Family Of Natural Products Research Articles

Bioinformatic Analysis Reveals both Oversampled and Underexplored Biosynthetic Diversity in Nonribosomal Peptides.

The traditional natural product discovery approach has accessed only a fraction of the chemical diversity in nature. The use of bioinformatic tools to interpret the instructions encoded in microbial biosynthetic genes has the potential to circumvent the existing methodological bottlenecks and greatly expand the scope of discovery. Structural prediction algorithms for nonribosomal peptides (NRPs), the largest family of microbial natural products, lie at the heart of this new approach. To understand the scope and limitation of the existing prediction algorithms, we evaluated their performances on NRP synthetase biosynthetic gene clusters. Our systematic analysis shows that the NRP biosynthetic landscape is uneven. Phenylglycine and its derivatives as a group of NRP building blocks (BBs), for example, have been oversampled, reflecting an extensive historical interest in the glycopeptide antibiotics family. In contrast, the benzoyl BB, including 2,3-dihydroxybenzoate (DHB), has been the most underexplored, hinting at the possibility of a reservoir of as yet unknown DHB containing NRPs with functional roles other than a siderophore. Our results also suggest that there is still vast unexplored biosynthetic diversity in nature, and the analysis presented herein shall help guide and strategize future natural product discovery campaigns. We also discuss possible ways bioinformaticians and biochemists could work together to improve the existing prediction algorithms.

New Nostocyclophanes from Nostoc linckia.

Six new nostocyclophanes and four known compounds have been isolated from Nostoc linckia (Nostocaceae) cyanobacterial strain UTEX B1932. The new compounds, nostocyclophanes E-J (1-6), were characterized by NMR and MS techniques. The known compounds were nostocyclophanes B-D, previously isolated from this strain, and dedichloronostocyclophane D. Structural modifications on the new [7.7]paracyclophane analogs 1-5, isolated from the 80% methanol fraction, range from simple changes such as the lack of methylation or halogenation to more unusual modifications such as those seen in nostocyclophane H (4), in which the exocyclic alkyl chains are of different length; this is the first time this modification has been observed in this family of natural products. In addition, nostocyclophane J (6) is a linear analog in which C-20 is chlorinated in preparation for the presumed enzymatic Friedel-Craft cyclization needed to form the final ring structure, analogous to the biosynthesis of the related cylindrocyclophanes. Nostocyclophane D, dedichloronostocyclophane D, and nostocyclophanes E-J demonstrated moderate to weak growth inhibition against MDA-MB-231 breast cancer cells.

A Survey of Didemnin Depsipeptide Production in Tistrella.

As one of the first families of marine natural products to undergo clinical trials, the didemnin depsipeptides have played a significant role in inspiring the discovery of marine drugs. Originally developed as anticancer therapeutics, the recent re-evaluation of these compounds including synthetically derived dehydrodidemnin B or Aplidine, has led to their advancement towards antiviral applications. While conventionally associated with production in colonial tunicates of the family Didemnidae, recent studies have identified their biosynthetic gene clusters from the marine-derived bacteria Tistrella mobilis. While these studies confirm the production of didemnin X/Y, the low titer and general lack of understanding of their biosynthesis in Tistrella currently prevents the development of effective microbial or synthetic biological approaches for their production. To this end, we conducted a survey of known species of Tistrella and report on their ability to produce the didemnin depsipeptides. These data were used to develop conditions to produce didemnin B at titers over 15 mg/L.

Genome mining of cryptic bisabolenes that were biosynthesized by intramembrane terpene synthases from Antrodia cinnamomea.

