The 6/5/6 to 6/6/5 ring system rearrangement from indole alkaloids strictosidine (1) and strictosamide (2) to quinoline alkaloid pumiloside (3) is an essential but unelucidated process in the biosynthesis of camptothecin, especially, the intermediates involved in the process have not been identified. Herein, the nonenzymatic conversions including spontaneous reaction from 1 to 2, and light- or flavin-catalyzed skeletal rearrangement from 2 to 3 have been discovered. Particularly, the crucial biosynthetic intermediate strictosamide ketolactam (2a) with dicarbonyl nine-membered ring involved in the conversion from 2 to 3 is obtained and structurally characterized. Moreover, the successive conversion from 1 to 3 is achieved in Nicotiana benthamiana. These findings not only provide a new insight into and better understanding for the biosynthesis of camptothecin with rare nonenzymatic reactions, also lay the foundation for further elucidation of the biosynthetic pathway of camptothecin.

Pyranocoumarins are a characteristic class of pyranophenolic compounds predominantly found in plants of the Moraceae, Umbelliferae, and Rutaceae families. These pyranophenolics not only exhibit diverse physiological activities, but also have a wide range of pharmacological activities such as anti-cancer, anti-spasmatic, and anticoagulant. However, the biosynthetic mechanism of pyranocoumarins, especially the formation mechanism of the pyran core in the final step, remains unclear. It was hypothesized the pyran core is formed by dehydration of decursinol after oxidation and cyclization of demethylsuberosin. Here we characterized a berberine bridge enzyme (BBE)-like enzyme namely FcBBElike4 from Ficus carica. FcBBElike4 could catalyze direct cyclization of 6- and 8-isoprenyl coumarins into corresponding pyranocoumarins, which functionally characterized as an oxidocyclase (OC). This finding elucidated the final step in the biosynthetic pathway for pyranocoumarins. The catalytic mechanism of FcBBElike4 was investigated and a general key active site of aspartic acid residue determining the cyclization activity of BBE-like enzymes was identified. Diverse BBE-like enzymes with diene synthesis activities were mined and further engineered into rare OCs with high catalytic activity and broad substrate spectra. What is more, an enzymatic approach to synthesize pyranophenolics was constructed based on engineered OCs and applied in the synthesis of drug molecules. This study not only elucidates the key biosynthetic steps of pyranocoumarins but also offers insights into engineering common BBE-like enzymes into rare and useful OCs.

Herein, enzymatic synthesis of maclurin directly from protocatechuic acid was designed and investigated in onepot multi-enzyme cascade including 3-hydroxybenzoyl-CoA ligase Ta3HBL, malonyl-CoA synthetase AAE13 and benzophenone synthase GmBPS. Protein engineering of GmBPS was explored to improve the catalytic capability for maclurin, and the mutant T133A exhibited the highest production of 13.2 mg/L with protocatechuoyl-CoA as the substrate, approximately 14-fold higher than that of wild-type. Moreover, a C-glycosyltransferase MiCGT was combined with the multienzyme cascade to produce maclurin 3-C-beta-D-glucoside with 22.8 mg/L. In addition, the titer of maclurin was achieved with 20.1 mg/L after 16 h incubation when 1 mM protocatechuic acid was used as the starting substrate. These findings not only provided practical synthetic alternative for the maclurin biosynthesis, and also paved the way for constructing efficient biosynthetic process in microbial hosts.

Abstract The prevalence of naturally occurring C13-acetoxy taxanes, together with the presence of a native C13-acetyltransferase in yew trees, suggests that the natural biosynthetic pathway for paclitaxel may involve a cryptic C13α- O -deacetylation step. However, whether a putative taxane C13α- O -deacetylase (T13dA) acts in the pathway of paclitaxel biosynthesis remains elusive. Herein, we functionally characterized two novel taxane C13α- O -deacetylases (T13dA1 and T13dA2) from Taxus × media cell cultures, providing experimental evidence for the molecular and biochemical plausibility of C13α- O -deacetylation in paclitaxel biosynthesis in Taxus species. Furthermore, T7dA1, a novel taxane C7β- O -deacetylase with higher activity than the reported T7dA was discovered and characterized here. Notably, we reconstituted a new 19-gene pathway enabled by the integration of a C13α- O -acetylation-deacetylation module for the de novo biosynthesis of baccatin III in Nicotiana benthamiana leaves, reaching a yield of 71 □ μg □ g −1 DW, much higher than that obtained from the 17 □ gene pathway lacking this module. These findings not only demonstrate the functional importance of the C13α- O -acetylation-deacetylation module as a branch within the paclitaxel biosynthetic network, but also expand the multi-branched taxane metabolic network in Taxus and provide novel enzymatic tools and engineering strategies to facilitate heterologous pathway reconstitution and efficient paclitaxel production.

