Heterologous expression confirms that the cyanobacterial mic gene cluster is responsible for the biosynthesis of the protease inhibitors microginins Diana Sousa 1,2 , Nádia Eusébio 2 , Raquel Castelo-Branco 2 , Marco Preto 2 , Paul D’Agostino 3 , Tobias M. Gulder 3 and Pedro N. Leão 2 1 Faculty of Sciences, University of Porto, Portugal, 2 (CIIMAR/CIMAR), University of Porto, Portugal, 3 Technical University of Dresden, Germany A vast array of cyanobacterial natural products with distinct biological activities have been described to date, but, for most, we lack experimental evidence for the correspondent biosynthetic gene cluster (BGC). This is the case of the lipopeptide protease inhibitors microginins. In this study, we established an experimental link between the microginins and their biosynthetic genes through heterologous expression of the 25 kb hybrid polyketide synthase (PKS)/non-ribosomal peptide synthase (NRPS) BGC ( mic ) encountered in the genome of the cyanobacterium Microcystis aeruginosa LEGE 91341. The mic BGC had been proposed to be responsible for the biosynthesis of microginins due to: i) its presence in microginin-producing strains and ii) encoded functionalities that are in line with microginin structures. In this work, analysis of a M. aeruginosa LEGE 91341 extract revealed the production of twelve new microginins. We applied Direct Pathway Cloning together with Sequence- and Ligation-Independent Cloning (DiPaC-SLIC) to clone and heterologously express the mic BGC in E. coli . This led to the production of several microginins. MS/MS analysis confirmed the relation between the microginins found in the M. aeruginosa LEGE 91341 extract and those produced in E. coli through heterologous expression. Our research strongly confirms the mic BGC origin of microginins. Moreover, our approach validates the suitability of DiPaC-SLIC to uncover new lipopeptide protease inhibitors from cyanobacterial genomes.
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© The Author(s), 2022
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