In vivo characterization of the activities of novel cyclodipeptide oxidases: new tools for increasing chemical diversity of bioproduced 2,5-diketopiperazines in Escherichia coli

• Published in: Microbial cell factories Vol. 19; no. 1; pp. 1 - 178
• Main Authors: Le Chevalier, Fabien, Correia, Isabelle, Matheron, Lucrèce, Babin, Morgan, Moutiez, Mireille, Canu, Nicolas, Gondry, Muriel, Lequin, Olivier, Belin, Pascal
• Format: Journal Article
• Language: English
• Published: London BioMed Central Ltd 07.09.2020; BioMed Central; BMC
• Subjects: 2,5-diketopiperazine; Bacteria; Biosynthesis; Chassis; Collateralized debt obligations; Combinatorial analysis; Combinatorial biosynthesis; Cyclodipeptide oxidase; Cyclodipeptide synthase; Dehydrogenation; E coli; Enzymes; Escherichia coli; Gene clusters; Genes; Genomes; Life Sciences; Microorganisms; Natural products; Oxidases; Physiological aspects; Synthetic biology; Toolkits; Cyclodipeptide synthase; 2; Natural products; Combinatorial biosynthesis; Synthetic biology; Cyclodipeptide oxidase; 5-diketopiperazine
• ISSN: 1475-2859; 1475-2859
• DOI: 10.1186/s12934-020-01432-y

Cyclodipeptide oxidases (CDOs) are enzymes involved in the biosynthesis of 2,5-diketopiperazines, a class of naturally occurring compounds with a large range of pharmaceutical activities. CDOs belong to cyclodipeptide synthase (CDPS)-dependent pathways, in which they play an early role in the chemical diversification of cyclodipeptides by introducing C[alpha]-C[beta] dehydrogenations. Although the activities of more than 100 CDPSs have been determined, the activities of only a few CDOs have been characterized. Furthermore, the assessment of the CDO activities on chemically-synthesized cyclodipeptides has shown these enzymes to be relatively promiscuous, making them interesting tools for cyclodipeptide chemical diversification. The purpose of this study is to provide the first completely microbial toolkit for the efficient bioproduction of a variety of dehydrogenated 2,5-diketopiperazines. We mined genomes for CDOs encoded in biosynthetic gene clusters of CDPS-dependent pathways and selected several for characterization. We co-expressed each with their associated CDPS in the pathway using Escherichia coli as a chassis and showed that the cyclodipeptides and the dehydrogenated derivatives were produced in the culture supernatants. We determined the biological activities of the six novel CDOs by solving the chemical structures of the biologically produced dehydrogenated cyclodipeptides. Then, we assessed the six novel CDOs plus two previously characterized CDOs in combinatorial engineering experiments in E. coli. We co-expressed each of the eight CDOs with each of 18 CDPSs selected for the diversity of cyclodipeptides they synthesize. We detected more than 50 dehydrogenated cyclodipeptides and determined the best CDPS/CDO combinations to optimize the production of 23. Our study establishes the usefulness of CDPS and CDO for the bioproduction of dehydrogenated cyclodipeptides. It constitutes the first step toward the bioproduction of more complex and diverse 2,5-diketopiperazines.