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. 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.

中文翻译：

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体内新型环二肽氧化酶活性的表征：增加大肠杆菌中生物产生的2,5-二酮哌嗪类药物化学多样性的新工具。

环二肽氧化酶（CDO）是参与2,5-二酮哌嗪类生物合成的酶，2,5-二酮哌嗪类是一类具有多种药物活性的天然存在的化合物。CDO属于环二肽合酶（CDPS）依赖性途径，在其中它们通过引入Cα-Cβ脱氢作用在环二肽的化学多样化中起早期作用。尽管已经确定了100多个CDPS的活动，但是仅对少数CDO的活动进行了表征。此外，对化学合成的环二肽的CDO活性的评估显示这些酶相对混杂，使其成为用于环二肽化学多样化的有趣工具。这项研究的目的是提供第一个完整的微生物工具包，以有效地生物生产各种脱氢的2,5-二酮哌嗪。我们针对CDPS依赖性途径的生物合成基因簇中编码的CDO挖掘了基因组，并选择了几种进行表征。我们使用大肠杆菌作为底物在通路中共表达了它们各自的相关CDPS，并表明在培养上清液中产生了环二肽和脱氢衍生物。我们通过解决生物产生的脱氢环二肽的化学结构，确定了六种新型CDO的生物活性。然后，我们在大肠杆菌的组合工程实验中评估了六种新颖的CDO以及两个先前表征的CDO。我们共表达了八个CDO中的每一个，并根据它们合成的环二肽的多样性选择了18个CDPS中的每一个。我们检测了50多个脱氢环二肽，并确定了最佳的CDPS / CDO组合以优化23种化合物的生产。我们的研究确定了CDPS和CDO对于脱氢环二肽生物生产的有用性。它是朝着更复杂和多样化的2,5-二酮哌嗪生物生产迈出的第一步。