Functional characterization of triterpene synthases in Cibotium barometz.

Cibotium barometz (Linn.) J. Sm., a tree fern in the Dicksoniaceae family, is an economically important industrial exported plant in China and widely used in Traditional Chinese Medicine. C. barometz produces a range of bioactive triterpenes and their metabolites. However, the biosynthetic pathway of triterpenes in C. barometz remains unknown. To clarify the origin of diverse triterpenes in C. barometz, we conducted de novo transcriptome sequencing and analysis of C. barometz rhizomes and leaves to identify the candidate genes involved in C. barometz triterpene biosynthesis. Three C. barometz triterpene synthases (CbTSs) candidate genes were obtained. All of them were highly expressed in C. barometz rhizomes, consisting of the accumulation pattern of triterpenes in C. barometz. To characterize the function of these CbTSs, we constructed a squalene- and oxidosqualene-overproducing yeast chassis by overexpressing all the enzymes in the MVA pathway under the control of GAL-regulated promoter and disrupted the GAL80 gene in Saccharomyces cerevisiae simultaneously. Heterologous expressing CbTS1, CbTS2, and CbTS3 in engineering yeast strain produced cycloartenol, dammaradiene, and diploptene, respectively. Phylogenetic analysis revealed that CbTS1 belongs to oxidosqualene cyclase, while CbTS2 and CbTS3 belong to squalene cyclase. These results decipher enzymatic mechanisms underlying the origin of diverse triterpene in C. barometz.

Discovering a uniform functional trade-off of the CBC-type 2,3-oxidosqualene cyclases and deciphering its chemical logic.

Many functionally promiscuous plant 2,3-oxidosqualene cyclases (OSCs) have been found, but complete functional reshaping is rarely reported. In this study, we have identified two new plant OSCs: a unique protostadienol synthase (AoPDS) and a common cycloartenol synthase (AoCAS) from Alisma orientale (Sam.) Juzep. Multiscale simulations and mutagenesis experiments revealed that threonine-727 is an essential residue responsible for protosta-13 (17),24-dienol biosynthesis in AoPDS and that the F726T mutant completely reshapes the native function of AoCAS into a PDS function to yield almost exclusively protosta-13 (17),24-dienol. Unexpectedly, various native functions were uniformly reshaped into a PDS function by introducing the phenylalanine → threonine substitution at this conserved position in other plant and non-plant chair-boat-chair-type OSCs. Further computational modeling elaborated the trade-off mechanisms of the phenylalanine → threonine substitution that leads to the PDS activity. This study demonstrates a general strategy for functional reshaping by using a plastic residue based on the decipherment of the catalytic mechanism.

Characterization and identification of chemical constituents in Corni Fructus and effect of storage using UHPLC-LTQ-Orbitrap-MS.

Corni Fructus (CF) has been widely used as both traditional medicine and food; however, systematic studies on its chemical profile and the impact of storage periods on the indicative components are lacking. In this study, UHPLC-LTQ-Orbitrap-MS was used to investigate the fragmentation behaviors of multiple compounds from CF and the content variety of its indicative components for different storage periods. The major basic components of CF were determined to be iridoid glucosides, pentacyclic triterpenoids, phenolic acids, tannins and flavonoids. The characteristic cleavage pathways of the iridoid glucosides, pentacyclic triterpenoids, phenolic acids, tannins and flavonoids were further investigated and elaborated, which could assist in identifying the structures of similar components of other Chinese herbal medicines. Using accurate mass measurements for each precursor ion and the subsequent fragmented ions, and then comparing with standards and literature data, a total of 130 components, including 69 iridoid glucosides, 9 pentacyclic triterpenoids, 16 phenolic acids, 20 tannins and 16 flavonoids, 47 of which are potentially new compounds, were identified. The storage period studies indicated that the contents of 19 indicative components in CF changed differently with the prolongation of the storage period. Among them, morroniside, loganin, sweroside, cornuside, gallic acid, oleanolic acid and ursolic acid were the most important. These results provide abundant information for the identification and improved understanding of the chemical constituents in CF to clarify the content variety of its indicative components for different storage periods.

