Reprogramming a module of the 6-deoxyerythronolide B synthase for iterative chain elongation

Multimodular polyketide synthases (PKSs) have an assembly line architecture in which a set of protein domains, known as a module, participates in one round of polyketide chain elongation and associated chemical modifications, after which the growing chain is translocated to the next PKS module. The ability to rationally reprogram these assembly lines to enable efficient synthesis of new polyketide antibiotics has been a long-standing goal in natural products biosynthesis. We have identified a ratchet mechanism that can explain the observed unidirectional translocation of the growing polyketide chain along the 6-deoxyerythronolide B synthase. As a test of this model, module 3 of the 6-deoxyerythronolide B synthase has been reengineered to catalyze two successive rounds of chain elongation. Our results suggest that high selectivity has been evolutionarily programmed at three types of protein-protein interfaces that are present repetitively along naturally occurring PKS assembly lines.     

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

The polyketide signaling metabolites bacillaene and dihydrobacillaene are biosynthesized in Bacillus subtilis on an enzymatic assembly line with both nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) modules acting along with catalytic domains servicing the assembly line in trans. These signaling metabolites possess the unusual starter unit α-hydroxyisocaproate (α-HIC). We show here that it arises from initial activation of α-ketoisocaproate (α-KIC) by the first adenylation domain of PksJ (a hybrid PKS/NRPS) and installation on the pantetheinyl arm of the adjacent thiolation (T) domain. The α-KIC unit is elongated to α-KIC-Gly by the second NRPS module in PksJ as demonstrated by mass spectrometric analysis. The third module of PksJ uses PKS logic and contains an embedded ketoreductase (KR) domain along with two adjacent T domains. We show that this KR domain reduces canonical 3-ketobutyryl chains but also the α-keto group of α-KIC-containing intermediates on the PksJ T-domain doublet. This KR activity accounts for the α-HIC moiety found in the dihydrobacillaene/bacillaene pair and represents an example of an assembly-line dual-function α- and β-KR acting on disparate positions of a growing chain intermediate.     

Unraveling the molecular basis of subunit specificity in P pilus assembly by mass spectrometry

P pili are multisubunit fibers essential for the attachment of uropathogenic Escherichia coli to the kidney. These fibers are formed by the noncovalent assembly of six different homologous subunit types in an array that is strictly defined in terms of both the number and order of each subunit type. Assembly occurs through a mechanism termed “donor-strand exchange (DSE)” in which an N-terminal extension (Nte) of one subunit donates a β-strand to an adjacent subunit, completing its Ig fold. Despite structural determination of the different subunits, the mechanism determining specificity of subunit ordering in pilus assembly remained unclear. Here, we have used noncovalent mass spectrometry to monitor DSE between all 30 possible pairs of P pilus subunits and their Ntes. We demonstrate a striking correlation between the natural order of subunits in pili and their ability to undergo DSE in vitro. The results reveal insights into the molecular mechanism by which subunit ordering during the assembly of this complex is achieved.     

Reproductive clonality of pathogens: A perspective on pathogenic viruses,bacteria, fungi,and parasitic protozoa

We propose that clonal evolution in micropathogens be defined as restrained recombination on an evolutionary scale, with genetic exchange scarce enough to not break the prevalent pattern of clonal population structure, a definition already widely used for all kinds of pathogens, although not clearly formulated by many scientists and rejected by others. The two main manifestations of clonal evolution are strong linkage disequilibrium (LD) and widespread genetic clustering (“near-clading”). We hypothesize that this pattern is not mainly due to natural selection, but originates chiefly from in-built genetic properties of pathogens, which could be ancestral and could function as alternative allelic systems to recombination genes (“clonality/sexuality machinery”) to escape recombinational load. The clonal framework of species of pathogens should be ascertained before any analysis of biomedical phenotypes (phylogenetic character mapping). In our opinion, this model provides a conceptual framework for the population genetics of any micropathogen.     

