Bioactivity-driven fungal metabologenomics identifies antiproliferative stemphone analogs and their biosynthetic gene cluster.

INTRODUCTION: Fungi biosynthesize chemically diverse secondary metabolites with a wide range of biological activities. Natural product scientists have increasingly turned towards bioinformatics approaches, combining metabolomics and genomics to target secondary metabolites and their biosynthetic machinery. We recently applied an integrated metabologenomics workflow to 110 fungi and identified more than 230 high-confidence linkages between metabolites and their biosynthetic pathways. OBJECTIVES: To prioritize the discovery of bioactive natural products and their biosynthetic pathways from these hundreds of high-confidence linkages, we developed a bioactivity-driven metabologenomics workflow combining quantitative chemical information, antiproliferative bioactivity data, and genome sequences. METHODS: The 110 fungi from our metabologenomics study were tested against multiple cancer cell lines to identify which strains produced antiproliferative natural products. Three strains were selected for further study, fractionated using flash chromatography, and subjected to an additional round of bioactivity testing and mass spectral analysis. Data were overlaid using biochemometrics analysis to predict active constituents early in the fractionation process following which their biosynthetic pathways were identified using metabologenomics. RESULTS: We isolated three new-to-nature stemphone analogs, 19-acetylstemphones G (1), B (2) and E (3), that demonstrated antiproliferative activity ranging from 3 to 5 µM against human melanoma (MDA-MB-435) and ovarian cancer (OVACR3) cells. We proposed a rational biosynthetic pathway for these compounds, highlighting the potential of using bioactivity as a filter for the analysis of integrated-Omics datasets. CONCLUSIONS: This work demonstrates how the incorporation of biochemometrics as a third dimension into the metabologenomics workflow can identify bioactive metabolites and link them to their biosynthetic machinery.

Persistent and Bioaccumulative Halogenated Natural Products in Various Tropical Reef Fish Species from the Seychelles, Western Indian Ocean.

Gas chromatography with electron capture negative ion mass spectrometry (GC/ECNI-MS) was used to quantify and compare halogenated natural products (HNPs) and selected anthropogenic persistent organic pollutants (POPs) in individual samples of 17 fish species from the Seychelles (Western Indian Ocean). The sum-HNP amounts (9.5-1100 ng/g lipid mass (lm)) were between 1 and 2 orders of magnitude higher than those of the sum of seven abundant polychlorinated biphenyl (PCB) congeners (0.2-15 ng/g lm) and dichlorodiphenyltrichloroethane-related compounds (DDTs) (<1.1-43 ng/g lm). Within the group of HNPs, the two tetrabrominated phenoxyanisoles (aka methoxylated diphenyl ethers, MeO-BDEs), 2'-MeO-BDE 68 ≫ 6-MeO-BDE 47, were predominant in most cases. Pearson correlation analysis showed that MeO-BDE levels were positively correlated with less abundant HNPs (2,2'-diMeO-BB 80, 2',6-diMeO-BDE 68, and Br6-DBP) (p < 0.01). Accordingly, HNPs, rather than PCBs and DDTs, were the predominant polyhalogenated contaminants in the current species.

Plant Tissue Culture and Metabolite Profiling for High-Value Natural Product Synthesis.

The engineering of plant cell cultures to produce high-value natural products is suggested to be a safe, low-cost, and environmentally friendly route to produce a wide range of chemicals. Given that the expression of heterologous biosynthetic pathways in plant tissue culture is limited by a lack of detailed protocols, the biosynthesis of high-value metabolites in plant cell culture is constrained compared with that in microbes. However, both Arabidopsis thaliana and Nicotiana benthamiana can be efficiently transformed with multigene constructs to produce high-value natural products in stable plant cell cultures. This chapter provides a detailed protocol as to how to engineer the plant cell culture as bio-factories for metabolite biosynthesis.

Illuminating a biologics development challenge: systematic characterization of CHO cell-derived hydrolases identified in monoclonal antibody formulations.