Two new metabolites, 29-demethyl-3-ene-cycloastragenol (2) and 1α,12α-dihydroxy-cycloastragenone (3), along with six known compounds (4-9) were obtained from the microbial transformation of cycloastragenol (1) by Penicillium spinulosum AS3.149. The structures of these metabolites were determined by extensive spectroscopic (IR, UV, HRESIMS, 1D and 2D NMR) data analyses. Compound 2 features a 29-nor-triterpenic skeleton, possibly formed through sequential oxidation, decarboxylation, and dehydration reactions. Biological assays revealed that compound 6 exhibited significant cytotoxic activity against the K562 cell line with an IC50 value of 1.0 μM.

Limonoids are a structurally diverse class of highly oxidized triterpenoids produced by Meliaceae and Rutaceae species. They exhibit remarkable biological activities but are difficult to produce owing to their complex biosynthesis. Plants have evolved ecologically adaptive specialized metabolites through the amplification and functional differentiation of gene families, but our understanding of the molecular mechanisms that underlie this process remains very limited. A chromosome-level genome assembly of Melia toosendan (219.5 Mb) shows no recent whole-genome duplications and contains 14 genes predicted to encode oxidosqualene cyclases (OSCs), key enzymes in triterpenoid biosynthesis. Transient expression of these OSCs in Nicotiana benthamiana produced 11 distinct triterpene skeletons, including eupha-7,24-dien-3β-ol, the product of MtOSC10. Phylogenetic analysis and examination of the ratio of non-synonymous to synonymous substitution rates (dN/dS) suggested that this OSC lineage may have undergone neofunctionalization correlated with signals of strong positive selection. Ancestral sequence reconstruction traced the divergence of an ancestral β-amyrin synthase into two evolutionary lineages: one lineage has retained the conserved activity of tirucalla-7,24-dien-3β-ol synthase, which produces the canonical limonoid precursor, and the other has diversified into novel eupha-7,24-dien-3β-ol synthases that produce a hypothetical alternative precursor. Strikingly, four key amino acid substitutions (W258L, T413S, M730Y, and L735H) in tirucalla-7,24-dien-3β-ol synthase are sufficient to switch the product specificity of MtOSC1 from tirucalla-7,24-dien-3β-ol to eupha-7,24-dien-3β-ol. Our findings identify a potential alternative pathway for limonoid biosynthesis and reveal the molecular basis of triterpene skeleton diversification in Meliaceae. More broadly, they illustrate how neofunctionalization of OSCs under positive selection has driven metabolic innovation across plant lineages. These findings also provide a foundation for engineering plant-derived insecticides through synthetic biology approaches.

Mediataxanes A-C (1-3), three unusual 4-oxo-20-nor-taxanes with 6/8/6 ring system were isolated from Taxus x media cell cultures. Their structures were determined by extensive spectroscopic data, and the absolute configurations were confirmed by X-ray crystallographic analysis (1 and 2) and NMR data comparison (3). Biological assay revealed that 1 exhibited moderate cytotoxicity against gastric carcinoma BGC823 and cervical cancer HeLa cells. In addition, a plausible mechanism for the oxidative cleavage of 4(20)-exocyclic-double bond and the formation of 4-carbonyl group in 1-3 is proposed.

