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

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.

Production of Diketopiperazine Derivatives by Pathway Engineering with Different Cyclodipeptide Synthases from Various Streptomyces Strains

“…Structures of already described compounds, 2a , 2b , 3a , 3b , 4a , 4b , 5a , and 6a , were proven by comparison of their 1 H NMR data with those reported previously (Tables S2, S4, S5, S7, and S8 and Figures S2, S3, S11–S18, S20, and S26). ,− Structures of newly described compounds were elucidated by interpretation of their NMR data including 1 H, 13 C, HSQC, HMBC, and 1 H– 1 H COSY. If necessary, NOESY measurements were also included (Tables S3, S5, S6, and S7 and Figures S4–S10, S19, and S21–S25).…”

“…Due to the low amount, the structure of 6b could not be elucidated by NMR and was deduced from its MS, MS 2 , and UV data (Figures S27 and S30). 31 An ion at m/z 130.0660 was observed as a daughter ion of [M + H] + in 6a. A comparable mass was detected at m/z 130.0659 in that of 6b.…”

Genome Mining and Metabolic Profiling Reveal Cytotoxic Cyclodipeptides in Streptomyces hygrospinosus var. Beijingensis

“…Cyclodipeptide oxidases (CDOs) AlbA and AlbB usually catalyze the dehydrogenation of cyclodipeptides to form dehydrogenated cyclodipeptide derivatives [ 31 ]. Whether the hydroxyl group in 1 and pyrazinone in 2 are catalyzed by CDO candidates CdpA and CdpB still awaits discovery.…”

Cyclodipeptide oxidase is an enzyme filament

“…The substrates of CDPS-associated CDOs are most likely cyclic dipeptides and their derivatives containing a 2,5-DKP scaffold. A number of CDOs found in CDPS gene clusters have been characterized to date and they often display some degree of substrate promiscuity 50,52,91 . Usually, CDOs display a preference for cyclic dipeptides containing aromatic or aliphatic amino acids over those containing charged or hydrophilic ones.…”