Monoclonal antibodies (mAb) and other biological drugs are affected by enzymatic polysorbate (PS) degradation that reduces product stability and jeopardizes the supply of innovative medicines. PS represents a critical surfactant stabilizing the active pharmaceutical ingredients, which are produced by recombinant Chinese hamster ovary (CHO) cell lines. While the list of potential PS-degrading CHO host cell proteins (HCPs) has grown over the years, tangible data on industrially relevant HCPs are still scarce. By means of a highly sensitive liquid chromatography-tandem mass spectrometry method, we investigated seven different mAb products, resulting in the identification of 12 potentially PS-degrading hydrolases, including the strongly PS-degrading lipoprotein lipase (LPL). Using an LPL knockout CHO host cell line, we were able to stably overexpress and purify the remaining candidate hydrolases through orthogonal affinity chromatography methods, enabling their detailed functional characterization. Applying a PS degradation assay, we found nine mostly secreted, PS-active hydrolases with varying hydrolytic activity. All active hydrolases showed a serine-histidine-aspartate/glutamate catalytical triad. Further, we subjected the active hydrolases to pH-screenings and revealed a diverse range of activity optima, which can facilitate the identification of residual hydrolases during bioprocess development. Ultimately, we compiled our dataset in a risk matrix identifying PAF-AH, LIPA, PPT1, and LPLA2 as highly critical hydrolases based on their cellular expression, detection in purified antibodies, active secretion, and PS degradation activity. With this work, we pave the way toward a comprehensive functional characterization of PS-degrading hydrolases and provide a basis for a future reduction of PS degradation in biopharmaceutical drug products.

Iron-sulphur protein catalysed [4+2] cycloadditions in natural product biosynthesis.

To the best of our knowledge, enzymes that catalyse intramolecular Diels-Alder ([4+2] cycloaddition) reactions are frequently reported in natural product biosynthesis; however, no native enzymes utilising Lewis acid catalysis have been reported. Verticilactam is a representative member of polycyclic macrolactams, presumably produced by spontaneous cycloaddition. We report that the intramolecular [4+2] cycloadditions can be significantly accelerated by ferredoxins (Fds), a class of small iron-sulphur (Fe-S) proteins. Through iron atom substitution by Lewis acidic gallium (Ga) iron and computational calculations, we confirm that the ubiquitous Fe-S cluster efficiently functions as Lewis acid to accelerate the tandem [4+2] cycloaddition and Michael addition reactions by lowering free energy barriers. Our work highlights Nature's ingenious strategy to generate complex molecule structures using the ubiquitous Fe-S protein. Furthermore, our study sheds light on the future design of Fd as a versatile Lewis acid catalyst for [4+2] cycloaddition reactions.

Integrating bacterial molecular genetics with chemical biology for renewed antibacterial drug discovery.

The application of dyes to understanding the aetiology of infection inspired antimicrobial chemotherapy and the first wave of antibacterial drugs. The second wave of antibacterial drug discovery was driven by rapid discovery of natural products, now making up 69% of current antibacterial drugs. But now with the most prevalent natural products already discovered, ∼107 new soil-dwelling bacterial species must be screened to discover one new class of natural product. Therefore, instead of a third wave of antibacterial drug discovery, there is now a discovery bottleneck. Unlike natural products which are curated by billions of years of microbial antagonism, the vast synthetic chemical space still requires artificial curation through the therapeutics science of antibacterial drugs - a systematic understanding of how small molecules interact with bacterial physiology, effect desired phenotypes, and benefit the host. Bacterial molecular genetics can elucidate pathogen biology relevant to therapeutics development, but it can also be applied directly to understanding mechanisms and liabilities of new chemical agents with new mechanisms of action. Therefore, the next phase of antibacterial drug discovery could be enabled by integrating chemical expertise with systematic dissection of bacterial infection biology. Facing the ambitious endeavour to find new molecules from nature or new-to-nature which cure bacterial infections, the capabilities furnished by modern chemical biology and molecular genetics can be applied to prospecting for chemical modulators of new targets which circumvent prevalent resistance mechanisms.

Microbial allies: exploring fungal endophytes for biosynthesis of terpenoid indole alkaloids.