Quaternary structure of the human Cdt1-Geminin complex regulates DNA replication licensing

All organisms need to ensure that no DNA segments are rereplicated in a single cell cycle. Eukaryotes achieve this through a process called origin licensing, which involves tight spatiotemporal control of the assembly of prereplicative complexes (pre-RCs) onto chromatin. Cdt1 is a key component and crucial regulator of pre-RC assembly. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent DNA rereplication. Here, we address the mechanism of DNA licensing inhibition by Geminin, by combining X-ray crystallography, small-angle X-ray scattering, and functional studies in Xenopus and mammalian cells. Our findings show that the Cdt1:Geminin complex can exist in two distinct forms, a “permissive” heterotrimer and an “inhibitory” heterohexamer. Specific Cdt1 residues, buried in the heterohexamer, are important for licensing. We postulate that the transition between the heterotrimer and the heterohexamer represents a molecular switch between licensing-competent and licensing-defective states.     

Direct evidence that the rifamycin polyketide synthase assembles polyketide chains processively

The assembly of the polyketide backbone of rifamycin B on the type I rifamycin polyketide synthase (PKS), encoded by the rifA-rifE genes, is terminated by the product of the rifF gene, an amide synthase that releases the completed undecaketide as its macrocyclic lactam. Inactivation of rifF gives a rifamycin B nonproducing mutant that still accumulates a series of linear polyketides ranging from the tetra- to a decaketide, also detected in the wild type, demonstrating that the PKS operates in a processive manner. Disruptions of the rifD module 8 and rifE module 9 and module 10 genes also result in accumulation of such linear polyketides as a consequence of premature termination of polyketide assembly. Whereas the tetraketide carries an unmodified aromatic chromophore, the penta- through decaketides have undergone oxidative cyclization to the naphthoquinone, suggesting that this modification occurs during, not after, PKS assembly. The structure of one of the accumulated compounds together with (18)O experiments suggests that this oxidative cyclization produces an 8-hydroxy-7, 8-dihydronaphthoquinone structure that, after the stage of proansamycin X, is dehydrogenated to an 8-hydroxynaphthoquinone.     

Deciphering the mechanism for the assembly of aromatic polyketides by a bacterial polyketide synthase.

Aromatic polyketides are assembled by a type 11 (iterative) polyketide synthase (PKS) in bacteria. Understanding the enzymology of such enzymes should provide the information needed for the synthesis of novel polyketides through the genetic engineering of PKSs. Using a previously described cell-free system [B.S. & C.R.H. (1993) Science 262, 1535-1540], we studied a PKS enzyme whose substrate is not directly available and purified the TcmN polyketide cyclase from Streptomyces glaucescens. TcmN is a bifunctional protein that catalyzes the regiospecific cyclization of the Tcm PKS-bound linear decaketide to Tcm F2 and the 0-methylation of Tcm D3 to Tcm B3. In the absence of TcmN, the decaketide formed by the minimal PKS consisting of the TcmJKLM proteins undergoes spontaneous cyclization to form some Tcm F2 as well as SEK15 and many other aberrant shunt products. Addition of purified TcmN to a mixture of the other Tcm PKS components both restores and enhances Tcm F2 production. Interestingly, Tcm F2 but none of the aberrant products was bound tightly to the PKS. The results described support the notion that the polyketide cyclase, not the minimal PKS, dictates the regiospecificity for the cyclization of the linear polyketide intermediate. Furthermore, because the addition of TcmN to the TcmJKLM proteins results in a significant increase of the total yield of decaketide, interactions among the individual components of the Tcm PKS complex must give rise to the optimal PKS activity.     

Structural and evolutionary relationships of “AT-less” type I polyketide synthase ketosynthases

Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representation of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs.Acyltransferase (AT)-less type I polyketide synthases (PKSs) violate the paradigm for canonical type I PKS architecture (1, 2). Canonical type I PKSs have an assembly line architecture with modules minimally composed of a ketosynthase (KS), an acyltransferase (AT), and an acyl carrier protein (ACP) (Fig. 1). AT-less type I PKSs have modules that lack AT domains; instead, AT is a discrete enzyme that acts in trans to iteratively load malonate units onto the ACPs (3). This activity was first demonstrated with domains from the leinamycin (LNM) biosynthetic pathway, in which the discrete AT is able to load all of the ACPs with malonate (4).Open in a separate windowFig. 1.Canonical and AT-less type I PKS module architecture and activity. The domain labeled “?” represents any number of modification domains. (A) Canonical type I PKS module activation, with the AT domain acting in cis. (B) AT-less type I PKS module activation, with a discrete AT acting in trans. (C) Canonical activities for KS domains catalyzing transthiolation and decarboxylative condensation. Magenta oval represents the ATd or KS/AT adapter. See Fig. 2 for representative KS transthiolation substrates.In the report of the LNM gene cluster, bioinformatics analysis of the regions C terminal to the KS domains revealed conserved regions that were hypothesized to be “AT-docking domains” (ATds) (5). Structural studies of canonical type I PKS KS-AT didomains revealed that KSs form a dimeric core structure that is flanked by AT domains connected through structured linkers or “KS/AT adapters” (6). Recent structural studies of AT-less type I PKSs have revealed that the KS domains indeed have a structured ATd or “flanking subdomain” that is equivalent in structure to the “KS/AT adapter” (Fig. 1) (7). Whether the ATd has function or is vestigial, and whether AT-less type I PKSs evolved from canonical type I PKSs or vice versa, remains to be determined.The products of AT-less type I PKSs are typically more complex than those of the type I PKSs because of the presence of unique AT-less type I PKS domains, producing diverse substrates for the KS domains. KS domains carry out two half-reactions in the process of polyketide chain elongation (Fig. 1C). The first reaction, transthiolation, is transfer of a substrate acyl chain carried by the pantetheine moiety of an ACP onto the KS active site cysteine. The second reaction is a decarboxylative Claisen-condensation of a malonyl-S-ACP on the acyl-S-KS thioester to generate β-keto-acyl-S-ACP. Common polyketide intermediates are ACP-tethered thioesters with β-keto, β-hydroxy, α/β-unsaturated (alkene), and alkane functionalities that are generated by KS, ketoreductase, dehydratase, and enoylreductase domains, respectively (SI Appendix, Fig. S1A). The α-carbon of β-keto-acyl-S-ACP can be alkylated by additional methyltransferase domains. Another common modification to β-keto-acyl-S-ACP is β-alkylation, which is carried out by hydroxy-methylglutaryl-CoA synthase homologs, and the resulting β-hydroxy-acyl-S-ACP is dehydrated and decarboxylated by enoyl-CoA-hydratase homologs (SI Appendix, Fig. S1B) (8). Two other modifications found within AT-less type I PKSs are pyran/furan formation by pyran synthase domains (9) and migration of α/β alkenes to β/γ alkenes by enoyl-isomerases (SI Appendix, Fig. S1 A and C) (10).AT-less type I PKSs are rich in domains catalyzing the biosynthesis of hybrid peptide–polyketide natural products. Two general strategies are known for hybrid peptide–polyketide natural product biosynthesis: hybrid nonribosomal peptide synthetase (NRPS)/PKS modules containing a KS domain that accepts N-acyl amino acid or peptide from a peptidyl carrier protein and performs C–C bond formation with a malonyl-S-ACP, or hybrid PKS/NRPS modules containing a condensation domain that forms an amide bond between acyl-S-ACP and the amine of an aminoacyl-S-peptidyl carrier protein (SI Appendix, Fig. S2 A and D) (11). We previously revealed that hybrid NRPS/PKS KS domains are phylogenetically distinct from canonical type I PKS KS domains (11); however, their relationships to AT-less type I PKS KSs were not examined.Some AT-less type I PKS KS domains have activities other than β-keto-acyl-S-ACP synthesis. The rhizoxin AT-less type I PKS contains a β-branching KS with an appended B domain, which produces a β-branch lactone (SI Appendix, Fig. S1C) (12, 13). In analogy, the glutarimide ring of iso-migrastatin (MGS) is installed by a β-branching KS performing similar β-branching chemistry (14). Although most KS domains possess a conserved active site cysteine and two histidines, some have mutated histidine residues, making them catalytically incompetent for C–C bond formation, yet capable of transthiolation activity, and are referred to as KS⁰. Recently, one of these KS⁰ domains from the {"type":"entrez-nucleotide","attrs":{"text":"FR901465","term_id":"525229800","term_text":"FR901465"}}FR901465 biosynthetic pathway was confirmed to have “gatekeeping” activity and would only transfer the product of the upstream module if correctly processed (SI Appendix, Fig. S2B) (15).A feature of canonical type I PKS architecture is colinearity; that is, the PKS amino acid sequence correlates with the linear extension of the polyketide product. Colinearity allows prediction of polyketide structures from PKS sequence and vice versa. Conversely, AT-less type I PKSs frequently deviate from colinearity as a result of the presence of unique domains, noncanonical KS activities, and cryptic modifying domain activities. Phylogenetic analysis of the canonical type I PKS KS domains reveals that they share high homology and mainly clade with members of their biosynthetic pathway (16). In stark contrast, phylogenetic analysis of the AT-less type I PKS KS domains reveals that they form clades that correlate with the functional groups of the α- and β-carbons of the acyl-S-ACP substrate (17). This phylogenetic substrate correlation has been used to predict the products of AT-less type I PKSs, as colinearity analysis often fails (18). These observations have led to the hypothesis that AT-less KS domains have specificity for their substrates. Characterization of the substrate specificity of six AT-less type I PKS KSs from the psymberin and bacillaene pathways demonstrate that the KSs have substrate preference (7, 19–21). Furthermore, a structure of a bacillaene KS, with the natural substrate or a mimic bound, reveals that there are specific interactions between the KS and natural substrate that further strengthen the argument for AT-less type I PKS KS substrate specificity (7).To obtain a more detailed understanding of how features in amino acid sequences relate to structure–function relationships, we subjected the KS domains from the LNM, oxazolomycin (OZM) (22), and MGS (23) biosynthetic pathways to high-throughput structural genomics analysis. This effort resulted in four structures from the OZM and three structures from the MGS biosynthetic pathways. Sequence similarity network analysis (SSA) of more than 600 KS domains was used to reveal sets of KSs with similarity to the structurally characterized members. The structures and SSA reveal the molecular details of the evolutionary relationships between the KS domains of canonical, hybrid NRPS/PKS, and AT-less type I PKSs. Structural and sequence alignment analysis gives further insight into the molecular details accounting for AT-less type I PKS KS substrate specificity. The structural details revealed here can be leveraged toward redesign of the KS active sites (by protein engineering) to enable combinatorial biosynthesis of AT-less type I PKS products.     

