Constructing a Myxobacterial Natural Product Database to Facilitate NMR-Based Metabolomics Bioprospecting of Myxobacteria.

Myxobacteria are fascinating prokaryotes featuring a potent capacity for producing a wealth of bioactive molecules with intricate chemical topology as well as intriguing enzymology, and thus it is critical to developing an efficient pipeline for bioprospecting. Herein, we construct the database MyxoDB, the first public compendium solely dedicated to myxobacteria, which enabled us to provide an overview of the structural diversity and taxonomic distribution of known myxobacterial natural products. Moreover, we demonstrated that the cutting-edge NMR-based metabolomics was effective to differentiate the biosynthetic priority of myxobacteria, whereby MyxoDB could greatly streamline the dereplication of multifarious known compounds and accordingly speed up the discovery of new compounds. This led to the rapid identification of a class of linear di-lipopeptides (archangimins) and a rare rearranged sterol (corasterol) that were endowed with unique chemical architectures and/or biosynthetic enzymology. We also showcased that NMR-based metabolomics, MyxoDB, and genomics can also work concertedly to accelerate the targeted discovery of a polyketidic compound pyxipyrrolone C. All in all, this study sets the stage for the discovery of many more novel natural products from underexplored myxobacterial resources.

Astellolides R-W, Drimane-Type Sesquiterpenoids from an Aspergillus parasiticus Strain Associated with an Isopod.

Sesquiterpenoids with a cage-like multiring frame are rarely found in nature. Mining of the isopod-derived fungus Aspergillus parasiticus SDU001 by the one strain-many compounds (OSMAC) strategy unexpectedly led to the discovery of fungal drimane-type sesquiterpenoids astellolide R (1), featuring an unusual cage-like 6/6/5/6/5 pentacyclic ring system, astellolide S (2), possessing a rare nicotinic acid building block, and astellolides T-W (3-6). Their structures were comprehensively assigned by spectroscopic data analysis, single-crystal X-ray diffraction, and electronic circular dichroism calculations. Furthermore, compounds 3 and 5 exhibited anti-inflammatory activity by inhibiting the lipopolyssacharide-induced NO production in RAW264.7 macrophages with IC50 values of 6.1 ± 0.8 and 6.8 ± 0.8 µM, respectively. A putative biosynthetic pathway for 1 is proposed. Our results enlarge the chemical space of the drimane-type sesquiterpenoids generated from endophytic fungi.

Silica Capsules Templated from Metal-Organic Frameworks for Enzyme Immobilization and Catalysis.

Inspired by the unique biological microenvironments of eukaryotic cells, hollow capsules are promising to immobilize enzymes due to their advantages for physical protection and improved activity of enzymes. Herein, we report a facile method to fabricate silica (SiO2) capsules using zeolitic imidazole framework-8 nanoparticles (ZIF-8 NPs) as templates for enzyme immobilization and catalysis. Enzyme-encapsulated SiO2 capsules are obtained by encapsulation of enzymes in ZIF-8 NPs and subsequent coating of silica layers, followed by the removal of templates in a mild condition (i.e., ethylenediaminetetraacetic acid (EDTA) solution). The enzyme (i.e., horseradish peroxidase, HRP) activity in SiO2 capsules is improved more than 15 times compared to that of enzyme-loaded ZIF-8 NPs. Enzymes in SiO2 capsules maintain a high relative activity after being subjected to high temperature, enzymolysis, and recycling compared to free enzymes. In addition, multienzymes (e.g., glucose oxidase and HRP) can also be coencapsulated within SiO2 capsules to show a reaction with a high cascade catalytic efficacy. This work provides a versatile strategy for enzyme immobilization and protection with potential applications in biocatalysis.

The Discovery and Biosynthesis of Nicotinic Myxochelins from an Archangium sp. SDU34.

Myxobacteria are a prolific source of structurally diverse natural products, and one of the best-studied myxobacterial products is the siderophore myxochelin. Herein, we report two new compounds, myxochelins N (1) and O (2), that are nicotinic paralogs of myxochelin A, from the terrestrial myxobacterium Archangium sp. SDU34; 2 is functionalized with a rare 2-oxazolidinone. A precursor-feeding experiment implied that the biosynthesis of 1 or 2 was due to altered substrate specificity of the loading module of MxcE, which likely accepts nicotinic acid and benzoic acid instead of more conventional 2,3-dihydroxybenzoic acid. We also employed a phylogenomic approach to map the evolutionary relationships of the myxochelin biosynthetic gene clusters (BGCs) in all the available myxobacterial genomes, to pave the way for the future discovery of potentially hidden myxochelin derivatives. Although the biological function of 1 and 2 is unclear yet, this work underpins that even extensively studied BGCs in myxobacteria can still produce new chemistry.

Saccharochelins A-H, Cytotoxic Amphiphilic Siderophores from the Rare Marine Actinomycete Saccharothrix sp. D09.

Siderophores are secreted by microorganisms to survive in iron-depleted conditions, and they also possess tremendous therapeutic potential. Genomic-inspired isolation facilitated the identification of eight amphiphilic siderophores, saccharochelins A-H (1-8), from a rare marine-derived Saccharothrix species. Saccharochelins feature a series of fatty acyl groups appended to the same tetrapeptide skeleton. With the help of gene disruption and heterologous expression, we identified the saccharochelin biosynthetic pathway. The diversity of saccharochelins originates from the flexible specificity of the starter condensation (CS) domain at the beginning of the nonribosomal peptide synthetase (NRPS) toward various fatty acyl substrates. Saccharochelins showed cytotoxicity against several human tumor cell lines, with IC50 values ranging from 2.3 to 17 µM. Additionally, the fatty acid side chains of the saccharochelins remarkably affected the cytotoxicity, suggesting changing the N-terminal acyl groups of lipopeptides may be a promising approach to produce more potent derivatives.