Terpenoid indole alkaloids (TIAs) are natural compounds found in medicinal plants that exhibit various therapeutic activities, such as antimicrobial, anti-inflammatory, antioxidant, anti-diabetic, anti-helminthic, and anti-tumor properties. However, the production of these alkaloids in plants is limited, and there is a high demand for them due to the increasing incidence of cancer cases. To address this research gap, researchers have focused on optimizing culture media, eliciting metabolic pathways, overexpressing genes, and searching for potential sources of TIAs in organisms other than plants. The insufficient number of essential genes and enzymes in the biosynthesis pathway is the reason behind the limited production of TIAs. As the field of natural product discovery from biological species continues to grow, endophytes are being investigated more and more as potential sources of bioactive metabolites with a variety of chemical structures. Endophytes are microorganisms (fungi, bacteria, archaea, and actinomycetes), that exert a significant influence on the metabolic pathways of both the host plants and the endophytic cells. Bio-prospection of fungal endophytes has shown the discovery of novel, high-value bioactive compounds of commercial significance. The discovery of therapeutically significant secondary metabolites has been made easier by endophytic entities' abundant but understudied diversity. It has been observed that fungal endophytes have better intermediate processing ability due to cellular compartmentation. This paper focuses on fungal endophytes and their metabolic ability to produce complex TIAs, recent advancements in this area, and addressing the limitations and future perspectives related to TIA production.

Advances in the development of synthetic biology tools for the bioproduction of natural product analogues / Postepy w rozwoju narzedzi biologii syntetycznej do bioprodukcji analogów produktów naturalnych.

Chemical compounds of natural origin, the so-called natural products and their derivatives, constitute the basis of medicines. They are widely used, among others, in the agricultural, veterinary, food and cosmetics industries. The article presents the division of natural products, with particular emphasis on bioactive secondary metabolites, and progress in the development of synthetic biology tools for their bioproduction.

Sophisticated natural products as antibiotics.

In this Review, we explore natural product antibiotics that do more than simply inhibit an active site of an essential enzyme. We review these compounds to provide inspiration for the design of much-needed new antibacterial agents, and examine the complex mechanisms that have evolved to effectively target bacteria, including covalent binders, inhibitors of resistance, compounds that utilize self-promoted entry, those that evade resistance, prodrugs, target corrupters, inhibitors of 'undruggable' targets, compounds that form supramolecular complexes, and selective membrane-acting agents. These are exemplified by ß-lactams that bind covalently to inhibit transpeptidases and ß-lactamases, siderophore chimeras that hijack import mechanisms to smuggle antibiotics into the cell, compounds that are activated by bacterial enzymes to produce reactive molecules, and antibiotics such as aminoglycosides that corrupt, rather than merely inhibit, their targets. Some of these mechanisms are highly sophisticated, such as the preformed ß-strands of darobactins that target the undruggable ß-barrel chaperone BamA, or teixobactin, which binds to a precursor of peptidoglycan and then forms a supramolecular structure that damages the membrane, impeding the emergence of resistance. Many of the compounds exhibit more than one notable feature, such as resistance evasion and target corruption. Understanding the surprising complexity of the best antimicrobial compounds provides a roadmap for developing novel compounds to address the antimicrobial resistance crisis by mining for new natural products and inspiring us to design similarly sophisticated antibiotics.

Exploring the secrets of marine microorganisms: Unveiling secondary metabolites through metagenomics.

Marine microorganisms are increasingly recognized as primary producers of marine secondary metabolites, drawing growing research interest. Many of these organisms are unculturable, posing challenges for study. Metagenomic techniques enable research on these unculturable microorganisms, identifying various biosynthetic gene clusters (BGCs) related to marine microbial secondary metabolites, thereby unveiling their secrets. This review comprehensively analyses metagenomic methods used in discovering marine microbial secondary metabolites, highlighting tools commonly employed in BGC identification, and discussing the potential and challenges in this field. It emphasizes the key role of metagenomics in unveiling secondary metabolites, particularly in marine sponges and tunicates. The review also explores current limitations in studying these metabolites through metagenomics, noting how long-read sequencing technologies and the evolution of computational biology tools offer more possibilities for BGC discovery. Furthermore, the development of synthetic biology allows experimental validation of computationally identified BGCs, showcasing the vast potential of metagenomics in mining marine microbial secondary metabolites.

Analysis of Co-localized Biosynthetic Gene Clusters Identifies a Membrane-Permeabilizing Natural Product.

