Targeted Discovery of Glycosylated Natural Products by Tailoring Enzyme-Guided Genome Mining and MS-Based Metabolome Analysis.

Glycosides make up a biomedically important class of secondary metabolites. Most naturally occurring glycosides were isolated from plants and bacteria; however, the chemical diversity of glycosylated natural products in fungi remains largely unexplored. Herein, we present a paradigm to specifically discover diverse and bioactive glycosylated natural products from fungi by combining tailoring enzyme-guided genome mining with mass spectrometry (MS)-based metabolome analysis. Through in vivo genes deletion and heterologous expression, the first fungal C-glycosyltransferase AuCGT involved in the biosynthesis of stromemycin was identified from Aspergillus ustus. Subsequent homology-based genome mining for fungal glycosyltransferases by using AuCGT as a probe revealed a variety of biosynthetic gene clusters (BGCs) containing its homologues in diverse fungi, of which the glycoside-producing capability was corroborated by high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis. Consequently, 28 fungal aromatic polyketide C/O-glycosides, including 20 new compounds, were efficiently discovered and isolated from the three selected fungi. Moreover, several novel fungal C/O-glycosyltransferases, especially three novel α-pyrone C-glycosyltransferases, were functionally characterized and verified in the biosynthesis of these glycosides. In addition, a proof of principle for combinatorial biosynthesis was applied to design the production of unnatural glycosides in Aspergillus nidulans. Notably, the newly discovered glycosides exhibited significant antiviral, antibacterial, and antidiabetic activities. Our work demonstrates the promise of tailoring enzyme-guided genome-mining approach for the targeted discovery of fungal glycosides and promotes the exploration of a broader chemical space for natural products with a target structural motif in microbial genomes.

A regio-specific 4'-O-methyltransferase from Epimedium pseudowushanense regiospecifically catalyzing 8-prenylkeampferol to icaritin.

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.

Exploring the catalytic diversity of two short-chain dehydrogenases/reductases from Stachybotrys chartarum.

Short-chain dehydrogenase/reductases (SDRs) belong to the NAD(P)(H)-dependent oxidoreductase superfamily, which have various functions of catalyzing oxidation/reduction reactions and have been generally used as powerful biocatalysts in the production of pharmaceuticals. In this study, ScSDR1 and ScSDR2, two new SDRs have been identified and characterized from Stachybotrys chartarum 3.5365. Substrate scope investigation revealed that both of the enzymes possessed the ability to oxidize ß-OH to ketone specifically, and exhibited substrate promiscuity and high stereo-selectivity for efficiently catalyzing the structurally different prochiral ketones to chiral alcohols. These findings not only suggest that ScSDR1 and ScSDR2 might be potent synthetic tools in drug research and development, but also provide good examples for further engineered enzymes with higher efficiency and stereo-selectivity.

A Cytochrome P450 Enzyme Catalyses Oxetane Ring Formation in Paclitaxel Biosynthesis.

Oxetane synthase (TmCYP1), a novel cytochrome P450 enzyme from Taxus×media cell cultures, has been functionally characterized to efficiently catalyse the formation of the oxetane ring in tetracyclic taxoids. Transient expression of TmCYP1 in Nicotiana benthamiana using 2α,5α,7ß,9α,10ß,13α-hexaacetoxytaxa-4(20),11(12)-diene (1) as a substrate led to the production of a major oxetane derivative, 1ß-dehydroxybaccatin IV (1 a), and a minor 4ß,20-epoxide derivative, baccatin I (1 b). However, feeding the substrate decinnamoyltaxinine J (2), a 5-deacetylated derivative of 1, yielded only 5α-deacetylbaccatin I (2 b), a 4ß,20-epoxide. A possible reaction mechanism was proposed on the basis of substrate-feeding, 2H and 18O isotope labelling experiments, and density functional theory calculations. This reaction could be an intramolecular oxidation-acetoxyl rearrangement and the construction of the oxetane ring may occur through a concerted process; however, the 4ß,20-epoxide might be a shunt product. In this process, the C5-O-acetyl group in substrate is crucial for the oxetane ring formation but not for the 4(20)-epoxy ring formation by TmCYP1. These findings provide a better understanding of the enzymatic formation of the oxetane ring in paclitaxel biosynthesis.

Characterization and Engineering of Two Highly Paralogous Sesquiterpene Synthases Reveal a Regioselective Reprotonation Switch.

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.

