P450-Modified Multicyclic Cyclophane-Containing Ribosomally Synthesized and Post-Translationally Modified Peptides.

Cyclic peptides with cyclophane linkers are an attractive compound type owing to the fine-tuned rigid three-dimensional structures and unusual biophysical features. Cytochrome P450 enzymes are capable of catalyzing not only the C-C and C-O oxidative coupling reactions found in vancomycin and other nonribosomal peptides (NRPs), but they also exhibit novel catalytic activities to generate cyclic ribosomally synthesized and post-translationally modified peptides (RiPPs) through cyclophane linkage. To discover more P450-modified multicyclic RiPPs, we set out to find cryptic and unknown P450-modified RiPP biosynthetic gene clusters (BGCs) through genome mining. Synergized bioinformatic analysis reveals that P450-modified RiPP BGCs are broadly distributed in bacteria and can be classified into 11 classes. Focusing on two classes of P450-modified RiPP BGCs where precursor peptides contain multiple conserved aromatic amino acid residues, we characterized 11 novel P450-modified multicyclic RiPPs with different cyclophane linkers through heterologous expression. Further mutation of the key ring-forming residues and combinatorial biosynthesis study revealed the order of bond formation and the specificity of P450s. This study reveals the functional diversity of P450 enzymes involved in the cyclophane-containing RiPPs and indicates that P450 enzymes are promising tools for rapidly obtaining structurally diverse cyclic peptide derivatives.

P450-Modified Ribosomally Synthesized Peptides with Aromatic Cross-Links.

Cyclization of linear peptides is an effective strategy to convert flexible molecules into rigid compounds, which is of great significance for enhancing the peptide stability and bioactivity. Despite significant advances in the past few decades, Nature and chemists' ability to macrocyclize linear peptides is still quite limited. P450 enzymes have been reported to catalyze macrocyclization of peptides through cross-linkers between aromatic amino acids with only three examples. Herein, we developed an efficient workflow for the identification of P450-modified RiPPs in bacterial genomes, resulting in the discovery of a large number of P450-modified RiPP gene clusters. Combined with subsequent expression and structural characterization of the products, we have identified 11 novel P450-modified RiPPs with different cross-linking patterns from four distinct classes. Our results greatly expand the structural diversity of P450-modified RiPPs and provide new insights and enzymatic tools for the production of cyclic peptides.

Building Streptomyces albus as a chassis for synthesis of bacterial terpenoids.

Terpenoids comprise the most chemically and structurally diverse family of natural products. In contrast to the huge numbers of terpenoids discovered from plants and fungi, only a relatively small number of terpenoids were reported from bacteria. Recent genomic data in bacteria suggest that a large number of biosynthetic gene clusters encoding terpenoids remain uncharacterized. In order to enable the functional characterization of terpene synthase and relevant tailoring enzymes, we selected and optimized an expression system based on a Streptomyces chassis. Through genome mining, 16 distinct bacterial terpene biosynthetic gene clusters were selected and 13 of them were successfully expressed in the Streptomyces chassis, leading to characterization of 11 terpene skeletons including three new ones, representing an ∼80% success rate. In addition, after functional expression of tailoring genes, 18 novel distinct terpenoids were isolated and characterized. This work demonstrates the advantages of a Streptomyces chassis which not only enabled the successful production of bacterial terpene synthases, but also enabled functional expression of tailoring genes, especially P450, for terpenoid modification.

Total Biosynthesis of Mutaxanthene Unveils a Flavoprotein Monooxygenase Catalyzing Xanthene Ring Formation.

Flavoprotein monooxygenases (FPMOs) play important roles in generating structural complexity and diversity in natural products biosynthesized by type II polyketide synthases (PKSs). In this study, we used genome mining to discover novel mutaxanthene analogues and investigated the biosynthesis of these aromatic polyketides and their unusual xanthene framework. We determined the complete biosynthetic pathway of mutaxathene through in vivo gene deletion and in vitro biochemical experiments. We show that a multifunctional FPMO, MtxO4, catalyzes ring rearrangement and generates the required xanthene ring through a multistep transformation. In addition, we successfully obtained all necessary enzymes for in vitro reconstitution and completed the total biosynthesis of mutaxanthene in a stepwise manner. Our results revealed the formation of a rare xanthene ring in type II polyketide biosynthesis, and demonstrate the potential of using total biosynthesis for the discovery of natural products synthesized by type II PKSs.

Cytochrome†P450 Catalyzes Benzene Ring Formation in the Biosynthesis of Trialkyl-Substituted Aromatic Polyketides.

