Genomic and structural basis for evolution of tropane alkaloid biosynthesis.

The tropane alkaloids (TAs) cocaine and hyoscyamine have been used medicinally for thousands of years. To understand the evolutionary origins and trajectories of serial biosynthetic enzymes of TAs and especially the characteristic tropane skeletons, we generated the chromosome-level genome assemblies of cocaine-producing Erythroxylum novogranatense (Erythroxylaceae, rosids clade) and hyoscyamine-producing Anisodus acutangulus (Solanaceae, asterids clade). Comparative genomic and phylogenetic analysis suggested that the lack of spermidine synthase/N-methyltransferase (EnSPMT1) in ancestral asterids species contributed to the divergence of polyamine (spermidine or putrescine) methylation in cocaine and hyoscyamine biosynthesis. Molecular docking analysis and key site mutation experiments suggested that ecgonone synthases CYP81AN15 and CYP82M3 adopt different active-site architectures to biosynthesize the same product ecgonone from the same substrate in Erythroxylaceae and Solanaceae. Further synteny analysis showed different evolutionary origins and trajectories of CYP81AN15 and CYP82M3, particularly the emergence of CYP81AN15 through the neofunctionalization of ancient tandem duplication genes. The combination of structural biology and comparative genomic analysis revealed that ecgonone methyltransferase, which is responsible for the biosynthesis of characteristic 2-substituted carboxymethyl group in cocaine, evolved from the tandem copies of salicylic acid methyltransferase by the mutations of critical E216 and S153 residues. Overall, we provided strong evidence for the independent origins of serial TA biosynthetic enzymes on the genomic and structural level, underlying the chemotypic convergence of TAs in phylogenetically distant species.

Identification and characterization of camptothecin tailoring enzymes in Nothapodytes tomentosa.

Camptothecin is a complex monoterpenoid indole alkaloid with remarkable antitumor activity. Given that two C-10 modified camptothecin derivatives, topotecan and irinotecan, have been approved as potent anticancer agents, there is a critical need for methods to access other aromatic ring-functionalized congeners (e.g., C-9, C-10, etc.). However, contemporary methods for chemical oxidation are generally harsh and low-yielding when applied to the camptothecin scaffold, thereby limiting the development of modified derivatives. Reported herein, we have identified four tailoring enzymes responsible for C-9 modifications of camptothecin from Nothapodytes tomentosa, via metabolomic and transcriptomic analysis. These consist of a cytochrome P450 (NtCPT9H) which catalyzes the regioselective oxidation of camptothecin to 9-hydroxycamptothecin, as well as two methyltransferases (NtOMT1/2, converting 9-hydroxycamptothecin to 9-methoxycamptothecin), and a uridine diphosphate-glycosyltransferase (NtUGT5, decorating 9-hydroxycamptothecin to 9-ß-D-glucosyloxycamptothecin). Importantly, the critical residues that contribute to the specific catalytic activity of NtCPT9H have been elucidated through molecular docking and mutagenesis experiments. This work provides a genetic basis for producing camptothecin derivatives through metabolic engineering. This will hasten the discovery of novel C-9 modified camptothecin derivatives, with profound implications for pharmaceutical manufacture.

O-Alkylazoxymycins A-F, Naturally Occurring Azoxy-Aromatic Compounds from Streptomyces sp. Py50.

Six new azoxy-aromatic compounds (o-alkylazoxymycins A-F, 1-6) and two new nitrogen-bearing phenylvaleric/phenylheptanoic acid derivatives (o-alkylphemycins A and B, 7 and 8) were isolated from Streptomyces sp. Py50. Their structures were elucidated based on HRESIMS, NMR, UV spectroscopic analyses, and X-ray crystallographic data. O-Alkylazoxymycins A-F (1-6) are the first natural examples of azoxy compounds with the azoxy bond attached to the ortho-position of the phenylheptanoic acid or phenylvaleric acid moiety. Compounds 1, 5, and 6 were active against Epidermophyton floccosum with MIC50 values ranging from 10.1 to 51.2 µM. A plausible biosynthetic pathway of 2 and 3 was proposed.

