(M1230-03-18) Whole Genome Sequencing and Analysis of Manzamine Producer Micromonospora sp. M42

Purpose: The purpose of this study is to conduct genomic analysis to identify biosynthetic gene clusters for secondary metabolites including Manzamine A

Methods: Micromonospora sp. M42 was cultured in ISP2 medium. Genomic DNA from the strain was isolated following standard protocol. Whole genome sequencing was carried out at Molecular Research, Texas. Assignment of open reading frames (ORFs) and their functional annotations were carried out using the bioinformatic tool RAST. Putative biosynthetic gene clusters in the genome were identified using AntiSMASH. Standard cloning protocols were followed to clone and heterologous expression of select genes in E. coli BL21. Protein purifications were carried out using immobilized metal affinity chromatography (IMAC). Enzyme assays were carried out in 50 mM phosphate buffer using purified enzyme, L-tryptophan (1 mM) and pyridoxal-5-phosphate (0.1 mM). The reactions were quenched using 10% (vol/vol) of trifluoroacetic acid. The assay products were filtered using syringe filter (2 µm pore size) and the filtrates were subjected to UPLC-ESI-MS analysis.

Results: Micromonospora sp. M42 is known to produce manzamine A that exhibits a broad range of biological activities including antimalarial, anticancer and antihyperlipidemic. With an aim to elucidate biosynthetic processes implicated in manzamine A production, we sequenced the whole genome that resulted in a contiguous sequence of 6,744,853 base pairs. RAST analysis identified a total of 6,145 genes, and of those, 4,123 had a predicted function (Aziz et al., 2008). AntiSMASH analysis produced a total of 21 biosynthetic gene clusters (Blin et al., 2023). These predicted gene clusters included Type I, II and III-polyketides, non-ribosomal peptides, enediynes, ribosomal peptides, alkaloids and mixture of two or more classes of natural products. Since the strain is known to produce manzamine A, we developed a hypothetical biosynthetic pathway founded on a thorough literature review support, and searched for key hypothesized biosynthetic enzymes in the genome. A pictet-spenglerases that attaches tryptamine moiety to the ircinal A core represents the key biosynthetic step of the proposed pathway. When searching the genome for homologous protein using known pictet-spenglerase, no hit could be identified, indicating a possibility of a novel enzyme. When testing a putative aromatic decarboxylase for its role in the conversion of L-tryptophan into tryptamine (one of the anticipated substrates for the pictet-spenglerase), no decarboxylative activity were observed, further suggesting for an alternative biosynthetic route for the biosynthesis of manzamine A.

Conclusion: We have sequenced and analyzed the whole genome of manzamine A producer Micromonospora sp.M42 strain. Bioinformatics revealed over a dozen putative gene clusters for biosynthesis of secondary metabolites. Although we were unable to pinpoint the biosynthetic enzymes implicated in Manzamine A biosynthesis, the results uncovered untapped biosynthetic potential of the strain for the discovery of new metabolites. Our preliminary results show that manzamine A biosynthesis potentially involved novel biosynthetic mechanisms, which are yet to be discovered.

References: Aziz, R. K., Bartels, D., Best, A. A., DeJongh, M., Disz, T., Edwards, R. A., Formsma, K., Gerdes, S., Glass, E. M., Kubal, M., Meyer, F., Olsen, G. J., Olson, R., Osterman, A. L., Overbeek, R. A., McNeil, L. K., Paarmann, D., Paczian, T., Parrello, B., … Zagnitko, O. (2008). The RAST Server: Rapid Annotations using Subsystems Technology. BMC Genomics, 9(1), 75. https://doi.org/10.1186/1471-2164-9-75
Blin, K., Shaw, S., Augustijn, H. E., Reitz, Z. L., Biermann, F., Alanjary, M., Fetter, A., Terlouw, B. R., Metcalf, W. W., Helfrich, E. J. N., van Wezel, G. P., Medema, M. H., & Weber, T. (2023). antiSMASH 7.0: New and improved predictions for detection, regulation, chemical structures and visualisation. Nucleic Acids Research, 51(W1), W46–W50. https://doi.org/10.1093/nar/gkad344
Grant, J. R., Enns, E., Marinier, E., Mandal, A., Herman, E. K., Chen, C., Graham, M., Van Domselaar, G., & Stothard, P. (2023). Proksee: In-depth characterization and visualization of bacterial genomes. Nucleic Acids Research, 51(W1), W484–W492. https://doi.org/10.1093/nar/gkad326

Acknowledgements: This work was supported by the National Science Foundation HBCU-EiR award 2302454.


Whole genome sequence map of Micromonospora sp.M42 strain. The rings represent six reading frames and each strip represents a putative open reading frame.