-
Methods in Molecular Biology (Clifton,... 2022The enzymes that comprise type II polyketide synthases (PKSs) are powerful biocatalysts that, once well-understood and strategically applied, could enable cost-effective...
The enzymes that comprise type II polyketide synthases (PKSs) are powerful biocatalysts that, once well-understood and strategically applied, could enable cost-effective and sustainable access to a range of pharmaceutically relevant molecules. Progress toward this goal hinges on gaining ample access to materials for in vitro characterizations and structural analysis of the components of these synthases. A central component of PKSs is the acyl carrier protein (ACP), which serves as a hub during the biosynthesis of type II polyketides. Herein, we share methods for accessing type II PKS ACPs via heterologous expression in E. coli . We also share how the installation of reactive and site-specific spectroscopic probes can be leveraged to study the conformational dynamics and interactions of type II PKS ACPs.
Topics: Acyl Carrier Protein; Escherichia coli; Polyketide Synthases
PubMed: 35524054
DOI: 10.1007/978-1-0716-2273-5_13 -
Nature Chemistry Sep 2022Modification of polyketides with fluorine offers a promising approach to develop new pharmaceuticals. While synthetic chemical methods for site-selective incorporation...
Modification of polyketides with fluorine offers a promising approach to develop new pharmaceuticals. While synthetic chemical methods for site-selective incorporation of fluorine in complex molecules have improved in recent years, approaches for the biosynthetic incorporation of fluorine in natural compounds are still rare. Here, we report a strategy to introduce fluorine into complex polyketides during biosynthesis. We exchanged the native acyltransferase domain of a polyketide synthase, which acts as the gatekeeper for the selection of extender units, with an evolutionarily related but substrate tolerant domain from metazoan type I fatty acid synthase. The resulting polyketide-synthase/fatty-acid-synthase hybrid can utilize fluoromalonyl coenzyme A and fluoromethylmalonyl coenzyme A for polyketide chain extension, introducing fluorine or fluoro-methyl units in polyketide scaffolds. We demonstrate the feasibility of our approach in the chemoenzymatic synthesis of fluorinated 12- and 14-membered macrolactones and fluorinated derivatives of the macrolide antibiotics YC-17 and methymycin.
Topics: Acyltransferases; Animals; Coenzyme A; Fluorine; Polyketide Synthases; Polyketides
PubMed: 35879443
DOI: 10.1038/s41557-022-00996-z -
ACS Synthetic Biology Nov 2023Polyketide retrobiosynthesis, where the biosynthetic pathway of a given polyketide can be reversibly engineered due to the colinearity of the polyketide synthase (PKS)... (Review)
Review
Polyketide retrobiosynthesis, where the biosynthetic pathway of a given polyketide can be reversibly engineered due to the colinearity of the polyketide synthase (PKS) structure and function, has the potential to produce millions of organic molecules. Mixing and matching modules from natural PKSs is one of the routes to produce many of these molecules. Evolutionary analysis of PKSs suggests that traditionally used module boundaries may not lead to the most productive hybrid PKSs and that new boundaries around and within the ketosynthase domain may be more active when constructing hybrid PKSs. As this is still a nascent area of research, the generality of these design principles based on existing engineering efforts remains inconclusive. Recent advances in structural modeling and synthetic biology present an opportunity to accelerate PKS engineering by re-evaluating insights gained from previous engineering efforts with cutting edge tools.
Topics: Polyketide Synthases; Polyketides
PubMed: 37871264
DOI: 10.1021/acssynbio.3c00282 -
Natural Product Reports Oct 2018Covering: up to the end of 2018 Polyketides are a valuable source of bioactive and clinically important molecules. The biosynthesis of these chemically complex molecules... (Review)
Review
Covering: up to the end of 2018 Polyketides are a valuable source of bioactive and clinically important molecules. The biosynthesis of these chemically complex molecules has led to the discovery of equally complex polyketide synthase (PKS) pathways. Crystallography has yielded snapshots of individual catalytic domains, di-domains, and multi-domains from a variety of PKS megasynthases, and cryo-EM studies have provided initial views of a PKS module in a series of defined biochemical states. Here, we review the structural and biochemical results that shed light on the protein-protein interactions critical to catalysis by PKS systems with an embedded acyltransferase. Interactions include those that occur both within and between PKS modules, as well as with accessory enzymes.