Combination therapy is an effective strategy to combat antibiotic resistance. Multiple synergistic antimicrobial combinations are produced by enzymes encoded in biosynthetic gene clusters (BGCs) that co-localize on the bacterial genome. This phenomenon led to the hypothesis that mining co-localized BGCs will reveal new synergistic combinations of natural products. Here, we bioinformatically identified 38 pairs of co-localized BGCs, which we predict to produce natural products that are related to known compounds, including polycyclic tetramate macrolactams (PoTeMs). We further showed that ikarugamycin, a PoTeM, increases the membrane permeability of Acinetobacter baumannii and Staphylococcus aureus, which suggests that ikarugamycin might be an adjuvant that facilitates the entry of other natural products. Our work outlines a promising avenue to discover synergistic combinations of natural products by mining bacterial genomes.

Microbiota, natural products, and human health: exploring interactions for therapeutic insights.

The symbiotic relationship between the human digestive system and its intricate microbiota is a captivating field of study that continues to unfold. Comprising predominantly anaerobic bacteria, this complex microbial ecosystem, teeming with trillions of organisms, plays a crucial role in various physiological processes. Beyond its primary function in breaking down indigestible dietary components, this microbial community significantly influences immune system modulation, central nervous system function, and disease prevention. Despite the strides made in microbiome research, the precise mechanisms underlying how bacterial effector functions impact mammalian and microbiome physiology remain elusive. Unlike the traditional DNA-RNA-protein paradigm, bacteria often communicate through small molecules, underscoring the imperative to identify compounds produced by human-associated bacteria. The gut microbiome emerges as a linchpin in the transformation of natural products, generating metabolites with distinct physiological functions. Unraveling these microbial transformations holds the key to understanding the pharmacological activities and metabolic mechanisms of natural products. Notably, the potential to leverage gut microorganisms for large-scale synthesis of bioactive compounds remains an underexplored frontier with promising implications. This review serves as a synthesis of current knowledge, shedding light on the dynamic interplay between natural products, bacteria, and human health. In doing so, it contributes to our evolving comprehension of microbiome dynamics, opening avenues for innovative applications in medicine and therapeutics. As we delve deeper into this intricate web of interactions, the prospect of harnessing the power of the gut microbiome for transformative medical interventions becomes increasingly tantalizing.

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.

Gut microbiome-derived hydrolases-an underrated target of natural product metabolism.

In recent years, there has been increasing interest in studying gut microbiome-derived hydrolases in relation to oral drug metabolism, particularly focusing on natural product drugs. Despite the significance of natural product drugs in the field of oral medications, there is a lack of research on the regulatory interplay between gut microbiome-derived hydrolases and these drugs. This review delves into the interaction between intestinal microbiome-derived hydrolases and natural product drugs metabolism from three key perspectives. Firstly, it examines the impact of glycoside hydrolases, amide hydrolases, carboxylesterase, bile salt hydrolases, and epoxide hydrolase on the structure of natural products. Secondly, it explores how natural product drugs influence microbiome-derived hydrolases. Lastly, it analyzes the impact of interactions between hydrolases and natural products on disease development and the challenges in developing microbial-derived enzymes. The overarching goal of this review is to lay a solid theoretical foundation for the advancement of research and development in new natural product drugs and personalized treatment.

Discovering type I cis-AT polyketides through computational mass spectrometry and genome mining with Seq2PKS.

Type 1 polyketides are a major class of natural products used as antiviral, antibiotic, antifungal, antiparasitic, immunosuppressive, and antitumor drugs. Analysis of public microbial genomes leads to the discovery of over sixty thousand type 1 polyketide gene clusters. However, the molecular products of only about a hundred of these clusters are characterized, leaving most metabolites unknown. Characterizing polyketides relies on bioactivity-guided purification, which is expensive and time-consuming. To address this, we present Seq2PKS, a machine learning algorithm that predicts chemical structures derived from Type 1 polyketide synthases. Seq2PKS predicts numerous putative structures for each gene cluster to enhance accuracy. The correct structure is identified using a variable mass spectral database search. Benchmarks show that Seq2PKS outperforms existing methods. Applying Seq2PKS to Actinobacteria datasets, we discover biosynthetic gene clusters for monazomycin, oasomycin A, and 2-aminobenzamide-actiphenol.

Planning biosynthetic pathways of target molecules based on metabolic reaction prediction and AND-OR tree search.

