DS0304 -

The use of cyclodipeptide synthase-dependent biosynthetic pathways for novel bioactive 2,5-diketopiperazines – DKP-COMBIBIO

Submission summary

2,5-diketopiperazines (2,5-DKPs) are secondary metabolites found in many natural products (NPs). They have attracted much attention because of the diversity of their pharmacological properties, ranging from antibacterial to immunosuppressive activities. However, the bioavailability of DKPs in NPs is variable (quantity, conditional production complex mixtures), which constitutes an obstacle to the study of their properties. The increasing volume of genome sequence data and synthetic biology offer new avenues of study of the NP’s chemical diversity that involve the knowledge of the DKPs biosynthetic pathways and their engineering. In recent years, the three project partners have participated in the identification and characterization of DKPs biosynthetic pathways involving cyclodipeptide synthases (CDPSs). These CDPS-dependent biosynthetic pathways are also constituted by various tailoring enzymes whose function is to chemically modify cyclodipeptides synthesized by CDPSs. While five of the CDPS-dependent biosynthetic pathways have been deciphered, the bioinformatic analysis of genomes shows nearly 500 CDPSs (March 2016), many of them being genetically linked to tailoring enzymes. The aim of our DKP-COMBIBIO project is to utilize the enzymes of the CDPS-dependent biosynthesis pathways to produce a variety of DKPs that will be evaluated in biological tests. The success of this project depends on the realization of precise objectives in the required time, namely:
- the biochemical characterization of the activities of the enzymes constituting the CDPS-dependent pathways identified in databases;
- the utilization of the CDPSs and tailoring enzymes to create chemical diversity by different means —in vivo combinatorial biosynthesis; incorporation of non-proteinogenic amino acids; in vitro assays in chemoenzymatic approaches using chemically synthesized cyclodipeptides (from commercial sources or present in our laboratories) and purified recombinant enzymes; —, leading to the establishment of a library of biologically synthesized DKPs;
- the assessment of the DKPs library by testing biological activities.

The implementation of the project involves three partners over a period of three years. Partner 1 has extensive experience in the production and handling of CDPSs and tailoring enzymes. He also developed tools to characterize the activity of these enzymes in vivo (in Escherichia coli) and in vitro. Partner 2 has recognized expertise in microbiology and molecular biology, with a particular interest in the secondary metabolites biosynthetic pathways from Streptomyces bacteria. He is also interested in the development of synthetic biology approaches. Partner 3 is a specialist in the chemical characterization of peptides and small molecules using nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). So the three partners have complementary expertise to complete successfully the entire project. The different expected results of this project (characterization of a large number of biosynthetic pathways, the establishment of a library of biosynthesized DKPs, new bioactive molecules) will be directly beneficial for the 3 partners (intellectual property, publications, conferences, educational training). They will also have a broader impact on the scientific community, in particular by opening a number of research areas related to the roles of a DKP biosynthetic pathway in a specific host and the biological or pharmacological activity of novel DKPs.

Project coordination

Pascal BELIN (Service de Biologie Intégrative et Génétique Moléculaire)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


CEA SACLAY Service de Biologie Intégrative et Génétique Moléculaire
LBM - UPMC Laboratoire des Biomolécules
I2BC - CNRS IdeF SUD Institut de Biologie Intégrative de la Cellule

Help of the ANR 455,920 euros
Beginning and duration of the scientific project: January 2017 - 36 Months

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