Distribution and diversity of dimetal-carboxylate halogenases in cyanobacteria

Abstract

Background Halogenation is a recurring feature in natural products, especially those from marine organisms. The selectivity with which halogenating enzymes act on their substrates renders halogenases interesting targets for biocatalyst development. Recently, CylC – the first predicted dimetal-carboxylate halogenase to be characterized – was shown to regio- and stereoselectively install a chlorine atom onto an unactivated carbon center during cylindrocyclophane biosynthesis. Homologs of CylC are also found in other characterized cyanobacterial secondary metabolite biosynthetic gene clusters. Due to its novelty in biological catalysis, selectivity and ability to perform C-H activation, this halogenase class is of considerable fundamental and applied interest. The study of CylC-like enzymes will provide insights into substrate scope, mechanism and catalytic partners, and will also enable engineering these biocatalysts for similar or additional C-H activating functions. Still, little is known regarding the diversity and distribution of these enzymes. Results In this study, we used both genome mining and PCR-based screening to explore the genetic diversity of CylC homologs and their distribution in bacteria. While we found non-cyanobacterial homologs of these enzymes to be rare, we identified a large number of genes encoding CylC-like enzymes in publicly available cyanobacterial genomes and in our in-house culture collection of cyanobacteria. Genes encoding CylC homologs are widely distributed throughout the cyanobacterial tree of life, within biosynthetic gene clusters of distinct architectures (combination of unique gene groups). These enzymes are found in a variety of biosynthetic contexts, which include fatty-acid activating enzymes, type I or type III polyketide synthases, dialkylresorcinol-generating enzymes, monooxygenases or Rieske proteins. Our study also reveals that dimetal-carboxylate halogenases are among the most abundant types of halogenating enzymes in the phylum Cyanobacteria. Conclusions Our data show that dimetal-carboxylate halogenases are widely distributed throughout the Cyanobacteria phylum and that BGCs encoding CylC homologs are diverse and mostly uncharacterized. This work will help guide the search for new halogenating biocatalysts and natural product scaffolds.

Discovery and Heterologous Expression of Microginins from Microcystis aeruginosa LEGE 91341

“…DNA extraction from M. aeruginosa LEGE 91341 and genome analysis were described previously . The first step of the cloning strategy was to identify putative terminators in the mic BGC using the ARNold tool, , but none were detected.…”

“…Our group recently detected a putative mic BGC in the cyanobacterium M. aeruginosa LEGE 91341 (NCBI: JADEXY000000000) encoding a dimetal carboxylate halogenase homolog (Figure a). Here, we report the discovery of 12 new microginins (compounds 1 – 12 , Figure b) from this strain, including mono- and di-chlorinated as well as non-halogenated congeners.…”

Discovery and heterologous expression of microginins from Microcystis aeruginosa LEGE 91341

“…3b). A monochlorinated version of 13 (m/z 692.378, [M+H] + ) could also be detected (16). LC-HRESI-MS/MS analysis of the most abundant compounds 13, 14 and 16 (Fig.…”

“…DNA extraction from M. aeruginosa LEGE 91341 and genome analysis was described previously. 16 The first step of the cloning strategy was to identify putative terminators in the mic BGC using the ARNold tool, 28,29 but none were detected. Because after several attempts, we were unable to amplify the entire 25 kb mic BGC, we split it in two fragments (Figure 3a) to facilitate their insertion into pET28-ptetO::gfpv2.…”

“…Our group recently detected a putative mic BGC in the cyanobacterium Microcystis aeruginosa LEGE 91341 (NCBI: JADEXY000000000) encoding a dimetal carboxylate halogenase homolog 16 (Fig. 1a).…”