Biosynthesis Of Aromatic Polyketides Research Articles

Metabolic Engineering and Synthetic Biology Approaches for the Heterologous Production of Aromatic Polyketides.

Polyketides are a diverse set of natural products with versatile applications as pharmaceuticals, nutraceuticals, and cosmetics, to name a few. Of several types of polyketides, aromatic polyketides comprising type II and III polyketides contain many chemicals important for human health such as antibiotics and anticancer agents. Most aromatic polyketides are produced from soil bacteria or plants, which are difficult to engineer and grow slowly in industrial settings. To this end, metabolic engineering and synthetic biology have been employed to efficiently engineer heterologous model microorganisms for enhanced production of important aromatic polyketides. In this review, we discuss the recent advancement in metabolic engineering and synthetic biology strategies for the production of type II and type III polyketides in model microorganisms. Future challenges and prospects of aromatic polyketide biosynthesis by synthetic biology and enzyme engineering approaches are also discussed.

Genomic and Metabolite Profiling Reveal a Novel Streptomyces Strain, QHH-9511, from the Qinghai-Tibet Plateau.

The prevalence of superbugs, represented by methicillin-resistant Staphylococcus aureus (MRSA), has become a serious clinical and public safety concern with rising incidence in hospitals. Polyketides with diverse chemical structures harbor many antimicrobial activities, including those of rifampin and rapamycin against MRSA. Streptomyces sp. QHH-9511 was isolated from a niche habitat in the Qinghai-Tibet Plateau and used to produce antibacterial metabolites. Herein, an integrated approach combining genome mining and metabolic analysis were employed to decipher the chemical origin of the antibacterial components with pigmented properties in strain QHH-9511, a novel Streptomyces species from a lichen symbiont on the Qinghai-Tibet Plateau. Genomic phylogeny assembled at the chromosome level revealed its unique evolutionary state. Further genome mining uncovered 36 candidate gene clusters, most of which were uncharacterized. Meanwhile, based on liquid chromatography coupled to diode array detection mass spectrometry, a series of granaticins, BSMs, chromones, phaeochromycins, and related molecules were discovered by using the Global Natural Product Social molecular networking platform. Subsequently, several pigment compounds were isolated and identified by high-resolution mass spectrometry and/or nuclear magnetic resonance, among which the structure-activity relationships of seven aromatic polyketides showed that the fused lactone ring of the C-2 carboxyl group could increase antibacterial activity. Genetic experiments indicated that all seven aromatic polyketides are a series of metabolic shunts produced by a single type II polyketide synthase (PKS) cluster. Comparative genomic analysis of granaticin producers showed that the granaticin gene cluster is widely distributed. This study provides an efficient method to combine genome mining and metabolic profiling techniques to uncover bioactive metabolites derived from specific habitats, while deepening our understanding of aromatic polyketide biosynthesis. IMPORTANCE Undescribed microorganisms from special habitats are being screened for anti-superbug drug molecules. In a project to screen actinomycetes for anti-MRSA activity, we isolated a Streptomyces strain from Qinghai Lake lichens. The phylogeny based on the genome assembled at the chromosome level revealed this strain's unique evolutionary state. The chemical origins of the antibacterial components with pigment properties in strain QHH-9511 were determined using an integrated approach combining genome mining and metabolic analysis. Further genome mining uncovered 36 secondary metabolite gene clusters, the majority of which were previously unknown. A series of aromatic compounds were discovered using molecular network analysis, separation, and extraction. Genetic experiments revealed that all seven aromatic polyketides are a series of metabolic shunts produced by a single cluster of type II PKSs. This study describes a method for identifying novel Streptomyces from specific habitats by combining genome mining with metabolic profiling techniques.

Cytochrome P450 Catalyzes Benzene Ring Formation in the Biosynthesis of Trialkyl-Substituted Aromatic Polyketides.

