Identification, cloning and heterologous expression of the gene cluster encoding globomycin biosynthesis Daniel Oves-Costales 1 , Tetiana Gren 2 , Jesús Martín 1 , Xinglin Jiang 2 , Eva Baggesgaard Sterndorff 2 , Fernando Román-Hurtado 1 , Francisco Javier Ortiz-López 1 , Tue S. Jørgensen 2 , Fernando Reyes 1 , Olga Genilloud 1 , Tilmann Weber 2 1 Fundacion MEDINA, Spain, 2 Technical University of Denmark, Denmark Globomycin and congeners are a family of cyclic lipodepsipeptides with strong activity against Gram-negative pathogens. 1 Their mode of action is rather unusual and is based on blocking the active site of LspA, thus interfering with the lipoprotein secretion pathway. Surprisingly, the biosynthetic gene cluster (BGC) encoding their biosynthesis has not been described. During our ongoing efforts to discover new bioactive microbial natural products we identified strain Streptomyces sp. CA-278952 as a producer of globomycins and we set ourselves the goal to identify the gene cluster responsible for their biosynthesis. The genome of the strain was subjected to PacBio and Illumina sequencing and a single 8.2 Mbp contig was obtained. AntiSMASH analysis afforded 23 regions potentially encoding secondary metabolite gene cluster. A retro biosynthetic analysis of globomycin allowed us to identify a hybrid PKS-NRPS cluster as the most promising candidate for its biosynthesis. CRISPR-cBEST 2 was employed to obtain a W2935* replacement in the PKS/NRPS gene globH , which was hypothesized to be essential for the globomycins biosynthesis. The mutant was then grown in 16 different fermentation conditions, together with the wild type as positive control, and the microbial broth extracts were then analyzed by LC-HRMS. Neither globomycin or any of its congeners could be detected in any of the fermentation conditions of the null mutant. To further confirm the identity of the globomycin BGC, the putative BGC was cloned using a modified CATCH procedure 3 and transferred by biparental conjugation to eight different Streptomyces sp. heterologous hosts. All the strains and their corresponding negative controls were grown in 8 different media. The microbial broths were then analyzed by LC-HRMS, which showed production of globomycin and congeners in recombinant S. albus J1074 and S. coelicolor M1146, thus proving that the cluster is indeed responsible of globomycin biosynthesis. Our work paves the way for futher studies in the biosynthesis of globomycin (specifically the mechanism controlling the lenght of the aliphatic chain) and opens the door to the generation of novel unnatural globomycin
derivatives. References
1. Inukai, R. Enokita, A. Torikata, M. Nakahara, S. Iwado, M. Arai, J. Antibiot . 1978 , 31 , 410-420. 2. Toing, C. M. Whitford, K. Blin, T. S. Jørgensen, T. Weber, Nat. Protoc . 2020 , 15 , 2470-2502 3. Jiang, X. Zhao, T. Gabrieli, C. Lou, Y. Ebenstein, T. F. Zhu. Nat. Commun . 2015 , 6:8101
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