Biosynthetic Pathways Of Natural Products Research Articles

Theoretical Study of Natural Product Biosynthesis Using Computational Chemistry.

The biosynthetic pathways of natural products are complicated, and it is difficult to fully elucidate their details using experimental chemistry alone. In recent years, efforts have been made to elucidate the biosynthetic reaction mechanisms by combining computational and experimental methods. In this review, we will discuss the biosynthetic studies using computational chemistry for various terpene compounds such as cyclooctatin, sesterfisherol, quiannulatene, trichobrasilenol, asperterpenol, preasperterpenoid, spiroviolene, and mangicol.

Molecular Basis of the Unusual Seven-Membered Methylenedioxy Bridge Formation Catalyzed by Fe(II)/α-KG-Dependent Oxygenase CTB9

Methylenedioxy bridges (MDBs) are architecturally important motifs in natural products and bioactive molecules. Cercosporin, a typical perylenequinone pigment, contains an unusual seven-membered MDB, which has versatile biological and photocatalytic activities. Although cercosporin has been isolated, characterized, and studied for several decades, its biosynthetic pathway, especially the formation of the seven-membered MDB, has remained unclear. Here, we show that the formation of the seven-membered MDB is catalyzed by Fe(II)/α-ketoglutaric acid (α-KG)-dependent dioxygenase CTB9 in cercosporin biosynthesis. Moreover, crystal structures of CTB9 in complex with an α-KG analogue NOG (CTB9·Cu·NOG) and its substrate pre-cercosporin with NOG (CTB9·Cu·NOG·pre-cercosporin) were determined. These structures, together with site-directed mutagenesis studies and quantum mechanics calculations, help define the mechanism of the unique seven-membered MDB in cercosporin biosynthesis. In summary, these results provide molecular insights into other biosynthetic pathways of natural products containing MDBs.

Bacterial-Like Nonribosomal Peptide Synthetases Produce Cyclopeptides in the Zygomycetous Fungus Mortierella alpina.

Fungi are traditionally considered a reservoir of biologically active natural products. However, an active secondary metabolism has long not been attributed to early-diverging fungi such as Mortierella Here, we report on the biosynthesis of two series of cyclic pentapeptides, the malpicyclins and malpibaldins, as products of Mortierella alpina ATCC 32222. The molecular structures of malpicyclins were elucidated by high-resolution tandem mass spectrometry (HR-MS/MS), Marfey's method, and one-dimensional (1D) and 2D nuclear magnetic resonance (NMR) spectroscopy. In addition, malpibaldin biosynthesis was confirmed by HR-MS. Genome mining and comparative quantitative real-time PCR (qRT-PCR) expression analysis pointed at two pentamodular nonribosomal peptide synthetases (NRPSs), malpicyclin synthetase MpcA and malpibaldin synthetase MpbA, as candidate biosynthetic enzymes. Heterologous production of the respective adenylation domains and substrate specificity assays proved promiscuous substrate selection and confirmed their respective biosynthetic roles. In stark contrast to known fungal NRPSs, MpbA and MpcA contain bacterial-like dual epimerase/condensation domains allowing the racemization of enzyme-tethered l-amino acids and the subsequent incorporation of d-amino acids into the metabolites. Phylogenetic analyses of both NRPS genes indicated a bacterial origin and a horizontal gene transfer into the fungal genome. We report on the as-yet-unexplored nonribosomal peptide biosynthesis in basal fungi which highlights this paraphylum as a novel and underrated resource of natural products.IMPORTANCE Fungal natural compounds are industrially produced, with application in antibiotic treatment, cancer medications, and crop plant protection. Traditionally, higher fungi have been intensively investigated concerning their metabolic potential, but reidentification of already known compounds is frequently observed. Hence, alternative strategies to acquire novel bioactive molecules are required. We present the genus Mortierella as representative of the early-diverging fungi as an underestimated resource of natural products. Mortierella alpina produces two families of cyclopeptides, designated malpicyclins and malpibaldins, respectively, via two pentamodular nonribosomal peptide synthetases (NRPSs). These enzymes are much more closely related to bacterial than to other fungal NRPSs, suggesting a bacterial origin of these NRPS genes in Mortierella Both enzymes were biochemically characterized and are involved in as-yet-unknown biosynthetic pathways of natural products in basal fungi. Hence, this report establishes early-diverging fungi as prolific natural compound producers and sheds light on the origin of their biosynthetic capacity.

Biocatalysts from Biosynthetic Pathways: Enabling Stereoselective, Enzymatic Cycloether Formation on a Gram Scale

Biosynthetic pathways of natural products contain many enzymes that contribute to the rapid assembly of molecular complexity. Enzymes that form complex structural elements with multiple stereocente...

