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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 -
MSystems Jun 2023Microbial polyketide synthase (PKS) genes encode the biosynthesis of many biomedically or otherwise commercially important natural products. Despite extensive discovery...
Microbial polyketide synthase (PKS) genes encode the biosynthesis of many biomedically or otherwise commercially important natural products. Despite extensive discovery efforts, metagenomic analyses suggest that only a small fraction of nature's polyketide biosynthetic potential has been realized. Much of this potential originates from type I PKSs (T1PKSs), which can be further delineated based on their domain organization and the structural features of the compounds they encode. Notably, phylogenetic relationships among ketosynthase (KS) domains provide an effective method to classify the larger and more complex T1PKS genes in which they occur. Increased access to large metagenomic data sets from diverse habitats provides opportunities to assess T1PKS biosynthetic diversity and distributions through their smaller and more tractable KS domain sequences. Here, we used the web tool NaPDoS2 to detect and classify over 35,000 type I KS domains from 137 metagenomic data sets reported from eight diverse, globally distributed biomes. We found biome-specific separation with soils enriched in KSs from modular -acetyltransferase (AT) and hybrid -AT KSs relative to other biomes and marine sediments enriched in KSs associated with polyunsaturated fatty acid and enediyne biosynthesis. We linked the phylum Actinobacteria to soil-derived enediyne and -AT KSs while marine-derived KSs associated with enediyne and monomodular PKSs were linked to phyla from which the compounds produced by these biosynthetic enzymes have not been reported. These KSs were phylogenetically distinct from those associated with experimentally characterized PKSs suggesting they may be associated with novel structures or enzyme functions. Finally, we employed our metagenome-extracted KS domains to evaluate the PCR primers commonly used to amplify type I KSs and identified modifications that could increase the KS sequence diversity recovered from amplicon libraries. IMPORTANCE Polyketides are a crucial source of medicines, agrichemicals, and other commercial products. Advances in our understanding of polyketide biosynthesis, coupled with the increased availability of metagenomic sequence data, provide new opportunities to assess polyketide biosynthetic potential across biomes. Here, we used the web tool NaPDoS2 to assess type I polyketide synthase (PKS) diversity and distributions by detecting and classifying ketosynthase (KS) domains across 137 metagenomes. We show that biomes are differentially enriched in type I KS domains, providing a roadmap for future biodiscovery strategies. Furthermore, KS phylogenies reveal biome-specific clades that do not include biochemically characterized PKSs, highlighting the biosynthetic potential of poorly explored environments. The large metagenome-derived KS data set allowed us to identify regions of commonly used type I KS PCR primers that could be modified to capture a larger extent of environmental KS diversity. These results facilitate both the search for novel polyketides and our understanding of the biogeographical distribution of PKSs across Earth's major biomes.
Topics: Polyketide Synthases; Metagenome; Phylogeny; Polyketides; Enediynes
PubMed: 37272717
DOI: 10.1128/msystems.00012-23 -
Proceedings. Biological Sciences Sep 2022The soil is a rich ecosystem where many ecological interactions are mediated by small molecules, and in which amoebae are low-level predators and also prey. The social...
The soil is a rich ecosystem where many ecological interactions are mediated by small molecules, and in which amoebae are low-level predators and also prey. The social amoeba has a high genomic potential for producing polyketides to mediate its ecological interactions, including the unique 'Steely' enzymes, consisting of a fusion between a fatty acid synthase and a chalcone synthase. We report here that further increases its polyketide potential by using the StlB Steely enzyme, and a downstream chlorinating enzyme, to make both a chlorinated signal molecule, DIF-1, during its multi-cellular development, and a set of abundant polyketides in terminally differentiated stalk cells. We identify one of these as a chlorinated dibenzofuran with potent anti-bacterial activity. To do this, StlB switches expression from prespore to stalk cells in late development and is cleaved to release the chalcone synthase domain. Expression of this domain alone in StlB null cells allows synthesis of the stalk-associated, chlorinated polyketides. Thus, by altered expression and processing of StlB, cells make first a signal molecule, and then abundant secondary metabolites, which we speculate help to protect the mature spores from bacterial infection.