Intimate bacterial–fungal interaction triggers biosynthesis of archetypal polyketides in Aspergillus nidulans

Fungi produce numerous low molecular weight molecules endowed with a multitude of biological activities. However, mining the full-genome sequences of fungi indicates that their potential to produce secondary metabolites is greatly underestimated. Because most of the biosynthesis gene clusters are silent under laboratory conditions, one of the major challenges is to understand the physiological conditions under which these genes are activated. Thus, we cocultivated the important model fungus Aspergillus nidulans with a collection of 58 soil-dwelling actinomycetes. By microarray analyses of both Aspergillus secondary metabolism and full-genome arrays and Northern blot and quantitative RT-PCR analyses, we demonstrate at the molecular level that a distinct fungal-bacterial interaction leads to the specific activation of fungal secondary metabolism genes. Most surprisingly, dialysis experiments and electron microscopy indicated that an intimate physical interaction of the bacterial and fungal mycelia is required to elicit the specific response. Gene knockout experiments provided evidence that one induced gene cluster codes for the long-sought after polyketide synthase (PKS) required for the biosynthesis of the archetypal polyketide orsellinic acid, the typical lichen metabolite lecanoric acid, and the cathepsin K inhibitors F-9775A and F-9775B. A phylogenetic analysis demonstrates that orthologs of this PKS are widespread in nature in all major fungal groups, including mycobionts of lichens. These results provide evidence of specific interaction among microorganisms belonging to different domains and support the hypothesis that not only diffusible signals but intimate physical interactions contribute to the communication among microorganisms and induction of otherwise silent biosynthesis genes.     

Expanding the biosynthetic repertoire of plant type III polyketide synthases by altering starter molecule specificity

Type III polyketide synthases (PKS) generate an array of natural products by condensing multiple acetyl units derived from malonyl-CoA to thioester-linked starter molecules covalently bound in the PKS active site. One strategy adopted by Nature for increasing the functional diversity of these biosynthetic enzymes involves modifying polyketide assembly by altering the preference for starter molecules. Chalcone synthase (CHS) is a ubiquitous plant PKS and the first type III PKS described functionally and structurally. Guided by the three-dimensional structure of CHS, Phe-215 and Phe-265, which are situated at the active site entrance, were targeted for site-directed mutagenesis to diversify CHS activity. The resulting mutants were screened against a panel of aliphatic and aromatic CoA-linked starter molecules to evaluate the degree of starter molecule specificity in CHS. Although wild-type CHS accepts a number of natural CoA thioesters, it does not use N-methylanthraniloyl-CoA as a substrate. Substitution of Phe-215 by serine yields a CHS mutant that preferentially accepts this CoA-thioester substrate to generate a novel alkaloid, namely N-methylanthraniloyltriacetic acid lactone. These results demonstrate that a point mutation in CHS dramatically shifts the molecular selectivity of this enzyme. This structure-based approach to metabolic redesign represents an initial step toward tailoring the biosynthetic activity of plant type III PKS.     