Terpenoids represent the largest structural family of natural products (NPs) and have various applications in the pharmaceutical, food and fragrance industries. Their diverse scaffolds are generated via a multi-step cyclization cascade of linear isoprene substrates catalysed by terpene synthases (TPSs). Bisabolene NPs, which are sesquiterpenes (C15), have wide applications in medicines and biofuels and serve as bioactive substances in ecology. Despite the discovery of some canonical class I TPSs that synthesize bisabolenes from plants, bacteria and insects, it remained unknown whether any bisabolene synthases from fungi could produce bisabolenes as a main product. Antrodia cinnamomea, a Basidiomycota fungus, is a medicinal mushroom indigenous to Taiwan and a known prolific producer of bioactive terpenoids, but little is known regarding the enzymes involved in the biosynthetic pathways. Here, we applied a genome mining approach against A. cinnamomea and discovered two non-canonical UbiA-type TPSs that both synthesize (+)-(S,Z)-α-bisabolene (1). It was determined that two tailoring enzymes, a P450 monooxygenase and a methyltransferase, install a C14-methyl ester on the bisabolene scaffold. In addition, four new bisabolene derivatives, 2 and 4-6, were characterized from heterologous reconstitution in Saccharomyces cerevisiae. Our study uncovered enzymatic tools to generate structurally diverse bisabolene NPs. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Newest perspectives of glycopeptide antibiotics: biosynthetic cascades, novel derivatives, and new appealing antimicrobial applications.

Glycopeptide antibiotics (GPAs) are a family of non-ribosomal peptide natural products with polypeptide skeleton characteristics, which are considered the last resort for treating severe infections caused by multidrug-resistant Gram-positive pathogens. Over the past few years, an increasing prevalence of Gram-positive resistant strain "superbugs" has emerged. Therefore, more efforts are needed to study and modify the GPAs to overcome the challenge of superbugs. In this mini-review, we provide an overview of the complex biosynthetic gene clusters (BGCs), the ingenious crosslinking and tailoring modifications, the new GPA derivatives, the discoveries of new natural GPAs, and the new applications of GPAs in antivirus and anti-Gram-negative bacteria. With the development and interdisciplinary integration of synthetic biology, next-generation sequencing (NGS), and artificial intelligence (AI), more GPAs with new chemical structures and action mechanisms will constantly be emerging.

First trans-eunicellane terpene synthase in bacteria

Terpenoids are the largest family of natural products, but prokaryotes are vastly underrepresented in this chemical space. However, genomics supports vast untapped biosynthetic potential for terpenoids in bacteria. We discovered the first trans-eunicellane terpene synthase (TS), AlbS from Streptomyces albireticuli NRRL B-1670, in nature. Mutagenesis, deuterium labeling studies, and quantum chemical calculations provided extensive support for its cyclization mechanism. In addition, parallel stereospecific labeling studies with Bnd4, a cis-eunicellane TS, revealed a key mechanistic distinction between these two enzymes. AlbS highlights bacteria as a valuable source of novel terpenoids, expands our understanding of the eunicellane family of natural products and the enzymes that biosynthesize them, and provides a model system to address fundamental questions about the chemistry of 6,10-bicyclic ring systems.

Direct pathway cloning and expression of the radiosumin biosynthetic gene cluster.

Radiosumins are a structurally diverse family of low molecular weight natural products that are produced by cyanobacteria and exhibit potent serine protease inhibition. Members of this family are dipeptides characterized by the presence of two similar non-proteinogenic amino acids. Here we used a comparative bioinformatic analysis to identify radiosumin biosynthetic gene clusters from the genomes of 13 filamentous cyanobacteria. We used direct pathway cloning to capture and express the entire 16.8 kb radiosumin biosynthetic gene cluster from Dolichospermum planctonicum UHCC 0167 in Escherichia coli. Bioinformatic analysis demonstrates that radiosumins represent a new group of chorismate-derived non-aromatic secondary metabolites. High-resolution liquid chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy and chemical degradation analysis revealed that cyanobacteria produce a cocktail of novel radiosumins. We report the chemical structure of radiosumin D, an N-methyl dipeptide, containing a special Aayp (2-amino-3-(4-amino-2-cyclohexen-1-ylidene) propionic acid) with R configuration that differs from radiosumin A-C, an N-Me derivative of Aayp (Amyp) and two acetyl groups. Radiosumin C inhibits all three human trypsin isoforms at micromolar concentrations with preference for trypsin-1 and -3 (IC50 values from 1.7 μM to >7.2 μM). These results provide a biosynthetic logic to explore the genetic and chemical diversity of the radiosumin family and suggest that these natural products may be a source of drug leads for selective human serine proteases inhibitors.

