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.

Four unreported aporphine alkaloids with antifungal activities from Artabotrys hexapetalus.

In this study, the extract from Artabotrys hexapetalus showed strong antifungal activity against phytopathogenic fungi in vitro. Four unreported aporphine alkaloids, hexapetalusine A-D (1-4), were isolated from stems and roots of Artabotrys hexapetalus (L.f.) Bhandari, along with six known aporphine alkaloids (5-10). Their chemical structures were elucidated by extensive spectroscopic analysis. The absolute configurations of 1-3 were determined using single-crystal X-ray diffractions and ECD calculations. Hexapetalusine A-C (1-3) were special amidic isomers. Additionally, all isolated compounds were evaluated for their antifungal activity against four phytopathogenic fungi in vitro. Hexapetalusine D (4) exhibited weak antifungal activity against Curvularia lunata. Liriodenine (5) displayed significant antifungal activity against Fusarium proliferatum and Fusarium oxysporum f. sp. vasinfectum, which is obviously better than positive control nystatin, suggesting that it had great potential to be developed into an effective and eco-friendly fungicide.

Chromosome level genome assembly of endangered medicinal plant Anisodus tanguticus.

Anisodus tanguticus is a medicinal herb that belongs to the Anisodus genus of the Solanaceae family. This endangered herb is mainly distributed in Qinghai-Tibet Plateau. In this study, we combined the Illumina short-read, Nanopore long-read and high-throughput chromosome conformation capture (Hi-C) sequencing technologies to de novo assemble the A. tanguticus genome. A high-quality chromosomal-level genome assembly was obtained with a genome size of 1.26 Gb and a contig N50 of 25.07 Mb. Of the draft genome sequences, 97.47% were anchored to 24 pseudochromosomes with a scaffold N50 of 51.28 Mb. In addition, 842.14 Mb of transposable elements occupying 66.70% of the genome assembly were identified and 44,252 protein-coding genes were predicted. The genome assembly of A. tanguticus will provide genetic repertoire to understand the adaptation strategy of Anisodus species in the plateau, which will further promote the conservation of endangered A. tanguticus resources.

Development of an amplicon-based sequencing approach in response to the global emergence of mpox.

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.

Characterization of two putative norlaudanosoline methyltransferases from Aristolochia debilis.

In view of the nephrotoxicity, hepatotoxicity, and carcinogenicity of aristolochic acids (AAs), the removal of AAs from plants becomes an urgent priority for ensuring the safety of Aristolochia herbal materials. In this study, based on the root-predominant distribution of aristolochic acid I (AAI) in Aristolochia debilis, transcriptome sequencing, in combination with phylogenetic analyses, and gene expression pattern analysis together provided five candidate genes for investigating AAI biosynthesis. Comprehensive in vitro and in vivo enzymatic assays revealed that Ab6OMT1 (6-O-methyltransferase) and AbNMT1 (N-methyltransferase) exhibit promiscuity in substrate recognition, and they could act in a cooperative fashion to achieve conversion of norlaudanosoline, a predicted intermediate in AAI biosynthetic route, into 3'-hydroxy-N-methylcoclaurine through two different methylation reaction sequences. These results shed light on the molecular basis for AAI biosynthesis in Aristolochia herbs. More importantly, Ab6OMT1 and AbNMT1 may be employed as targets for the metabolic engineering of AAI biosynthesis to produce AAs-free Aristolochia herbal materials.

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.

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.

Discovery and biosynthesis of karnamicins as angiotensin converting enzyme inhibitors.