Engineered biosynthesis of bacterial aromatic polyketides in Escherichia coli

Bacterial aromatic polyketides are important therapeutic compounds including front line antibiotics and anticancer drugs. It is one of the last remaining major classes of natural products of which the biosynthesis has not been reconstituted in the genetically superior host Escherichia coli. Here, we demonstrate the engineered biosynthesis of bacterial aromatic polyketides in E. coli by using a dissected and reassembled fungal polyketide synthase (PKS). The minimal PKS of the megasynthase PKS4 from Gibberella fujikuroi was extracted by using two approaches. The first approach yielded a stand-alone Ketosynthase (KS)_malonyl-CoA:ACP transferase (MAT) didomain and an acyl-carrier protein (ACP) domain, whereas the second approach yielded a compact PKS (PKS_WJ) that consists of KS, MAT, and ACP on a single polypeptide. Both minimal PKSs produced nonfungal polyketides cyclized via different regioselectivity, whereas the fungal-specific C2-C7 cyclization mode was not observed. The kinetic properties of the two minimal PKSs were characterized to confirm both PKSs can synthesize polyketides with similar efficiency as the parent PKS4 megasynthase. Both minimal PKSs interacted effectively with exogenous polyketide cyclases as demonstrated by the synthesis of predominantly PK8 3 or NonaSEK4 6 in the presence of a C9-C14 or a C7-C12 cyclase, respectively. When PKS_WJ and downstream tailoring enzymes were expressed in E. coli, the expected nonaketide anthraquinone SEK26 was recovered in good titer. High-cell density fermentation was performed to demonstrate the scale-up potential of the in vivo platform for the biosynthesis of bacterial polyketides. Using engineered fungal PKSs can therefore be a general approach toward the heterologous biosynthesis of bacterial aromatic polyketides in E. coli.     

General mechanism for modulating immunoglobulin effector function

Immunoglobulins recognize and clear microbial pathogens and toxins through the coupling of variable region specificity to Fc-triggered cellular activation. These proinflammatory activities are regulated, thus avoiding the pathogenic sequelae of uncontrolled inflammation by modulating the composition of the Fc-linked glycan. Upon sialylation, the affinities for Fcγ receptors are reduced, whereas those for alternative cellular receptors, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN)/CD23, are increased. We demonstrate that sialylation induces significant structural alterations in the Cγ2 domain and propose a model that explains the observed changes in ligand specificity and biological activity. By analogy to related complexes formed by IgE and its evolutionarily related Fc receptors, we conclude that this mechanism is general for the modulation of antibody-triggered immune responses, characterized by a shift between an “open” activating conformation and a “closed” anti-inflammatory state of antibody Fc fragments. This common mechanism has been targeted by pathogens to avoid host defense and offers targets for therapeutic intervention in allergic and autoimmune disorders.     

Evolution of chemical diversity by coordinated gene swaps in type II polyketide gene clusters

