Exploiting the mosaic structure of trans-acyltransferase polyketide synthases for natural product discovery and pathway dissection

Abstract

Modular polyketide synthases (PKSs) are giant bacterial enzymes that synthesize many polyketides of therapeutic value. In contrast to PKSs that provide acyltransferase (AT) activities in cis, trans-AT PKSs lack integrated AT domains and exhibit unusual enzymatic features with poorly understood functions in polyketide assembly. This has retarded insight into the assembly of products such as mupirocin, leinamycin and bryostatin 1. We show that trans-AT PKSs evolved in a fundamentally different fashion from cis-AT systems, through horizontal recruitment and assembly of substrate-specific ketosynthase (KS) domains. The insights obtained from analysis of these KS mosaics will facilitate both the discovery of novel polyketides by genome mining, as we demonstrate for the thailandamides of Burkholderia thailandensis, and the extraction of chemical information from short trans-AT PCR products, as we show using metagenomic DNA of marine sponges. Our data also suggest new strategies for dissecting polyketide biosynthetic pathways and engineering polyketide assembly.

Metagenomic natural product discovery in lichen provides evidence for a family of biosynthetic pathways in diverse symbioses

“…1B; SI Appendix, Table S3). Although trans-AT PKS systems have been found in a wide range of bacteria (16,17), none have been reported for cyanobacteria, which are otherwise rich sources of cis-AT PKSs (17,18). These observations suggested the possibility of metabolic products that might be novel, not only from structural but also from ecological and evolutionary perspectives.…”

“…This observation indicated that a large part of the polyketide product would resemble pederin and onnamides. The remainder of the core structure was more difficult to predict, because two of the four KSs (KS6 and KS8) fell into clades consisting of KS 0 s, which are nonelongating KS variants that usually show little consistency between phylogeny and substrate structure (16). KS 0 6 was positioned in a small subclade containing homologs from the rhizoxin and bacillaene PKSs that are involved in shifting double bonds from the α,β-to the β,γ-position (24,25).…”

“…Detailed examination of the ketosynthase (KS) domains in PKS gene clusters using phylogenetic methods and comparisons of module architecture in pathways with similar products can often facilitate prediction of natural product structures generated by trans-AT PKSs (16,19). When the Nsp KS sequences (KS1-KS9, referring to the module number in which the domain occurs) were aligned and compared with 494 homologs using KSs from cis-AT systems as an outgroup, the resulting clades were generally consistent with respect to KS functions (SI Appendix, Fig.…”

High-throughput platform for the discovery of elicitors of silent bacterial gene clusters

“…Production of the thailandamides (Fig. 1, 5), a family of polyenes comprising multiple geometric isomers generated by the tha gene cluster (25,26), was increased 8-36-fold, depending on the isomer (Fig. 4A).…”

A ketoreductase domain in the PksJ protein of the bacillaene assembly line carries out both α- and β-ketone reduction during chain growth

“…Blastp searches of the dual-function PksJ-KR revealed numerous orthologs predominantly in trans-AT clusters, including those responsible for mupirocin, bryostatin, and rhizoxin biosynthesis (19,20), with 44%, 44%, and 43% sequence identity, respectively. Notably, many of these possible dual-function ␣/␤ KRs are predicted to act on C 2 -extended ␣-ketoacyl-Gly-S-T thioesters, perhaps reflecting a preference for conformationally flexible substrates (21). For example, the first KR domain in Ta1 from the myxovirescin biosynthetic cluster is 38% identical to PksJ-KR.…”