Phytochemical Analysis of Nothapodytes tomentosa and Distribution and Content of Camptothecin and its Analogues in Four Plants.

Camptothecin (CPT) and its derivatives have attracted worldwide attention because of their notable anticancer activity. However, the growing demand for CPT in the global pharmaceutical industry has caused a severe shortage of CPT-producing plant resources. In this study, phytochemical analysis of Nothapodytes tomentosa results in the isolation and identification of CPT (13: ) and 16 analogues (1:  - 12, 14:  - 17: ), including a new (1: ) and five known (9, 10, 12, 15: , and 17: ) CPT analogues with an open E-ring. In view of the potential anticancer activity of CPT analogues with an open E-ring, the fragmentation pathways and mass spectra profiles of these six CPT analogues (1, 9, 10, 12, 15: , and 17: ) are investigated, providing a reference for the rapid detection of these compounds in other plants. Furthermore, based on the fragmentation patterns of CPT (13: ) and known analogues (2:  - 8, 11, 14, 16, 18:  - 26: ), the distribution and content of these compounds in different tissues of N. tomentosa, N. nimmoniana, Camptotheca acuminata, and Ophiorrhiza japonica are further studied. Our findings not only provide an alternative plant resource for further expanding the development and utilization of CPT and its analogues, but also lay a foundation for improving the utilization of known CPT-producing plant resources.

Discovery and Engineering of the Cocaine Biosynthetic Pathway.

Cocaine, the archetypal tropane alkaloid from the plant genus Erythroxylum, has recently been used clinically as a topical anesthesia of the mucous membranes. Despite this, the key biosynthetic step of the requisite tropane skeleton (methylecgonone) from the identified intermediate 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid (MPOA) has remained, until this point, unknown. Herein, we identify two missing enzymes (EnCYP81AN15 and EnMT4) necessary for the biosynthesis of the tropane skeleton in cocaine by transient expression of the candidate genes in Nicotiana benthamiana. Cytochrome P450 EnCYP81AN15 was observed to selectively mediate the oxidative cyclization of S-MPOA to yield the unstable intermediate ecgonone, which was then methylated to form optically active methylecgonone by methyltransferase EnMT4 in Erythroxylum novogranatense. The establishment of this pathway corrects the long-standing (but incorrect) biosynthetic hypothesis of MPOA methylation first and oxidative cyclization second. Notably, the de novo reconstruction of cocaine was realized in N. benthamiana with the two newly identified genes, as well as four already known ones. This study not only reports a near-complete biosynthetic pathway of cocaine and provides new insights into the metabolic networks of tropane alkaloids (cocaine and hyoscyamine) in plants but also enables the heterologous synthesis of tropane alkaloids in other (micro)organisms, entailing significant implications for pharmaceutical production.

Isolation and Biosynthesis of Phenazine-Polyketide Hybrids from Streptomyces sp. KIB-H483.

A phenazine-polyketide hybrid compound, nexphenazine A (1), was isolated from Streptomyces sp. KIB-H483. The bioinformatic analysis of the draft genome of the producing strain and gene inactivation experiments revealed that the biosynthesis of 1 involves a phenazine-polyketide hybrid gene cluster. The abolished production of 1 as well as the accumulation of shunt metabolites 4-7 in mutant strain ΔnpzI revealed the key role of the npzI gene, which encodes an NAD(P)H-dependent ketoreductase, in nexphenazine biosynthesis. The structures and absolute configurations of the isolated intermediates were established on the basis of spectroscopic data analysis, single-crystal X-ray diffraction, chiral chromatography, and chemical conversion experiments. NpzI exhibited stereochemical selectivity in reducing the carbonyl group of 4. Nexphenazine biosynthesis is proposed to involve a condensation of the carboxyl group of phenazine with one molecule of methylmalonyl-CoA by a type I PKS, followed by a ketone reduction by NpzI and an unknown methylation reaction.