Lorneic acid and related natural products are characterized by a trialkyl-substituted benzene ring. The formation of the aromatic core in the middle of the polyketide chain is unusual. We characterized a cytochrome P450 enzyme that can catalyze the hallmark benzene ring formation from an acyclic polyene substrate through genetic and biochemical analysis. Using this P450 as a beacon for genome mining, we obtained 12 homologous type I polyketide synthase (PKS) gene clusters, among which two gene clusters are activated and able to produce trialkyl-substituted aromatic polyketides. Quantum chemical calculations were performed to elucidate the plausible mechanism for P450-catalyzed benzene ring formation. Our work expands our knowledge of the catalytic diversity of cytochrome P450.

Programmable polyketide biosynthesis platform for production of aromatic compounds in yeast

To accelerate the shift to bio-based production and overcome complicated functional implementation of natural and artificial biosynthetic pathways to industry relevant organisms, development of new, versatile, bio-based production platforms is required. Here we present a novel yeast-based platform for biosynthesis of bacterial aromatic polyketides. The platform is based on a synthetic polyketide synthase system enabling a first demonstration of bacterial aromatic polyketide biosynthesis in a eukaryotic host.

Synthesis of Enantioenriched Tetra-ortho-3,3′-substituted Biaryls by Small-Molecule-Catalyzed Noncanonical Polyketide Cyclizations

The arene-forming aldol condensation is a fundamental reaction in the biosynthesis of aromatic polyketides. Precisely controlled by the polyketide synthases, the highly reactive poly-β-carbonyl substrates are diverged into numerous aromatic natural products by selective cyclization reactions; a fascinating biosynthetic strategy that sparked our interest to investigate atroposelective aldol condensations. In this Account, we contextualize and highlight the ability of small-molecule catalysts to selectively convert noncanonical hexacarbonyl substrates in a double arene-forming aldol condensation resulting in the atroposelective synthesis of tetra-ortho-3,3′-substituted biaryls. The hexacarbonyl substrates were obtained by a fourfold ozonolysis enabling a late-stage introduction of all carbonyl functions in one step. Secondary amine catalysts capable of forming an extended hydrogen-bonding network triggered the noncanonical polyketide cyclization in order to form valuable biaryls in high yields and with stereocontrol of up to 98:2 er.1 Biosynthesis of Aromatic Polyketides2 Rotationally Restricted Aromatic Polyketides3 3,3′-Substituted Binaphthalenes in Catalysis4 Stereoselective Synthesis of Atropisomers5 Synthesis of Enantioenriched Tetra-ortho-3,3′-Substituted Biaryls by the Atroposelective Arene-Forming Aldol Condensation6 Conclusion

A role for antibiotic biosynthesis monooxygenase domain proteins in fidelity control during aromatic polyketide biosynthesis

The formicamycin biosynthetic gene cluster encodes two groups of type 2 polyketide antibiotics: the formicamycins and their biosynthetic precursors the fasamycins, both of which have activity against methicillin-resistant Staphylococcus aureus. Here, we report the formicapyridines which are encoded by the same gene cluster and are structurally and biosynthetically related to the fasamycins and formicamycins but comprise a rare pyridine moiety. These compounds are trace-level metabolites formed by derailment of the major biosynthetic pathway. Inspired by evolutionary logic we show that rational mutation of a single gene in the biosynthetic gene cluster encoding an antibiotic biosynthesis monooxygenase (ABM) superfamily protein leads to a significant increase both in total formicapyridine production and their enrichment relative to the fasamycins/formicamycins. Our observations broaden the polyketide biosynthetic landscape and identify a non-catalytic role for ABM superfamily proteins in type II polyketide synthase assemblages for maintaining biosynthetic pathway fidelity.

Assembling a plug-and-play production line for combinatorial biosynthesis of aromatic polyketides in Escherichia coli.

