Modular Polyketide Synthases Research Articles

Modular oxime formation by a trans-AT polyketide synthase.

Modular trans-acyltransferase polyketide synthases (trans-AT PKSs) are enzymatic assembly lines that biosynthesize complex polyketide natural products. Relative to their better studied cis-AT counterparts, the trans-AT PKSs introduce remarkable chemical diversity into their polyketide products. A notable example is the lobatamide A PKS, which incorporates a methylated oxime. Here we demonstrate biochemically that this functionality is installed on-line by an unusual oxygenase-containing bimodule. Furthermore, analysis of the oxygenase crystal structure coupled with site-directed mutagenesis allows us to propose a model for catalysis, as well as identifying key protein-protein interactions that support this chemistry. Overall, our work adds oxime-forming machinery to the biomolecular toolbox available for trans-AT PKS engineering, opening the way to introducing such masked aldehyde functionalities into diverse polyketides.

Docking Domain Engineering in a Modular Polyketide Synthase and Its Impact on Structure and Function.

Modular polyketide synthases (PKSs) are attractive targets for the directed, biosynthetic production of platform chemicals and pharmaceuticals by protein engineering. In this study, we analyze docking domains from the 6-deoxyerythronolide B synthase, SYNZIP domains, and the SpyCatcher:SpyTag complex as engineering tools to couple the polypeptides VemG and VemH to functional venemycin synthases. Our data show that the high-affinity interaction or covalent connection of modules, enabled by SYNZIP domains and the SpyCatcher:SpyTag complex, can be advantageous, e.g., in synthesis at low protein concentrations, but their rigidity and steric demand decrease synthesis rates. However, we also show that efficiency can be recovered when inserting a hinge region distant from the rigid interface. This study demonstrates that engineering approaches should take the conformational properties of modular PKSs into account and establishes a three-polypeptide split venemycin synthase as an exquisite in vitro platform for the analysis and engineering of modular PKSs.

Structure of a modular polyketide synthase reducing region

The chemical scaffolds of numerous therapeutics are polyketide natural products, many formed by bacterial modular polyketide synthases (PKS). The large and flexible dimeric PKS modules have distinct extension and reducing regions. Structures are known for all individual enzyme domains and several extension regions. Here, we report the structure of the full reducing region from a modular PKS, the ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains of module 5 of the juvenimicin PKS. The modular PKS-reducing region has a different architecture than the homologous fatty acid synthase (FAS) and iterative PKS systems in its arrangement of domains and dimer interface. The structure reveals a critical role for linker peptides in the domain interfaces, leading to discovery of key differences in KR domains dependent on module composition. Finally, our studies provide insight into the mechanism underlying modular PKS intermediate shuttling by carrier protein (ACP) domains.

Boosting titers of engineered triketide and tetraketide synthases to record levels through T7 promoter tuning

Modular polyketide synthases (PKS's) are promising platforms for the rational engineering of designer polyketides and commodity chemicals, yet their low productivities are a barrier to the practical biosynthesis of these compounds. Previously, we engineered triketide lactone synthases such as Pik167 using the recently updated module definition and showed they generate hundreds of milligrams of product per liter of Escherichia coli K207-3 shake flask culture. As the molar ratio between the 2 polypeptides of Pik167 is highly skewed, we sought to attenuate the strength of the T7 promoter controlling the production of the smaller, better-expressing polypeptide and thereby increase production of the first polypeptide under the control of an unoptimized T7 promoter. Through this strategy, a 1.8-fold boost in titer was obtained. After a further 1.5-fold boost obtained by increasing the propionate concentration in the media from 20 to 80 mM, a record titer of 791 mg L−1 (627 mg L−1 isolated) was achieved, a 2.6-fold increase overall. Spurred on by this result, the tetraketide synthase Pik1567 was engineered and the T7 promoter attenuation strategy was applied to its second and third genes. A 5-fold boost, from 20 mg L−1 to 100 mg L−1, in the titer of its tetraketide product was achieved.