Characterization of Multifunctional and Non-stereoselective Oxidoreductase RubE7/IstO, Expanding the Functional Diversity of the Flavoenzyme Superfamily.

Flavin-dependent enzymes enable a broad range of redox transformations and generally act as monofunctional and stereoselective catalysts. Herein, we report the investigation of a multifunctional and non-stereoselective FMN-dependent oxidoreductase RubE7 from the rubrolone biosynthetic pathway. Our study outlines a single RubE7-catalysed sequential reduction of three spatially distinct bonds in a tropolone ring and a reversible double-bond reduction and dehydrogenation. The crystal structure of IstO (a RubE7 homologue) with 2.0 Šresolution reveals the location of the active site at the interface of two monomers, and the size of active site is large enough to permit both flipping and free rotation of the substrate, resulting in multiple nonselective reduction reactions. Molecular docking and site mutation studies demonstrate that His106 is oriented towards the substrate and is important for the reverse dehydrogenation reaction.

Isolation and biosynthesis of daturamycins from Streptomyces sp. KIB-H1544.

Two novel diarylcyclopentenones daturamycin A and B (1 and 2), and one new p-terphenyl daturamycin C (3), along with three known congeners (4-6), were isolated from a rhizosphere soil-derived Streptomyces sp. KIB-H1544. The structures of new compounds were elucidated via a joint use of spectroscopic analyses and single-crystal X-ray diffractions. Compounds 1 and 2 belong to a rare class of tricyclic 6/5/6 diarylcyclopentenones, and compounds 3-6 possess a C-18 tricyclic aromatic skeleton. The biosynthetic gene cluster of daturamycins was identified through gene knockout and biochemical characterization experiments and the biosynthetic pathway of daturamycins was proposed.

Tropane alkaloid biosynthesis: a centennial review.

Covering: 1917 to 2020Tropane alkaloids (TAs) are a remarkable class of plant secondary metabolites, which are characterized by an 8-azabicyclo[3.2.1]octane (nortropane) ring. Members of this class, such as hyoscyamine, scopolamine, and cocaine, are well known for their long history as poisons, hallucinogens, and anaesthetic agents. Since the structure of the tropane ring system was first elucidated in 1901, organic chemists and biochemists have been interested in how these mysterious tropane alkaloids are assembled in vitro and in vivo. However, it was only in 2020 that the complete biosynthetic route of hyoscyamine and scopolamine was clarified, and their de novo production in yeast was also achieved. The aim of this review is to present the innovative ideas and results in exploring the story of tropane alkaloid biosynthesis in plants from 1917 to 2020. This review also highlights that Robinson's classic synthesis of tropinone, which is one hundred years old, is biomimetic, and underscores the importance of total synthesis in the study of natural product biosynthesis.

Bisaspochalasins D and E: Two Heterocycle-Fused Cytochalasan Homodimers from an Endophytic Aspergillus flavipes.

Two heterocycle-fused cytochalasan homodimers, bisaspochalasins D (1) and E (2), were isolated from an endophytic Aspergillus flavipes. Their chemical structures were elucidated using a combination of HRESIMS, NMR, theoretical calculations, and crystallographic techniques. Bisaspochalasin D (1) is dimerized by the first reported naturally occurring triple heterobridged 3,8-dioxa-6-azabicyclo[3.2.1]octane framework, while bisaspochalasin E (2) employs a pyrrole ring as the linking moiety. Possible dimerization mechanisms of bisaspochalasins D and E were proposed. The bioassay screening revealed that bisaspochalasin D showed cytotoxic activities against five cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480) with IC50 values ranging from 4.45 to 22.99 µM. Additionally, bisaspochalasin D exhibited neurotrophic activities in a PC12 cell-based assay. At a concentration of 10 µM, bisaspochalasin D can promote neurite growth by inducing a differentiation rate of 12.52% for PC12 cells.