Oxidosqualene Cyclases Involved in the Biosynthesis of Diverse Triterpenes in Camellia sasanqua.

Camellia sasanqua is an important economic plant that is rich in lipophilic triterpenols with pharmacological activities including antiallergic, anti-inflammatory, and anticancer activities. However, the key enzymes related to triterpene biosynthesis have seldom been studied in C. sasanqua. Oxidosqualene cyclases (OSCs) are the rate-limiting enzymes related to triterpene biosynthesis. In this study, seven putative OSC genes (CsOSC1-7) were mined from the C. sasanqua transcriptome. Six CsOSCs were characterized for the biosynthesis of diverse triterpene skeletons, including α-amyrin, ß-amyrin, δ-amyrin, dammarenediol-II, ψ-taraxasterol, taraxasterol, and cycloartenol by the heterologous expression system. CsOSC3 was a multiple functional α-amyrin synthase. Three key residues, Trp260, Tyr262, and Phe415, are critical to the catalytic performance of CsOSC3 judging from the results of molecular docking and site-directed mutagenesis. These findings provide important insights into the biosynthesis pathway of triterpenes in C. sasanqua.

Exploring the catalytic function and active sites of a novel C-glycosyltransferase from Anemarrhena asphodeloides.

Anemarrhena asphodeloides is an immensely popular medicinal herb in China, which contains an abundant of mangiferin. As an important bioactive xanthone C-glycoside, mangiferin possesses a variety of pharmacological activities and is derived from the cyclization reaction of a benzophenone C-glycoside (maclurin). Biosynthetically, C-glycosyltransferases are critical for the formation of benzophenone C-glycosides. However, the benzophenone C-glycosyltransferases from Anemarrhena asphodeloides have not been discovered. Herein, a promiscuous C-glycosyltransferase (AaCGT) was identified from Anemarrhena asphodeloides. It was able to catalyze efficiently mono-C-glycosylation of benzophenone, together with di-C-glycosylation of dihydrochalcone. It also exhibited the weak O-glycosylation or potent S-glycosylation capacities toward 12 other types of flavonoid scaffolds and a simple aromatic compound with -SH group. Homology modeling and mutagenesis experiments revealed that the glycosylation reaction of AaCGT was initiated by the conserved residue H23 as the catalytic base. Three critical residues H356, W359 and D380 were involved in the recognition of sugar donor through hydrogen-bonding interactions. In particular, the double mutant of F94W/L378M led to an unexpected enzymatic conversion of mono-C- to di-C-glycosylation. This study highlights the important value of AaCGT as a potential biocatalyst for efficiently synthesizing high-value C-glycosides.

Functional Characterization of a Novel Glycosyltransferase (UGT73CD1) from Iris tectorum Maxim. for the Substrate promiscuity.

Glycosylflavonoids are a class of natural products with multiple pharmacological activities and a lot of glycosyltransferases from various plant species have been reported that they were involved in the biosynthesis of these phytochemicals. However, no corresponding glycosyltransferase has been identified from the famous horticultural and medicinal plant Iris tectorum Maxim. Here, UGT73CD1, a novel glycosyltransferase, was identified from I. tectorum. based on transcriptome analysis and functional identification. Phylogenetic analysis revealed that UGT73CD1 grouped into the clade of flavonoid 7-OH OGTs. Biochemical analysis showed that UGT73CD1 was able to glycosylate tectorigenin at 7-OH to produce tectoridin, and thus assigned as a 7-O-glycosyltransferase. In addition, it also possessed robust catalytic promiscuity toward 12 structurally diverse flavonoid scaffolds and 3, 4-dichloroaniline, resulting in forming O- and N-glycosides. This work will provide insights into efficient biosynthesis of structurally diverse flavonoid glycosides for drug discovery.