Angiotensin-converting enzyme inhibitors are widely used for treatment of hypertension and related diseases. Here, six karnamicins E1-E6 (1-6), which bear fully substituted hydroxypyridine and thiazole moieties are characterized from the rare actinobacterium Lechevalieria rhizosphaerae NEAU-A2. Through a combination of isotopic labeling, genome mining, and enzymatic characterization studies, the programmed assembly of the fully substituted hydroxypyridine moiety in karnamicin is proposed to be due to sequential operation of a hybrid polyketide synthase-nonribosomal peptide synthetase, two regioselective pyridine ring flavoprotein hydroxylases, and a methyltransferase. Based on AlphaFold protein structures predictions, molecular docking, and site-directed mutagenesis, we find that two pyridine hydroxylases deploy active site residues distinct from other flavoprotein monooxygenases to direct the chemo- and regioselective hydroxylation of the pyridine nucleus. Pleasingly, karnamicins show significant angiotensin-converting enzyme inhibitory activity with IC50 values ranging from 0.24 to 5.81 µM, suggesting their potential use for the treatment of hypertension and related diseases.

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.

Development of an amplicon-based sequencing approach in response to the global emergence of human monkeypox virus.

The 2022 multi-country monkeypox (mpox) outbreak concurrent with the ongoing COVID-19 pandemic has further highlighted the need for genomic surveillance and rapid pathogen whole genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for SARS-CoV-2. Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical samples that tested presumptive positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR cycle threshold below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.

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.

Catalytic innovation underlies independent recruitment of polyketide synthases in cocaine and hyoscyamine biosynthesis.

Tropane alkaloids such as hyoscyamine and cocaine are of importance in medicinal uses. Only recently has the hyoscyamine biosynthetic machinery become complete. However, the cocaine biosynthesis pathway remains only partially elucidated. Here we characterize polyketide synthases required for generating 3-oxo-glutaric acid from malonyl-CoA in cocaine biosynthetic route. Structural analysis shows that these two polyketide synthases adopt distinctly different active site architecture to catalyze the same reaction as pyrrolidine ketide synthase in hyoscyamine biosynthesis, revealing an unusual parallel/convergent evolution of biochemical function in homologous enzymes. Further phylogenetic analysis suggests lineage-specific acquisition of polyketide synthases required for tropane alkaloid biosynthesis in Erythroxylaceae and Solanaceae species, respectively. Overall, our work elucidates not only a key unknown step in cocaine biosynthesis pathway but also, more importantly, structural and biochemical basis for independent recruitment of polyketide synthases in tropane alkaloid biosynthesis, thus broadening the understanding of conservation and innovation of biosynthetic catalysts.

Advances in chemistry and bioactivity of the genus Erythroxylum.

Erythroxylum P. Browne is the largest and most representative genus of Erythroxylaceae family. It contains approximately 230 species that are mainly distributed in tropical and subtropical regions. Some species in this genus, such as E. monogynum and E. coca, have been used as folk medicines in India or South America for a long history. It is well known that Erythroxylum plants are rich in tropane alkaloids, and the representative member cocaine shows remarkable activity in human central nervous system. However, many other types of active compounds have also been found in Erythroxylum along with the broadening and deepening of phytochemical research. To date, a total of 383 compounds from Erythroxylum have been reported, among which only 186 tropane alkaloids have been reviewed in 2010. In this review, we summarized all remained 197 compounds characterized from 53 Erythroxylum species from 1960 to 2021, which include diterpenes, triterpenes, alkaloids, flavonoids, and other derivates, providing a comprehensive overview of phytoconstituents profile of Erythroxylum plants. In addition, the biological activities of representative phytochemicals and crude extracts were also highlighted.

[Effects of miR-335-5p targeting G6PD on proliferation and apoptosis of colon cancer cells].