Polyketides are a class of specialised metabolites synthesised by both eukaryotes and prokaryotes. These chemically and structurally diverse molecules are heavily used in the clinic and include frontline antimicrobial and anticancer drugs such as erythromycin and doxorubicin. To replenish the clinicians’ diminishing arsenal of bioactive molecules, a promising strategy aims at transferring polyketide biosynthetic pathways from their native producers into the biotechnologically desirable host Escherichia coli. This approach has been successful for type I modular polyketide synthases (PKSs); however, despite more than 3 decades of research, the large and important group of type II PKSs has until now been elusive in E. coli. Here, we report on a versatile polyketide biosynthesis pipeline, based on identification of E. coli–compatible type II PKSs. We successfully express 5 ketosynthase (KS) and chain length factor (CLF) pairs—e.g., from Photorhabdus luminescens TT01, Streptomyces resistomycificus, Streptoccocus sp. GMD2S, Pseudoalteromonas luteoviolacea, and Ktedonobacter racemifer—as soluble heterodimeric recombinant proteins in E. coli for the first time. We define the anthraquinone minimal PKS components and utilise this biosynthetic system to synthesise anthraquinones, dianthrones, and benzoisochromanequinones (BIQs). Furthermore, we demonstrate the tolerance and promiscuity of the anthraquinone heterologous biosynthetic pathway in E. coli to act as genetically applicable plug-and-play scaffold, showing it to function successfully when combined with enzymes from phylogenetically distant species, endophytic fungi and plants, which resulted in 2 new-to-nature compounds, neomedicamycin and neochaetomycin. This work enables plug-and-play combinatorial biosynthesis of aromatic polyketides using bacterial type II PKSs in E. coli, providing full access to its many advantages in terms of easy and fast genetic manipulation, accessibility for high-throughput robotics, and convenient biotechnological scale-up. Using the synthetic and systems biology toolbox, this plug-and-play biosynthetic platform can serve as an engine for the production of new and diversified bioactive polyketides in an automated, rapid, and versatile fashion.

Stereoselective Arene-Forming Aldol Condensation: Catalyst-Controlled Synthesis of Axially Chiral Compounds.

The fundamental role that aldol chemistry adopts in various disciplines, such as stereoselective catalysis or the biosynthesis of aromatic polyketides, illustrates its exceptional versatility. On the one hand, numerous aldol addition reactions reliably transfer the stereochemical information from catalysts into various valuable products. On the other hand, countless aromatic polyketide natural products are produced by an ingenious biosynthetic machinery based on arene-forming aldol condensations. With the aim of complementing aldol methodology that controls stereocenter configuration, we recently combined these two tenets by investigating small-molecule-catalyzed aldol condensation reactions that stereoselectively form diverse axially chiral compounds through the construction of a new aromatic ring.

Chemical shift assignments of polyketide cyclase_like protein CGL2373 from Corynebacterium glutamicum.

Protein CGL2373 from Corynebacterium glutamicum, which is 155 amino acids long and 17.7kDa, is a member of the polyketide_cyc2 family. As a potential polyketide cyclase, it may play an important role in the biosynthesis of aromatic polyketides that are the source of many bioactive molecules. Here we report the complete 1H, 13C and 15N chemical shift assignments of CGL2373, which lays a foundation for further structural and functional research.

Formation of an Angular Aromatic Polyketide from a Linear Anthrene Precursor via Oxidative Rearrangement

Bacterial aromatic polyketides are a group of natural products synthesized by polyketide synthases (PKSs) that show diverse structures and biological activities. They are structurally subclassified into linear, angular, and discoid aromatic polyketides, the formation of which is commonly determined by the shaping and folding of the poly-β-keto intermediates under the concerted actions of the minimal PKSs, cyclases and ketoreductases. Murayaquinone, found in several streptomycetes, possesses an unusual tricyclic angular aromatic polyketide core containing a 9,10-phenanthraquinone. In this study, genes essential for murayaquinone biosynthesis were identified, and a linear anthraoxirene intermediate was discovered. A unique biosynthetic model for the angular aromatic polyketide formation was discovered and confirmed through invivo and invitro studies. Three oxidoreductases, MrqO3, MrqO6, and MrqO7, were identified to catalyze the conversion of the linear aromatic polyketide intermediate into the final angularly arranged framework, which exemplifies a novel strategy for the biosynthesis of angular aromatic polyketides.

Aromatic Polyketide GTRI-02 is a Previously Unidentified Product of the act Gene Cluster in Streptomyces coelicolor A3(2).

