The l-Alanosine Gene Cluster Encodes a Pathway for Diazeniumdiolate Biosynthesis

N-Nitroso-containing natural products are bioactive metabolites with antibacterial and anticancer properties. In particular, compounds containing the diazeniumdiolate (N-nitrosohydroxylamine) group display a wide range of bioactivities ranging from cytotoxicity to metal chelation. Despite the importance of this structural motif, knowledge of its biosynthesis is limited. Herein we describe the discovery of a biosynthetic gene cluster in Streptomyces alanosinicus ATCC 15710 responsible for producing the diazeniumdiolate natural product l -alanosine. Gene disruption and stable isotope feeding experiments identified essential biosynthetic genes and revealed the source of the N-nitroso group. Additional biochemical characterization of the biosynthetic enzymes revealed that the non-proteinogenic amino acid l -2,3-diaminopropionic acid (l -Dap) is synthesized and loaded onto a free-standing peptidyl carrier protein (PCP) domain in l -alanosine biosynthesis, which we propose may be a mechanism of handling unstable intermediates generated en route to the diazeniumdiolate. These discoveries will facilitate efforts to determine the biochemistry of diazeniumdiolate formation.     

Biosynthesis and Structure–Activity Relationship Investigations of the Diazeniumdiolate Antifungal Agent Fragin

Only a few natural products incorporating a diazeniumdiolate moiety have been isolated, and these compounds usually display a broad range of biological activities. Only recently has the first diazeniumdiolate natural product biosynthetic gene cluster been identified in Burkholderia cenocepacia H111, which produces the fungicide (−)-fragin and the signal molecule rac-valdiazen. In this study, l -valine was identified as the initial substrate of (−)-fragin biosynthesis with the aid of feeding experiments using isotopically labelled amino acid. The formation of the diazeniumdiolate was chemically studied with several proposed intermediates. Our results indicate that the functional group is formed during an early stage of the biosynthesis. Furthermore, an oxime compound was identified as a degradation product of (−)-fragin and was also observed in the crude extract of the wild-type strain. Moreover, a structure–activity relationship analysis revealed that each moiety of (−)-fragin is essential for its biological activity.     

Synthesis of Functionalized δ-Hydroxy-β-keto Esters and Evaluation of Their Anti-inflammatory Properties

δ-Hydroxy-β-keto esters and δ,β-dihydroxy esters are characteristic structural motifs of statin-type natural products and drug candidates. Here, we describe the synthesis of functionalized δ-hydroxy-β-keto esters in good yields and excellent enantioselectivities using Chan's diene and modified Mukaiyama-aldol reaction conditions. Diastereoselective reduction of δ,β-dihydroxy esters afforded the respective syn- and anti-diols, and saponification yielded the corresponding acids. All products were evaluated for their anti-inflammatory properties, which uncovered a surprising structure-activity relationship.     

An Iterative O‐Methyltransferase Catalyzes 1,11‐Dimethylation of Aspergillus fumigatus Fumaric Acid Amides

S‐adenosyl‐l ‐methionine (SAM)‐dependent methyltransfer is a common biosynthetic strategy to modify natural products. We investigated the previously uncharacterized Aspergillus fumigatus methyltransferase FtpM, which is encoded next to the bimodular fumaric acid amide synthetase FtpA. Structure elucidation of two new A. fumigatus natural products, the 1,11‐dimethyl esters of fumaryl‐l ‐tyrosine and fumaryl‐l ‐phenylalanine, together with ftpM gene disruption suggested that FtpM catalyzes iterative methylation. Final evidence that a single enzyme repeatedly acts on fumaric acid amides came from an in vitro biochemical investigation with recombinantly produced FtpM. Size‐exclusion chromatography indicated that this methyltransferase is active as a dimer. As ftpA and ftpM homologues are found clustered in other fungi, we expect our work will help to identify and annotate natural product biosynthesis genes in various species.     

Characterization of the Calicheamicin Orsellinate C2‐O‐Methyltransferase CalO6

Although bacterial iterative type I polyketide synthases are now known to participate in the biosynthesis of a small set of diverse natural products, the subsequent downstream modification of the resulting polyketide products is poorly understood. We report the functional characterization of the putative orsellinic acid C2‐O‐methyltransferase, which is involved in calicheamicin biosynthesis. This study suggests that C2‐O‐methylation precedes C3‐hydroxylation/methylation and C5‐iodination and requires a coenzyme A‐ or acyl carrier protein‐bound substrate.     