Natural product biosynthetic pathways generate molecules of enormous structural complexity and exquisitely tuned biological activities. Studies of natural products have led to the discovery of many pharmaceutical agents, particularly antibiotics. Attempts to harness the catalytic prowess of biosynthetic enzyme systems, for both compound discovery and engineering, have been limited by a poor understanding of the evolution of the underlying gene clusters. We developed an approach to study the evolution of biosynthetic genes on a cluster-wide scale, integrating pairwise gene coevolution information with large-scale phylogenetic analysis. We used this method to infer the evolution of type II polyketide gene clusters, tracing the path of evolution from the single ancestor to those gene clusters surviving today. We identified 10 key gene types in these clusters, most of which were swapped in from existing cellular processes and subsequently specialized. The ancestral type II polyketide gene cluster likely comprised a core set of five genes, a roster that expanded and contracted throughout evolution. A key C24 ancestor diversified into major classes of longer and shorter chain length systems, from which a C20 ancestor gave rise to the majority of characterized type II polyketide antibiotics. Our findings reveal that (i) type II polyketide structure is predictable from its gene roster, (ii) only certain gene combinations are compatible, and (iii) gene swaps were likely a key to evolution of chemical diversity. The lessons learned about how natural selection drives polyketide chemical innovation can be applied to the rational design and guided discovery of chemicals with desired structures and properties.Microorganisms produce structurally diverse secondary metabolites, many of which have been successfully repurposed by mankind as pharmaceutical agents. These molecules are manufactured by multienzyme assemblies, many of which are encoded by biosynthetic gene clusters. Elucidating the history of how gene clusters evolved to produce a powerhouse of structurally diverse and biologically active molecules could reveal how synthases can be engineered to produce new therapeutic agents. Phylogenetic analyses have revealed evolutionary histories of individual biosynthetic genes, but the mechanisms of evolution of entire gene clusters are not well understood (1–4).Here, we present an approach to study gene cluster evolution on a cluster-wide scale, and we apply it to type II polyketide gene clusters. In their native bacterial hosts, type II polyketides are thought to confer a selective advantage by serving important roles in chemical defense, signaling, and virulence (5). This class is rich in pharmacologically relevant compounds, including potent antibiotics (e.g., tetracycline) and anticancer agents (e.g., doxorubicin) (5, 6). The historical success of type II polyketides in the clinic, coupled with the need for new antibiotics, has spurred great interest in identifying and engineering new compounds in this class (7). Type II polyketide gene clusters encode discrete and dissociable polyketide synthase (PKS) enzyme assemblies. The core proteins of type II PKS gene clusters are a ketosynthase (KS)-alpha subunit and a KS-beta subunit, also known as a chain length factor (CLF), which collaborate with the acyl carrier protein (ACP) to construct a nascent polyketide chain. Reactive beta-keto chains are converted into structurally diverse molecules by the action of tailoring enzymes, including cyclases and reductases, giving rise to the final branching, oxidation state, and cyclization pattern of the polyaromatic product. The remarkable chemical diversity observed in this class of molecules is thought to originate from variations in chain length and tailoring reactions. Previous phylogenetic studies have revealed the role of the CLF in controlling the chain length of type II polyketides (8–15), but the evolution of the KS-CLF within the context of the entire protein assembly is not well understood.Our analyses trace the evolution of type II PKS gene clusters, from the initial divergence of an ancestral KS into the homologous KS-CLF pair, and the gain of several key classes of accessory enzymes. We identified 544 putative type II PKSs in public genome databases, ∼15% of which encode a product that has been structurally characterized. Our studies revealed that the ancient pairing of the KS and CLF coincided with the gain of two accessory genes responsible for ring cyclization, an evolutionary shift that likely resulted in the introduction of the characteristic polyaromatic structure of type II polyketides. Subsequent gene swaps of accessory enzymes were highly coordinated with mutations to the KS-CLF, thereby enabling PKSs to diversify the chain length, oxidation state, and overall shape of their molecular products. These findings provide an unprecedented glimpse into the mechanisms by which evolution has led to the chemical diversity of natural products. The application of these methods to other gene collectives could unveil additional modes of chemical diversity generation in nature.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:GRINS: Genetic elements that recode assembly-line polyketide synthases and accelerate their diversification

Assembly-line polyketide synthases (PKSs) are large and complex enzymatic machineries with a multimodular architecture, typically encoded in bacterial genomes by biosynthetic gene clusters. Their modularity has led to an astounding diversity of biosynthesized molecules, many with medical relevance. Thus, understanding the mechanisms that drive PKS evolution is fundamental for both functional prediction of natural PKSs as well as for the engineering of novel PKSs. Here, we describe a repetitive genetic element in assembly-line PKS genes which appears to play a role in accelerating the diversification of closely related biosynthetic clusters. We named this element GRINS: genetic repeats of intense nucleotide skews. GRINS appear to recode PKS protein regions with a biased nucleotide composition and to promote gene conversion. GRINS are present in a large number of assembly-line PKS gene clusters and are particularly widespread in the actinobacterial genus Streptomyces. While the molecular mechanisms associated with GRINS appearance, dissemination, and maintenance are unknown, the presence of GRINS in a broad range of bacterial phyla and gene families indicates that these genetic elements could play a fundamental role in protein evolution.

