In vivo characterization of the activities of novel cyclodipeptide oxidases: new tools for increasing chemical diversity
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RESEARCH
In vivo characterization of the activities of novel cyclodipeptide oxidases: new tools for increasing chemical diversity of bioproduced 2,5‑diketopiperazines in Escherichia coli Fabien Le Chevalier1, Isabelle Correia3, Lucrèce Matheron2, Morgan Babin1, Mireille Moutiez1, Nicolas Canu1, Muriel Gondry1, Olivier Lequin3 and Pascal Belin1*
Abstract Background: 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 Cα-Cβ 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. Results: 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. Conclusions: 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. Keywords: Synthetic biology, Combinatorial biosynthesis, Natural products, 2,5-diketopiperazine, Cyclodipeptide synthase, Cyclodipeptide oxidase
*Correspondence: [email protected] 1 Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif‑sur‑Yvette, France Full list of author information is available at the end of the article
Background 2,5-diketopiperazines (2,5-DKPs) are a large class of molecules characterized by a 2,5-DKP ring, resulting from the condensation of two α-amino acids [1]. Originally
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