Anti-inflammatory and Neuroprotective α-Pyrones from a Marine-Derived Strain of the Fungus Arthrinium arundinis and Their Heterologous Expression.

Fungal linear polyketides, such as α-pyrones with a 6-alkenyl chain, have been a rich source of biologically active compounds. Two new (1 and 2) and four known (3-6) 6-alkenylpyrone polyketides were isolated from a marine-derived strain of the fungus Arthrinium arundinis. Their structures were determined based on extensive spectroscopic analysis. The biosynthetic gene cluster (alt) for alternapyrones was identified from A. arundinis ZSDS-F3 and validated by heterologous expression in Aspergillus nidulans A1145 ΔSTΔEM, which revealed that the cytochrome P450 monooxygenase Alt2' could convert the methyl group 26-CH3 to a carboxyl group to produce 4 from 3. Another cytochrome P450 monooxygenase, Alt3', catalyzed successive hydroxylation, epoxidation, and oxidation steps to produce 1, 2, 5, and 6 from 4. Alternapyrone G (1) not only suppressed M1 polarization in lipopolysaccharide (LPS)-stimulated BV2 microglia but also stimulated dendrite regeneration and neuronal survival after Aß treatment, suggesting alternapyrone G may be utilized as a privileged scaffold for Alzheimer's disease drug discovery.

An Enzymatic Carbon-Carbon Bond Cleavage and Aldol Reaction Cascade Converts an Angular Scaffold into the Linear Tetracyclic Core of Ochraceopones.

Meroterpenoids of the ochraceopones family featuring a linear tetracyclic scaffold exhibit exceptional antiviral and anti-inflammatory activities. The biosynthetic pathway and chemical logic to generate this linear tetracycle, however, remain unknown. In this study, we identified and characterized all biosynthetic enzymes to afford ochraceopones and elucidated the complete biosynthetic pathway. We demonstrated that the linear tetracyclic scaffold of ochraceopones was derived from an angular tetracyclic precursor. A multifunctional cytochrome P450 OchH was validated to catalyze the free-radical-initiated carbon-carbon bond cleavage of the angular tetracycle. Then, a new carbon-carbon bond was verified to be constructed using a new aldolase OchL, which catalyzes an intramolecular aldol reaction to form the linear tetracycle. This carbon-carbon bond fragmentation and aldol reaction cascade features an unprecedented strategy for converting a common angular tetracycle to a distinctive linear tetracyclic scaffold in meroterpenoid biosynthesis.

Discovery of Aburatubolactams Reveals Biosynthetic Logic for Distinct 5/5-Type Polycyclic Tetramate Macrolactams.

A known polycyclic tetramate macrolactam (aburatubolactam C, 3) and three new ones (aburatubolactams D-F, 4-6, respectively) were isolated from the marine-derived Streptomyces sp. SCSIO 40070. The absolute configuration of 3 was established by X-ray analysis. A combinatorial biosynthetic approach unveiled biosynthetic enzymes dictating the formation of distinct 5/5-type ring systems (such as C7-C14 cyclization by AtlB1 in 5 and C6-C13 cyclization by AtlB2 in 6) in aburatubolactams.

Naturally Occurring and Widespread Resistance to Bioactive Natural Products.

Naturally occurring resistances diminish the effectiveness of antibiotics, and present significant challenges to human health. Human activities are usually considered as the main drivers of the dissemination of antibiotic resistance, however, the origin of the clinical antibiotic resistance can be traced to the environmental microbes, and the clinically relevant resistance determinants have already pre-existed in nature before the antibiotics come into clinic. In this concept, we present the naturally occurring and widespread resistance determinants recently discovered during the biosynthesis study of bioactive compounds. These widely prevalent resistances in environmental microbes, including antibiotic producers and non-producers, advance the understanding of the origin of resistance, and provide prediction for the clinically relevant resistance to aid in the rational design of more effective drug analogues to combat resistance.