The biosynthesis of aromatic polyketides derived from type II polyketide synthases (PKSs) is complex, and it is not uncommon that highly similar gene clusters give rise to diverse structural architectures. The act biosynthetic gene cluster (BGC) of the model actinomycete Streptomyces coelicolor A3(2) is an archetypal type II PKS. Here we show that the act BGC also specifies the aromatic polyketide GTRI-02 (1) and propose a mechanism for the biogenesis of its 3,4-dihydronaphthalen-1(2H)-one backbone. Polyketide 1 was also produced by Streptomyces sp. MBT76 after activation of the act-like qin gene cluster by overexpression of the pathway-specific activator. Mining of this strain also identified dehydroxy-GTRI-02 (2), which most likely originated from dehydration of 1 during the isolation process. This work shows that even extensively studied model gene clusters such as act of S. coelicolor can still produce new chemistry, offering new perspectives for drug discovery.

New insights into bacterial type II polyketide biosynthesis.

Bacterial aromatic polyketides, exemplified by anthracyclines, angucyclines, tetracyclines, and pentangular polyphenols, are a large family of natural products with diverse structures and biological activities and are usually biosynthesized by type II polyketide synthases (PKSs). Since the starting point of biosynthesis and combinatorial biosynthesis in 1984–1985, there has been a continuous effort to investigate the biosynthetic logic of aromatic polyketides owing to the urgent need of developing promising therapeutic candidates from these compounds. Recently, significant advances in the structural and mechanistic identification of enzymes involved in aromatic polyketide biosynthesis have been made on the basis of novel genetic, biochemical, and chemical technologies. This review highlights the progress in bacterial type II PKSs in the past three years (2013–2016). Moreover, novel compounds discovered or created by genome mining and biosynthetic engineering are also included.

Comparative Analyses of Gene Clusters and Ks-alpha Genes Involved in the Biosynthesis of Chromomycin A3 and Mithramycin

Chromomycin A3 and mithramycin are tricyclic antitumor compounds of aureolic acid class of polyketides, which are structurally related. Both polyketides differ in their glycosylation profiles and substitutions of sugars in their functional groups. Genetic organizations for the biosynthetic gene clusters of both polyketides were studied through antibiotics and secondary metabolite analysis shell and compared the genes involved in their polyketide and post-polyketide biosynthesis steps. Mauve application was also used for further visualization. 3D models of KSα gene for both polyketides along with two structurally similar polyketides of two different classes were also predicted and analyzed. Superimpositions of each model with template showed very low root-mean-square deviation values of 0.399 Å for chromomycin and chlortetracycline, 0.191 Å for mithramycin and polyketomycin, and 0.395 Å for chromomycin and mithramycin, respectively. Very low root-mean-square deviation values proved that there are high similarities between each query and template. It was also found that mithramycin and polyketomycin (tetracyclic quinone) have better superimposition hence more structural similarities as compared to mithramycin and chromomycin A3 and these discrepancies strongly suggest the horizontal transfer of aromatic polyketide biosynthesis genes of aureolic acid class. The current study will surely help in better classification of different classes of bacterial type II polyketides in future using KSα domains.

The Distant Siblings-A Phylogenomic Roadmap Illuminates the Origins of Extant Diversity in Fungal Aromatic Polyketide Biosynthesis.

In recent years, the influx of newly sequenced fungal genomes has enabled sampling of secondary metabolite biosynthesis on an unprecedented scale. However, explanations of extant diversity which take into account both large-scale phylogeny reconstructions and knowledge gained from multiple genome projects are still lacking. We analyzed the evolutionary sources of genetic diversity in aromatic polyketide biosynthesis in over 100 model fungal genomes. By reconciling the history of over 400 nonreducing polyketide synthases (NR-PKSs) with corresponding species history, we demonstrate that extant fungal NR-PKSs are clades of distant siblings, originating from a burst of duplications in early Pezizomycotina and thinned by extensive losses. The capability of higher fungi to biosynthesize the simplest precursor molecule (orsellinic acid) is highlighted as an ancestral trait underlying biosynthesis of aromatic compounds. This base activity was modified during early evolution of filamentous fungi, toward divergent reaction schemes associated with biosynthesis of, for example, aflatoxins and fusarubins (C4–C9 cyclization) or various anthraquinone derivatives (C6–C11 cyclization). The functional plasticity is further shown to have been supplemented by modularization of domain architecture into discrete pieces (conserved splice junctions within product template domain), as well as tight linkage of key accessory enzyme families and divergence in employed transcriptional factors. Although the majority of discord between species and gene history is explained by ancient duplications, this landscape has been altered by more recent duplications, as well as multiple horizontal gene transfers. The 25 detected transfers include previously undescribed events leading to emergence of, for example, fusarubin biosynthesis in Fusarium genus. Both the underlying data and the results of present analysis (including alternative scenarios revealed by sampling multiple reconciliation optima) are maintained as a freely available web-based resource: http://cropnet.pl/metasites/sekmet/nrpks_2014.