Characterization of a Malabaricane-Type Triterpene Synthase from Astragalus membranaceus and Enzymatic Synthesis of Astramalabaricosides.

Triterpenoids are a large and medicinally important group of natural products with a wide range of biological and pharmacological effects. Among them, malabaricane-type triterpenoids are a rare group of terpenoids with a 6,6,5-tricyclic ring system, and a few malabaricane triterpene synthases have been characterized to date. Here, an arabidiol synthase AmAS for the formation of the malabaricane-type 6,6,5-tricyclic triterpenoid skeleton in astramalabaricosides biosynthesis was characterized from Astragalus membranaceus. Multiple sequence alignment, site-directed mutagenesis, and molecular docking of AmAS reveal that residues Q256 and Y258 are essential for AmAS activity, and the triad motif IIH725-727 was the critical residue necessary for its product specificity. Mutation of IIH725-727 with VFN led to the formation of seven tricyclic, tetracyclic, and pentacyclic triterpenoids (1-7). Glycosylation of malabaricane-type triterpenoids in the biosynthesis of astramalabaricosides was also explored. Three triterpenoids (1, 5, and 6) displayed potent inhibitory effects against influenza A virus in vitro. These findings provide insights into malabaricane-type triterpenoids biosynthesis in A. membranaceus and access to diverse bioactive triterpenoids for drug discovery.

Diphenyl Ether Derivative Rhexocerins and Rhexocercosporins from the Endophytic Fungus Rhexocercosporidium sp. Dzf14 Active against Gram-Positive Bacteria with Multidrug-Resistance.

Ten new diphenyl ether polyketides, including rhexocerins A-D (1-4) and rhexocercosporins A-F (5-10), together with three known congeners (11-13), were isolated from the endophytic fungus Rhexocercosporidium sp. Dzf14 obtained from Dioscorea zingiberensis. Their structures were elucidated by analysis of NMR and HRESIMS data, and their absolute configurations were determined by quantum chemical ECD calculations and X-ray crystallography. Compounds 1-4 featured an unprecedented tetracyclic carbon skeleton (6/7/5/6). Among them, compounds 1 and 5-9 showed antibacterial activities against methicillin-resistant S. aureus T144 and vancomycin-resistant E. faecalis 10.

Enzymatic synthesis of anhydroicaritin, baohuoside and icariin.

Anhydroicaritin (1a), baohuoside (1b) and icariin (1c) were recognized as major pharmacologically active ingredients of Epimedium plants. Their primary means of acquisition were chemical isolation from plants. However, it suffers from low yield, environmental pollution and shortage of plants. Herein, to remedy these problems, biosynthesis was explored to obtain the three active ingredients. Fortunately, with SfFPT as 8-prenyltransferase, EpPF3RT and Ep7GT as glycosyltransferases, kaempferide (1) was transferred to 1a, 1b and 1c enzymatically. Thus, we report the details of this method. This approach represents a promising environmental friendly alternative for the production of these compounds from an abundant analogue.

Functional Characterization and Cyclization Mechanism of a Diterpene Synthase Catalyzing the Skeleton Formation of Cephalotane-Type Diterpenoids.

CsCTS, a new diterpene synthase from Cephalotaxus sinensis responsible for forming cephalotene, the core skeleton of cephalotane-type diterpenoids with a highly rigid 6/6/5/7 tetracyclic ring system, was functionally characterized. The stepwise cyclization mechanism is proposed mainly based on structural investigation of its derailment products, and further demonstrated through isotopic labeling experiments and density functional theory calculations. Homology modeling and molecular dynamics simulation combined with site-directed mutagenesis revealed the critical amino acid residues for the unique carbocation-driven cascade cyclization mechanism of CsCTS. Altogether, this study reports the discovery of the diterpene synthase that catalyzes the first committed step of cephalotane-type diterpenoid biosynthesis and delineates its cyclization mechanism, laying the foundation to decipher and artificially construct the complete biosynthetic pathway of this type diterpenoids.

Two new 5/6/6 polyketide-amino acid hybrids from a genetic mutant of Periconia sp. F-31.

Peridecalins C and D (1 and 2), one decalin and one oxygen-decalin containing polyketide-amino acid hybrids with 5/6/6 ring system, was isolated from a genetic mutant of Periconia sp. F-31. Their structures were elucidated through extensive spectroscopic data analysis, including 1 D/2D NMR and HR-MS spectra. Biosynthetically, two proposed Diels-Alder reactions are supposed to be involved in the skeleton construction of 1 and 2.