Deciphering Deoxynybomycin Biosynthesis Reveals Fe(II)/α-Ketoglutarate-Dependent Dioxygenase-Catalyzed Oxazoline Ring Formation and Decomposition.

The antibacterial agents deoxynybomycin (DNM) and nybomycin (NM) have a unique tetracyclic structure featuring an angularly fused 4-oxazoline ring. Here, we report the identification of key enzymes responsible for forming the 4-oxazoline ring in Embleya hyalina NBRC 13850 by comparative bioinformatics analysis of the biosynthetic gene clusters encoding structurally similar natural products DNM, deoxynyboquinone (DNQ), and diazaquinomycins (DAQs). The N-methyltransferase DnmS plays a crucial role in catalyzing the N-dimethylation of a tricyclic precursor prenybomycin to generate NM D; subsequently, the Fe(II)/α-ketoglutarate-dependent dioxygenase (Fe/αKGD) DnmT catalyzes the formation of a 4-oxazoline ring from NM D to produce DNM; finally, a second Fe/αKGD DnmU catalyzes the C-12 hydroxylation of DNM to yield NM. Strikingly, DnmT is shown to display unexpected functions to also catalyze the decomposition of the 4-oxazoline ring and the N-demethylation, thereby converting DNM back to prenybomycin, to putatively serve as a manner to control the intracellular yield of DNM. Structure modeling, site-directed mutagenesis, and quantum mechanics calculations provide mechanistic insights into the DnmT-catalyzed reactions. This work expands our understanding of the functional diversity of Fe/αKGDs in natural product biosynthesis.

A Mechanistic Understanding of the Distinct Regio- and Chemoselectivity of Multifunctional P450s by Structural Comparison of IkaD and CftA Complexed with Common Substrates.

Regio- and chemoselective C-H activation at multi-positions of a single molecule is fascinating but chemically challenging. The homologous cytochrome P450 enzymes IkaD and CftA catalyze multiple C-H oxidations on the same polycyclic tetramate macrolactam (PoTeM) ikarugamycin, with distinct regio- and chemoselectivity. Herein we provide mechanistic understanding of their functional differences by solving crystal structures of IkaD and CftA in complex with ikarugamycin and unnatural substrates. Distinct conformations of the F/G region in IkaD and CftA are found to differentiate the orientation of PoTeM substrates, by causing different binding patterns with polar moieties to determine site selection, oxidation order, and chemoselectivity. Fine-tuning the polar subpocket altered the regioselectivity of IkaD, indicating that substrate re-orientation by mutating residues distal to the oxidation site could serve as an important method in future engineering of P450 enzymes.

Genome Mining and Biosynthetic Reconstitution of Fungal Depsidone Mollicellins Reveal a Dual Functional Cytochrome P450 for Ether Formation.

Depsidones are significant in structural diversity and broad in biological activities; however, their biosynthetic pathways have not been well understood and have attracted considerable attention. Herein, we heterologously reconstituted a depsidone encoding gene cluster from Ovatospora sp. SCSIO SY280D in Aspergillus nidulans A1145, leading to production of mollicellins, a representative family of depsidones, and discovering a bifunctional P450 monooxygenase that catalyzes both ether formation and hydroxylation in the biosynthesis of the mollicellins. The functions of a decarboxylase and an aromatic prenyltransferase are also characterized to understand the tailoring modification steps. This work provides important insights into the biosynthesis of mollicellins.

Discovery of Tetronate-Containing Kongjuemycins from a Coral-Associated Actinomycete and Elucidation of Their Biosynthetic Origin.

Tetronate antibiotics make up a growing family of natural products with a wide variety of biological activities. Herein, we report four new tetronates kongjuemycins (KJMs, 5-8) from a coral-associated actinomycete Pseudonocardia kongjuensis SCSIO 11457, and the identification and characterization of the KJM biosynthetic gene cluster (kjm) by heterologous expression, comparative genomic analysis, isotope labeling, and gene knockout studies. The biosynthesis of KJMs is demonstrated to harness diverse precursors from primary metabolism for building secondary metabolites.