An ATP-dependent ligase catalyzes the fourth ring cyclization in tetracycline biosynthesis

Cyclization steps in biosynthesis of aromatic polyketides are typically directed by specific enzymes known as cyclases. The fourth ring cyclization step that forms the fully aromatic pretetramid during tetracycline biosynthesis has not been resolved. Herein we report in vitro and in vivo studies on the fourth ring cyclization step in biosynthesis of SF2575 and related tetracyclic natural products. We demonstrate that an enzyme belonging to the ATP-dependent acyl-CoA ligase family catalyzes the C1–C18 Claisen condensation step that forms the A ring and yields the linear, tetracyclic aromatic compound that is the precursor to tetracyclic natural products. The enzyme, SsfL2, is well-conserved in all tetracycline biosynthetic gene clusters discovered to date. It is proposed that SsfL2 directly adenylates the tricyclic carboxylic acid to facilitate the final carbon-carbon bond formation, and coenzyme A is not required.

Organocatalytic atroposelective aldol condensation: synthesis of axially chiral biaryls by arene formation.

Axially chiral compounds are of significant importance in modern synthetic chemistry and particularly valuable in drug discovery and development. Nonetheless, current approaches for the preparation of pure atropisomers often prove tedious. We demonstrate here a synthetic method that efficiently transfers the stereochemical information of a secondary amine organocatalyst into the axial chirality of tri-ortho-substituted biaryls. An aromatic ring is formed during the dehydration step of the described aldol condensation cascade, leading to highly enantioenriched binaphthyl derivatives. The fundamental course of the reaction is related to the biosynthesis of aromatic polyketides.

Biosynthesis of pyranonaphthoquinone polyketides reveals diverse strategies for enzymatic carbon–carbon bond formation

Pyranonaphthoquinones synthesized by Streptomyces bacteria via type II polyketide pathways are aromatic compounds build around a common three-ring structure, which is composed of pyran, quinone and benzene rings. Over the years, actinorhodin in particular has served as a model compound for studying the biosynthesis of aromatic polyketides, while some of the other metabolites such as granaticin, medermycin, frenolicin and alnumycin A have enabled comparative studies that complement our understanding how these complex biological systems function and have evolved. In addition, despite the similarity of the aglycone units, pyranonaphthoquinones in effect display remarkable diversity in tailoring reactions, which include numerous enzymatic carbon-carbon bond forming reactions. This review focuses on the current status of molecular genetic, biochemical and structural investigations on this intriguing family of natural products.

Insight into the Molecular Basis of Aromatic Polyketide Cyclization: Crystal Structure and in Vitro Characterization of WhiE-ORFVI

Aromatic polyketides are biologically active natural products. Many important pharmaceuticals are derived from aromatic polyketides. Especially important in aromatic polyketide biosynthesis is the regiospecific cyclization of a linear, preassembled polyketide chain catalyzed by aromatase/cyclase (ARO/CYC), which serves as a key control point in aromatic ring formation. How different ARO/CYCs promote different cyclization patterns is not well understood. The whiE locus of Streptomyces coelicolor A3(2) is responsible for the biosynthesis of an aromatic polyketide precursor to the gray spore pigment. The WhiE ARO/CYC catalyzes the regiospecific C9-C14 and C7-C16 cyclization and aromatization of a 24-carbon polyketide chain. WhiE ARO/CYC shares a high degree of similarity to another nonreducing PKS ARO/CYC, TcmN ARO/CYC. This paper presents the apo crystal structure of WhiE ARO/CYC, and cocrystal structures of WhiE and TcmN ARO/CYCs bound with polycyclic aromatic compounds that mimic the respective ARO/CYC products. Site-directed mutagenesis coupled with in vitro PKS reconstitution assays was used to characterize the interior pocket residues of WhiE ARO/CYC. The results confirmed that the interior pocket of ARO/CYCs is a critical determinant of polyketide cyclization specificity. A unified ARO/CYC-mediated cyclization mechanism is proposed on the basis of these structural and functional results.