Polyketide synthases (PKSs) are large enzymatic machines that synthesize structurally diverse natural products, many of which are used as antibiotics, immunosuppressants, anticancer agents, and other types of medicines. In bacteria, a substantial fraction of polyketides is synthesized by multimodular PKSs, where each module consists of a set of domains that collectively catalyze one round of elongation and chemical modification of the growing polyketide chain (1) (Fig. 1A). The homologous modules of each PKS operate in a defined assembly-line manner. The emergence of this multimodular architecture and its subsequent diversification has led to an astounding complexity and variety of polyketide natural products. However, the underlying evolutionary processes that drive the evolution of assembly-line PKSs are not well understood (2).Open in a separate windowFig. 1.Gene conversion in assembly-line PKSs. (A) Typical architecture of an assembly-line PKS, exemplified by closely related angolamycin synthase (ANGS) and tylactone synthase (TYLS), which contain the same set of biosynthetic domains and produce the same polyketide, tylactone. Domains: KS, ketosynthase; KS^Q, decarboxylative ketosynthase; AT, acyltransferase; DH, dehydratase; DHt, inactive dehydratase; ER, enoyl-reductase; KR, ketoreductase; KR°, ketoreductase-inactive epimerase; ACP, acyl carrier protein; TE, thioesterase. (B) Phylogenetic tree of protein sequences of angolamycin and tylactone synthase modules. Some orthologous modules clade together, as is expected for closely related PKSs that only recently diverged from a common ancestor through point mutations. However, in many cases paralogous modules clade more closely together, which can be explained by gene conversion between these modules. Sequence alignment performed using ClustalOmega (10); phylogenetic tree constructed using RAxML (30). (C) Gene conversion is a process in which a DNA sequence is nonreciprocally transferred from one homologous region to another and was implicated in the evolution of assembly-line PKSs (6, 7).According to one model, present-day assembly-line PKSs mainly arose through successive duplications of a parent module, whereafter each prototypical multimodular PKS evolved into a family of distinct but functionally related contemporary PKSs. However, this model has several discordances. For instance, it predicts that orthologous modules of closely related PKSs should cluster together in phylogenetic trees, which is often not the case (Fig. 1B). An alternative model proposes that assembly-line PKSs arose through recombination between different modules in a mosaic-like manner (3), whereafter present-day PKSs evolved through a combination of point mutations, recombination, and gene conversion (2).Gene conversion is a process in which a DNA sequence is nonreciprocally transferred from one homologous region to another, thereby homogenizing these homologous sequences (Fig. 1C). It is common in eukaryotic genomes, where it frequently occurs during mitosis, meiosis, and double-strand-break repair and has not only been implicated in the evolution of many gene families but also identified as the mechanism causing certain genetic diseases (4). In bacteria, gene conversion has been described only in a few systems, but its overall evolutionary role in prokaryotic genomes is not well understood (5). Gene conversion has also been implicated in assembly-line PKS evolution (6, 7), although its extent, role, and mechanism remain unclear.We recently proposed that extensive gene conversion between paralogous modules of assembly-line PKSs could explain why paralogous modules are often more similar to each other than orthologous ones (Fig. 1B) (2). In this work we sought to quantify the prevalence of gene conversion in assembly-line PKSs and investigate whether it might confer an evolutionary advantage. We discovered not only that gene conversion is widespread in assembly-line PKSs but also that it is frequently associated with the presence of a genetic element which recodes PKS genes and undergoes gene conversion. This association is particularly strong within Streptomyces bacteria, suggesting a major role in the diversification of assembly-line PKSs and possibly other gene families.     

A structure-based mechanism for benzalacetone synthase from Rheum palmatum

Benzalacetone synthase (BAS), a plant-specific type III polyketide synthase (PKS), catalyzes a one-step decarboxylative condensation of malonyl-CoA and 4-coumaroyl-CoA to produce the diketide benzalacetone. We solved the crystal structures of both the wild-type and chalcone-producing I207L/L208F mutant of Rheum palmatum BAS at 1.8 Å resolution. In addition, we solved the crystal structure of the wild-type enzyme, in which a monoketide coumarate intermediate is covalently bound to the catalytic cysteine residue, at 1.6 Å resolution. This is the first direct evidence that type III PKS utilizes the cysteine as the nucleophile and as the attachment site for the polyketide intermediate. The crystal structures revealed that BAS utilizes an alternative, novel active-site pocket for locking the aromatic moiety of the coumarate, instead of the chalcone synthase’s coumaroyl-binding pocket, which is lost in the active-site of the wild-type enzyme and restored in the I207L/L208F mutant. Furthermore, the crystal structures indicated the presence of a putative nucleophilic water molecule which forms hydrogen bond networks with the Cys-His-Asn catalytic triad. This suggested that BAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.     