Co-crystal structure provides insights on transaminase CrmG recognition amino donor L-Arg.

ω-transaminase has attracted growing attention for chiral amine synthesis, although it commonly suffers from severe by-product inhibition. ω-transaminase CrmG is critical for the biosynthesis of Caerulomycin A, a natural product that possesses broad bioactivity, including immunosuppressive and anti-cancer. Compared to L-Arg, amino donor L-Glu, L-Gln or L-Ala is more preferred by CrmG. In this study, we determined the crystal structure of CrmG in complex with amino donor L-Arg, unveiling the detailed binding mode. Specifically, L-Arg exhibits an extensive contact with aromatic residues F207 and W223 on the roof of CrmG active site via cation-π network. This interaction may render the deamination by-product of L-Arg to be an inhibitor against PMP-bound CrmG by stabilizing its flexible roof, thus reducing the reactivity of L-Arg as an amino donor for CrmG. These data provide further evidence to support our previous proposal that CrmG can overcome inhibition from those by-products that are not able to stabilize the flexible roof of active site in PMP-bound CrmG. Thus, our result can not only facilitate the biosynthesis of CRM A but also be beneficial for the rational design of ω-transaminase to bypass by-product inhibition.

Biosynthesis and Engineered Overproduction of Everninomicins with Promising Activity against Multidrug-Resistant Bacteria.

Ribosome-targeting oligosaccharides, everninomicins (EVNs), are promising drug leads with a unique mode of action distinct from that of currently used antibiotics in human therapy. However, the low yields in natural microbial producers hamper an efficient preparation of EVNs for detailed structure-activity relationship analysis. Herein, we enhance the production of EVNs by duplicating the biosynthetic gene cluster (BGC) in Micromonospora sp. SCSIO 07395 and thus obtain multiple EVNs that are sufficient for bioactivity evaluation. EVNs (1-5) are shown to significantly inhibit the growth of multidrug-resistant Gram-positive staphylococcal, enterococcal, and streptococcal strains and Gram-negative pathogens Acinetobacter baumannii and Vibrio cholerae, with micromolar to nanomolar potency, which are comparable or superior to vancomycin, linezolid, and daptomycin. Furthermore, the BGC duplication strategy is proven effective in stepwisely improving titers of the bioactive EVN M (5) from the trace amount to 98.6 mg L-1. Our findings demonstrate the utility of a bioengineering approach for enhanced production and chemical diversification of the medicinally promising EVNs.

A Widespread Glycosidase Confers Lobophorin Resistance and Host-Dependent Structural Diversity.

Identifying new environmental resistance determinants is significant to combat rising antibiotic resistance. Herein we report the unexpected correlation of a lobophorin (LOB) resistance-related glycosidase KijX with the host-dependent chemical diversity of LOBs, by a process of glycosylation, deglycosylation and reglycosylation. KijX homologues are widespread among bacteria, archaea and fungi, and encode the same glycohydrolytic activity on LOBs. The crystal structure of AcvX (a KijX homologue) shows a similar fold to that of the glycoside hydrolase family 113 and a special negatively charged groove to accommodate and deglycosylate LOBs. Antagonistic assays indicate kijX as a defense weapon of actinomycetes to combat LOB producers in environment, reflecting an elegant coevolution relationship. Our study provides insight into the KijX-related glycosidases as preexisting resistance determinants and represents an example of resistance genes accidentally integrated into natural product assembly.

Biosynthetic Diversification of Fidaxomicin Aglycones by Heterologous Expression and Promoter Refactoring.