Bioinspired chemical synthesis of monomeric and dimeric stephacidin A congeners.

Stephacidin A and its congeners are a collection of secondary metabolites that possess intriguing structural motifs. They stem from unusual biosynthetic sequences that lead to the incorporation of a prenyl or reverse-prenyl group into a bicyclo[2.2.2]diazaoctane framework, a chromene unit or the vestige thereof. To complement biosynthetic studies, which normally play a significant role in unveiling the biosynthetic pathways of natural products, here we demonstrate that chemical synthesis can provide important insights into biosynthesis. We identify a short total synthesis of congeners in the reverse-prenylated indole alkaloid family related to stephacidin A by taking advantage of a direct indole C6 halogenation of the related ketopremalbrancheamide. This novel strategic approach has now made possible the syntheses of several natural products, including malbrancheamides B and C, notoamides F, I and R, aspergamide B, and waikialoid A, which is a heterodimer of avrainvillamide and aspergamide B. Our approach to the preparation of these prenylated and reverse-prenylated indole alkaloids is bioinspired, and may also inform the as-yet undetermined biosynthesis of several congeners.

Elucidation of Biosynthetic Pathways of Natural Products.

During the last decade, we have revealed biosynthetic pathways responsible for the formation of important and chemically complex natural products isolated from various organisms through genetic manipulation. Detailed in vivo and in vitro characterizations enabled elucidation of unexpected mechanisms of secondary metabolite biosynthesis. This personal account focuses on our recent efforts in identifying the genes responsible for the biosynthesis of spirotryprostatin, aspoquinolone, Sch 210972, pyranonigrin, fumagillin and pseurotin. We exploit heterologous reconstitution of biosynthetic pathways of interest in our study. In particular, extensive involvement of oxidation reactions is discussed. Heterologous hosts employed here are Saccharomyces cerevisiae, Aspergillus nidulans and A. niger that can also be used to prepare biosynthetic intermediates and product analogs by engineering the biosynthetic pathways using the knowledge obtained by detailed characterizations of the enzymes. (998 char.).

Strategies of elucidation of biosynthetic pathways of natural products

Elucidation of the biosynthetic pathways of natural products is not only the major goal of herb genomics, but also the solid foundation of synthetic biology of natural products. Here, this paper reviewed recent advance in this field and put forward strategies to elucidate the biosynthetic pathway of natural products. Firstly, a proposed biosynthetic pathway should be set up based on well-known knowledge about chemical reactions and information on the identified compounds, as well as studies with isotope tracer. Secondly, candidate genes possibly involved in the biosynthetic pathway were screened out by co-expression analysis and/or gene cluster mining. Lastly, all the candidate genes were heterologously expressed in the host and then the enzyme involved in the biosynthetic pathway was characterized by activity assay. Sometimes, the function of the enzyme in the original plant could be further studied by RNAi or VIGS technology. Understanding the biosynthetic pathways of natural products will contribute to supply of new leading compounds by synthetic biology and provide "functional marker" for herbal molecular breeding, thus but boosting the development of traditional Chinese medicine agriculture.

Bioactive Natural Products from Plants and Biotechnological Approaches for their Production

Bioactive natural products are economically important as drugs, fragrances, pigments, food additives and pesticides. The biotechnological tools are important to select, multiply, improve and analyze medicinal plants for production of such products. The utilization of medicinal plant cells for the production of natural or recombinant compounds of commercial interest has gained increasing attention over the past decades. Plant tissue culture systems are possible source of valuable medicinal compounds, fragrances and colorants, which cannot be produced by microbial cells or chemical synthesis. In vitro production of bioactive natural products in plant cell suspension culture has been reported from various medicinal plants and bioreactors are the key step towards commercial production. Genetic transformation is a powerful tool for enhancing the productivity of novel products; especially by Agrobacterium tumefacians . Combinatorial biosynthesis is another approach in the generation of novel natural products and for the production of rare and expensive natural products. Recent advances in the molecular biology, enzymology and bioreactor technology of plant cell culture suggest that these systems may become a viable source of important secondary metabolites. Genetic fingerprinting could be a powerful tool in the field of medicinal plants to be used for correct germplasm identification. In addition, when linked to emerging tools such as metabolomics and proteomics, providing fingerprints of the plant’s metabolites or protein composition, it gives data on phenotypic variation, caused by growth conditions or environmental factors, and also yield data on the genes involved in the biosynthesis. DNA profiling techniques like DNA microarrays serve as suitable high throughput tools for the simultaneous analysis of multiple genes and analysis of gene expression that becomes necessary for providing clues about regulatory mechanisms, biochemical pathways and broader cellular functions. New and powerful tools in functional genomics can be used in combination with metabolomics to elucidate biosynthetic pathways of natural products.