How does selection reconcile individual advantage with the good of the group?

An individual's advantage often conflicts with the good of its group, as when an allele spreads by meiotic drive through a population whose death rate it increases, or when an asexual genotype derives immediate advantage at the expense of future adaptability. We show how selection within populations may reconcile individual and group advantage, as in the evolution of “honest meioses” resistant to segregation distortion, and the avoidance of the “cost of sex.” Selection between species, whose evolutionary importance we document, presumably favors the survival and multiplication of species whose genetic systems or social organizations favor the evolution of mechanisms reconciling individual with group advantage: in other words, species with genetic or social systems where a gene's long-term selective advantage most nearly matches its contribution to the good of the species.     

A singular enzymatic megacomplex from Bacillus subtilis

Nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and hybrid NRPS/PKS are of particular interest, because they produce numerous therapeutic agents, have great potential for engineering novel compounds, and are the largest enzymes known. The predicted masses of known enzymatic assembly lines can reach almost 5 megadaltons, dwarfing even the ribosome (approximately 2.6 megadaltons). Despite their uniqueness and importance, little is known about the organization of these enzymes within the native producer cells. Here we report that an 80-kb gene cluster, which occupies approximately 2% of the Bacillus subtilis genome, encodes the subunits of approximately 2.5 megadalton active hybrid NRPS/PKS. Many copies of the NRPS/PKS assemble into a single organelle-like membrane-associated complex of tens to hundreds of megadaltons. Such an enzymatic megacomplex is unprecedented in bacterial subcellular organization and has important implications for engineering novel NRPS/PKSs.     

Lack of evolvability in self-sustaining autocatalytic networks constraints metabolism-first scenarios for the origin of life

A basic property of life is its capacity to experience Darwinian evolution. The replicator concept is at the core of genetics-first theories of the origin of life, which suggest that self-replicating oligonucleotides or their similar ancestors may have been the first “living” systems and may have led to the evolution of an RNA world. But problems with the nonenzymatic synthesis of biopolymers and the origin of template replication have spurred the alternative metabolism-first scenario, where self-reproducing and evolving proto-metabolic networks are assumed to have predated self-replicating genes. Recent theoretical work shows that “compositional genomes” (i.e., the counts of different molecular species in an assembly) are able to propagate compositional information and can provide a setup on which natural selection acts. Accordingly, if we stick to the notion of replicator as an entity that passes on its structure largely intact in successive replications, those macromolecular aggregates could be dubbed “ensemble replicators” (composomes) and quite different from the more familiar genes and memes. In sharp contrast with template-dependent replication dynamics, we demonstrate here that replication of compositional information is so inaccurate that fitter compositional genomes cannot be maintained by selection and, therefore, the system lacks evolvability (i.e., it cannot substantially depart from the asymptotic steady-state solution already built-in in the dynamical equations). We conclude that this fundamental limitation of ensemble replicators cautions against metabolism-first theories of the origin of life, although ancient metabolic systems could have provided a stable habitat within which polymer replicators later evolved.     

Rates and patterns of great ape retrotransposition

We analyzed 83 fully sequenced great ape genomes for mobile element insertions, predicting a total of 49,452 fixed and polymorphic Alu and long interspersed element 1 (L1) insertions not present in the human reference assembly and assigning each retrotransposition event to a different time point during great ape evolution. We used these homoplasy-free markers to construct a mobile element insertions-based phylogeny of humans and great apes and demonstrate their differential power to discern ape subspecies and populations. Within this context, we find a good correlation between L1 diversity and single-nucleotide polymorphism heterozygosity (r² = 0.65) in contrast to Alu repeats, which show little correlation (r² = 0.07). We estimate that the “rate” of Alu retrotransposition has differed by a factor of 15-fold in these lineages. Humans, chimpanzees, and bonobos show the highest rates of Alu accumulation—the latter two since divergence 1.5 Mya. The L1 insertion rate, in contrast, has remained relatively constant, with rates differing by less than a factor of three. We conclude that Alu retrotransposition has been the most variable form of genetic variation during recent human–great ape evolution, with increases and decreases occurring over very short periods of evolutionary time.