Topics: Acyltransferases; Catalytic Domain; Polyketide Synthases; Protein Interaction Domains and Motifs; Protein Multimerization
PubMed: 30188553
DOI: 10.1039/c8np00058a -
Angewandte Chemie (International Ed. in... Apr 2017Modular redefinition: A long-standing paradigm in modular polyketide synthase enzymology, namely the definition of a module, has been challenged by Abe and co-workers in...
Modular redefinition: A long-standing paradigm in modular polyketide synthase enzymology, namely the definition of a module, has been challenged by Abe and co-workers in their recent study. With this new understanding has emerged renewed hope for engineering these assembly lines to produce new materials and medicines.
Topics: Acyl Carrier Protein; Acyltransferases; Animals; Bacteria; Biocatalysis; Computational Biology; Evolution, Molecular; Fatty Acid Synthases; Humans; Polyketide Synthases; Polyketides; Protein Domains; Protein Engineering; Substrate Specificity
PubMed: 28322495
DOI: 10.1002/anie.201701281 -
Microbial Cell Factories Aug 2019Actinobacteria are characterized as the most prominent producer of natural products (NPs) with pharmaceutical importance. The production of NPs from these actinobacteria... (Review)
Review
Actinobacteria are characterized as the most prominent producer of natural products (NPs) with pharmaceutical importance. The production of NPs from these actinobacteria is associated with particular biosynthetic gene clusters (BGCs) in these microorganisms. The majority of these BGCs include polyketide synthase (PKS) or non-ribosomal peptide synthase (NRPS) or a combination of both PKS and NRPS. Macrolides compounds contain a core macro-lactone ring (aglycone) decorated with diverse functional groups in their chemical structures. The aglycon is generated by megaenzyme polyketide synthases (PKSs) from diverse acyl-CoA as precursor substrates. Further, post-PKS enzymes are responsible for allocating the structural diversity and functional characteristics for their biological activities. Macrolides are biologically important for their uses in therapeutics as antibiotics, anti-tumor agents, immunosuppressants, anti-parasites and many more. Thus, precise genetic/metabolic engineering of actinobacteria along with the application of various chemical/biological approaches have made it plausible for production of macrolides in industrial scale or generation of their novel derivatives with more effective biological properties. In this review, we have discussed versatile approaches for generating a wide range of macrolide structures by engineering the PKS and post-PKS cascades at either enzyme or cellular level in actinobacteria species, either the native or heterologous producer strains.
Topics: Actinobacteria; Biological Products; Genetic Engineering; Macrolides; Multigene Family; Polyketide Synthases; Polyketides
PubMed: 31409353
DOI: 10.1186/s12934-019-1184-z -
Nature Structural & Molecular Biology Mar 2023The mycolic acid layer of the Mycobacterium tuberculosis cell wall is essential for viability and virulence, and the enzymes responsible for its synthesis are targets...
The mycolic acid layer of the Mycobacterium tuberculosis cell wall is essential for viability and virulence, and the enzymes responsible for its synthesis are targets for antimycobacterial drug development. Polyketide synthase 13 (Pks13) is a module encoding several enzymatic and transport functions that carries out the condensation of two different long-chain fatty acids to produce mycolic acids. We determined structures by cryogenic-electron microscopy of dimeric multi-enzyme Pks13 purified from mycobacteria under normal growth conditions, captured with native substrates. Structures define the ketosynthase (KS), linker and acyl transferase (AT) domains at 1.8 Å resolution and two alternative locations of the N-terminal acyl carrier protein. These structures suggest intermediate states on the pathway for substrate delivery to the KS domain. Other domains, visible at lower resolution, are flexible relative to the KS-AT core. The chemical structures of three bound endogenous long-chain fatty acid substrates were determined by electrospray ionization mass spectrometry.
Topics: Polyketide Synthases; Mycobacterium tuberculosis; Mycolic Acids; Fatty Acids
PubMed: 36782050
DOI: 10.1038/s41594-022-00918-0 -
The Plant Journal : For Cell and... Oct 2022Type III polyketide synthases (PKSs) are key enzymes involved in the biosynthesis of a variety of plant specialized metabolites, including flavonoids, stilbenes, and...