Topics: Dibenzofurans, Polychlorinated; Dictyostelium; Ecosystem; Fatty Acid Synthases; Polyketide Synthases; Polyketides; Soil
PubMed: 36126683
DOI: 10.1098/rspb.2022.1176 -
Applied Microbiology and Biotechnology Jan 2016Modular polyketide synthases (type I PKSs) in bacteria are responsible for synthesizing a significant percentage of bioactive natural products. This group of synthases... (Review)
Review
Modular polyketide synthases (type I PKSs) in bacteria are responsible for synthesizing a significant percentage of bioactive natural products. This group of synthases has a characteristic modular organization, and each module within a PKS carries out one cycle of polyketide chain elongation; thus each module is non-iterative in function. It was possible to predict the basic structure of a polyketide product from the module organization of the PKSs, since there generally existed a co-linearity between the number of modules and the number of chain elongations. However, more and more bacterial modular PKSs fail to conform to the canonical rules, and a particularly noteworthy group of non-canonical PKSs is the bacterial iterative type I PKSs. This review covers recent examples of iteratively used modular PKSs in bacteria. These non-canonical PKSs give rise to a large array of natural products with impressive structural diversity. The molecular mechanism behind the iterations is often unclear, presenting a new challenge to the rational engineering of these PKSs with the goal of generating new natural products. Structural elucidation of these synthase complexes and better understanding of potential PKS-PKS interactions as well as PKS-substrate recognition may provide new prospects and inspirations for the discovery and engineering of new bioactive polyketides.
Topics: Amino Acid Sequence; Bacteria; Biological Products; Biosynthetic Pathways; Polyketide Synthases; Polyketides; Secondary Metabolism; Substrate Specificity
PubMed: 26549236
DOI: 10.1007/s00253-015-7093-0 -
ACS Chemical Biology Dec 2018Modular type I polyketide synthases (PKSs) produce some of the most chemically complex metabolites in nature through a series of multienzyme modules. Each module...
Modular type I polyketide synthases (PKSs) produce some of the most chemically complex metabolites in nature through a series of multienzyme modules. Each module contains a variety of catalytic domains to selectively tailor the growing molecule. PKS O-methyltransferases ( O-MTs) are predicted to methylate β-hydroxyl or β-keto groups, but their activity and structure have not been reported. We determined the domain boundaries and characterized the catalytic activity and structure of the StiD and StiE O-MTs, which methylate opposite β-hydroxyl stereocenters in the myxobacterial stigmatellin biosynthetic pathway. Substrate stereospecificity was demonstrated for the StiD O-MT. Key catalytic residues were identified in the crystal structures and investigated in StiE O-MT via site-directed mutagenesis and further validated with the cyanobacterial CurL O-MT from the curacin biosynthetic pathway. Initial structural and biochemical analysis of PKS O-MTs supplies a new chemoenzymatic tool, with the unique ability to selectively modify hydroxyl groups during polyketide biosynthesis.
Topics: Bacterial Proteins; Catalytic Domain; Cyanobacteria; Methylation; Methyltransferases; Mutagenesis, Site-Directed; Mutation; Myxococcales; Polyketide Synthases; Polyketides; Protein Conformation; Protein Domains; Substrate Specificity
PubMed: 30489068
DOI: 10.1021/acschembio.8b00687 -
Journal of the American Chemical Society Feb 2018Like many complex natural products, the intricate architecture of saxitoxin (STX) has hindered full exploration of this scaffold's utility as a tool for studying...
Like many complex natural products, the intricate architecture of saxitoxin (STX) has hindered full exploration of this scaffold's utility as a tool for studying voltage-gated sodium ion channels and as a pharmaceutical agent. Established chemical strategies can provide access to the natural product; however, a chemoenzymatic route to saxitoxin that could provide expedited access to related compounds has not been devised. The first step toward realizing a chemoenzymatic approach toward this class of molecules is the elucidation of the saxitoxin biosynthetic pathway. To date, a biochemical link between STX and its putative biosynthetic enzymes has not been demonstrated. Herein, we report the first biochemical characterization of any enzyme involved in STX biosynthesis. Specifically, the chemical functions of a polyketide-like synthase, SxtA, from the cyanobacteria Cylindrospermopsis raciborskii T3 are elucidated. This unique megasynthase is comprised of four domains: methyltransferase (MT), GCN5-related N-acetyltransferase (GNAT), acyl carrier protein (ACP), and the first example of an 8-amino-7-oxononanoate synthase (AONS) associated with a multidomain synthase. We have established that this single polypeptide carries out the formation of two carbon-carbon bonds, two decarboxylation events and a stereospecific protonation to afford the linear biosynthetic precursor to STX (4). The synthetic utility of the SxtA AONS is demonstrated by the synthesis of a suite of α-amino ketones from the corresponding α-amino acid in a single step.
Topics: Cylindrospermopsis; Molecular Structure; Polyketide Synthases; Saxitoxin
PubMed: 29390180
DOI: 10.1021/jacs.7b13297 -
The Journal of General and Applied... Feb 2021Acrocarpospora is a rare, recently established actinomycete genus of the family Streptosporangiaceae. In the present study, we sequenced whole genomes of the type...