Structure-Based Analysis of Transient Interactions between Ketosynthase-like Decarboxylase and Acyl Carrier Protein in a Loading Module of Modular Polyketide Synthase.

Ketosynthase-like decarboxylase (KSQ) domains are widely distributed in the loading modules of modular type I polyketide synthases (PKSs) and catalyze the decarboxylation of the (alkyl-)malonyl unit bound to the acyl carrier protein (ACP) in the loading module for the construction of the PKS starter unit. Previously, we performed a structural and functional analysis of the GfsA KSQ domain involved in the biosynthesis of macrolide antibiotic FD-891. We furthermore revealed the recognition mechanism for the malonic acid thioester moiety of the malonyl-GfsA loading module ACP (ACPL) as a substrate. However, the exact recognition mechanism for the GfsA ACPL moiety remains unclear. Here, we present a structural basis for the interactions between the GfsA KSQ domain and GfsA ACPL. We determined the crystal structure of the GfsA KSQ-acyltransferase (AT) didomain in complex with ACPL (ACPL=KSQAT complex) by using a pantetheine crosslinking probe. We identified the key amino acid residues involved in the KSQ domain-ACPL interactions and confirmed the importance of these residues by mutational analysis. The binding mode of ACPL to the GfsA KSQ domain is similar to that of ACP to the ketosynthase domain in modular type I PKSs. Furthermore, comparing the ACPL=KSQAT complex structure with other full-length PKS module structures provides important insights into the overall architectures and conformational dynamics of the type I PKS modules.

Expanding Extender Substrate Selection for Unnatural Polyketide Biosynthesis by Acyltransferase Domain Exchange within a Modular Polyketide Synthase

Modular polyketide synthases (PKSs) are polymerases that employ α-carboxyacyl-CoAs as extender substrates. This enzyme family contains several catalytic modules, where each module is responsible for a single round of polyketide chain extension. Although PKS modules typically use malonyl-CoA or methylmalonyl-CoA for chain elongation, many other malonyl-CoA analogues are used to diversify polyketide structures in nature. Previously, we developed a method to alter an extension substrate of a given module by exchanging an acyltransferase (AT) domain while maintaining protein folding. Here, we report in vitro polyketide biosynthesis by 13 PKSs (the wild-type PKS and 12 AT-exchanged PKSs with unusual ATs) and 14 extender substrates. Our ∼200 in vitro reactions resulted in 13 structurally different polyketides, including several polyketides that have not been reported. In some cases, AT-exchanged PKSs produced target polyketides by >100-fold compared to the wild-type PKS. These data also indicate that most unusual AT domains do not incorporate malonyl-CoA and methylmalonyl-CoA but incorporate various rare extender substrates that are equal to in size or slightly larger than natural substrates. We developed a computational workflow to predict the approximate AT substrate range based on active site volumes to support the selection of ATs. These results greatly enhance our understanding of rare AT domains and demonstrate the benefit of using the proposed PKS engineering strategy to produce novel chemicals in vitro.

Enzymology of assembly line synthesis by modular polyketide synthases.

Modular polyketide synthases (PKSs) run catalytic reactions over dozens of steps in a highly orchestrated manner. To accomplish this synthetic feat, they form megadalton multienzyme complexes that are among the most intricate proteins on earth. Polyketide products are of elaborate chemistry with molecular weights of usually several hundred daltons and include clinically important drugs such as erythromycin (antibiotic), rapamycin (immunosuppressant) and epothilone (anticancer drug). The term 'modular' refers to a hierarchical structuring of modules and domains within an overall assembly line arrangement, in which PKS organization is colinearly translated into the polyketide structure. New structural information obtained during the past few years provides substantial direct insight into the orchestration of catalytic events within a PKS module and leads to plausible models for synthetic progress along assembly lines. In light of these structural insights, the PKS engineering field is poised to enter a new era of engineering.