Ischemic stroke is one of the most common forms of stroke. There are no effective pharmacological agents to promote recovery yet. Dendrobium is a valuable herb in traditional Chinese medicine, which has shown antiviral, anti-inflammatory, antioxidant and immunomodulatory activities. We previously used dendrobium to synthesize a novel bibenzyl compound NPB-1575 with the ability to scavenge free radicals in vitro. Increasing evidence shows that IRS2 has biological functions other than participating in the insulin signaling pathway. In this study we utilized NPB-1575 to explore the role of IRS2 in neuroinflammation and ferroptosis during cerebral ischemia. Rats were subjected to permanent middle cerebral artery occlusion (pMCAO) surgery. NPB-1575 (25 mg/kg) was orally administered to the rats 5 min to 4 h after the surgery. We showed that NPB-1575 administration significantly reduced the infarct volume and improved neurological outcome at different stages of ischemic stroke in pMCAO rats. In the brain tissue of pMCAO rats and LPS-stimulated BV2 cells, we demonstrated that NPB-1575 exerted the anti-inflammatory effect on microglia through upregulating Nrf2 and inhibiting FOXO1 via IRS2. NPB-1575 might effectively interact with IRS2 to enhance the stability of IRS2 protein. Knockdown of IRS2 in BV2 cells reversed the protective effect of NPB-1575 against cerebral ischemic injury, manifested by upregulated NF-κB and inactivated ferroptosis defense system. In conclusion, we demonstrate that IRS2 might be a novel target in regulation of neuroinflammation and played an indispensable role in anti-ischemic injury of the brain. NPB-1575 mitigates neuroinflammation and resists ferroptosis through the IRS2/Nrf2/NF-κB axis, demonstrating its potential therapeutic effects on ischemic stroke.

There is no doubt that breakthroughs in the enzyme-mediated formation of the oxetane ring in paclitaxel biosynthesis constitute significant milestones in the biosynthesis of complex natural products. In this review, we summarize the understanding of the biosynthesis of the oxetane ring of paclitaxel from different viewpoints. Generally, it covers five aspects, (1) a different understanding of the mechanistic formation of the oxetane ring on the basis of sound chemical reasoning, (2) a reasonable speculation of the biosynthetic pathways and suitable surrogate substrates for oxetane ring formation based on the natural and chemical logical analysis, (3) Taxus genome-enabled enzymes identification, (4) the discovery of different enzymes that mediate oxetane ring formation, and (5) a mechanistic investigation involving the use of isotopic labelling experiments and quantum chemical calculations. This review provides a detailed overview of the history of the studies on the oxetane ring formation in paclitaxel biosynthesis, which may be useful for a better understanding this process in combined view of nature, chemistry and biology logics, also for efficient heterologous reconstruction of the paclitaxel biosynthetic pathway in the future.

Ustiloxins are a group of cyclopeptide mycotoxins produced by rice false smut pathogen Villosiclava virens (anamorph: Ustilaginoidea virens) which seriously threaten the safety production of rice and the health of humans and livestock. Ustiloxin A, accounting for 60% of the total ustiloxins, is the main toxic component. Biotransformation, a process of modifying the functional groups of compounds by means of regio- or stereo-specific reactions catalyzed by the enzymes produced by organisms, has been considered as an efficient way to detoxify mycotoxins. In this study, the endophytic fungus Petriella setifera Nitaf10 was found to be able to detoxify ustiloxin A through biotransformation. Two transformed products were obtained by using the cell-free extract (CFE) containing intracellular enzymes of P. setifera Nitaf10. They were structurally characterized as novel ustiloxin analogs named ustiloxins A1 (1) and A2 (2) by analysis of the 1D and 2D NMR and HRESIMS spectra as well as by comparison with known ustiloxins. The cytotoxic activity of ustiloxins A1 (1) and A2 (2) was much weaker than that of ustiloxin A. The biotransformation of ustiloxin A was found to proceed via oxidative deamination and decarboxylation and was possibly catalyzed by the intracellular amine oxidase and oxidative decarboxylase in the CFE. An appropriate bioconversion was achieved by incubating ustiloxin A with the CFE prepared in 0.5 mol/L phosphate buffer (pH 7.0) for 24 to 48 h. The optimum initial pH values for the bioconversion of ustiloxin A were 7–9. Among eight metal ions (Co2+, Cu2+, Fe3+, Zn2+, Ba2+, Ca2+, Mg2+ and Mn2+) tested at 5 mmol/L, Cu2+, Fe3+ and Zn2+ totally inhibited the conversion of ustiloxin A. In conclusion, detoxification of ustiloxin A through oxidative deamination and decarboxylation is an efficient strategy.