Streptomyces typhae sp. nov., a novel endophytic actinomycete with antifungal activity isolated the root of cattail (Typha angustifolia L.).

A novel endophytic actinomycete with antagonistic activity against various phytopathogenic fungi, designated strain p1417T, was isolated from the root of cattail (Typha angustifolia L.) collected from Yunnan Province, Southwest China. A polyphasic taxonomic study was carried out to establish the taxonomic status of this strain. Strain p1417T was found to have morphological and chemotaxonomic characteristics typical of the genus Streptomyces. The diamino acid present in the cell wall was LL-diaminopimelic acid. Xylose and arabinose occurred in whole cell hydrolysates. The menaquinones were identified as MK-9(H8), MK-9(H6) and MK-9(H4). The polar lipid profile was found to contain diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. The major fatty acids were found to be iso-C16:0, anteiso-C15:0, iso-C15:0 and C16:0. The genomic DNA G + C content of strain p1417T based on the genome sequence was 72.0 mol%. Based on 16 S rRNA gene, five housekeeping genes and whole genome sequences analysis, strain p1417T was most closely related to Streptomyces flavofungini JCM 4753T (99.4% 16 S rRNA gene sequence similarity), Streptomyces alboflavus JCM 4615T (98.8%) and Streptomyces aureoverticillatus JCM 4347T (98.2%). However, the average nucleotide identity values, the digital DNA-DNA hybridization values and the multilocus sequence analysis evolutionary distances between this strain and its closely related strains showed that it belonged to one distinct species. In addition, these results were also supported by differences in the phenotypic and chemotaxonomic characteristics between strain p1417T and three closely related type strains. Therefore, it is concluded that strain p1417T represents a novel species of the genus of Streptomyces, for which the name Streptomyces typhae sp. nov. is proposed. The type strain is p1417T (= CCTCC AA 2019091T = DSM 110636T).

Functional genomics analysis reveals two novel genes required for littorine biosynthesis.

Some medicinal plants of the Solanaceae produce pharmaceutical tropane alkaloids (TAs), such as hyoscyamine and scopolamine. Littorine is a key biosynthetic intermediate in the hyoscyamine and scopolamine biosynthetic pathways. However, the mechanism underlying littorine formation from the precursors phenyllactate and tropine is not completely understood. Here, we report the elucidation of littorine biosynthesis through a functional genomics approach and functional identification of two novel biosynthesis genes that encode phenyllactate UDP-glycosyltransferase (UGT1) and littorine synthase (LS). UGT1 and LS are highly and specifically expressed in Atropa belladonna secondary roots. Suppression of either UGT1 or LS disrupted the biosynthesis of littorine and its TA derivatives (hyoscyamine and scopolamine). Purified His-tagged UGT1 catalysed phenyllactate glycosylation to form phenyllactylglucose. UGT1 and LS co-expression in tobacco leaves led to littorine synthesis if tropine and phenyllactate were added. This identification of UGT1 and LS provides the missing link in littorine biosynthesis. The results pave the way for producing hyoscyamine and scopolamine for medical use by metabolic engineering or synthetic biology.

Antibacterial Pentacyclic Polyketides from a Soil-Derived Streptomyces.

Nine new pentacyclic polyketides, fasamycins G-K (1-5) and formicamycins N-Q (6-9), along with 10 known analogues (10-19), were isolated from a rhizospheric soil-derived Streptomyces sp. KIB-1414. Their structures and absolute configurations were elucidated by interpretation of NMR and HRMS data and comparisons of CD data. The compounds were active against methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus, Bacillus subtilis, and Escherichia coli strains, with MIC values ranging from 0.20 to 50.00 µg/mL.

Benwamycins A-G, Trialkyl-Substituted Benzene Derivatives from a Soil-Derived Streptomyces.