Lorneic acid and related natural products are characterized by a trialkyl-substituted benzene ring. The formation of the aromatic core in the middle of the polyketide chain is unusual. We characterized a cytochrome P450 enzyme that can catalyze the hallmark benzene ring formation from an acyclic polyene substrate through genetic and biochemical analysis. Using this P450 as a beacon for genome mining, we obtained 12 homologous type I polyketide synthase (PKS) gene clusters, among which two gene clusters are activated and able to produce trialkyl-substituted aromatic polyketides. Quantum chemical calculations were performed to elucidate the plausible mechanism for P450-catalyzed benzene ring formation. Our work expands our knowledge of the catalytic diversity of cytochrome P450.

New antibacterial depsidones from an ant-derived fungus Spiromastix sp. MY-1.

Six new (1-6) and seven known depsidones (7-13) were isolated from the culture of an ant (Monomorium chinensis)-derived fungus Spiromastix sp. MY-1. Their structures were elucidated by extensive spectroscopic analysis including high resolution MS, 1D and 2D NMR data. The new bromide depsidones were obtained through supplementing potassium bromide in the fermentation medium of Spiromastix sp. MY-1. All isolated compounds showed various bioactivities against the tested phytopathogenic bacteria. Particularly, new bromide compound 4, named spiromastixone S, exhibited the strongest activity against Xanthomonas oryzae pv. oryzae with a MIC value of 5.2 µmol·-1.

AvmM catalyses macrocyclization through dehydration/Michael-type addition in alchivemycin A biosynthesis.

Macrocyclization is an important process that affords morphed scaffold in biosynthesis of bioactive natural products. Nature has adapted diverse biosynthetic strategies to form macrocycles. In this work, we report the identification and characterization of a small enzyme AvmM that can catalyze the construction of a 16-membered macrocyclic ring in the biosynthesis of alchivemycin A (1). We show through in vivo gene deletion, in vitro biochemical assay and isotope labelling experiments that AvmM catalyzes tandem dehydration and Michael-type addition to generate the core scaffold of 1. Mechanistic studies by crystallography, DFT calculations and MD simulations of AvmM reveal that the reactions are achieved with assistance from the special tenuazonic acid like moiety of substrate. Our results thus uncover an uncharacterized macrocyclization strategy in natural product biosynthesis.

Biosynthesis of Sordarin Revealing a Diels-Alderase for the Formation of the Norbornene Skeleton.

Sordarin (1) is a fungal diterpene glycoside that displays potent antifungal bioactivity through inhibition of elongation factor 2. The structures of sordarin and related compounds feature a highly rearranged tetracyclic diterpene core. In this study, we identified a concise pathway in the biosynthesis of sordarin. A diterpene cyclase (SdnA) generates the 5/8/5 cycloaraneosene framework, which is decorated by a set of P450s that catalyze a series of oxidation reactions, including hydroxylation, desaturation, and C-C bond oxidative cleavage, to give a carboxylate intermediate with a terminal alkene and a cyclopentadiene moiety. A novel Diels-Alderase SdnG catalyzes an intramolecular Diels-Alder (IMDA) reaction on this intermediate to forge the sordarin core structure. Subsequent methyl hydroxylation and glycosylation complete the biosynthesis of sordarin. Our work discloses a new strategy used by nature for the formation of the rearranged diterpene skeleton.

Spirocitromycetin, a Fungal Polyketide with an Antiosteoporotic Pharmacophore.

Spirocitromycetin, an antiosteoporotic polyketide bearing a unique spirocycle, was characterized from a human mucus sputum-derived Penicillium velutinum. Its structure and absolute configuration were elucidated spectrally, with its biosynthetic pathway likely mediated via polivione, a reported heptaketide. Spirocitromycetin was shown to be antiosteoporotic at 0.1 µM in the prednisolone-induced osteoporotic zebrafish model. A combination of spirocitromycetin variant synthesis and bioassay has identified 5'-methyl-3'H-spiro[chromane-3,2'-furan]-3',4-dione as an unreported antiosteroporotic pharmacophore. Collectively, this work offers new starting (sub)structures that may be of significance for antiosteoporotic drug discovery.

In Vitro Reconstitution of Cinnamoyl Moiety Reveals Two Distinct Cyclases for Benzene Ring Formation.

Cinnamoyl-containing natural products (CCNPs) are a small class of bacterial metabolites with notable bioactivities. The biosynthesis of cinnamoyl moiety has been proposed to be assembled by an unusual highly reducing (HR) type II polyketide synthases (PKS). However, the biosynthetic route, especially the cyclization step for the benzene ring formation, remains unclear. In this work, we successfully reconstituted the pathway of cinnamoyl moiety in kitacinnamycin biosynthesis through a step-wise approach in vitro and demonstrated that a three-protein complex, Kcn17-Kcn18-Kcn19, can catalyze 6π-electrocyclization followed by dehydrogenation to form the benzene ring. We found that the three-protein homologues were widely distributed among 207 HR type II PKS biosynthetic gene clusters including five known CCNPs. In contrast, in the biosynthesis of youssoufene, a cinnamoyl-containing polyene, we identified that the benzene ring formation was accomplished by a distinct orphan protein. Thus, our work resolved the long-standing mystery in cinnamoyl biosynthesis and revealed two distinct enzymes that can synthesize benzene rings via polyene precursors.