Heterocomplex structure of a polyketide synthase component involved in modular backbone halogenation

Bacterial modular polyketide synthases (PKSs) generate diverse, complex and bioactive natural products that are constructed mainly based on principles of fatty acid biosynthesis. The cytotoxic oocydin-type polyketides contain a vinyl chloride moiety introduced during polyketide chain elongation. Required for modular polyketide backbone halogenation are a non-heme iron and ɑ-ketoglutarate-dependent halogenase OocP and OocQ lacking characterized homologs. This work provides structural insights into these unusual PKS components and their interactions via a high-resolution X-ray crystallography structure of the heterocomplex. By mapping the protein-protein interactions and comparison with structures of similar halogenases, we illustrate the potential of this heterodimer complex as a replacement for the conserved homodimeric structure of homologous enzymes. The OocPQ protein pair has thus evolved as a means of stabilizing the halogenase and facilitating chemical transformations with great synthetic utility.

Elucidation of the Late Steps during Hexacosalactone A Biosynthesis in Streptomyces samsunensis OUCT16-12.

Hexacosalactone A (1) is a polyene macrolide compound featuring a 2-amino-3-hydroxycyclopent-2-enone (C5N)-fumaryl moiety. While compound 1 has been proposed to be assembled via a type I modular polyketide synthase (PKS) system, most of the putative biosynthetic steps lack experimental evidence. In this study, we elucidated the post-PKS tailoring steps of compound 1 through in vivo gene inactivation and in vitro biochemical assays. We demonstrated that the amide synthetase HexB and O-methyltransferase HexF are responsible for the installations of the C5N moiety and the methyl group at 15-OH of compound 1, respectively; two new hexacosalactone analogs, named hexacosalactones B (4) and C (5), were purified and structurally characterized, followed by anti-multidrug resistance (anti-MDR) bacterial assays, revealing that the C5N ring and the methyl group are necessary for the antibacterial bioactivities. Through database mining of C5N-forming proteins HexABC, six uncharacterized biosynthetic gene clusters (BGCs), putatively encoding compounds with different types of backbones, were identified, providing potentials to discover novel bioactive compounds with C5N moiety. IMPORTANCE In this study, we elucidate the post-PKS tailoring steps during the biosynthesis of compound 1 and demonstrate that both C5N and 15-OMe groups are critical for the antibacterial activities of compound 1, paving the way for generation of hexacosalactone derivatives via synthetic biology strategy. In addition, mining of HexABC homologs from the GenBank database revealed their wide distribution across the bacterial world, facilitating the discovery of other bioactive natural products with C5N moiety.

Insights into azalomycin F assembly-line contribute to evolution-guided polyketide synthase engineering and identification of intermodular recognition

Modular polyketide synthase (PKS) is an ingenious core machine that catalyzes abundant polyketides in nature. Exploring interactions among modules in PKS is very important for understanding the overall biosynthetic process and for engineering PKS assembly-lines. Here, we show that intermodular recognition between the enoylreductase domain ER1/2 inside module 1/2 and the ketosynthase domain KS3 inside module 3 is required for the cross-module enoylreduction in azalomycin F (AZL) biosynthesis. We also show that KS4 of module 4 acts as a gatekeeper facilitating cross-module enoylreduction. Additionally, evidence is provided that module 3 and module 6 in the AZL PKS are evolutionarily homologous, which makes evolution-oriented PKS engineering possible. These results reveal intermodular recognition, furthering understanding of the mechanism of the PKS assembly-line, thus providing different insights into PKS engineering. This also reveals that gene duplication/conversion and subsequent combinations may be a neofunctionalization process in modular PKS assembly-lines, hence providing a different case for supporting the investigation of modular PKS evolution.

Structural Basis of Transient Interactions of Acyltransferase VinK with the Loading Acyl Carrier Protein of the Vicenistatin Modular Polyketide Synthase.