Camptothecin, a plant-derived pentacyclic pyrroloquinoline alkaloid, and its derivatives like topotecan and irinotecan have been used as clinical anticancer agents for decades. However, the complete biosynthetic pathway of camptothecin still remains unelucidated due to the unknown complex formation processes and corresponding enzymes for the downstream biosynthetic pathway including the committed hydrolysis of glycosides. Herein, a novel glycoside hydrolase (CaGH1) responsible for the deglycosylation of biosynthetic glycoside intermediates including both quinoline-type alkaloids pumiloside (1), (3S)-deoxypumiloside (2) and indole-type alkaloid strictosamide (3) has been functionally identified. Moreover, CaGH1 exhibits the highly strict stereoselectivity towards the substrates with 3S configuration. Furthermore, a combined strategy for the discovery of the unknown biosynthetic enzyme by employing activity-guided enzyme verification, transcriptome-based gene mining, biochemical assay in vitro, and structurally characterizing the unstable enzymatic products by derivatization, is reported. These findings not only provide a better understanding of the deglycosylation in camptothecin biosynthesis, also lay the foundation for the complete elucidation of camptothecin biosynthetic pathway and biological production of camptothecin.

Background and PurposeChronic pain affects nearly 30% of the global population. Because of significant adverse effects of opioids, alternative therapies are urgently needed. In a drug discovery project, we identified grifolic acid (GA) as a potent NaV1.7 antagonist. Here, we have evaluated its biophysical properties and efficacy in animal pain models.Experimental ApproachA mechanistic investigation of GA was carried out on dorsal root ganglion (DRG) neurons, and various stable cell lines, using whole-cell patch clamp techniques. Site-directed mutagenesis and molecular docking analyses also were performed to identify the binding pocket of GA on NaV1.7. The antinociceptive efficacy of GA was evaluated in inflammatory pain models.Key ResultsGA exhibited state-dependent blockade of NaV1.7 channels and modulated channel gating kinetics. It suppressed native NaV currents and action potential (AP) firing in DRG neurons. GA inhibited the increase in action potential firing frequency in DRG neurons induced by inflammatory mediators. Mutational and molecular docking studies revealed that GA and bupivacaine targeted anaesthetics binding sites, with their use-dependent properties almost abolished in the F1737A mutant. In formalin and CFA-induced inflammatory pain models in male mice, GA demonstrated analgesic effects comparable to, or exceeding, those of the indomethacin, lidocaine and carbamazepine. GA showed minimal effects on skeletal muscle function but exhibited an inhibitory effect on the CaV2.2 channel.Conclusions and ImplicationsGA is a state-dependent sodium channel and CaV2.2 channel antagonist with potent analgesic effects. These findings support its potential as an antinociceptive agent in the treatment of chronic pain conditions.

Plant cell cultures of Taxus is a noteworthy resource of structurally novel taxanes. Here, five new 14‑oxygenated 6/8/6-type taxanes (1-5, mediataxanes DH) were isolated and their structures with absolute configurations were elucidated on the basis of comprehensive spectroscopic data and single-crystal X-ray diffraction analyses. The in vitro biological activity assays showed that mediataxane D (1) exhibited potent cytotoxicity against cervical carcinoma HeLa cells with an IC50 value of 4.5 μM.

Here, we report the discovery and functional characterization of one novel taxane C1β-hydroxylase (TmT1βH), belonging to the α-ketoglutarate (α-KG)/Fe(II)-dependent dioxygenase family from Taxus × media cell cultures. The incubation of recombinant TmT1βH with 1β-dehydroxybaccatin IV (1) as a substrate led to the production of a major C1-hydroxylated product, baccatin IV (1a), and a minor product, 15-hydroxy-11(15→1)abeo-baccatin IV (1b), a non-classical 5/7/6-type taxane. Moreover, in vitro biochemical assays, molecular docking, and molecular dynamics simulation combined with site-directed mutagenesis revealed the critical amino acid residues for TmT1βH catalysis. Substrate specificity investigations revealed that TmT1βH preferred taxoids with high oxygenation level. Notably, we have also discovered a novel specific enzyme (Tm576) belonging to α-KG/Fe(II)-dependent dioxygenase that was able to convert 1 to 1b independently. A mechanism that the 5/7/6-membered carbon framework arises from prototypical 6/8/6-type taxane skeleton via radical rearrangement was proposed based on DFT calculations. More importantly, we artificially reconstructed the biosynthetic pathway of two important taxanes, baccatin IV, and baccatin VI, from GGPP in tobacco system. This work not only fully characterizes the role of C1β-hydroxylase of taxoids, but also offered new insights into the formation of taxane structural diversity.