Comparative Characterization of Fungal Anthracenone and Naphthacenedione Biosynthetic Pathways Reveals an α-Hydroxylation-Dependent Claisen-like Cyclization Catalyzed by a Dimanganese Thioesterase

The linear tetracyclic TAN-1612 (1) and BMS-192548 (2) were isolated from different filamentous fungal strains and have been examined as potential neuropeptide Y and neurokinin-1 receptor antagonists, respectively. Although the biosynthesis of fungal aromatic polyketides has attracted much interest in recent years, the biosynthetic mechanism for such naphthacenedione-containing products has not been established. Using a targeted genome mining approach, we first located the ada gene cluster responsible for the biosynthesis of 1 in Aspergillus niger ATCC 1015. The connection between 1 and the ada pathway was verified through overexpression of the Zn(2)Cys(6)-type pathway-specific transcriptional regulator AdaR and subsequent gene expression analysis. The enzymes encoded in the ada gene cluster share high sequence similarities to the known apt pathway linked to the biosynthesis of anthraquinone asperthecin 3. Subsequent comparative investigation of these two highly homologous gene clusters by heterologous pathway reconstitution in Saccharomyces cerevisiae revealed a novel α-hydroxylation-dependent Claisen cyclization cascade, which involves a flavin-dependent monooxygenase that hydroxylates the α-carbon of an acyl carrier protein-bound polyketide and a bifunctional metallo-β-lactamase-type thioesterase (MβL-TE). The bifunctional MβL-TE catalyzes the fourth ring cyclization to afford the naphthacenedione scaffold upon α-hydroxylation, whereas it performs hydrolytic release of an anthracenone product in the absence of α-hydroxylation. Through in vitro biochemical characterizations and metal analyses, we verified that the apt MβL-TE is a dimanganese enzyme and requires both Mn(2+) cations for the observed activities. The MβL-TE is the first example of a thioesterase in polyketide biosynthesis that catalyzes the Claisen-like condensation without an α/β hydrolase fold and forms no covalent bond with the substrate. These mechanistic features should be general to the biosynthesis of tetracyclic naphthacenedione compounds in fungi.

Structural and Biochemical Characterization of ZhuI Aromatase/Cyclase from the R1128 Polyketide Pathway

Aromatic polyketides comprise an important class of natural products that possess a wide range of biological activities. The cyclization of the polyketide chain is a critical control point in the biosynthesis of aromatic polyketides. The aromatase/cyclases (ARO/CYCs) are an important component of the type II polyketide synthase (PKS) and help fold the polyketide for regiospecific cyclizations of the first ring and/or aromatization, promoting two commonly observed first-ring cyclization patterns for the bacterial type II PKSs: C7-C12 and C9-C14. We had previously reported the crystal structure and enzymological analyses of the TcmN ARO/CYC, which promotes C9-C14 first-ring cyclization. However, how C7-C12 first-ring cyclization is controlled remains unresolved. In this work, we present the 2.4 Å crystal structure of ZhuI, a C7-C12-specific first-ring ARO/CYC from the type II PKS pathway responsible for the production of the R1128 polyketides. Though ZhuI possesses a helix-grip fold shared by TcmN ARO/CYC, there are substantial differences in overall structure and pocket residue composition that may be important for directing C7-C12 (rather than C9-C14) cyclization. Docking studies and site-directed mutagenesis coupled to an in vitro activity assay demonstrate that ZhuI pocket residues R66, H109, and D146 are important for enzyme function. The ZhuI crystal structure helps visualize the structure and putative dehydratase function of the didomain ARO/CYCs from KR-containing type II PKSs. The sequence-structure-function analysis described for ZhuI elucidates the molecular mechanisms that control C7-C12 first-ring polyketide cyclization and builds a foundation for future endeavors into directing cyclization patterns for engineered biosynthesis of aromatic polyketides.