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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 -
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 -
MedChemComm Aug 2019Polyketide natural products possess diverse biological activities including antibiotic, anticancer, and immunosuppressive. Their equally varied and complex structures... (Review)
Review
Polyketide natural products possess diverse biological activities including antibiotic, anticancer, and immunosuppressive. Their equally varied and complex structures arise from head-to-tail condensation of simple carboxyacyl monomers. Since the seminal discovery that biosynthesis of polyketides such as the macrolide erythromycin is catalyzed by uncharacteristically large, multifunctional enzymes, termed modular type I polyketide synthases, chemists and biologists alike have been inspired to harness the apparent modularity of the synthases to further diversify polyketide structures. Yet, initial attempts to perform "combinatorial biosynthesis" failed due to challenges associated with maintaining the structural and catalytic integrity of large, chimeric synthases. Fast forward nearly 30 years, and advancements in our understanding of polyketide synthase structure and function have allowed the field to make significant progress toward effecting desired modifications to polyketide scaffolds in addition to engineering small, chiral fragments. This review highlights selected examples of polyketide diversification control of monomer selection, oxidation state, stereochemistry, and cyclization. We conclude with a perspective on the present and future of polyketide structure diversification and hope that the examples presented here will encourage medicinal chemists to embrace polyketide synthetic biology as a means to revitalize polyketide drug discovery.
PubMed: 32180918
DOI: 10.1039/c9md00141g -
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 -
Journal of Industrial Microbiology &... Jun 2021Polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) multienzymes produce numerous high value metabolites. The protein subunits which constitute these... (Review)
Review
Polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) multienzymes produce numerous high value metabolites. The protein subunits which constitute these megasynth(et)ases must undergo ordered self-assembly to ensure correct organisation of catalytic domains for the biosynthesis of a given natural product. Short amino acid regions at the N- and C-termini of each subunit, termed docking domains (DDs), often occur in complementary pairs, which interact to facilitate substrate transfer and maintain pathway fidelity. This review details all structurally characterised examples of NRPS and PKS DDs to date and summarises efforts to utilise DDs for the engineering of biosynthetic pathways.
Topics: Biological Products; Humans; Peptide Synthases; Polyketide Synthases
PubMed: 33640957
DOI: 10.1093/jimb/kuab018 -
Journal of Natural Medicines Jun 2021It has been proposed that biosyntheses of many natural products involve pericyclic reactions, including Diels-Alder (DA) reaction. However, only a small set of enzymes... (Review)
Review
It has been proposed that biosyntheses of many natural products involve pericyclic reactions, including Diels-Alder (DA) reaction. However, only a small set of enzymes have been proposed to catalyze pericyclic reactions. Most surprisingly, there has been no formal identification of natural enzymes that can be defined to catalyze DA reactions (DAases), despite the wide application of the reaction in chemical syntheses of complex organic compounds. However, recent studies began to accumulate a growing body of evidence that supports the notion that enzymes that formally catalyze DA reactions, in fact exist. In this review, I will begin by describing a short history behind the discovery and characterization of macrophomate synthase, one of the earliest enzymes that was proposed to catalyze an intermolecular DA reaction during the biosynthesis of a substituted benzoic acid in a phytopathogenic fungus Macrophoma commelinae. Then, I will discuss representative enzymes that have been chemically authenticated to catalyze DA reactions, with emphasis on more recent discoveries of DAases involved mainly in fungal secondary metabolite biosynthesis except for one example from a marine streptomycete. The current success in identification of a series of DAases and enzymes that catalyze other pericyclic reactions owes to the combined efforts from both the experimental and theoretical approaches in discovering natural products. Such efforts typically involve identifying the chemical features derived from cycloaddition reactions, isolating the biosynthetic genes that encode enzymes that generate such chemical features and deciphering the reaction mechanisms for the enzyme-catalyzed pericyclic reactions.