Seven new trialkyl-substituted benzene derivatives named benwamycins A-G (1-7), together with three known congeners, 8-10, were isolated from culture broth of the soil-derived Streptomyces sp. KIB-H1471. Their structures were elucidated by using 1D and 2D NMR analyses in combination with HRESIMS data. The absolute configurations of 1-9 were determined by chemical conversion and comparison of circular dichroism spectra and confirmed for 1 by single-crystal X-ray crystallography. Compounds 6 and 7 have a unique γ-pyrone-like ring on one side chain. Compounds 2 and 6 inhibited human T cell proliferation with IC50 values of 14.3 and 12.5 µM, respectively, without obvious cytotoxicity for naïve human T cells. Compounds 3 and 6 could weakly enhance insulin-stimulated glucose uptake.

A Hydrolase-Catalyzed Cyclization Forms the Fused Bicyclic ß-Lactone in Vibralactone.

Vibralactone is isolated from the basidiomycete fungus Boreostereum vibrans as one of the strongest lipase inhibitors. Its unusual ß-lactone-fused bicycle is derived from an aryl ring moiety by an oxidative ring-expansion prior to an intramolecular cyclization. Herein, we report the discovery of the cyclase VibC which belongs to the α/ß-hydrolase superfamily and is involved in the vibralactone biosynthesis. Biochemical and crystal studies suggest that VibC may catalyze an aldol or an electrocyclic reaction initiated by the Ser-His-Asp catalytic triad. For the aldol and pericyclic chemistry in living cells, VibC is a unique hydrolase performing the carbocycle formation of an oxepinone to a fused bicyclic ß-lactone. This presents a naturally occurring, new enzymatic reaction in both aldol and hydrolase (bio)chemistry that will guide future exploitation of these enzymes in synthetic biology for chemical-diversity expansion of natural products.

Functional Genome Mining Reveals a Class V Lanthipeptide Containing a d-Amino Acid Introduced by an F420 H2 -Dependent Reductase.

Lantibiotics are a type of ribosomally synthesized and post-translationally modified peptides (termed lanthipeptides) with often potent antimicrobial activity. Herein, we report the discovery of a new lantibiotic, lexapeptide, using the library expression analysis system (LEXAS) approach. Lexapeptide has rare structural modifications, including N-terminal (N,N)-dimethyl phenylalanine, C-terminal (2-aminovinyl)-3-methyl-cysteine, and d-Ala. The characteristic lanthionine moiety in lexapeptide is formed by three proteins (LxmK, LxmX, and LxmY), which are distinct from enzymes known to be involved in lanthipeptide biosynthesis. Furthermore, a novel F420 H2 -dependent reductase (LxmJ) from the lexapeptide biosynthetic gene cluster (BGC) is identified to catalyze the reduction of dehydroalanine to install d-Ala. Our findings suggest that lexapeptide is the founding member of a new class of lanthipeptides that we designate as class V. We also identified further class V lanthipeptide BGCs in actinomycetes and cyanobacteria genomes, implying that other class V lantibiotics await discovery.

Characterization of the N-methyltransferases involved in the biosynthesis of toxoflavin, fervenulin and reumycin from Streptomyces hiroshimensis ATCC53615.

Toxoflavin (1), fervenulin (2), and reumycin (3), known to be produced by plant pathogen Burkholderia glumae BGR1, are structurally related 7-azapteridine antibiotics. Previous biosynthetic studies revealed that N-methyltransferase ToxA from B. glumae BGR1 catalyzed the sequential methylation at N6 and N1 in pyrimido[5,4-e]-as-triazine-5,7(6H,8H)-dione (4) to generate 1. However, the N8 methylation of 4 in the biosynthesis of fervenulin remains unclear. To explore the N-methyltransferases required for the biosynthesis of 1 and 2, we identified and characterized the fervenulin and toxoflavin biosynthetic gene clusters in S. hiroshimensis ATCC53615. On the basis of the structures of intermediates accumulated from the four N-methyltransferase gene inactivation mutants and systematic enzymatic methylation reactions, the tailoring steps for the methylation order in the biosynthesis of 1 and 2 were proposed. The N-methylation order and routes for the biosynthesis of fervenulin and toxoflavin in S. hiroshimensis are more complex and represent an obvious departure from those in B. glumae BGR1.