Acyltransferase (AT) recognizes its cognate acyl carrier protein (ACP) for functional transfer of an acyl unit in polyketide biosynthesis. However, structural characterization of AT-ACP complexes is limited because of the weak and transient interactions between them. In the biosynthesis of macrolactam polyketide vicenistatin, the trans-acting loading AT VinK transfers a dipeptidyl unit from the stand-alone ACP VinL to the ACP domain (VinP1ACPL) of the loading module of modular polyketide synthase VinP1. Although the previously determined structure of the VinK-VinL complex clearly illustrates the VinL recognition mechanism of VinK, how VinK recognizes VinP1ACPL remains unclear. Here, the crystal structure of a covalent VinK-VinP1ACPL complex formed with a pantetheine-type cross-linking probe is reported at 3.0 Å resolution. The structure of the VinK-VinP1ACPL complex provides detailed insights into the transient interactions between VinK and VinP1ACPL. The importance of residues in the binding interface was confirmed by site-directed mutational analyses. The binding interface between VinK and VinP1ACPL is similar to that between VinK and VinL, although some of the interface residues are different. However, the ACP orientation and interaction mode observed in the VinK-VinP1ACPL complex are different from those observed in other AT-ACP complexes such as the disorazole trans-AT-ACP complex and cis-AT-ACP complexes of modular polyketide synthases. Thus, AT-ACP binding interface interactions are different in each type of AT-ACP pair.

ClusterCAD 2.0: an updated computational platform for chimeric type I polyketide synthase and nonribosomal peptide synthetase design.

Megasynthase enzymes such as type I modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) play a central role in microbial chemical warfare because they can evolve rapidly by shuffling parts (catalytic domains) to produce novel chemicals. If we can understand the design rules to reshuffle these parts, PKSs and NRPSs will provide a systematic and modular way to synthesize millions of molecules including pharmaceuticals, biomaterials, and biofuels. However, PKS and NRPS engineering remains difficult due to a limited understanding of the determinants of PKS and NRPS fold and function. We developed ClusterCAD to streamline and simplify the process of designing and testing engineered PKS variants. Here, we present the highly improved ClusterCAD 2.0 release, available at https://clustercad.jbei.org. ClusterCAD 2.0 boasts support for PKS-NRPS hybrid and NRPS clusters in addition to PKS clusters; a vastly enlarged database of curated PKS, PKS-NRPS hybrid,and NRPS clusters; a diverse set of chemical 'starters' and loading modules; the new Domain Architecture Cluster Search Tool; and an offline Jupyter Notebook workspace, among other improvements. Together these features massively expand the chemical space that can be accessed by enzymes engineered with ClusterCAD.

Discovery of venediols by activation of a silent type I polyketide biosynthetic gene cluster in Streptomyces venezuelae ATCC 15439

The genus of Streptomyces plays an indispensable role in discovery of bioactive secondary metabolites. As a model strain, Streptomyces venezuelae ATCC 15439 possesses more than 30 biosynthetic gene clusters (BGCs) in its genome; however, many of which are transcriptionally silent and hence uncharacterized. Here, we apply CRISPR/Cas9 genome editing technology to activate a cryptic type I polyketide biosynthetic gene cluster by site-specific insertion of the constitutive promoter kasOp∗, leading to the discovery of two new polyketide metabolites including venediols A (1) and B (2). The elucidated structures and heterologous expression of this BGC suggest an unusual loss of colinearity of the modular polyketide synthases during their biosynthesis.

