Discovery and heterologous expression of new microviridins from a Portuguese Culture Collection Raquel Castelo-Branco 1,2 , Paul M. D’Agostino 3 , Tobias A. M. Gulder 3 , Vitor Vasconcelos 1,2 , David P. Fewer 4 and Pedro N. Leão 1
1 CIIMAR/FCUP, Portugal, 2 Faculty of Sciences, University of Porto, Portugal, 3 Technische Universität Dresden, Germany, 4 Department of Microbiology, University of Helsinki, Finland
Microviridins are peptides with a unique tricyclic cage-like architecture that confers therapeutically relevant including serine protease inhibition. These metabolites are produced by cyanobacteria through the post- translational modification of precursor peptides (RiPPs). Here, we used genome mining to identify several novel microviridin biosynthetic gene clusters (BGCs) in cyanobacterial genomes from strains of LEGE Culture Collection. The corresponding mature peptide was searched for in methanolic extracts of these strains. We then selected a microviridin BGC encoding two precursor peptides with unusual core peptides from a Microcystis aeruginosa strain for further study. One of the microviridins was found in very low abundance in the native producer. Therefore, we applied direct pathway cloning (DiPaC) to heterologously express the BGC in a in a surrogate host. Long amplicon PCR and DNA assembly by sequence- and ligation-independent cloning (SLIC) enabled the capture of the bidirectional 6.5 Kb mdnABCADE microviridin BGC. Expression of the microviridin BGC in Escherichia coli BL21(DE3) using temperature and culture media optimized conditions increased the production yields compared to the native host for both microviridins. The new metabolites were isolated and their chemical structures elucidated. Our results highlight the potential of screening cyanobacterial genomes for novel chemical diversity and the usefulness of heterologous expression to overcome the typically low RiPP yields in cyanobacteria. We acknowledge funding from Fundação para a Ciência e a Tecnologia through strategic Funding UIDP/04423/2020 and UIDB/04565/2020, RCB scholarship (SFRH/BD/136367/2018) and through the project ATLANTIDA (ref. NORTE-01–0145-FEDER-000040), supported by the Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement and through the European Regional Development Fund (ERDF). This project has also received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 952374.
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© The Author(s), 2022
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