Genome-Guided Discovery of Highly Oxygenated Aromatic Polyketides, Saccharothrixins D-M, from the Rare Marine Actinomycete Saccharothrix sp. D09.

Angucyclines and angucyclinones are aromatic polyketides with intriguing structures and therapeutic value. Genome mining of the rare marine actinomycete Saccharothrix sp. D09 led to the identification of a type II polyketide synthase biosynthetic gene cluster, sxn, which encodes several distinct subclasses of oxidoreductases, implying that this strain has the potential to produce novel polycyclic aromatic polyketides with unusual redox modifications. The "one strain-many compounds" (OSMAC) strategy and comparative metabolite analysis facilitated the discovery of 20 angucycline derivatives from the D09 strain, including six new highly oxygenated saccharothrixins D-I (1-6), four new glycosylated saccharothrixins J-M (7-10), and 10 known analogues (11-20). Their structures were elucidated based on detailed HRESIMS, NMR spectroscopic, and X-ray crystallographic analysis. With the help of gene disruption and heterologous expression, we proposed their plausible biosynthetic pathways. In addition, compounds 3, 4, and 8 showed antibacterial activity against Helicobacter pylori with MIC values ranging from 16 to 32 µg/mL. Compound 3 also revealed anti-inflammatory activity by inhibiting the production of NO with an IC50 value of 28 µM.

Dual pH-Responsive Polymer Nanogels with a Core-Shell Structure for Improved Cell Association.

We report the fabrication of polymer nanogels with a pH-responsive core and a pH-sheddable shell and investigate the pH-dependent cell association of the pH-responsive polymer nanogels. The pH-responsive core composed of poly(2-diisopropylaminoethyl methacrylate) (PDPA) with a pKa ≈ 6.2 was synthesized by using polymerization in emulsion droplets. The pH-sheddable poly(ethylene glycol) (PEG) shell was coated on the amine-modified PDPA nanogels by an acid-degradable amide bond. The PEG shell is cleavable in response to the acidic tumor microenvironment, and subsequently, the surface charge of the nanogels can be reversed, which effectively enhances cellular association of these nanogels. The reported pH-responsive polymer nanogels provide a promising way for the better understanding of bio-nano interactions and potentially enrich the application of therapeutic delivery for cancer therapy.

Ribosomal Incorporation of Lithocholic Acid into Peptides for the De Novo Discovery Of Peptide-Lithocholic Acid Hybrid Macrocyclic Peptides.

Peptide-bile acid hybrids offer promising drug candidates due to enhanced pharmacological properties, such as improved protease resistance and oral bioavailability. However, it remains unknown whether bile acids can be incorporated into peptide chains by the ribosome to produce a peptide-bile acid hybrid macrocyclic peptide library for target-based de novo screening. In this study, we achieved the ribosomal incorporation of lithocholic acid (LCA)-d-tyrosine into peptide chains. This led to the construction of a peptide-LCA hybrid macrocyclic peptide library, which enabled the identification of peptides TP-2C-4L3 (targeting Trop2) and EP-2C-4L5 (targeting EphA2) with strong binding affinities. Notably, LCA was found to directly participate in binding to EphA2 and confer on the peptides improved stability and resistance to proteases. Cell staining experiments confirmed the high specificity of the peptides for targeting Trop2 and EphA2. This study highlights the benefits of LCA in peptides and paves the way for de novo discovery of stable peptide-LCA hybrid drugs.

Biosynthesis of trans-AT PKS-Derived Shuangdaolides Featuring a trans-acting Enzyme for Online Epoxidation.

Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) synthesize natural products with intricate structures and potent biological activities. They generally contain various unusual modules or trans-acting enzymes. Herein, we report the trans-AT PKS-derived biosynthetic pathway of the shuangdaolide with a rare internal 2-hydroxycyclopentenone moiety. The multidomain protein SdlR catalyzes the synthesis of 16,17-epoxide during polyketide chain elongation. The SdlR contains a ketoreductase, an acyl carrier protein, a flavoprotein monooxygenase, and a serine hydrolase domain. This online epoxidation occurs at unusual positions away from the thioester. Then, two tailoring enzymes, SdlB and SdlQ, convert a methylene to a carbonyl group and oxidize a hydroxyl group to a carbonyl group, respectively. The following spontaneous opening of 16,17-epoxide induces the formation of a new C-C bond to generate the 2-hydroxycyclopentenone moiety. The characterization of the shuangdaolide pathway extends the understanding of the trans-AT PKSs, facilitating the mining and identification of this class of natural products.

Combining NMR-Based Metabolic Profiling and Genome Mining for the Accelerated Discovery of Archangiumide, an Allenic Macrolide from the Myxobacterium Archangium violaceum SDU8.

An unprecedented 19-membered allenic macrolide archangiumide (1) was discovered from the myxobacterium Archangium violaceum SDU8 by integrating NMR-based metabolic profiling and genome mining. Its biosynthesis pathway was proposed based on the architectural analysis of the encoding trans-AT PKS genes and validated by isotope labeling. The methodology of combing 2D NMR-based metabolic profiling and bioinformatics-aided structure prediction, as exemplified by this study, is anticipated to improve discovery efficiency of a broader range of microbial "dark matter".