Construction of a Chimeric Biosynthetic Pathway for the De Novo Biosynthesis of Rosmarinic Acid in Escherichia coli

Hydroxycinnamic acid esters (HCEs) are widely‐distributed phenylpropanoid‐derived plant natural products. Rosmarinic acid (RA), the most well‐known HCE, shows promise as a treatment for cancer and neurological disorders. In contrast to extraction from plant material or plant cell culture, microbial production of HCEs could be a sustainable, controlled means of production. Through the overexpression of a six‐enzyme chimeric bacterial and plant pathway, we show the de novo biosynthesis of RA, and the related HCE isorinic acid (IA), in Escherichia coli. Probing the pathway through precursor supplementation showed several potential pathway bottlenecks. We demonstrated HCE biosynthesis using three plant rosmarinic acid synthase (RAS) orthologues, which exhibited different levels of HCE biosynthesis but produced the same ratio of IA to RA. This work serves as a proof‐of‐concept for a microbial production platform for HCEs by using a modular biosynthetic approach to access diverse natural and non‐natural HCEs.     

Identifying the Biosynthetic Gene Cluster for Triacsins with an N-Hydroxytriazene Moiety

Triacsins are a family of natural products having in common an N-hydroxytriazene moiety not found in any other known secondary metabolites. Though many studies have examined the biological activity of triacsins in lipid metabolism, their biosynthesis has remained unknown. Here we report the identification of the triacsin biosynthetic gene cluster in Streptomyces aureofaciens ATCC 31442. Bioinformatic analysis of the gene cluster led to the discovery of the tacrolimus producer Streptomyces tsukubaensis NRRL 18488 as a new triacsin producer. In addition to targeted gene disruption to identify necessary genes for triacsin production, stable isotope feeding was performed in vivo to advance the understanding of N-hydroxytriazene biosynthesis.     

Characterization of an Orphan Type III Polyketide Synthase Conserved in Uncultivated “Entotheonella” Sponge Symbionts

Uncultivated bacterial symbionts from the candidate genus “Entotheonella” have been shown to produce diverse natural products previously attributed to their sponge hosts. In addition to these known compounds, “Entotheonella” genomes contain rich sets of biosynthetic gene clusters that lack identified natural products. Among these is a small type III polyketide synthase (PKS) cluster, one of only three clusters present in all known “Entotheonella” genomes. This conserved “Entotheonella” PKS (cep) cluster encodes the type III PKS CepA and the putative methyltransferase CepB. Herein, the characterization of CepA as an enzyme involved in phenolic lipid biosynthesis is reported. In vitro analysis showed a specificity for alkyl starter substrates and the production of tri- and tetraketide pyrones and tetraketide resorcinols. The conserved distribution of the cep cluster suggests an important role for the phenolic lipid polyketides produced in “Entotheonella” variants.     

Rapid Reconstitution of Biosynthetic Machinery for Fungal Metabolites in Aspergillus oryzae: Total Biosynthesis of Aflatrem

Reconstitution of the biosynthetic machinery for fungal secondary metabolites in Aspergillus oryzae provides an opportunity both for stepwise determination of the biosynthetic pathways and the total biosynthesis of fungal natural products. However, to maximize the utility of the reconstitution system, a simple and rapid strategy for the introduction of heterologous genes into A. oryzae is required. In this study, we demonstrated an effective method for introducing multiple genes involved in the biosynthesis of fungal metabolites by using the expression vectors pUARA2 and pUSA2, each of which contains two cloning sites. The successful introduction of all the aflatrem biosynthetic genes (seven genes in total) after two rounds of transformation enabled the total biosynthesis of aflatrem. This rapid reconstitution strategy will facilitate the functional analysis of the biosynthetic machinery of fungal metabolites.     

植物天然产物的微生物合成与转化

植物天然产物是一类结构复杂、性能多样的次级代谢产物,广泛应用于食品、药品、化妆品等多个领域。目前植物天然产物的主要来源依赖于从植物中提取,这种生产方式周期长且占用大量耕地。微生物细胞因其生长周期短、操作简便、环境友好、大规模发酵可控等优势而被广泛研究用以替代传统的植物提取法。目前利用微生物细胞工厂合成和转化植物天然产物已成为研究的热点,实现了萜类、黄酮、生物碱、皂苷等多种植物天然产物的合成和转化。本文分别从从头合成和生物转化的角度综述了微生物细胞工厂在植物天然产物合成中的应用,为更加系统、深入地研究植物天然产物的微生物合成与转化提供参考。     

Regio‐ and Stereospecific Prenylation of Flavonoids by Sophora flavescens Prenyltransferase