Maleidride biosynthesis - construction of dimeric anhydrides - more than just heads or tails.

Covering: up to early 2022Maleidrides are a family of polyketide-based dimeric natural products isolated from fungi. Many maleidrides possess significant bioactivities, making them attractive pharmaceutical or agrochemical lead compounds. Their unusual biosynthetic pathways have fascinated scientists for decades, with recent advances in our bioinformatic and enzymatic understanding providing further insights into their construction. However, many intriguing questions remain, including exactly how the enzymatic dimerisation, which creates the diverse core structure of the maleidrides, is controlled. This review will explore the literature from the initial isolation of maleidride compounds in the 1930s, through the first full structural elucidation in the 1960s, to the most recent in vivo, in vitro, and in silico analyses.

Bioinformatic prediction and experimental validation of RiPP recognition elements.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a family of natural products for which discovery efforts have rapidly grown over the past decade. There are currently 38 known RiPP classes encoded by prokaryotes. Half of the prokaryotic RiPP classes include a protein domain called the RiPP Recognition Element (RRE) for successful installation of post-translational modifications on a RiPP precursor peptide. In most cases, the RRE domain binds to the N-terminal "leader" region of the precursor peptide, facilitating enzymatic modification of the C-terminal "core" region. The prevalence of the RRE domain renders it a theoretically useful bioinformatic handle for class-independent RiPP discovery; however, first-in-class RiPPs have yet to be isolated and experimentally characterized using an RRE-centric strategy. Moreover, with most known RRE domains engaging their cognate precursor peptide(s) with high specificity and nanomolar affinity, evaluation of the residue-specific interactions that govern RRE:substrate complexation is a necessary first step to leveraging the RRE domain for various bioengineering applications. This chapter details protocols for developing custom bioinformatic models to predict and annotate RRE domains in a class-specific manner. Next, we outline methods for experimental validation of precursor peptide binding using fluorescence polarization binding assays and in vitro enzyme activity assays. We anticipate the methods herein will guide and enhance future critical analyses of the RRE domain, eventually enabling its future use as a customizable tool for molecular biology.

Divergent total syntheses of ITHQ-type bis-β-carboline alkaloids by regio-selective formal aza-[4 + 2] cycloaddition and late-stage C-H functionalization.

We herein report the first total syntheses of several bis-β-carboline alkaloids, picrasidines G, S, R, and T, and natural product-like derivatives in a divergent manner. Picrasidines G, S, and T feature an indolotetrahydroquinolizinium (ITHQ) skeleton, while picrasidine R possesses a 1,4-diketone linker between two β-carboline fragments. The synthesis of ITHQ-type bis-β-carboline alkaloids could be directly achieved by a late-stage regio-selective aza-[4 + 2] cycloaddition of vinyl β-carboline alkaloids, suggesting that this remarkable aza-[4 + 2] cycloaddition might be involved in the biosynthetic pathway. Computational studies revealed that such aza-[4 + 2] cycloaddition is a stepwise process and explained the unique regioselectivity (ΔΔG = 3.77 kcal mol-1). Moreover, the successful application of iridium-catalyzed C-H borylation on β-carboline substrates enabled the site-selective C-8 functionalization for efficient synthesis and structural diversification of this family of natural products. Finally, concise synthesis of picrasidine R by the thiazolium-catalyzed Stetter reaction was also accomplished.