Metabolic pathway assembly using docking domains from type I cis-AT polyketide synthases

Engineered metabolic pathways in microbial cell factories often have no natural organization and have challenging flux imbalances, leading to low biocatalytic efficiency. Modular polyketide synthases (PKSs) are multienzyme complexes that synthesize polyketide products via an assembly line thiotemplate mechanism. Here, we develop a strategy named mimic PKS enzyme assembly line (mPKSeal) that assembles key cascade enzymes to enhance biocatalytic efficiency and increase target production by recruiting cascade enzymes tagged with docking domains from type I cis-AT PKS. We apply this strategy to the astaxanthin biosynthetic pathway in engineered Escherichia coli for multienzyme assembly to increase astaxanthin production by 2.4-fold. The docking pairs, from the same PKSs or those from different cis-AT PKSs evidently belonging to distinct classes, are effective enzyme assembly tools for increasing astaxanthin production. This study addresses the challenge of cascade catalytic efficiency and highlights the potential for engineering enzyme assembly.

Diene incorporation by a dehydratase domain variant in modular polyketide synthases.

Modular polyketide synthases (PKSs) are biosynthetic assembly lines that construct structurally diverse natural products with wide-ranging applications in medicine and agriculture. Various mechanisms contribute to structural diversification during PKS-mediated chain assembly, including dehydratase (DH) domain-mediated elimination of water from R and S-configured 3-hydroxy-thioesters to introduce E- and Z-configured carbon-carbon double bonds, respectively. Here we report the discovery of a DH domain variant that catalyzes the sequential elimination of two molecules of water from a (3R, 5S)-3,5-dihydroxy thioester during polyketide chain assembly, introducing a conjugated E,Z-diene into various modular PKS products. We show that the reaction proceeds via a (2E, 5S)-2-enoyl-5-hydroxy-thioester intermediate and involves an additional universally conserved histidine residue that is absent from the active site of most conventional DH domains. These findings expand the diverse range of chemistries mediated by DH-like domains in modular PKSs, highlighting the catalytic versatility of the double hotdog fold.

Functional Studies and Revision of the NFAT-133/TM-123 Biosynthetic Pathway in Streptomyces pactum.

The biosynthetic gene cluster of NFAT-133, an inhibitor of the nuclear factor of activated T cells, was recently identified in Streptomyces pactum ATCC 27456. This cluster is conspicuous by its highly disordered noncollinear type I modular polyketide synthase (PKS) genes that encode PKSs with one module more than those expected for the heptaketide NFAT-133 biosynthesis. Thus, the major metabolite NFAT-133 was proposed to derive from an octaketide analogue, TM-123. Here, we report that further bioinformatic analysis and gene inactivation studies suggest that NFAT-133 is not derived from TM-123 but rather a product of programmed KS7 extension skipping of a nascent heptaketide from the PKS assembly line that produces TM-123. Furthermore, identification of NFAT-133/TM-123 analogues from mutants of the ATCC 27456 strain suggests that NftN (a putative dehydrogenase), NftE (a cytochrome P450), and NftG (a putative hydrolase/decarboxylase) function "in trans" during the polyketide chain assembly processes.

Priming enzymes from the pikromycin synthase reveal how assembly-line ketosynthases catalyze carbon-carbon chemistry

The first domain of modular polyketide synthases (PKSs) is most commonly a ketosynthase (KS)-like enzyme, KSQ, that primes polyketide synthesis. Unlike downstream KSs that fuse α-carboxyacyl groups to growing polyketide chains, it performs an extension-decoupled decarboxylation of these groups to generate primer units. When Pik127, a model triketide synthase constructed from modules of the pikromycin synthase, was studied by cryoelectron microscopy (cryo-EM), the dimeric didomain comprised of KSQ and the neighboring methylmalonyl-selective acyltransferase (AT) dominated the class averages and yielded structures at 2.5- and 2.8-Å resolution, respectively. Comparisons with ketosynthases complexed with their substrates revealed the conformation of the (2S)-methylmalonyl-S-phosphopantetheinyl portion of KSQ and KS substrates prior to decarboxylation. Point mutants of Pik127 probed the roles of residues in the KSQ active site, while an AT-swapped version of Pik127 demonstrated that KSQ can also decarboxylate malonyl groups. Mechanisms for how KSQ and KS domains catalyze carbon-carbon chemistry are proposed.