Prenylflavonoids are valuable natural products that are widely distributed in plants. They often possess divergent biological properties, including phytoestrogenic, anti‐bacterial, anti‐tumor, and anti‐diabetic activities. The reaction catalyzed by prenyltransferases represents a Friedel–Crafts alkylation of the flavonoid skeleton in the biosynthesis of natural prenylflavonoids and often contributes to the structural diversity and biological activity of these compounds. However, only a few plant flavonoid prenyltransferases have been identified thus far, and these prenyltransferases exhibit strict substrate specificity and low catalytic efficiency. In this article, a flavonoid prenyltransferase from Sophora flavescens, SfFPT, has been identified that displays high catalytic efficiency with high regiospecificity acting on C‐8 of structurally different types of flavonoid (i.e., flavanone, flavone, flavanonol, and dihydrochalcone, etc.). Furthermore, SfPFT exhibits strict stereospecificity for levorotatory flavanones to produce (2S)‐prenylflavanones. This study is the first to demonstrate the substrate promiscuity and stereospecificity of a plant flavonoid prenyltransferase in vitro. Given its substrate promiscuity and high catalytic efficiency, SfFPT can be used as an environmentally friendly and efficient biological catalyst for the regio‐ and stereospecific prenylation of flavonoids to produce bioactive compounds for potential therapeutic applications.     

Enzymatic Formation of Oxygen‐Containing Heterocycles in Natural Product Biosynthesis

Oxygen‐containing heterocycles are widely encountered in natural products that display diverse pharmacological properties and have potential benefits to human health. The formation of O‐heterocycles catalyzed by different types of enzymes in the biosynthesis of natural products not only contributes to the structural diversity of these compounds, but also enriches our understanding of nature's ability to construct complex molecules. This minireview focuses on the various modes of enzymatic O‐heterocyclization identified in natural product biosynthesis and summarizes the possible mechanisms involved in ring closure.     

An Engineered Tryptophan Synthase Opens New Enzymatic Pathways to β‐Methyltryptophan and Derivatives

β‐Methyltryptophans (β‐mTrp) are precursors in the biosynthesis of bioactive natural products and are used in the synthesis of peptidomimetic‐based therapeutics. Currently β‐mTrp is produced by inefficient multistep synthetic methods. Here we demonstrate how an engineered variant of tryptophan synthase from Salmonella (StTrpS) can catalyse the efficient condensation of l ‐threonine and various indoles to generate β‐mTrp and derivatives in a single step. Although l ‐serine is the natural substrate for TrpS, targeted mutagenesis of the StTrpS active site provided a variant (βL166V) that can better accommodate l ‐Thr as a substrate. The condensation of l ‐Thr and indole proceeds with retention of configuration at both α‐ and β‐positions to give (2S,3S)‐β‐mTrp. The integration of StTrpS (βL166V) with l ‐amino acid oxidase, halogenase enzymes and palladium chemocatalysts provides access to further d ‐configured and regioselectively halogenated or arylated β‐mTrp derivatives.     

Modeling natural anti-inflammatory compounds by molecular topology

One of the main pharmacological problems today in the treatment of chronic inflammation diseases consists of the fact that anti-inflammatory drugs usually exhibit side effects. The natural products offer a great hope in the identification of bioactive lead compounds and their development into drugs for treating inflammatory diseases. Computer-aided drug design has proved to be a very useful tool for discovering new drugs and, specifically, Molecular Topology has become a good technique for such a goal. A topological-mathematical model, obtained by linear discriminant analysis, has been developed for the search of new anti-inflammatory natural compounds. An external validation obtained with the remaining compounds (those not used in building up the model), has been carried out. Finally, a virtual screening on natural products was performed and 74 compounds showed actual anti-inflammatory activity. From them, 54 had been previously described as anti-inflammatory in the literature. This can be seen as a plus in the model validation and as a reinforcement of the role of Molecular Topology as an efficient tool for the discovery of new anti-inflammatory natural compounds.     

The Cyanobacterial “Nutraceutical” Phycocyanobilin Inhibits Cysteine Protease Legumain

The blue biliprotein phycocyanin, produced by photo-autotrophic cyanobacteria including spirulina (Arthrospira) and marketed as a natural food supplement or “nutraceutical,” is reported to have anti-inflammatory, antioxidant, immunomodulatory, and anticancer activity. These diverse biological activities have been specifically attributed to the phycocyanin chromophore, phycocyanobilin (PCB). However, the mechanism of action of PCB and the molecular targets responsible for the beneficial properties of PCB are not well understood. We have developed a procedure to rapidly cleave the PCB pigment from phycocyanin by ethanolysis and then characterized it as an electrophilic natural product that interacts covalently with thiol nucleophiles but lacks any appreciable cytotoxicity or antibacterial activity against common pathogens and gut microbes. We then designed alkyne-bearing PCB probes for use in chemical proteomics target deconvolution studies. Target identification and validation revealed the cysteine protease legumain (also known as asparaginyl endopeptidase, AEP) to be a target of PCB. Inhibition of this target may account for PCB's diverse reported biological activities.     