Dimeric Pyranonaphthoquinone Glycosides with Anti-HIV and Cytotoxic Activities from a Soil-Derived Streptomyces.

Eight new sulfur-bridged pyranonaphthoquinone (PNQ) dimers, naquihexcins C-J (1-8), a new PNQ monomer, naquihexcin K (10), and three known analogues (9, 11, and 12) were isolated from Streptomyces sp. KIB3133. The new structures were elucidated by interpretation of spectroscopic data. Dimer 4 was synthesized via a cascade SN2 reactions between two monomers and sodium sulfide, an approach motivated by the proposed biosynthetic pathway of dimeric pyranonaphthoquinones. Naquihexcin E (3) exhibited moderate HIV-1 inhibitory activity. Naquihexcins C (1), E (3), and I (7) showed inhibitory effects against two tumor cell lines (HL-60 and MCF-7) with IC50 values ranging from 1.4 to 16.1 µM.

Leinamycin E1 acting as an anticancer prodrug activated by reactive oxygen species.

Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140, featuring an unusual 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a thiazole-containing 18-membered lactam ring. Upon reductive activation in the presence of cellular thiols, LNM exerts its antitumor activity by an episulfonium ion-mediated DNA alkylation. Previously, we have cloned the lnm gene cluster from S. atroolivaceus S-140 and characterized the biosynthetic machinery responsible for the 18-membered lactam backbone and the alkyl branch at C3 of LNM. We now report the isolation and characterization of leinamycin E1 (LNM E1) from S. atroolivacues SB3033, a ΔlnmE mutant strain of S. atroolivaceus S-140. Complementary to the reductive activation of LNM by cellular thiols, LNM E1 can be oxidatively activated by cellular reactive oxygen species (ROS) to generate a similar episulfonium ion intermediate, thereby alkylating DNA and leading to eventual cell death. The feasibility of exploiting LNM E1 as an anticancer prodrug activated by ROS was demonstrated in two prostate cancer cell lines, LNCaP and DU-145. Because many cancer cells are under higher cellular oxidative stress with increased levels of ROS than normal cells, these findings support the idea of exploiting ROS as a means to target cancer cells and highlight LNM E1 as a novel lead for the development of anticancer prodrugs activated by ROS. The structure of LNM E1 also reveals critical new insights into LNM biosynthesis, setting the stage to investigate sulfur incorporation, as well as the tailoring steps that convert the nascent hybrid peptide-polyketide biosynthetic intermediate into LNM.

Cytotoxicity-Guided Isolation of Two New Phenolic Derivatives from Dryopteris fragrans (L.) Schott.

Dryopteris fragrans is a valuable medicinal plant resource with extensive biological activities including anti-cancer, anti-oxidation, and anti-inflammation activities. This work aims to study further the cytotoxic constituents from Dryopteris fragrans. In this work, two new phenolic derivatives known as dryofragone (1) and dryofracoumarin B (2) with six known compounds (3⁻8) were isolated from the petroleum ether fraction of the methanol extract of the aerial parts of Dryopteris fragrans (L.) Schott by two round cytotoxicity-guided tracking with the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and cell counting kit-8 (CCK-8) assay. Their structures were elucidated by the extensive spectroscopic analysis (¹H-NMR, 13C-NMR, and two dimensions NMR), chemical derivatization, and comparison with data reported in the literature. All the isolates were evaluated for their cytotoxicity against nine cancer cell lines as well as their in vitro immunomodulatory activity. The results showed that compounds have a modest cytotoxicity toward human HeLa cell line with IC50 value below 30 µM and compounds 4 and 5 may modulate immunity to affect the growth of tumor cells.