Structural visualization of transient interactions between the cis-acting acyltransferase and acyl carrier protein of the salinomycin modular polyketide synthase

Transient protein–protein interactions between cis-acting acyltransferase (AT) and acyl carrier protein (ACP) domains are critical for the catalysis and processivity of modular polyketide synthases (mPKSs), but are challenging for structural characterization due to the intrinsically weak binding affinity. Here, a stable complex of cis-acting AT and ACP domains from the ninth module of the salinomycin mPKS was obtained using a maleimide cross-linker and the structure of the complex was determined at 2.6 Å resolution. The crystal structure shows that the AT in combination with the ketosynthase (KS)-to-AT linker forms a C-shaped architecture to embrace the ACP. The large hydrolase subdomain of the AT serves as a major binding platform for the ACP, while the small ferredoxin-like subdomain of the AT and the KS-to-AT linker cooperate with each other to constrain binding of the ACP. The importance of interface residues in cis-acting AT–ACP interactions was confirmed by mutagenesis assays. The interaction mode observed in the cis-acting AT–ACP complex is completely different from those observed in trans-acting AT–ACP complexes, where the ACP primarily contacts the small domain of the AT. The complex structure provides detailed mechanistic insights into AT–ACP recognition in cis-AT mPKSs.

Protein-Protein Recognition Involved in the Intermodular Transacylation Reaction in Modular Polyketide Synthase in the Biosynthesis of Vicenistatin.

The ketosynthase (KS) domain is a core domain found in modular polyketide synthases (PKSs). To maintain the polyketide biosynthetic fidelity, the KS domain must only accept an acyl group from the acyl carrier protein (ACP) domain of the immediate upstream module even when they are separated into different polypeptides. Although it was reported that both the docking domain‐based interactions and KS‐ACP compatibility are important for the interpolypeptide transacylation reaction in 6‐deoxyerythronolide B synthase, it is not clear whether these findings are broadly applied to other modular PKSs. Herein, we describe the importance of protein‐protein recognition in the intermodular transacylation between VinP1 module 3 and VinP2 module 4 in vicenistatin biosynthesis. We compared the transacylation activity and crosslinking efficiency of VinP2 KS4 against the cognate VinP1 ACP3 with the noncognate one. As a result, it appeared that VinP2 KS4 distinguishes the cognate ACP3 from other ACPs.

Investigation of carbonyl amidation and O-methylation during biosynthesis of the pharmacophore pyridyl of antitumor piericidins

Piericidins are a large family of bacterial α-pyridone antibiotics with antitumor activities such as their anti-renal carcinoma activity exhibited recently in nude mice. The backbones of piericidins are derived from β, δ-diketo carboxylic acids, which are offloaded from a modular polyketide synthase (PKS) and putatively undergo a carbonyl amidation before α-pyridone ring formation. The tailoring modifications to the α-pyridone structure mainly include the verified hydroxylation and O-methylation of the C-4′ position and an unidentified C-5′ O-methylation. Here, we describe a piericidin producer, terrestrial Streptomyces conglobatus, which contains a piericidin biosynthetic gene cluster in two different loci. Deletion of the amidotransferase gene pieD resulted in the accumulation of two fatty acids that should be degraded from the nascent carboxylic acid released by the PKS, supporting the carbonyl amidation function of PieD during α-pyridone ring formation. Deletion of the O-methyltransferase gene pieB1 led to the production of three piericidin analogues lacking C-5′ O-methylation, therefore confirming that PieB1 specifically catalyses the tailoring modification. Moreover, bioactivity analysis of the mutant-derived products provided clues regarding the structure-function relationship for antitumor activity. The work addresses two previously unidentified steps involved in pyridyl pharmacophore formation during piericidin biosynthesis, facilitating the rational bioengineering of the biosynthetic pathway towards valuable antitumor agents.