Phenylspirodrimanes are a class of structurally diverse meroterpenoids, including the bioactive dimer stachybocin A (1) and the high-reactivity monomer stachybotrydial (2), which are isolated from the genus Stachybotrys. Whereas the biosynthetic pathway of these phenylspirodrimane meroterpenoids has remained elusive. Herein, we deciphered the complete biosynthetic pathway of 2 with unprecedented two gene clusters and five discrete genes by genome mining, gene inactivation, heterologous expression, biochemical experiments, and especially combining with transcriptome-based hierarchical clustering and expression correlation analyses. Totally, 11 genes for the phenylspirodrimane core skeleton formation, 8'-methyl oxidation, and 3-OH epimerization were efficiently discovered and functionally characterized. Notably, these biosynthetic genes are distributed across seven distinct regions, with a rare combination of multiple gene clusters and genes outside the clusters. Bioactivity assays revealed that four intermediates 6-8, and 9a exhibited significant inhibitory effect on the inactivated state hNaV1.2 channels with IC50 values of 0.15, 0.04, 0.28, and 1.91 μmol/L, respectively. These findings expand our understanding of phenylspirodrimane-type meroterpenoid biosynthesis and underscore the utility of transcriptome-based hierarchical clustering and expression correlation analyses for identifying unclustered biosynthetic genes in fungi.

Cephalotane-type diterpenoids, a class of natural products exclusively found in Cephalotaxus plants, are well known for their attractive structures and potent biological activities. However, their low natural abundance and intricate cage-like structures hinder their accessibility. Recently, the identification of a cephalotene synthase (CsCTS) has addressed the first committed step in the biosynthesis. However, the enzymes involved in the complex post-modification of the cephalotene core into structurally diverse cephalotane-type diterpenoids remain obscure. In this study, we functionally characterised four novel cytochrome P450 enzymes from C. sinensis. These enzymes demonstrate multiple oxidative functions and cooperatively catalyse a cascade of oxidation reactions, including the formation of signature 13,17-lactone, 5,19-lactone, and tropone. We further co-expressed the characterised CYP450 enzymes in combination with CsCTS to produce a variety of cephalotane-type diterpenoids, including hainanolidol (2), mannolide C (3), mannolide A (4), and cephinoid H (5), in Nicotiana benthamiana. Subsequently, harringtonolide (1) was chemically converted from hainanolidol (3.9 µmol with 10 equiv of Pb(OAc)4) in 87.5% yield. In this study, the biosynthetic pathways of representative cephalotane-type diterpenoids were elucidated and reconstructed, thereby establishing a foundation for their sustainable production through biosynthesis and/or chemo-biosynthesis, highlighting the remarkable efficiency of merely five Cephalotaxus-specific enzymes in assembling such structurally complex natural products.

Sesquiterpene synthases (STPSs) catalyze carbocation-driven cyclization reactions that can generate structurally diverse hydrocarbons. The deprotonation-reprotonation process is widely used in STPSs to promote structural diversity, largely attributable to the distinct regio/stereoselective reprotonations. However, the molecular basis for reprotonation regioselectivity remains largely understudied. Herein, we analyzed two highly paralogous STPSs, Artabotrys hexapetalus (−)-cyperene synthase (AhCS) and ishwarane synthase (AhIS), which catalyze reactions that are distinct from the regioselective protonation of germacrene A (GA), resulting in distinct skeletons of 5/5/6 tricyclic (−)-cyperene and 6/6/5/3 tetracyclic ishwarane, respectively. Isotopic labeling experiments demonstrated that these protonations occur at C3 and C6 of GA in AhCS and AhIS, respectively. The cryo-electron microscopy-derived AhCS complex structure provided the structural basis for identifying different key active site residues that may govern their functional disparity. The structure-guided mutagenesis of these residues resulted in successful functional interconversion between AhCS and AhIS, thus targeting the three active site residues [L311-S419-C458]/[M311-V419-A458] that may act as a C3/C6 reprotonation switch for GA. These findings facilitate the rational design or directed evolution of STPSs with structurally diverse skeletons.

Epimedium is widely used in traditional Chinese medicine and contains rich bioactive compounds. These compounds often have a methyl group at their 4'-OH position catalyzed by methyltransferases. Therefore, studying methyltransferases in Epimedium plants is of great significance. In this study, a flavonol methyltransferase, EpOMT4, was isolated from Epimedium pseudowushanense B.L. Guo. The recombinant enzyme regiospecifically transferred a methyl group to the 4'-OH position of 8-prenylkaempferol forming icaritin. The study demonstrates that enzymatic methylation of flavonoids in Epimedium plants holds significant potential and could provide a promising alternative method for the biosynthetic production of bioactive methylated prenylflavonoids.