Discovery of a novel cyclic lipopeptide and its biosynthetic gene cluster from a BAC library of Kutzneria sp Francisco Javier Ortiz-López 1 , Daniel Oves-Costales 1 , Jaime Felipe Guerrero Garzón 2 , Tetiana Gren 2 , Eva Baggesgaard Sterndorff 2 , Tue Sparholt Jørgensen 2 , Xinglin Jiang 2 , José Rubén Tormo 1 , Jesús Martín 1 , Mercedes de la Cruz 1 , Fernando Reyes 1 , Tilmann Weber 2 , Olga Genilloud 1 1 Fundacion MEDINA, Spain, 2 Technical University of Denmark, Denmark The application of different strategies to exploit the biosynthetic potential of underexplored bacteria is particularly important in the field of microbial natural product chemistry. One established approach is the construction of a Bacterial Artificial Chromosome (BAC) library and its insertion into a heterologous host. 1 On the other hand, the OSMAC ( One Strain, Many Compounds ) 2 approach is an efficient way to induce the production of cryptic secondary metabolites with potential therapeutic applications. Previous work of our group with Kutzneria sp. CA-103260 3,4 prompted us to explore the potential of this rare genus. As a result of this study, we report here the discovery of a novel cyclic lipopeptide (MDN-0278) and its biosynthetic gene cluster (BGC) from a BAC library of this strain. First, a BAC library of CA-103260 was constructed and screened for interesting BGCs. Selected BACs were then separately inserted into S. coelicolor hosts and cultured using an OSMAC approach. Subsequent LC-MS analyses allowed us to identify a new compound (MDN-0278) differentially produced from one of the BACs under only one of the conditions. The fermentation was then scaled-up and targeted isolation of the new compound was achieved. The structure of MDN-0278 was elucidated by combining HRMS, 1D/2D NMR spectroscopy and chemical approaches. The novel cyclic lipopeptide contains two 2,3-diaminopropionic acid (Dap) residues, one of them linked through its beta-amino group to an O -methyl tyrosine residue. Remarkably, the structure shows an exotic N,N -acetonide ring that turned out to be an artifact derived from the extraction of the culture with acetone. The BGC responsible to produce the new lipopeptide was identified within the genome region contained in the BAC. The gene cluster encodes two NRPSs putatively accounting for the biosynthesis of the cyclic tetrapeptide core. Two enzymes likely involved in the biosynthesis of Dap are also encoded in the cluster, as well as an O -methyl transferase presumably responsible for the methylation of the Tyr residue. Finally, the BGC codes for a set of genes probably related to the synthesis and attachment of the unsaturated fatty acid chain. MDN-0278 was tested for its antimicrobial activity, showing significant activity against MRSA (MIC 4-8 ug/mL). These results illustrate the powerful combination of different approaches (BAC library, OSMAC), to produce novel secondary metabolites with potential therapeutic applications from rare actinobacteria. References 1. H. Shizuya et al, Proc. Natl. Acad. Sci. U. S. A. , 1992, 89 , 8794–8797. 2. H. B. Bode et al, ChemBioChem , 2002, 3 , 619. 3. E. E. Kontou, T. Gren, F. J. Ortiz-López, E. Thomsen, D. Oves-Costales, C. Díaz, M. de la Cruz, X. Jiang, T. S. Jørgensen, K. Blin, P. Charusanti, F. Reyes, O. Genilloud and T. Weber, ACS Chem. Biol. , 2021, 16 , 1456–1468. 4. T. S. Jørgensen, T. Gren, E. E. Kontou, I. González, F. Román-Hurtado, D. Oves-Costales, E. Thomsen, P. Charusanti, O. Genilloud and T. Weber, Microbiol. Resour. Announc. , 2021, 10 , 2647–2648.
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