Full-length transcriptome and metabolite analysis reveal reticuline epimerase-independent pathways for benzylisoquinoline alkaloids biosynthesis in Sinomenium acutum.

Benzylisoquinoline alkaloids (BIAs) are a large family of plant natural products with important pharmaceutical applications. Sinomenium acutum is a medicinal plant from the Menispermaceae family and has been used to treat rheumatoid arthritis for hundreds of years. Sinomenium acutum contains more than 50 BIAs, and sinomenine is a representative BIA from this plant. Sinomenine was found to have preventive and curative effects on opioid dependence. Despite the broad applications of S. acutum, investigation on the biosynthetic pathways of BIAs from S. acutum is limited. In this study, we comprehensively analyzed the transcriptome data and BIAs in the root, stem, leaf, and seed of S. acutum. Metabolic analysis showed a noticeable difference in BIA contents in different tissues. Based on the study of the full-length transcriptome, differentially expressed genes, and weighted gene co-expression network, we proposed the biosynthetic pathways for a few BIAs from S. acutum, such as sinomenine, magnoflorine, and tetrahydropalmatine, and screened candidate genes involved in these biosynthesis processes. Notably, the reticuline epimerase (REPI/STORR), which converts (S)-reticuline to (R)-reticuline and plays an essential role in morphine and codeine biosynthesis, was not found in the transcriptome data of S. acutum. Our results shed light on the biogenesis of the BIAs in S. acutum and may pave the way for the future development of this important medicinal plant.

Spirobisnaphthalenes from Fungi, Biological activities and Total Synthesis: A Research Review

AbstractSpirobisnaphthalenes are a family of natural products containing two naphthalenes connected with spiroketal or dinaphthalene spiroether. This family of natural compounds has a wide range of biological activities and complex structures, and has attracted great attention from synthetic chemists in recent years. Hundreds of novel spirobisnaphthalenes have been identified from the first separation in 1989. Despite the slow pace of research in recent years, since the latest review in 2017, 30 new natural products have been isolated from the endolichenic and endophytic fungus, including Type A, B, D and E, a new special skeleton with a spiro quaternary carbon center, as well as significant breakthroughs are made in the total synthesis. Here, the last research advances in the isolation of new spirobisnaphthalenes, their activities, mode of action and the total synthesis of spirobisnaphthalenes in the last five years were afforded based on our research in this field in this review.

Two distinct rotations of bithiazole DNA intercalation revealed by direct comparison of crystal structures of Co(III)•bleomycin A2 and B2 bound to duplex 5′-TAGTT sites

Bleomycins constitute a family of anticancer natural products that bind DNA through intercalation of a C-terminal tail/bithiazole moiety and hydrogen-bonding interactions between the remainder of the drug and the minor groove. The clinical utility of the bleomycins is believed to result from single- and double-strand DNA cleavage mediated by the HOO-Fe(III) form of the drug. The bleomycins also serve as a model system to understand the nature of complex drug-DNA interactions that may guide future DNA-targeted drug discovery. In this study, the impact of the C-terminal tail on bleomycin-DNA interactions was investigated. Toward this goal, we determined two crystal structures of HOO-Co(III)•BLMA2 “green” (a stable structural analogue of the active HOO-Fe(III) drug) bound to duplex DNA containing 5′-TAGTT, one in which the entire drug is bound (fully bound) and a second with only the C-terminal tail/bithiazole bound (partially bound). The structures reported here were captured by soaking HOO-Co(III)•BLMA2 into preformed host–guest crystals including a preferred DNA-binding site. While the overall structure of DNA-bound BLMA2 was found to be similar to those reported earlier at the same DNA site for BLMB2, the intercalated bithiazole of BLMB2 is “flipped” 180˚ relative to DNA-bound BLMA2. This finding highlights an unidentified role for the C-terminal tail in directing the intercalation of the bithiazole. In addition, these analyses identified specific bond rotations within the C-terminal domain of the drug that may be relevant for its reorganization and ability to carry out a double-strand DNA cleavage event.