Fidaxomicin (Dificid) is a commercial macrolide antibiotic for treating Clostridium difficile infection. Total synthesis of fidaxomicin and its aglycone had been achieved through different synthetic schemes. In this study, an alternative biological route to afford the unique 18-membered macrolactone aglycone of fidaxomicin was developed. The promoter refactored fidaxomicin biosynthetic gene cluster from Dactylosporangium aurantiacum was expressed in the commonly used host Streptomyces albus J1074, thereby delivering five structurally diverse fidaxomicin aglycones with the corresponding titers ranging from 4.9 to 15.0 mg L-1. In general, these results validated a biological strategy to construct and diversify fidaxomicin aglycones on the basis of promoter refactoring and heterologous expression.

Aromatic Polyketides from the Mangrove-Derived Streptomyces sp. SCSIO 40069.

A chemical investigation of Streptomyces sp. SCSIO 40069 resulted in the isolation of a series of aromatic polyketides with rare skeletons, including five new compounds RM18c-RM18g (1-5) and three known ones (6-8). Their structures and absolute configurations were determined by diverse methods, including HRMS and NMR spectra, chemical reaction, Snatzke's method, quantum mechanical-nuclear magnetic resonance (QM-NMR), and X-ray crystallographic analysis. Compounds 1, 2, 4b, and 8 displayed moderate or weak antibacterial activities.

Totopotensamide Congeners from a Halogenase-Inactivated Mutant.

The installation of halogen atoms into aromatic and less activated polyketide substrates by halogenases is a powerful strategy to tune the bioactivity, bioavailability, and reactivity of compounds. In the biosynthetic pathway of totopotensamide A (1), the halogenase TotH was confirmed in vivo to catalyze the C-4 chlorination to form the nonproteinogenic amino acid ClMeDPG. Herein, we report the isolation, structure elucidation, and bioactivity evaluation of six new deschloro totopotensamide (TPM) congeners TPMs H2-H7 (5-10) from the totH-inactivated strain and the proposed absolute configuration of the polyketide chain in TPMs using 4 as a model compound by a combination of the JBCA and bioinformatic analysis. Compounds 5, 6, 8, and 9 displayed cytotoxicity against the A549, PANC-1, Calu3, and BXPC3 cell lines with IC50 values ranging from 2.3 to 9.7 µM.

Genomics-Driven Discovery of Benzoxazole Alkaloids from the Marine-Derived Micromonospora sp. SCSIO 07395.

Benzoxazole alkaloids exhibit a diverse array of structures and interesting biological activities. Herein we report the identification of a benzoxazole alkaloid-encoding biosynthetic gene cluster (mich BGC) in the marine-derived actinomycete Micromonospora sp. SCSIO 07395 and the heterologous expression of this BGC in Streptomyces albus. This approach led to the discovery of five new benzoxazole alkaloids microechmycin A-E (1-5), and a previously synthesized compound 6. Their structures were elucidated by HRESIMS and 1D and 2D NMR data. Microechmycin A (1) showed moderate antibacterial activity against Micrococcus luteus SCSIO ML01 with the minimal inhibitory concentration (MIC) value of 8 µg mL-1.

Discovery of Efrotomycin Congeners and Heterologous Expression-Based Insights into the Self-Resistance Mechanism.

Four new efrotomycins, A1-A4 (1-4), were isolated from the salt mine-derived Amycolatopsis cihanbeyliensis DSM 45679 and structurally determined. Efrotomycins A3 (3) and A4 (4) feature a tetrahydrofuran ring configured distinctly from known elfamycins. Heterologous expression of the efrotomycin gene cluster (efr BGC) in Streptomyces lividans SBT18 led to efrotomycin B1 (5), the yield of which was improved several fold upon introduction of the transporter gene efrT, a putative self-resistance determinant outside of the efr BGC.

Biochemical and structural insights of multifunctional flavin-dependent monooxygenase FlsO1-catalyzed unexpected xanthone formation.