Bioretrosynthesis problem is to predict synthetic routes using substrates for given natural products (NPs). However, the huge number of metabolic reactions leads to a combinatorial explosion of searching space, which is high time-consuming and costly. Here, we propose a framework called BioRetro to predict bioretrosynthesis pathways using a one-step bioretrosynthesis network, termed HybridMLP combined with AND-OR tree heuristic search. The HybridMLP predicts precursors that will produce the target NPs, while the AND-OR tree generates the iterative multi-step biosynthetic pathways. The one-step bioretrosynthesis prediction experiments are conducted on MetaNetX dataset by using HybridMLP, which achieves 46.5%, 74.6%, 81.6% in terms of the top-1, top-5, top-10 accuracies. The great performance demonstrates the effectiveness of HybridMLP in one-step bioretrosynthesis. Besides, the evaluation of two benchmark datasets reveals that BioRetro can significantly improve the speed and success rate in predicting biosynthesis pathways. In addition, the BioRetro is further shown to find the synthetic pathway of compounds, such as ginsenoside F1 with the same substrates as reported but different enzymes, which may be the novel potential enzyme to have better catalytic performance.

A systematic comparison of natural product potential, with an emphasis on RiPPs, by mining of bacteria of three large ecosystems.

The implementation of several global microbiome studies has yielded extensive insights into the biosynthetic potential of natural microbial communities. However, studies on the distribution of several classes of ribosomally synthesized and post-translationally modified peptides (RiPPs), non-ribosomal peptides (NRPs) and polyketides (PKs) in different large microbial ecosystems have been very limited. Here, we collected a large set of metagenome-assembled bacterial genomes from marine, freshwater and terrestrial ecosystems to investigate the biosynthetic potential of these bacteria. We demonstrate the utility of public dataset collections for revealing the different secondary metabolite biosynthetic potentials among these different living environments. We show that there is a higher occurrence of RiPPs in terrestrial systems, while in marine systems, we found relatively more terpene-, NRP-, and PK encoding gene clusters. Among the many new biosynthetic gene clusters (BGCs) identified, we analyzed various Nif-11-like and nitrile hydratase leader peptide (NHLP) containing gene clusters that would merit further study, including promising products, such as mersacidin-, LAP- and proteusin analogs. This research highlights the significance of public datasets in elucidating the biosynthetic potential of microbes in different living environments and underscores the wide bioengineering opportunities within the RiPP family.

The biomolecules of Euglena gracilis: Harnessing biology for natural solutions to future problems.

Over the past decade, the autotrophic and heterotrophic protist Euglena gracilis (E. gracilis) has gained popularity across the studies of environmental science, biosynthesis experiments, and nutritional substitutes. The unique physiology and versatile metabolism of E. gracilis have been a recent topic of interest to many researchers who continue to understand the complexity and possibilities of using E. gracilis biomolecule production. In this review, we present a comprehensive representation of recent literature outlining the various uses of biomolecules derived from E. gracilis across the fields of natural product biosynthesis, as a nutritional substitute, and as bioremediation tools. In addition, we highlight effective strategies for altering metabolite production using abiotic stressors and growth conditions. To better understand metabolite biosynthesis and its role in E. gracilis, integrated studies involving genomics, metabolomics, and proteomics should be considered. Together, we show how the ongoing advancements in E. gracilis related research continue to broaden applications in the biosynthetic sector and highlight future works that would strengthen our understanding of overall Euglena metabolism.

Sorbremnoids A and B: NLRP3 Inflammasome Inhibitors Discovered from Spatially Restricted Crosstalk of Biosynthetic Pathways.

Crosstalk-oriented chemical evolution of natural products (NPs) is an efficacious strategy for generating novel skeletons through coupling reactions between NP fragments. In this study, two NOD-like receptor protein 3 (NLRP3) inflammasome inhibitors, sorbremnoids A and B (1 and 2), with unprecedented chemical architectures were identified from a fungus Penicillium citrinum. Compounds 1 and 2 exemplify rare instances of hybrid NPs formed via a major facilitator superfamily (MFS)-like enzyme by coupling reactive intermediates from two separate biosynthetic gene clusters (BGCs), pcisor and pci56. Both sorbremnoids A and B are NLRP3 inflammasome inhibitors. Sorbremnoid A demonstrated strong inhibition of IL-1ß by directly binding to the NLRP3 protein, inhibiting the assembly and activation of the NLRP3 inflammasome in vitro, with potential application in diabetic refractory wound healing through the suppression of excessive inflammatory responses. This research will inspire the development of anti-NLRP3 inflammasome agents as lead treatments and enhance knowledge pertaining to NPs derived from biosynthetic crosstalk.