EPA and DHA in microalgae: Health benefits,biosynthesis, and metabolic engineering advances

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are ω-3 very long-chain polyunsaturated fatty acids (VLC-PUFAs) that offer a wide range of human health benefits impacting cardiovascular, anti-inflammatory, and neurological health. It is widely known that humans inefficiently synthesize these compounds and as such rely on exogenous dietary sources, such as marine fish oils. Unfortunately, the production of marine fish oils is an unsustainable process and has suffered a dramatic fall in recent years due to overfishing and climate change, as the demand for EPA and DHA continues to rise. Therefore, there is an urgent need to develop alternative, sustainable sources for consumable EPA and DHA. Metabolic engineering of marine microalgae to improve their EPA and DHA productivity is regarded as a promising option that has received increasing commercial attention in recent years. In this mini-review, we describe several notable health benefits of EPA and DHA, summarize the natural sources and biosynthesis of VLC-PUFAS, as well as the recent advances in metabolic engineering of EPA and DHA production in representative microalgal and protist species, including Schizochytrium sp., Phaeodactylum tricornutum, and Nannochloropsis oceanica.     

Studies on the Biosynthesis of the Stephacidin and Notoamide Natural Products: A Stereochemical and Genetic Conundrum

The stephacidin and notoamide natural products belong to a group of prenylated indole alkaloids containing a bicyclo[2.2.2]diazaoctane core. Biosynthetically, this bicyclic core is believed to be the product of an intermolecular Diels–Alder (IMDA) cycloaddition of an achiral azadiene. Since all of the natural products in this family have been isolated in enantiomerically pure form to date, it is believed that an elusive Diels–Alderase enzyme mediates the IMDA reaction. Adding further intrigue to this biosynthetic puzzle is the fact that several related Aspergillus fungi produce a number of metabolites with the opposite absolute configuration, implying that these fungi have evolved enantiomerically distinct Diels–Alderases. We have undertaken a program to identify every step in the biogenesis of the stephacidins and notoamides, and by combining the techniques of chemical synthesis and biochemical analysis we have been able to identify the two prenyltransferases involved in the early stages of the stephacidin and notoamide biosyntheses. This has allowed us to propose a modified biosynthesis for stephacidin A, and has brought us closer to our goal of finding evidence for, or against, the presence of a Diels–Alderase in this biosynthetic pathway.     

Investigations into the Biosynthesis,Regulation, and Self‐Resistance of Toxoflavin in Pseudomonas protegens Pf‐5

Pseudomonas spp. are prolific producers of natural products from many structural classes. Here we show that the soil bacterium Pseudomonas protegens Pf‐5 is capable of producing trace levels of the triazine natural product toxoflavin ( 1 ) under microaerobic conditions. We evaluated toxoflavin production by derivatives of Pf‐5 with deletions in specific biosynthesis genes, which led us to propose a revised biosynthetic pathway for toxoflavin that shares the first two steps with riboflavin biosynthesis. We also report that toxM, which is not present in the well‐characterized cluster of Burkholderia glumae, encodes a monooxygenase that degrades toxoflavin. The toxoflavin degradation product of ToxM is identical to that of TflA, the toxoflavin lyase from Paenibacillus polymyxa. Toxoflavin production by P. protegens causes inhibition of several plant‐pathogenic bacteria, and introduction of toxM into the toxoflavin‐sensitive strain Pseudomonas syringae DC3000 results in resistance to toxoflavin.     

Marine Molecular Machines: Heterocyclization in Cyanobactin Biosynthesis

Natural products that contain amino‐acid‐derived (Cys, Ser, Thr) heterocycles are ubiquitous in nature, yet key aspects of their biosynthesis remain undefined. Cyanobactins are heterocyclic ribosomal peptide natural products from cyanobacteria, including symbiotic bacteria living with marine ascidians. In contrast to other ribosomal peptide heterocyclases that have been studied, the cyanobactin heterocyclase is a single protein that does not require an oxidase enzyme. Using this simplifying condition, we provide new evidence to support the hypothesis that these enzymes are molecular machines that use ATP in a product binding or orientation cycle. Further, we show that both protease inhibitors and ATP analogues inhibit heterocyclization and define the order of biochemical steps in the cyanobactin biosynthetic pathway. The cyanobactin pathway enzymes, PatD and TruD, are thiazoline and oxazoline synthetases.