A Collaborative Journey towards the Late‐Stage Functionalization of Added‐Value Chemicals Using Engineered Halogenases

AbstractIn 2017, two companies, Novartis AG and Syngenta AG, joined forces with the group of Prof. Buller, head of the Competence Center for Biocatalysis (CCBIO), to tackle the challenge of enzymatic late‐stage halogenation. This biocatalytic method was considered to provide a more sustainable approach to late‐stage halogenation of complex molecules than traditional synthetic approaches. Using machine‐learning guided protein engineering, α‐ketoglutarate dependent halogenases were evolved into versatile catalysts capable of selectively chlorinating inactivated C−H bonds. Structurally diverse molecules, namely an analogue of martinelline as well as two members of the soraphen natural product family, were enzymatically chlorinated at two distinct positions in a regio‐ and stereoselective manner, thus demonstrating the synthetic usefulness of such a strategy. As part of our three‐year collaboration, flavin‐dependent halogenases were also studied.

Thiamyxins: Structure and Biosynthesis of Myxobacterial RNA-Virus Inhibitors.

During our search for novel myxobacterial natural products, we discovered the thiamyxins: thiazole- and thiazoline-rich non-ribosomal peptide-polyketide hybrids with potent antiviral activity. We isolated four congeners of this unprecedented natural product family with the non-cyclized thiamyxin D fused to a glycerol unit at the C-terminus. Alongside their structure elucidation, we present a concise biosynthesis model based on biosynthetic gene cluster analysis and isotopically labelled precursor feeding. We report incorporation of a 2-(hydroxymethyl)-4-methylpent-3-enoic acid moiety by a GCN5-related N-acetyltransferase-like decarboxylase domain featuring polyketide synthase. The thiamyxins show potent inhibition of RNA viruses in cell culture models of corona, zika and dengue virus infection. Their potency up to a half maximal inhibitory concentration of 560 nM combined with milder cytotoxic effects on human cell lines indicate the potential for further development of the thiamyxins.

Structures, biosynthesis, and bioactivities of prodiginine natural products.

Prodiginines are a large family of microbial secondary metabolites with a core structure of tripyrrole rings. They exhibit not only diverse chemical structures but also rich biological activities, such as anti-cancer, anti-microbial, anti-algae, anti-parasitic, pesticides, and UV radiation resistance. The preferred cytotoxicity to cancer cells rather than normal cells indicates a good biological selectivity and safety, which makes the prodiginines promising candidates for drug development and novel additives for food processing. Until now, 33 prodiginine natural products have been identified in various bacteria, including Serratia, Hahella, Pseudoalteromonas, Vibrio, Zooshikella, Streptomyces, and Actinomadura. However, most efforts are still focused on the star molecule prodigiosin, while little yet is known about other prodiginine members, which retards the research and application of prodiginine compounds. To gain insight into the prodiginine family, we reviewed the recent discoveries on their chemical structures, biosynthesis, biological activities, and mechanisms of action. We believe this article will provide a guideline for new research on prodiginines, such as the discovery of new congeners and drug development. KEY POINTS: • The prodiginines are a large family of natural products with a core structure of tripyrrole rings and exhibit various bioactivities. • The prodiginines have a widespread distribution among many environmental microbes and diverse biosynthetic pathways, indicating important ecological roles and a great potential for new congeners. • The potent biological activities and good selectivity of action make prodiginines good lead compounds for drug development.

Biosynthesis of geranate via isopentenol utilization pathway in Escherichia coli.