Multiple Oxidative Modifications in the Ophiobolin Biosynthesis: P450 Oxidations Found in Genome Mining.

Heterologous expression of four candidate genes found in ophiobolin gene clusters from three fungal strains was employed to elucidate the late-stage biosynthetic pathway of phytotoxin ophiobolin. Expression of oblBAc (cytochrome P450) from the cryptic gene cluster gave unexpected products, and that of oblBBm/oblBEv from the gene cluster of ophiobolin producers, with oblDBm as the transporter, yielded intermediate ophiobolin C through an unusual four-step oxidation process. The observation made in this study may provide a useful guideline for the elucidation of genuine biosynthetic pathways of natural products.

Mass spectrometric dereplication of nitrogen-containing constituents of black cohosh (Cimicifuga racemosa L.)

Black cohosh preparations are popular dietary supplements among women seeking alternative treatments for menopausal complaints. For decades, triterpene glycosides and phenolic acids have dominated the phytochemical and biomedical research on this plant. In this study, we provide evidence that black cohosh contains an unexpected and highly diverse group of secondary nitrogenous metabolites previously unknown to exist in this plant. Using a dereplication approach that combines accurate mass measurements, database searches and general knowledge of biosynthetic pathways of natural products, we identified or tentatively identified 73 nitrogen-containing metabolites, many of which are new natural products. The identified compounds belong to several structural groups including alkaloids, amides or esters of hydroxycinnamic acids and betains. Among the alkaloids, several classes such as guanidino alkaloids, isoquinolines and β-carbolines were identified. Fragmentation patterns for major compound classes are discussed, which provides a framework for the discovery of these compounds from other sources. Identification of alkaloids as a well-known group of bioactive natural products represents an important advance in better understanding of the pharmacological profile of black cohosh.

Applications of Baeyer-Villiger Monooxygenases in Organic Synthesis

The knowledge about these Baeyer-Villiger monooxygenases has grown tremendously since the first discovery and fundamental progress in the understanding of structure, function, substrate specificities and other enzyme properties has been facilitated by the development of recombinant biocatalysts. Nature uses these biocatalysts in aerobic biodegradation pathways of cyclic and acyclic ketones and in the biosynthetic pathways of natural products. The excellent performance of Baeyer-Villiger monooxygenases in nature for the catalysis of Baeyer-Villiger oxidations with high chemo-, regio- and enantioselectivity is a role model for sustainable catalytic Baeyer- Villiger oxidations in organic synthesis. A broad range of biocatalytic conversions of cyclic ketones to lactones, linear ketones to esters, sulfoxidations and other oxidations is described. Applications in dynamic kinetic resolution as well as process and scale-up issues have been important in making this reaction platform attractive to industrial scale Baeyer-Villiger oxidations. New discoveries of Baeyer- Villiger monooxygenases in biosynthesis are promising for highly selective oxidations. Keywords: Biocatalytic Baeyer-Villiger Oxidation, Asymmetric Synthesis, Monooxygenases, Biocatalytic Asymmetric Oxidation, Lactones, Esters, Sulfoxides, Desymmetrization, Kinetic Resolution, Large-Scale Processes, Oxidation, Synthesis, Biocatalytic, Resolution, Large-Scale, BVMOs, FAD, FMN, CHMOAcineto, CHMOs, lactone, E value, ATCC 17453, bicyloketone, NCIMB, P.putida, CDPMO, CPDMO, HAPMO, CHMO, MeKA, kcat/KM, CCT 3119, AKMO, PAMO, Diels-Alder reactions, Xanthobacter sp, DKR process, HPLC, Mithramycin

The role of ATP in metabolism

Covering: up to the beginning of 2020Enzymes depending on cofactors are essential in many biosynthetic pathways of natural products. They are often involved in key steps: catalytic conversions that are difficult to achieve purely with synthetic organic chemistry. Hence, cofactor-dependent enzymes have great potential for biocatalysis, on the condition that a corresponding cofactor regeneration system is available. For some cofactors, these regeneration systems require multiple steps; such complex enzyme cascades/multi-enzyme systems are (still) challenging for in vitro biocatalysis. Further, artificial cofactor analogues have been synthesised that are more stable, show an altered reaction range, or act as inhibitors. The development of bio-orthogonal systems that can be used for the production of modified natural products in vivo is an ongoing challenge. In light of the recent progress in this field, this review aims to provide an overview of general strategies involving enzyme cofactors, cofactor analogues, and regeneration systems; highlighting the current possibilities for application of enzymes using some of the most common cofactors.