Xanthone-containing natural products display diverse pharmacological properties. The biosynthetic mechanisms of the xanthone formation have not been well documented. Here we show that the flavoprotein monooxygenase FlsO1 in the biosynthesis of fluostatins not only functionally compensates for the monooxygenase FlsO2 in converting prejadomycin to dehydrorabelomycin, but also unexpectedly converts prejadomycin to xanthone-containing products by catalyzing three successive oxidations including hydroxylation, epoxidation and Baeyer-Villiger oxidation. We also provide biochemical evidence to support the physiological role of FlsO1 as the benzo[b]-fluorene C5-hydrolase by using nenestatin C as a substrate mimic. Finally, we resolve the crystal structure of FlsO1 in complex with the cofactor flavin adenine dinucleotide close to the "in" conformation to enable the construction of reactive substrate-docking models to understand the basis of a single enzyme-catalyzed multiple oxidations. This study highlights a mechanistic perspective for the enzymatic xanthone formation in actinomycetes and sets an example for the versatile functions of flavoproteins.

Flavin-enabled reductive and oxidative epoxide ring opening reactions.

Epoxide ring opening reactions are common and important in both biological processes and synthetic applications and can be catalyzed in a non-redox manner by epoxide hydrolases or reductively by oxidoreductases. Here we report that fluostatins (FSTs), a family of atypical angucyclines with a benzofluorene core, can undergo nonenzyme-catalyzed epoxide ring opening reactions in the presence of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH). The 2,3-epoxide ring in FST C is shown to open reductively via a putative enol intermediate, or oxidatively via a peroxylated intermediate with molecular oxygen as the oxidant. These reactions lead to multiple products with different redox states that possess a single hydroxyl group at C-2, a 2,3-vicinal diol, a contracted five-membered A-ring, or an expanded seven-membered A-ring. Similar reactions also take place in both natural products and other organic compounds harboring an epoxide adjacent to a carbonyl group that is conjugated to an aromatic moiety. Our findings extend the repertoire of known flavin chemistry that may provide new and useful tools for organic synthesis.

Antifungal Macrolides Kongjuemycins from Coral-Associated Rare Actinomycete Pseudonocardia kongjuensis SCSIO 11457.

Four new macrolides, kongjuemycins A and B1-B3 (1-4), were isolated from a coral-associated actinomycete Pseudonocardia kongjuensis SCSIO 11457. Their structures were characterized by comprehensive spectroscopic analysis and single-crystal X-ray diffraction. The absolute configurations of 1 and 2 were established by electronic circular dichroism calculation and the modified Mosher's method. Kongjuemycins displayed antifungal activity against three phytopathogenic fungi.

Natural products from mangrove sediments-derived microbes: Structural diversity, bioactivities, biosynthesis, and total synthesis.

The mangrove forests are a complex ecosystem, and the microbial communities in mangrove sediments play a critical role in the biogeochemical cycles of mangrove ecosystems. Mangrove sediments-derived microbes (MSM), as a rich reservoir of natural product diversity, could be utilized in the exploration of new antibiotics or drugs. To understand the structural diversity and bioactivities of the metabolites of MSM, this review for the first time provides a comprehensive overview of 519 natural products isolated from MSM with their bioactivities, up to 2021. Most of the structural types of these compounds are alkaloids, lactones, xanthones, quinones, terpenoids, and steroids. Among them, 210 compounds are obtained from bacteria, most of which are from Streptomyces, while 309 compounds are from fungus, especially genus Aspergillus and Penicillium. The pharmacological mechanisms of some representative lead compounds are well studied, revealing that they have important medicinal potentials, such as piericidins with anti-renal cell cancer effects, azalomycins with anti-MRSA activities, and ophiobolins as antineoplastic agents. The biosynthetic pathways of representative natural products from MSM have also been summarized, especially ikarugamycin, piericidins, divergolides, and azalomycins. In addition, the total synthetic strategies of representative secondary metabolites from MSM are also reviewed, such as piericidin A and borrelidin. This review provides an important reference for the research status of natural products isolated from MSM and the lead compounds worthy of further development, and reveals that MSM have important medicinal values and are worthy of further development.