Isoprenoids are a large family of natural products with diverse structures, which allow them to play diverse and important roles in the physiology of plants and animals. They also have important commercial uses as pharmaceuticals, flavoring agents, fragrances, and nutritional supplements. Recently, metabolic engineering has been intensively investigated and emerged as the technology of choice for the production of isoprenoids through microbial fermentation. Isoprenoid biosynthesis typically originates in plants from acetyl-coA in central carbon metabolism, however, a recent study reported an alternative pathway, the isopentenol utilization pathway (IUP), that can provide the building blocks of isoprenoid biosynthesis from affordable C5 substrates. In this study, we expressed the IUP in Escherichia coli to efficiently convert isopentenols into geranate, a valuable isoprenoid compound. We first established a geraniol-producing strain in E. coli that uses the IUP. Then, we extended the geraniol synthesis pathway to produce geranate through two oxidation reactions catalyzed by two alcohol/aldehyde dehydrogenases from Castellaniella defragrans. The geranate titer was further increased by optimizing the expression of the two dehydrogenases and also parameters of the fermentation process. The best strain produced 764 mg/L geranate in 24 h from 2 g/L isopentenols (a mixture of isoprenol and prenol). We also investigated if the dehydrogenases could accept other isoprenoid alcohols as substrates.

Recent Developments with Icetexane Natural Products.

Icetexane diterpenoids are a diverse family of natural products sourced from several species of terrestrial plants. Icetexanes exhibit a broad array of biological activities and together with their complex 6-7-6 tricyclic scaffolds, they have piqued the interest of synthetic organic chemists, natural products chemists, and biological investigators over the past four decades and were reviewed 13 years ago. This review summarizes icetexane natural products isolated since 2009, provides an overview of new synthetic approaches to the icetexane problem, and proposes an additional classification of icetexanes based on novel structures that are unlike previously isolated materials.

Discovery of p-Terphenyl Metabolites as Potential Phosphodiesterase PDE4D Inhibitors from the Coral-Associated Fungus Aspergillus sp. ITBBc1.

Chemical investigation of the fermentation extract of the coral-associated fungus Aspergillus sp. ITBBc1 led to the discovery of five unreported p-terphenyl derivatives, sanshamycins A-E (1-5), together with five previously described analogues, terphenyllin (6), 3-hydroxyterphenyllin (7), candidusin A (8), 4,5-dimethoxycandidusin A (9), and candidusin C (10). Their structures were elucidated by HRESIMS data and NMR spectroscopic analysis. Compound 1 represents the first example of p-terphenyls with an aldehyde substitution on the benzene ring. Compounds 2-4 feature varying methoxyl and isopentenyl substitutions, while compound 5 features a five-membered lactone linked to a biphenyl. These findings expand the chemical diversity of the family of p-terphenyl natural products. Compounds 1-6 and 9 were evaluated for their inhibitory activity against type 4 phosphodiesterase (PDE4), which is a fascinating drug target for treatment of inflammatory, respiratory, and neurological diseases. Compound 3 was the most potent and exhibited PDE4D inhibitory activity with an IC50 value of 5.543 µM.

Expanded Sequence Space of Radical S‐Adenosylmethionine‐Dependent Enzymes Involved in Post‐translational Macrocyclization**

Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent one of the largest but primarily underexplored natural product families in bacteria. The genetically encoded nature of RiPPs simplifies the prediction and prioritization of their biosynthetic gene clusters (BGCs). We report a small peptide and enzyme co-occurrence analysis workflow (SPECO), which allowed us to identify 32 220 prospective rSAM-catalyzed RiPP BGCs from 161 954 bacterial genomes and prioritize 25 families with new biosynthetic architectures or precursor patterns. We characterized three new enzymes that respectively catalyze cysteine-glycine (BlaB), histidine-aliphatic side chain (ScaB), and tyrosine/histidine-arginine (VguB) cross-links. The cyclophane-forming enzyme ScaB exhibits broad substrate selectivity, allowing it to catalyze diverse triceptide formation. These results demonstrate the strength of the SPECO workflow in discovering new enzymes for peptide macrocyclization.