Genome Mining and Evolutionary Analysis Reveal Diverse Type III Polyketide Synthase Pathways in Cyanobacteria

Abstract

Cyanobacteria are prolific producers of natural products, including polyketides and hybrid compounds thereof. Type III polyketide synthases (PKSs) are of particular interest, due to their wide substrate specificity and simple reaction mechanism, compared with both type I and type II PKSs. Surprisingly, only two type III PKS products, hierridins, and (7.7)paracyclophanes, have been isolated from cyanobacteria. Here, we report the mining of 517 cyanobacterial genomes for type III PKS biosynthesis gene clusters. Approximately 17% of the genomes analyzed encoded one or more type III PKSs. Together with already characterized type III PKSs, the phylogeny of this group of enzymes was investigated. Our analysis showed that type III PKSs in cyanobacteria evolved into three major lineages, including enzymes associated with 1) (7.7)paracyclophane-like biosynthesis gene clusters, 2) hierridin-like biosynthesis gene clusters, and 3) cytochrome b5 genes. The evolutionary history of these enzymes is complex, with some sequences partitioning primarily according to speciation and others putatively according to their reaction type. Protein modeling showed that cyanobacterial type III PKSs generally have a smaller active site cavity (mean = 109.035 Å3) compared with enzymes from other organisms. The size of the active site did not correlate well with substrate size, however, the "Gatekeeper" amino acid residues within the active site were strongly correlated to enzyme phylogeny. Our study provides unprecedented insight into the distribution, diversity, and molecular evolution of cyanobacterial type III PKSs, which could facilitate the discovery, characterization, and exploitation of novel enzymes, biochemical pathways, and specialized metabolites from this biosynthetically talented clade of microorganisms.

Keywords: (7.7)paracyclophane; cyanobacteria; cytochrome b5; evolution; hierridin; type III PKS.

Fig. 1.

Compounds produced by cyanobacterial type III PKSs. Structures reproduced from Martins et al. (2019).

Fig. 2.

Distribution and diversity of type III PKS gene clusters from cyanobacteria. Clade numbers correspond to the type III PKS phylogenetic tree (fig. 3). Detailed comparison of gene cluster families using Clinker v 0.0.12 can be found in the Supplementary Material online.

Fig. 3.

Phylogenetic tree of type III PKSs found in cyanobacteria, other bacteria, plants, and fungi. The tree is based on the inferred primary peptide sequences of 142 type III PKSs. Clades are numbered in a hierarchical manner. Names of characterized enzymes are provided next to the species name. The tree is color-coded according to speciation; cyanobacteria (light blue), bacteria (purple), plants (green), and fungi (orange). The most common product for each characterized enzyme/family is indicated. Sequences used for protein modeling are indicated by an asterisk. The tree was generated using MrBayes with the LG + I + G model. Bootstrap values from 1,000,000 replicates above a threshold of 0.9 are represented on branches. The scale bar represents one amino acid substitution per amino acid site.

Fig. 4.

Protein models of representative type III PKSs from cyanobacteria, other bacteria, plants, and fungi. Structures were obtained through I-TASSER and visualized using PyMol2. A cross-section of the type III PKSs is displayed, with the active site cavity (dark gray) highlighted. Green residues are the conserved active site residues, Cys–His–Asn. Blue residues correspond to the so called “Gatekeeper” residues identified in CHS from Medicago sativa. The size of the active site cavity is given below the protein model. The crystal structures used to infer the models are listed in supplementary table 4, Supplementary Material online.