Abstract:[Background] The establishment of cyclodipeptide synthase (CDPS)-associated tailoring enzymes offers particular promise for the generation of diketopiperazines with novel structures and good bioactivities. In the previous studies, coded diketopiperazines of the dmtA3B3C3 in CDPS gene cluster-terpenoid drimentines (DMTs) was identified in Streptomyces aidingensis CGMCC 4.5739. It was speculated that dmtD3-E3 in the downstream of CDPS participated in the biosynthesis of DMTs. However, the function of dmtD3 _ E3 has not been characterized. [Objective] To characterize the cyclodipeptide oxidase dmtD3_E3 in the gene cluster dmt3 of CDPS in S. aidingensis CGMCC 4.5739, and provide functional elements for structural diversity study of diketopiperazines. [Methods] dmtD3_E3 was cloned from the genome of S. aidingensis CGMCC 4.5739, and the recombinant plasmid pWLI209 was constructed and expressed soluble in Escherichia coli BL21(DE3). In vitro enzymatic reactions were performed, and high performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) were used to ensure the structure of catalysate. [Results] The cyclodipeptide oxidase dmtD3_E3 catalyzed the oxidative dehydrogenation of C14-C17 in cyclo- (L-Trp-L-Leu) (cWL) to generate cWΔL, and the dehydrogenation of C10-C11 in cyclo -(L-Trp-L-Ala) (cWA) to generate cΔWA. The results showed that dmtD3_E3 demonstrated broad substrate specificity. [Conclusion] This study explored and characterized the novel cyclodipeptide oxidase dmtD3_E3 in the CDPS biosynthesis pathway, laying a foundation for the further generation of “non-natural” diketopiperazines through strategies of combinatorial biosynthesis and synthetic biology.