Objective: To investigate the effects of miR-335-5p targeting glucose-6-phosphate dehydrogenase (G6PD) on the proliferation and apoptosis of colon cancer cells. Methods: Normal colon cell group, blank control group, NC group and miRNA-335-5p mimic group were set up. Colonic epithelial cells (IEC) and human colon cancer cells SW480 were cultured in vitro, and the cells in the NC group and miRNA-335-5p mimic group cells were transfected. RT-qPCR was used to detect the expression levels of miR-335-5p and G6PD mRNA in each group of cells. The targeting effect of miR-335-5p on G6PD was verified by Double Luciferase Report experiment. MTT assay was used to detect cell proliferation. Flow cytometry was used to detect the apoptosis rate. The expressions of G6PD, Bax, Bcl-2 and caspase-3 were detected by Western blot. Results: Compared with normal colon cells, the relative expression levels of miR-335-5p in SW480 cells of colon cancer in the blank control group and NC group were decreased, and the relative expression level of G6PD mRNA was increased (P＜0.05); compared with the blank control group and NC group, the expression level of miR-335-5p in miR-335-5p mimic group was increased significantly, and the expression of G6PD mRNA was decreased significantly (P＜0.05). Compared with the blank control group and NC group, the proliferative activity of colon cancer SW480 cells in miR-335-5p mimic group was decreased significantly, and the apoptosis rate was increased significantly (P＜0.05). The relative activity of luciferase in miR-335-5p mimic + WT-G6PD 3 '- UTR group was lower than that in miR-335-5p NC + WT-G6PD 3' - UTR group (P＜0.05). Compared with the blank control group, the relative expression levels of G6PD and bcl-2 protein in miR-335-5p mimic group were decreased significantly, and the expression levels of Bax and caspase-3 protein were increased significantly (P＜0.05). Conclusion: MiR-335-5p may inhibit the proliferation and promote apoptosis of colon cancer cells by targeting G6PD.

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.

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.

Mono-/Bis-Alkenoic Acid Derivatives From an Endophytic Fungus Scopulariopsis candelabrum and Their Antifungal Activity.

During a screening for antifungal secondary metabolites, six new mono-/bis-alkenoic acid derivatives (2-7) and one known alkenoic acid derivative (1) were isolated from an endophytic fungi Scopulariopsis candelabrum. Their chemical structures were identified by 1H-NMR, 13C-NMR, 2D NMR, and high-resolution mass spectrometry, as well as comparisons with previously reported literatures. Among them, fusariumesters C‒F (2-5) are bis-alkenoic acid derivatives dimerized by an ester bond, while acetylfusaridioic acid A (6) and fusaridioic acid D (7) are alkenoic acid monomers. All the isolates were submitted to an antifungal assay against Candida albicans and the corn pathogen Exserohilum turcicum using the filter paper agar diffusion method. As a result, only compound 1 decorating with ß-lactone ring turned out to be active against these two tested fungi. The broth microdilution assay against Candida albicans showed the minimum inhibitory concentration (MIC) value of 1 to be 20 µg/ml, while the minimum inhibitory concentration value of the positive control (naystatin) was 10 µg/ml. And the half maximal inhibitory concentration (IC50) value (21.23 µg/ml) of 1 against Exserohilum turcicum was determined by analyzing its inhibition effect on the mycelial growth, using cycloheximide (IC50 = 46.70 µg/ml) as the positive control.

Bisaspochalasins A-C: Three Cytochalasan Homodimers with Highly Fused Ring System from an Endophytic Aspergillus flavipes.

Three unprecedented cytochalasan homodimers, bisaspochalasins A-C (1-3), and two known monomers, aspochalasins B and D (4 and 5), were isolated from an endophytic Aspergillus flavipes. Bisaspochalasin A (1) contains a 13-hydroxy-3,24-dioxatricyclo[11.10.11,13.02,15]tetracos-4-one cross-linkage, representing an unprecedented carbon skeleton. Bisaspochalasins B (2) and C (3) share a thioether bridge, while 3 has a peroxy modification at C-7, which may be generated by Schenck-ene photooxygenation. Their structures, including their absolute configurations, were elucidated by HRESIMS, NMR, chemical transformation, and X-ray crystallography. Bisaspochalasin A showed inhibitory activity against human T cell proliferation with an IC50 value of 15.8 µM while maintaining low cytotoxicity to T cells.

Corrigendum: Dimeric Pimprinine Alkaloids From Soil-Derived Streptomyces sp. NEAU-C99.

[This corrects the article DOI: 10.3389/fchem.2020.00095.].