Alternatones A and B, two polyketides possessing novel skeletons from entophyte Alternaria alternate L-10.

On our ongoing searching for bioactive natural products derived from entophytes, two polyketides possessing novel skeletons, alternatones A-B (1-2), were identified from the culture of Alternaria alternate L-10. Their structures were established by a combination of spectroscopic and single-crystal X-ray diffraction with Cu Ka radiation. Alternatone A (1) exhibited cytotoxic activity against human hepatoma carcinoma HepG-2 cell line. The putative biosynthetic pathways for compounds 1-2 were also proposed.

An NADPH-Dependent Ketoreductase Catalyses the Tetracyclic to Pentacyclic Skeletal Rearrangement in Chartreusin Biosynthesis.

Redox tailoring enzymes play key roles in generating structural complexity and diversity in type II polyketides. In chartreusin biosynthesis, the early 13 C-labeling experiments and bioinformatic analysis suggest the unusual aglycone is originated from a tetracyclic anthracyclic polyketide. Here, we demonstrated that the carbon skeleton rearrangement from a linear anthracyclic polyketide to an angular pentacyclic biosynthetic intermediate requires two redox enzymes. The flavin-dependent monooxygenase ChaZ catalyses a Baeyer-Villiger oxidation on resomycin C to form a seven-membered lactone. Subsequently, a ketoreductase ChaE rearranges the carbon skeleton and affords the α-pyrone containing pentacyclic intermediate in an NADPH-dependent manner via tandem reactions including the reduction of the lactone carbonyl group, Aldol-type reaction, followed by a spontaneous γ-lactone ring formation, oxidation and aromatization. Our work reveals an unprecedented function of a ketoreductase that contributes to generate structural complexity of aromatic polyketide.

Discovery and biosynthesis of guanipiperazine from a NRPS-like pathway.

Nonribosomal peptide synthetases (NRPSs) are modular enzymes that use a thiotemplate mechanism to assemble the peptide backbones of structurally diverse and biologically active natural products in bacteria and fungi. Unlike these canonical multi-modular NRPSs, single-module NRPS-like enzymes, which lack the key condensation (C) domain, are rare in bacteria, and have been largely unexplored to date. Here, we report the discovery of a gene cluster (gup) encoding a NRPS-like megasynthetase through genome mining. Heterologous expression of the gup cluster led to the production of two unprecedented alkaloids, guanipiperazines A and B. The NRPS-like enzyme activates two l-tyrosine molecules, reduces them to the corresponding amino aldehydes, and forms an unstable imine product. The subsequent enzymatic reduction affords piperazine, which can be morphed by a P450 monooxygenase into a highly strained compound through C-O bond formation. Further intermolecular oxidative coupling forming the C-C or C-O bond is catalyzed by another P450 enzyme. This work reveals the huge potential of NRPS-like biosynthetic gene clusters in the discovery of novel natural products.

Acautalides A-C, Neuroprotective Diels-Alder Adducts from Solid-State Cultivated Acaulium sp. H-JQSF.

The solid-state cultivation of Acaulium sp. H-JQSF isolated from Armadillidium vulgare produces acautalides A-C (1-3) as skeletally unprecedented Diels-Alder adducts of a 14-membered macrodiolide to an octadeca-9,11,13-trienoic acid. The acautalide structures, along with the intramolecular transesterifications of 1-acylglycerols, were elucidated by mass spectrometry, nuclear magnetic resonance, chemical transformation, and single-crystal X-ray diffraction. Compounds 1-3 were found to be neuroprotective with antiparkinsonic potential in the 1-methyl-4-phenylpyridinium-challenged nematode model, with the magnitude impacted by the glycerol esterification.

A BBE-like Oxidase, AsmF, Dictates the Formation of Naphthalenic Hydroxyl Groups in Ansaseomycin Biosynthesis.

Ansaseomycins are ansamycin-type natural products produced through expression of the asm gene cluster in a heterologous host. A rare berberine bridge enzyme (BBE) like oxidase, AsmF, is encoded in the asm gene cluster. Deletion of asmF led to the accumulation of a series of structurally diverse compounds, all of which lacked the 23-hydroxyl group in naphthalenic motif. Our work demonstrated that AsmF dictated the formation of the naphthalenic hydroxyl group in ansaseomycin biosynthesis.