Type III polyketide synthases (PKSs) are key enzymes involved in the biosynthesis of a variety of plant specialized metabolites, including flavonoids, stilbenes, and sporopollenin, to name a few. These enzymes likely played vital roles in plant adaptation during their transition from aquatic to terrestrial habitats and their colonization of specific ecological environments. Members of this supergene family have diverse functions, but how type III PKSs and their functions have evolved remains poorly understood. Here, we conducted comprehensive phylogenomics analysis of the type III PKS supergene family in 60 species representing the major plant lineages and elucidated the classification, origin, and evolutionary history of each class. Molecular evolutionary analysis of the typical chalcone synthase and stilbene synthase types revealed evidence for strong positive natural selection in both the Pinaceae and Fabaceae lineages. The positively selected sites of these proteins include residues at the catalytic tunnel entrance and homodimer interface, which might have driven the functional divergence between the two types. Our results also suggest that convergent evolution of enzymes involved in plant flavonoid biosynthesis is quite common. The results of this study provide new insights into the origin, evolution, and functional diversity of plant type III PKSs. In addition, they serve as a guide for the enzymatic engineering of plant polyketides.
Topics: Polyketide Synthases; Plants; Polyketides; Stilbenes; Flavonoids
PubMed: 36004534
DOI: 10.1111/tpj.15953 -
Natural Product Reports Oct 2018Covering: up to April 5, 2018 Metabolites from type II fatty acid synthase (FAS) and polyketide synthase (PKS) pathways differ broadly in their identities and functional... (Review)
Review
Covering: up to April 5, 2018 Metabolites from type II fatty acid synthase (FAS) and polyketide synthase (PKS) pathways differ broadly in their identities and functional roles. The former are considered primary metabolites that are linear hydrocarbon acids, while the latter are complex aromatic or polyunsaturated secondary metabolites. Though the study of bacterial FAS has benefitted from decades of biochemical and structural investigations, type II PKSs have remained less understood. Here we review the recent approaches to understanding the protein-protein and protein-substrate interactions in these pathways, with an emphasis on recent chemical biology and structural applications. New approaches to the study of FAS have highlighted the critical role of the acyl carrier protein (ACP) with regard to how it stabilizes intermediates through sequestration and selectively delivers cargo to successive enzymes within these iterative pathways, utilizing protein-protein interactions to guide and organize enzymatic timing and specificity. Recent tools that have shown promise in FAS elucidation should find new approaches to studying type II PKS systems in the coming years.
Topics: Crystallography, X-Ray; Cyclization; Fatty Acid Synthase, Type II; Helicobacter pylori; Mutagenesis; Nuclear Magnetic Resonance, Biomolecular; Polyketide Synthases; Protein Interaction Maps; Proteins; Substrate Specificity
PubMed: 30046786
DOI: 10.1039/c8np00040a -
The New Phytologist Oct 2022Plant polyketides are well-known for their crucial functions in plants and their importance in the context of human health. They are synthesized by type III polyketide...
Plant polyketides are well-known for their crucial functions in plants and their importance in the context of human health. They are synthesized by type III polyketide synthases (PKSs) and their final functional diversity is determined by post-PKS tailoring enzymes. Gerbera hybrida is rich in two defense-related polyketides: gerberin and parasorboside. Their synthesis is known to be initiated by GERBERA 2-PYRONE SYNTHASE 1 (G2PS1), but the polyketide reductases (PKRs) that determine their final structure have not yet been identified. We identified two PKR candidates in the pathway, GERBERA REDUCTASE 1 (GRED1) and GRED2. Gene expression and metabolite analysis of different gerbera tissues, cultivars, and transgenic gerbera plants, and in vitro enzyme assays, were performed for functional characterization of the enzymes. GRED1 and GRED2 catalyze the second reduction step in parasorboside biosynthesis. They reduce the proximal keto domain of the linear CoA bound intermediate before lactonization. We identified a crucial tailoring step in an important gerbera PKS pathway and show that plant polyketide biosynthesis shares processing strategies with fungi and bacteria. The two tailoring enzymes are recruited from the ancient sporopollenin biosynthetic pathway to a defense-related PKS pathway in gerbera. Our data provide an example of how plants recruit conserved genes to new functions in secondary metabolism that are important for environmental adaptation.
Topics: Asteraceae; Glucosides; Plants, Genetically Modified; Polyketide Synthases; Polyketides; Pyrones
PubMed: 35719102
DOI: 10.1111/nph.18328