Sesquiterpenes from the endophytic fungus Periconia sp. F-31.

One new eremophilane sesquiterpene periconianone L (1), together with four known guaiane-type sesquiterpenes 4,10,11-trihydroxyguaiane (2), (-)-guai-1(10)-ene-4α,11-diolhydroxymecuration (3), guaidiol A (4), and epi-guaidiol A (5) were isolated from the endophytic fungus Periconia sp. F-31. The structure of the new compound was established by spectroscopic methods, including UV, IR, HRESIMS, and extensive NMR techniques. Compound 3 was isolated as natural product for the first time.

Biosynthesis of polyketides by two type III polyketide synthases from Aloe barbadensis.

Various bioactive polyketides have been found in Aloe barbadensis. However, the polyketide synthases (PKSs), which participate in biosynthesis of polyketides in A. barbadensis remain unknown. In this study, two type III PKSs (AbPKS1 and AbPKS2) were identified from A. barbadensis. AbPKS1 and AbPKS2 were able to utilize malonyl-CoA to yield heptaketides (TW93a and aloesone) and octaketides (SEK4 and SEK4b), respectively. AbPKS1 also exhibited catalytic promiscuity in recognizing CoA thioesters of aromatics to produce unusual polyketides. What Is more, a whole cell biocatalysis system with the capability of producing 26.4 mg/L of SEK4/SEK4b and 2.1 mg/L of aloesone was successfully established.

Bistachybotrysin K, one new phenylspirodrimane dimer from Stachybotrys chartarum with potent cytotoxic activity.

Bistachybotrysin K (1), one new phenylspirodrimane dimer with a central 6/7 oxygen heterocycle core, was isolated from the fungus Stachybotrys chartarum CGMCC 3.5365. Its structure was elucidated by extensive spectroscopic data and single-crystal X-ray diffraction. Compound 1 showed significant cytotoxicity against human tumor cell lines HCT116, NCI-H460, BGC823, Daoy, and HepG2 with IC50 values in the range of 1.1-4.7 µM.

Molecular insights into the enzyme promiscuity of an aromatic prenyltransferase.

Aromatic prenyltransferases (aPTases) transfer prenyl moieties from isoprenoid donors to various aromatic acceptors, some of which have the rare property of extreme enzymatic promiscuity toward both a variety of prenyl donors and a large diversity of acceptors. In this study, we discovered a new aPTase, AtaPT, from Aspergillus terreus that exhibits unprecedented promiscuity toward diverse aromatic acceptors and prenyl donors and also yields products with a range of prenylation patterns. Systematic crystallographic studies revealed various discrete conformations for ligand binding with donor-dependent acceptor specificity and multiple binding sites within a spacious hydrophobic substrate-binding pocket. Further structure-guided mutagenesis of active sites at the substrate-binding pocket is responsible for altering the specificity and promiscuity toward substrates and the diversity of product prenylations. Our study reveals the molecular mechanism underlying the promiscuity of AtaPT and suggests an efficient protein engineering strategy to generate new prenylated derivatives in drug discovery applications.

Ep7GT, a glycosyltransferase with sugar donor flexibility from Epimedium pseudowushanense, catalyzes the 7-O-glycosylation of baohuoside.

Icariin (1a), a 7-O-glycosylated flavonoid glycoside, is recognized as the major pharmacologically active ingredient of Epimedium plants, which have been used in traditional Chinese medicine for thousands of years. However, no glycosyltransferase (GT) responsible for the 7-O-glycosylation of flavonoids has been identified from Epimedium plants to date. Herein, a GT, Ep7GT, was identified from E. pseudowushanense B. L. Guo, which can regiospecifically transfer a glucose moiety to baohuoside (1) at 7-OH to form icariin (1a). Ep7GT showed a rare broad donor substrate spectrum, including UDP-glucose, UDP-xylose, UDP-N-acetylglucosamine, UDP-rhamnose, UDP-galactose, UDP-glucuronic acid and TDP-glucose. Moreover, two new derivatives of icariin (1a), 7-O-ß-d-[2-(acetylamino)-2-deoxy-glucopyranosyl]-baohuoside (1b) and 7-O-ß-d-xylosyl-baohuoside (1c), were biosynthesized by using Ep7GT in vitro. Engineered Escherichia coli harbouring Ep7GT was constructed, and 10.1 µg mL-1 icariin (1a) was yielded by whole-cell biotransformation with baohuoside (1) as the substrate. The present work not only characterizes the GT responsible for the 7-O-glycosylation in the biosynthesis of icariin in Epimedium plants, but also indicates the significant potential of an enzymatic approach for the production of glycosylated baohuoside derivatives with different sugar moieties. What's more, these findings also provide a promising alternative for producing natural/unnatural bioactive flavonoid glycosides by metabolic engineering.