Acrocarpospora is a rare, recently established actinomycete genus of the family Streptosporangiaceae. In the present study, we sequenced whole genomes of the type strains of Acrocarpospora corrugate, Acrocarpospora macrocephala, and Acrocarpospora pleiomorpha to assess their potency as secondary metabolite producers; we then surveyed their nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) gene clusters. The genome sizes of A. corrugate NBRC 13972, A. macrocephala NBRC 16266, and A. pleiomorpha NBRC 16267 were 9.3 Mb, 12.1 Mb, and 11.8 Mb, respectively. Each genome contained 12-17 modular NRPS and PKS gene clusters. Among the 23 kinds of NRPS and PKS gene clusters identified from the three strains, eight clusters were conserved in all the strains, six were shared between A. macrocephala and A. pleiomorpha, and the remaining nine were strain-specific. We predicted the chemical structures of the products synthesized by these gene clusters based on bioinformatic analyses. Since the chemical structures are diverse, Acrocarpospora strains are considered an attractive source of diverse nonribosomal peptide and polyketide compounds.
Topics: Actinobacteria; Base Sequence; Genome, Bacterial; Multigene Family; Peptide Biosynthesis, Nucleic Acid-Independent; Peptide Synthases; Polyketide Synthases; Sequence Analysis, DNA; Whole Genome Sequencing
PubMed: 32801283
DOI: 10.2323/jgam.2020.01.001 -
Science Advances Sep 2022Polyketide synthases (PKSs) are predominantly microbial biosynthetic enzymes. They assemble highly potent bioactive natural products from simple carboxylic acid...
Polyketide synthases (PKSs) are predominantly microbial biosynthetic enzymes. They assemble highly potent bioactive natural products from simple carboxylic acid precursors. The most versatile families of PKSs are organized as assembly lines of functional modules. Each module performs one round of precursor extension and optional modification, followed by directed transfer of the intermediate to the next module. While enzymatic domains and even modules of PKSs are well understood, the higher-order modular architecture of PKS assembly lines remains elusive. Here, we visualize a PKS bimodule core using cryo-electron microscopy and resolve a two-dimensional meshwork of the bimodule core formed by homotypic interactions between modules. The sheet-like organization provides the framework for efficient substrate transfer and for sequestration of trans-acting enzymes required for polyketide production.
Topics: Biological Products; Carboxylic Acids; Cryoelectron Microscopy; Polyketide Synthases; Polyketides
PubMed: 36129979
DOI: 10.1126/sciadv.abo6918 -
Nature Communications Feb 2021Statins are effective cholesterol-lowering drugs. Lovastatin, one of the precursors of statins, is formed from dihydromonacolin L (DML), which is synthesized by...
Statins are effective cholesterol-lowering drugs. Lovastatin, one of the precursors of statins, is formed from dihydromonacolin L (DML), which is synthesized by lovastatin nonaketide synthase (LovB), with the assistance of a separate trans-acting enoyl reductase (LovC). A full DML synthesis comprises 8 polyketide synthetic cycles with about 35 steps. The assembling of the LovB-LovC complex, and the structural basis for the iterative and yet permutative functions of the megasynthase have remained a mystery. Here, we present the cryo-EM structures of the LovB-LovC complex at 3.60 Å and the core LovB at 2.91 Å resolution. The domain organization of LovB is an X-shaped face-to-face dimer containing eight connected domains. The binding of LovC laterally to the malonyl-acetyl transferase domain allows the completion of a L-shaped catalytic chamber consisting of six active domains. This architecture and the structural details of the megasynthase provide the basis for the processing of the intermediates by the individual catalytic domains. The detailed architectural model provides structural insights that may enable the re-engineering of the megasynthase for the generation of new statins.
Topics: Biocatalysis; Lovastatin; Models, Molecular; Naphthalenes; Oxidoreductases Acting on CH-CH Group Donors; Polyketide Synthases; Protein Domains; Substrate Specificity
PubMed: 33558520
DOI: 10.1038/s41467-021-21174-8 -
Nature Communications Feb 2023Modular polyketide synthase (PKS) is an ingenious core machine that catalyzes abundant polyketides in nature. Exploring interactions among modules in PKS is very...
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 ER inside module 1/2 and the ketosynthase domain KS inside module 3 is required for the cross-module enoylreduction in azalomycin F (AZL) biosynthesis. We also show that KS 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.
Topics: Polyketide Synthases; Macrolides; Polyketides
PubMed: 36739290
DOI: 10.1038/s41467-023-36213-9