Topics: Ascomycota; Biological Products; Cycloaddition Reaction; Multienzyme Complexes; Secondary Metabolism
PubMed: 33683566
DOI: 10.1007/s11418-021-01502-4 -
Applied Microbiology and Biotechnology Nov 2021Fluorinated compounds are widely used in the fields of molecular imaging, pharmaceuticals, and materials. Fluorinated natural products in nature are rare, and the... (Review)
Review
Fluorinated compounds are widely used in the fields of molecular imaging, pharmaceuticals, and materials. Fluorinated natural products in nature are rare, and the introduction of fluorine atoms into organic compound molecules can give these compounds new functions and make them have better performance. Therefore, the synthesis of fluorides has attracted more and more attention from biologists and chemists. Even so, achieving selective fluorination is still a huge challenge under mild conditions. In this review, the research progress of enzymatic synthesis of fluorinated compounds is summarized since 2015, including cytochrome P450 enzymes, aldolases, fluoroacetyl coenzyme A thioesterases, lipases, transaminases, reductive aminases, purine nucleoside phosphorylases, polyketide synthases, fluoroacetate dehalogenases, tyrosine phenol-lyases, glycosidases, fluorinases, and multienzyme system. Of all enzyme-catalyzed synthesis methods, the direct formation of the C-F bond by fluorinase is the most effective and promising method. The structure and catalytic mechanism of fluorinase are introduced to understand fluorobiochemistry. Furthermore, the distribution, applications, and future development trends of fluorinated compounds are also outlined. Hopefully, this review will help researchers to understand the significance of enzymatic methods for the synthesis of fluorinated compounds and find or create excellent fluoride synthase in future research.Key points• Fluorinated compounds are distributed in plants and microorganisms, and are used in imaging, medicine, materials science.• Enzyme catalysis is essential for the synthesis of fluorinated compounds.• The loop structure of fluorinase is the key to forming the C-F bond.
Topics: Aldehyde-Lyases; Catalysis; Fluorine; Halogenation; Transaminases
PubMed: 34625820
DOI: 10.1007/s00253-021-11608-0 -
Chemical Reviews Dec 2019Assembly-line polyketide synthases (PKSs) are among the most complex protein machineries known in nature, responsible for the biosynthesis of numerous compounds used in... (Review)
Review
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.
Topics: Catalysis; Evolution, Molecular; Models, Genetic; Polyketide Synthases; Protein Domains
PubMed: 31838842
DOI: 10.1021/acs.chemrev.9b00525 -
Research Square Jul 2023The modular nature of polyketide assembly lines and the significance of their products make them prime targets for combinatorial engineering. While short synthases...
The modular nature of polyketide assembly lines and the significance of their products make them prime targets for combinatorial engineering. While short synthases constructed using the recently updated module boundary have been shown to outperform those using the traditional boundary, larger synthases constructed using the updated boundary have not been investigated. Here we describe our design and implementation of a BioBricks-like platform to rapidly construct 5 triketide, 25 tetraketide, and 125 pentaketide synthases from the updated modules of the Pikromycin synthase. Every combinatorial possibility of modules 2-6 inserted between the first and last modules of the native synthase was constructed and assayed. Anticipated products were observed from 60% of the triketide synthases, 32% of the tetraketide synthases, and 6.4% of the pentaketide synthases. Ketosynthase gatekeeping and module-skipping were determined to be the principal impediments to obtaining functional synthases. The platform was also used to create functional hybrid synthases through the incorporation of modules from the Erythromycin, Spinosyn, and Rapamycin assembly lines. The relaxed gatekeeping observed from a ketosynthase in the Rapamycin synthase is especially encouraging in the quest to produce designer polyketides.
PubMed: 37546965
DOI: 10.21203/rs.3.rs-3157617/v1 -
Journal of Industrial Microbiology &... Dec 2021Microbial genome mining for drug discovery and development has been accelerating in recent years, driven by technical advancements in genome sequencing, bioinformatics,... (Review)
Review
Microbial genome mining for drug discovery and development has been accelerating in recent years, driven by technical advancements in genome sequencing, bioinformatics, metabolomics/metabologenomics, and synthetic biology. Microbial genome mining is a multistep process that starts with the sequencing of microbes that encode multiple secondary metabolites and identifying new and novel secondary metabolite biosynthetic gene clusters (BGCs) to pursue. The initial steps in the process are critical for the overall success, and they encompass the most innovative new technologies to revitalize natural product discovery. As microbial genome mining has matured in recent years, unvalidated conjectures about what microbes to pursue, how to identify legitimate secondary metabolite BGCs, and how to sequence DNA to satisfactory levels of completion have been identified. The solutions to correct the misconceptions around these topics are beginning to be implemented.
Topics: Biological Products; Computational Biology; Drug Discovery; Genome, Bacterial; Multigene Family
PubMed: 34279640
DOI: 10.1093/jimb/kuab044