Study on the secondary metabolites of grasshopper-derived fungi Arthrinium sp. NF2410.

Two new 2-carboxymethyl-3-hexyl-maleic anhydride derivatives, arthrianhydride A (1) and B (2), along with three known compounds 3-5, were isolated from the fermentation broth of a grasshopper-associated fungus Arthrinium sp. NF2410. The structures of new compounds 1 and 2 were determined based on the analysis of the HR-ESI-MS and NMR spectroscopic data. Furthermore, compounds 1 and 2 were evaluated on inhibitory activity against the enzyme SHP2 and both of them showed moderate inhibitory activity against SHP2.

Cytotoxic Xanthone Derivatives from the Mangrove-Derived Endophytic Fungus Peniophora incarnata Z4.

The mangrove-derived endophytic fungus Peniophora incarnata Z4 produced seven new xanthone derivatives, including four new tetrahydroxanthones (1-4), one new chromone (5), one new xanthone (6), and one new xanthone dimer (7), together with one known compound, globosuxanthone B (8). Their structures were determined by an extensive analysis of 1D and 2D NMR, HRESIMS, ECD, and single-crystal X-ray diffraction data. In cytotoxic activity assays, compound 2 showed cytotoxicity against three carcinoma cell lines with IC50 values less than 10 µM.

Discovery, Biosynthesis, and Heterologous Production of Loonamycin, a Potent Anticancer Indolocarbazole Alkaloid.

Genome mining of an indolocarbazole-type gene cluster from a marine-derived Nocardiopsis flavescens NA01583 strain led to the discovery of three new indolocarbazole alkaloids (1-3). Heterologous expression of the intact loo gene cluster in a surrogate host Streptomyces lividans K4-114 led to the successful production of 3. Notably, compound 1 showed potent cytotoxic activities toward eight cancer cell lines with IC50 values ranging from 41 to 283 nM.

Discovery and biosynthesis of bosamycins from Streptomyces sp. 120454.

Nonribosomal peptides (NRPs) that are synthesized by modular megaenzymes known as nonribosomal peptide synthetases (NRPSs) are a rich source for drug discovery. By targeting an unusual NRPS architecture, we discovered an unusual biosynthetic gene cluster (bsm) from Streptomyces sp. 120454 and identified that it was responsible for the biosynthesis of a series of novel linear peptides, bosamycins. The bsm gene cluster contains a unique monomodular NRPS, BsmF, that contains a cytochrome P450 domain at the N-terminal. BsmF (P450 + A + T) can selectively activate tyrosine with its adenylation (A) domain, load it onto the thiolation (T) domain, and then hydroxylate tyrosine to form 5-OH tyrosine with the P450 domain. We demonstrated a NRPS assembly line for the formation of bosamycins by genetic and biochemical analysis and heterologous expression. Our work reveals a genome mining strategy targeting a unique NRPS domain for the discovery of novel NRPs.

Pyridoxal-5'-phosphate-dependent bifunctional enzyme catalyzed biosynthesis of indolizidine alkaloids in fungi.

Indolizidine alkaloids such as anticancer drugs vinblastine and vincristine are exceptionally attractive due to their widespread occurrence, prominent bioactivity, complex structure, and sophisticated involvement in the chemical defense for the producing organisms. However, the versatility of the indolizidine alkaloid biosynthesis remains incompletely addressed since the knowledge about such biosynthetic machineries is only limited to several representatives. Herein, we describe the biosynthetic gene cluster (BGC) for the biosynthesis of curvulamine, a skeletally unprecedented antibacterial indolizidine alkaloid from Curvularia sp. IFB-Z10. The molecular architecture of curvulamine results from the functional collaboration of a highly reducing polyketide synthase (CuaA), a pyridoxal-5'-phosphate (PLP)-dependent aminotransferase (CuaB), an NADPH-dependent dehydrogenase (CuaC), and a FAD-dependent monooxygenase (CuaD), with its transportation and abundance regulated by a major facilitator superfamily permease (CuaE) and a Zn(II)Cys6 transcription factor (CuaF), respectively. In contrast to expectations, CuaB is bifunctional and capable of catalyzing the Claisen condensation to form a new C-C bond and the α-hydroxylation of the alanine moiety in exposure to dioxygen. Inspired and guided by the distinct function of CuaB, our genome mining effort discovers bipolamines A-I (bipolamine G is more antibacterial than curvulamine), which represent a collection of previously undescribed polyketide alkaloids from a silent BGC in Bipolaris maydis ATCC48331. The work provides insight into nature's arsenal for the indolizidine-coined skeletal formation and adds evidence in support of the functional versatility of PLP-dependent enzymes in fungi.