Discovery and Heterologous Expression of Microginins from Microcystis aeruginosa LEGE 91341

Abstract

Microginins are a large family of cyanobacterial lipopeptide protease inhibitors. A hybrid polyketide synthase/non-ribosomal peptide synthetase biosynthetic gene cluster (BGC) found in several microginin-producing strainsmicwas proposed to encode the production of microginins, based on bioinformatic analysis. Here, we explored a cyanobacterium, Microcystis aeruginosa LEGE 91341, which contains a mic BGC, to discover 12 new microginin variants. The new compounds contain uncommon amino acids, namely, homophenylalanine (Hphe), homotyrosine (Htyr), or methylproline, as well as a 3-aminodecanoic acid (Ada) residue, which in some variants was chlorinated at its terminal methyl group. We have used direct pathway cloning (DiPaC) to heterologously express the mic BGC from M. aeruginosa LEGE 91341 in Escherichia coli, which led to the production of several microginins. This proved that the mic BGC is, in fact, responsible for the biosynthesis of microginins and paves the way to accessing new variants from (meta)­genome data or through pathway engineering.

Direct pathway cloning and expression of the radiosumin biosynthetic gene cluster

“…Direct pathway cloning was recently developed to streamline the capture and heterologous expression of biosynthetic gene clusters allowing the rapid refactoring and expression of small to medium-sized natural product biosynthetic pathways into any vector system of choice. 2,[44][45][46] Here, we identified 13 complete putative radiosumin biosynthetic gene clusters from microbial genomes and experimentally confirmed our bioinformatic predictions through direct pathway cloning and heterologous expression of the entire radiosumin pathway in Escherichia coli BAP1. We report the discovery of a novel radiosumin chemical variant based on high-resolution liquid chromatography mass spectrometry (LC-HRMS), nuclear magnetic resonance (NMR) spectroscopy and chemical analysis.…”

Incorporation and modification of fatty acids in cyanobacterial natural products biosynthesis

“…23) was functional in E. coli and appeared to prefer bromine to generate dibromomethyl microginin analogs. 43 Using the cyanobacterium Nostoc sp. PCC 7120 as a heterologous host, a range of halogenated columbamides ( e.g.…”

“…36 BGCs that encode FAALs usually code for enzymes that modify and decorate acyl chains, including halogenases, cytochrome P450s, desaturases and other. 16,[40][41][42][43] Such modifications can influence the biological activity of the resulting natural product e.g. 44…”

“…9), 47 microginins (e.g. microginin 739, 23), 43,73 cylindrocyclophanes/ cylindrofridins (e.g. 1/10), 17,49,74 among others.…”

“…14,37 Another FAAL-containing BGCmic, associated with the microginin family of compounds -was again successfully expressed in E. coli using DiPaC-SLIC to generate natural and non-natural variants of these metabolites. 43 The dimetal carboxylate halogenase that generates the dichloromethyl terminus in several microginins (e.g. 23) was functional in E. coli and appeared to prefer bromine to generate dibromomethyl microginin analogs.…”