Evolution and Diversity of Assembly-Line Polyketide Synthases

Abstract

Assembly-line polyketide synthases (PKSs) are among the most complex protein machineries known in nature, responsible for the biosynthesis of numerous compounds used in the clinic. Their present-day diversity is the result of an evolutionary path that has involved the emergence of a multimodular architecture and further diversification of assembly-line PKSs. In this review, we provide an overview of previous studies that investigated PKS evolution and propose a model that challenges the currently prevailing view that gene duplication has played a major role in the emergence of multimodularity. We also analyze the ensemble of orphan PKS clusters sequenced so far to evaluate how large the entire diversity of assembly-line PKS clusters and their chemical products could be. Finally, we examine the existing techniques to access the natural PKS diversity in natural and heterologous hosts and describe approaches to further expand this diversity through engineering.

Figure 1

(A) The 6-deoxyerythronolide B synthase (DEBS), a prototypical assembly-line PKS, synthesizes 6-deoxyerythronolide B, the precursor of erythromycin A. (B–E) Reactions catalyzed by module 2 (M2) of DEBS. (B,C) Transacylation of the electrophilic and nucleophilic substrates of M2 from the ACP of module 1 (M1) and (2S)-methylmalonyl-CoA, respectively. (D,E) Polyketide chain elongation and ketoreduction. KS, ketosynthase; AT, acyltransferase; ACP, acyl carrier protein; KR, ketoreductase; KR⁰, redox-inactive KR with epimerase activity; DH, dehydratase; ER, enoylreductase; TE, thioesterase.

Figure 2

General model for PKS evolution. The multidomain architecture of type I PKS modules evidently arose through the fusion of genes encoding single-domain proteins of type II systems. The processes that led to the emergence of the multimodular architecture are less well understood. For instance, it is unclear whether assembly-line systems evolved from iterative PKSs that lost their ability to perform several consecutive condensation reactions on the same polyketide chain or from a separate subset of type II proteins. Once a set of assembly-line PKSs emerged, other processes allowed further diversification of these modular enzymes and their products.

Figure 3

Models of cis-AT and trans-AT PKS evolution. (A) It has been hypothesized that evolution of cis- versus trans-AT PKSs took distinct paths.^,, However, this dichotomy has some discordances. It does not explain the absence of iterative trans-AT PKSs, the convergence toward strikingly similar architectures despite different evolutionary paths, the presence of AT domain vestiges in trans-AT modules, or (B) the inconsistency of the phylogenetic tree of cis-AT KS domains with this hypothesis. The last inconsistency is exemplified by KS domains from four homologous 16-membered macrolide synthases (left; TYLS, tylactone synthase; CHMS, chalcomycin synthase; SRMS, spiramycin synthase; NIDS, niddamycin synthase). Under the current model, their KS domains would be expected to form groups of orthologous domains (center). In fact, most KS domains are grouped with paralogues from the same PKS (right). Protein sequence alignment was performed with ClustalOmega, and the dendrogram was constructed using UPGMA hierarchical clustering. (C) The discordance in KS sequence alignment is a result of concerted evolution and can be explained by gene conversion events between KS domains.^, Gene conversion leads to high sequence similarity between paralogous domains, causing them to cluster closer to each other than to their orthologues (e.g., teal square). Because gene conversion need not affect all domains within a PKS (e.g., red square), some of them maintain a phylogenetic pattern reflecting ancestral events that had led to the separation of homologous assembly-line PKSs. (D) An alternative model for assembly-line PKS evolution builds on the hypothesis that trans-AT PKSs evolved from cis-AT PKSs through loss of AT domains. In this model, the high sequence identity of KS domains in cis-AT PKSs would be explained by subsequent gene conversion events rather than ancestral gene duplications.

Figure 4

Summary of the workflow to generate the catalogue of distinct assembly-line PKSs. In the final clustering schematic, the red line represents a PKS sequence that scored higher than 90% in amino acid similarity to another sequence and was thus removed from the catalogue of distinct clusters.

Figure 5

(A) The discovery rate of distinct clusters is shown (blue; having less than 90% amino acid sequence similarity score to any other cluster). Also shown (in red) is the number of clusters with known products, determined using MIBiG database and NCBI annotations. For years 1994–2017, numbers reflect sequences deposited by December of that year. For 2018, only sequences deposited by May were taken into account. (B) Rediscovery rate among nucleotide sequences deposited to NCBI, determined as the percentage of redundant clusters (having more than 90% amino acid sequence similarity score to a previously sequenced cluster). (C) Distribution of sequence similarity scores between an orphan assembly-line PKS and its closest neighbor whose product has been characterized. PKSs with pairwise similarity scores above 50% probably make structurally similar polyketides, while orphan PKSs whose sequences show greater differences from those of any known PKS most likely produce novel chemotypes. (D) The red line plots the percentage of all distinct assembly-line PKSs that are chemically decoded. The blue line plots the percentage of orphan PKSs that are more than 50% similar to a chemically decoded assembly-line PKS.

Figure 6

Network of 3551 distinct assembly-line PKS clusters, visualized by Cytoscape 3.7.2. Nodes correspond to known (larger circles) and orphan (smaller circles) PKSs and are color-coded according to antiSMASH predictions (legend). Edges represent >50% sequence similarity between two clusters, calculated as described in ref (102).

Figure 7

(A) Distribution of assembly-line PKSs among the different phyla. (B) The nemamide PKS from C. elegans, described in ref (137). KS, ketosynthase; AT, acyltransferase; KR, ketoreductase; DH, dehydratase; C, condensation domain; A, adenylation domain; ACP, acyl carrier protein; PCP, peptidyl carrier protein; TE, thioesterase.

Figure 8

A general workflow for expressing assembly-line PKSs in heterologous hosts.

Figure 9

Over time, assembly-line PKS engineering has shifted from rational design (e.g., by domain swapping, module swapping, and module insertion) and combinatorial engineering (e.g., through in vitro combinatorial assembly) toward evolution-inspired approaches (e.g., use of natural splicing points, or inter- and intra-PKS recombination).

Figure 10

Alternative splice points for PKS engineering. (A) Two domain swapping strategies can lead to predictable changes in the structure of biosynthesized molecule: AT domain swaps affect the choice of starter or extender unit (malonyl-CoA, methylmalonyl-CoA, or other), whereas reductive loop swaps alter the configuration and oxidative state of the newly added extender unit. For both types of domain swaps, conserved regions were identified that can be used as splice points.^, (B) “Classical” module boundaries match the boundaries of unimodular proteins and correspond to the functional unit of chain elongation (KS and downstream ACP) and subsequent modification (reductive loop). “Alternative” module boundaries break the functional unit of chain elongation but preserve chain translocation unit (KS and upstream ACP), along with the reductive loop that determines the oxidative state of the translocated substrate. Both “classical” and “alternative” module boundaries have been successfully used for module deletion (shown here), as well as module swapping and insertion.^,, KS, ketosynthase; AT, acyltransferase; ACP, acyl carrier protein; KR, ketoreductase; DH, dehydratase.