquired through horizontal gene transfer (HGT) from related organisms 2 , phylogenetically divergent bacteria 3 , or even other kingdoms of life 4 . The sampling of untargeted mutations during natural BGC evolution contrasts with genetic refactoring methods used in synthetic biology that rely on regulatory element exchange and codon optimization to adapt BGCs and improve metabolite yield 5 .…”

A chromatogram-simplified Streptomyces albus host for heterologous production of natural products

“…The unsuccessful attempt to heterologously produce bicyclomycin and methylenomycin underscores a key consideration for heterologous expression studies – host selection. For instance, S. coelicolor is a native producer of methylenomycin and was recently used as a host for bicyclomycin production [34], however it will not produce neoantimycin/prunustatin unless the biosynthetic genes are artificially expressed from constitutive promoters [33]. Indeed, there is no panacea in terms of a heterologous expression host, each one being somewhat enigmatic with peculiarities rooted in primary metabolism (i.e.…”