Three new phenylspirodrimane derivatives with inhibitory effect towards potassium channel Kv1.3 from the fungus Stachybotrys chartarum.

Three new phenylspirodrimanes derivatives named stachybotrysins H and I (1 and 2) and stachybotrin E (3), together with one known compound stachybotrylactam (4), were isolated from Stachybotrys chartarum CGMCC 3.5365. Their structures were determined by extensive NMR data and mass spectroscopic analysis. Compounds 1 and 2 showed inhibitory effect towards potassium channel Kv1.3 with IC50 values of 13.4 and 10.9 µM, respectively.

Bistachybotrysins A-C, three phenylspirodrimane dimers with cytotoxicity from Stachybotrys chartarum.

Bistachybotrysins A-C (1-3), three phenylspirodrimane dimers representing an unusual [6,6,7,6]-tetracyclic skeleton with a central 2,10-dioxabicyclo[4.3.1]decan-7-ol core fused with two phenyl units, were isolated from a fungal strain, Stachybotrys chartarum CGMCC 3.5365. The structures of 1-3 were elucidated through extensive spectroscopic data analysis, including Mo2(AcO)4-induced and calculated electronic circular dichroism (ECD). 1 and 2 exhibited potent cytotoxicity against four human tumor cell lines with IC50 values in the range of 2.8-7.5 µM. Furthermore, a possible biogenesis for 1-3 is proposed.

A regiospecific rhamnosyltransferase from Epimedium pseudowushanense catalyzes the 3-O-rhamnosylation of prenylflavonols.

Epimedium is used in traditional Chinese medicine and contains flavonol glycosides that exhibit multiple biological activities. These bioactive flavonol glycosides usually have a rhamnose moiety at the 3-OH position of prenylflavonols, such as icariin (9), baohuoside I (1a) and baohuoside II (2a). However, to date, no rhamnosyltransferase has been reported to catalyze the 3-O-rhamnosylation of prenylflavonols. In this article, a flavonol rhamnosyltransferase, EpPF3RT, was identified from E. pseudowushanense B. L. Guo. The recombinant enzyme regiospecifically transfers a rhamnose moiety to 8-prenylkaempferol (1) and anhydroicaritin (2) at the 3-OH position to form baohuoside II (1a) and baohuoside I (2a) in vitro. In addition, a UDP-rhamnose synthase gene, EpRhS, from E. pseudowushanense was functionally characterized and used to produce the UDP-rhamnose sugar donor. Furthermore, an engineered Escherichia coli strain containing EpPF3RT and EpRhS was established to produce baohuoside II (1a) from whole cells. These studies indicate the significant potential of an enzymatic approach for the rhamnosylation of bioactive flavonoids in Epimedium plants and will provide a promising alternative for producing bioactive rhamnosylated flavonoids combined with other genes/enzymes by synthetic biology.

Enzymatic synthesis of glucuronidated metabolites of two neurological active agents using plant glucuronosyltransferases.

Glucuronidation is an important and popular metabolic reaction in vivo of drugs. The further evaluation of biological activity and toxicity of glucuronides is necessary in the course of the drug research and development. However, the synthesis of glucuronides is limited by the lack of efficient approach. Herein, we have developed a new glucuronide synthesis method using plant uridine diphosphate-dependent glucuronosyltransferases (UGTs), UGT88D4, UGT88D7, and EpGT8, enabling the convenient preparation for corresponding O-glucuronide metabolites (1a, 2a, 3a, and 3b) in milligram scale of two neurological active agents, IMM-H004 (1) and FLZ (2). Their structures were characterized by spectroscopic data analyses.

Three xanthone dimers from the Thai mangrove endophytic fungus Phomopsis sp. xy21.

Three new xanthone dimers, named phomoxanthones C-E (1-3), were obtained from the Thai mangrove fungus Phomopsis sp. xy21, together with four known ones. The structures of these compounds were elucidated by the analysis of HRESIMS and extensive NMR spectroscopic data. The absolute configuration of 1 was established by the analysis of single-crystal X-ray diffraction with Cu Kα radiation. Phomoxanthones C (1) and D (2) possess a highly oxidized hexahydroxanthone skeleton.