Natural biocombinatorics in the polyketide synthase genes of the actinobacterium Streptomyces avermitilis

Abstract

Modular polyketide synthases (PKSs) of bacteria provide an enormous reservoir of natural chemical diversity. Studying natural biocombinatorics may aid in the development of concepts for experimental design of genes for the biosynthesis of new bioactive compounds. Here we address the question of how the modularity of biosynthetic enzymes and the prevalence of multiple gene clusters in Streptomyces drive the evolution of metabolic diversity. The phylogeny of ketosynthase (KS) domains of Streptomyces PKSs revealed that the majority of modules involved in the biosynthesis of a single compound evolved by duplication of a single ancestor module. Using Streptomyces avermitilis as a model organism, we have reconstructed the evolutionary relationships of different domain types. This analysis suggests that 65% of the modules were altered by recombinational replacements that occurred within and between biosynthetic gene clusters. The natural reprogramming of the biosynthetic pathways was unambiguously confined to domains that account for the structural diversity of the polyketide products and never observed for the KS domains. We provide examples for natural acyltransferase (AT), ketoreductase (KR), and dehydratase (DH)-KR domain replacements. Potential sites of homologous recombination could be identified in interdomain regions and within domains. Our results indicate that homologous recombination facilitated by the modularity of PKS architecture is the most important mechanism underlying polyketide diversity in bacteria.

Figure 1. The Different Module Types of Modular PKSs and Their Influence on the Structure of the Polyketide Backbone

The numbers written between domains give the typical length of the respective interdomain region in terms of amino acid residues. ER, enoylreductase.

Figure 2. Representative Structures of Secondary Metabolites Classes Produced by the Large PKSs of S. avermitilis

The avermectin and oligomycin structures are examples of the respective compound groups. The exact structure of the polyene macrolide compound is not known.

Figure 3. Phylogeny of the KS Domains of Selected PKS Clusters from Streptomyces Strains

The tree was inferred by Bayesian estimation using amino acid sequences. The domains belonging to the three large PKS clusters of S. avermitilis are highlighted in red and marked by arrows. KS domains that are located outside the main oligomycin and polyene macrolide clades are labeled with a single asterisk and double asterisks, respectively.

Figure 4. Phylogenies of the Different PKS Domain Types from S. avermitilis Projected onto the Cluster Structure

Modules that show complete congruity in all their domains are marked by asterisks on the left. The different subtypes of AT as well as KR domains are represented by different colors. The module types specified on the right are as in Figure 1.

Figure 5. Replacement of an AT Domain

The incongruent phylogenetic clustering of the PteA2–2 AT domain is displayed in the miniaturized trees. AT–DH interdomain regions are highlighted in blue and yellow to show the hybrid character of the PteA2–2 interdomain region.

Figure 6. Reduction Level Changes by Recombinatorial Sequence Replacements

(A) Homologous sequence stretches in the interdomain linkers of the different module types. (B) Loss or gain of a KR domain. (C) Exchange of a DH–KR domain unit. (D) Creation of a mixed KR domain type by recombination. Partial amino acid sequences are depicted in blue and orange to show the hybrid character of the PteA1–2 KR domain.