Aided-efflux and high production of β-amyrin realized by β-cyclodextrin in situ synthesized on surface of Saccharomyces cerevisiae

As a plant-derived pentacyclic triterpenoid, β-amyrin has been heterogeneously synthesized in Saccharomyces cerevisiae. However, β-amyrin is intracellularly produced in a lower gram scale using recombinant S. cerevisiae, which limits the industrial applications. Although many strategies have been proven to be effective to improve the production of β-amyrin, the intracellularly accumulation is still a challenge in reaching higher titer and simplifying the extraction process. To solve this problem, the amphiphilic β-cyclodextrin (β-CD) has been previously employed to aid the efflux of β-amyrin out of the cells. Nevertheless, the supplemented β-CD in the medium is not consistent with β-amyrin synthesis and has the disadvantage of rather high cost. Therefore, an aided-efflux system based on in situ synthesis of β-CD was developed in this study to enhance the biosynthesis of β-amyrin and its efflux. The in situ synthesis of β-CD was started from starch by the surface displayed cyclodextrin glycosyltransferase (CGTase) on yeast cells. As a result, the synthesized β-CD could capture 16% of the intracellular β-amyrin and improve the total production by 77%. Furthermore, more strategies including inducing system remodeling, precursor supply enhancement, two-phase fermentation and lipid synthesis regulation were employed. Finally, the production of β-amyrin was increased to 73 mg/L in shake flask, 31 folds higher than the original strain, containing 31 mg/L of extracellular β-amyrin. Overall, this work provides novel strategies for the aided-efflux of natural products with high hydrophobicity in engineered S. cerevisiae.     

Biosynthesis of sterols and triterpenes in Pelargonium hortorum

Although all parts of the geranium plant (Pelargonium hortorum) are capable of synthesizing sterols and triterpenes and their esters in vitro from mevalonic acid-[2-¹⁴C], the aerial portions are more active than other tissues. All plant parts were shown to incorporate mevalonic acid-[2-¹⁴C] into isoprenoids for at least 3 days. The leaves and petioles had the greatest incorporation on a wet weight basis. Chopped preparations showed comparable incorporations of mevalonate whereby rootlets incorporated about one half as much as most parts; the flower petals incorporated five times the average amount. In leaves the principal sterol synthesized was sitosterol. Metabolic studies with isolated leaves indicated a fairly rapid conversion of free tetracyclic triterpenes to 4-desmethyl-sterols, while β-amyrin was synthesized at a different rate than α-amyrin. Esterified tetracyclic triterpenes exhibited only a slight amount of conversion to 4-desmethylsterols.     

The biosynthesis,metabolism and translocation of β-amyrin in Sorghum bicolor

The biosynthesis of [¹⁴C] β-amyrin and three other labelled 3β-hydroxypentacyclic triterpenes from [2-^14C] acetate in leaves of Sorghum bicolor was demonstrated. Evidence for the metabolism of [¹⁴C] β-amyrin to the corresponding C-3 ketone (β-amyrone) and for the transport of [¹⁴C] β-amyrin in leaves was also obtained.     

Triterpenoids and steroids of some Sapotaceae and their chemotaxonomic significance

Extraction of the bark and timber of four Madhuca and five Palaquium species has yielded β-amyrin, β-amyrin acetate, β-amyrin cinnamate,     

The Chemical Constituents of Myripnois dioica Bunge

Five compounds have been isolated from Myripnois dioica Bunge, a plant native to China. They are identified as nonacosane, taraxerol acetate, β-amyrin acetate, β-amyrin and β-sitosterol.     

An Acyl Migration in Acetohalogenoglucosamines

Two triterpenoid alcohols, i.e., α- and β-amyrin have been isolated from the unsaponifiable fraction of the ethereal extract of Ilex latifolia Thunberg (Aquifoliaceae). The ratio of the amyrin contents, i.e., α-amyrin to β-amyrin was about 4:1. Stearic acid has also been isolated from the fatty-acid fraction of the hydrolysates.     

杠柳枝皮的化学成分研究(英文)

将杠柳枝皮粉末的乙醇提取物悬浮于水后,依次用石油醚、乙酸乙酯和正丁醇萃取.对乙酸乙酯萃取物采用各种柱层析进行多次分离后得到4个化合物,经波谱分析,鉴定它们的结构分别为β-香树素乙酸酯(1)、β-香树素(2)、periplogenin(3)和perilocoside N(4),其中化合物1和2是首次从该植物中分离得到.     

Terpenoids of Canarium zeylanicum

The following terpenoids were identified in the oleoresin, bark and timber of Canarium zeylanicum: 3β-hydroxyurs-12-en-11-one, 3β-hydroxyolean-12-en-11-one, olean-12-en-3,11-dione, urs-12-en-3,11-dione, α- and β-amyrin, α- and β-amyrenone, taraxerol, sitosterol, canaric acid, elemene, elemol, α-pinene, α- and β-phellandrene, limonene, terpineol and carvone.     

β-amyrin-induced apoptosis in Candida albicans triggered by calcium

The emergence of drug-resistant pathogens has urged researchers to discover alternatives for traditional antibiotics. β-amyrin, which is included in the category of triterpenoids extracted from plants, is known for its antimicrobial activity, although the underlying mechanism has not yet been revealed. This study was conducted to elucidate the antifungal mode of action of β-amyrin against Candida albicans. Based on the relevance between triterpenoids and oxidative molecules, reactive oxygen species (ROS) concentrations were detected, which showed a noticeable increment. Disruption of Ca²⁺ homeostasis in the cytosol was additionally analyzed, which was supported by interactions between two. Subsequently, decrease in mitochondrial membrane potential, increment of mitochondrial Ca²⁺, and ROS concentration were monitored, which suggested mitochondrial dysfunction modulated by Ca²⁺. Further investigation confirmed oxidative damage through glutathione reduction and DNA fragmentation. Accumulation of lethal damages resulted in the activation of caspases and externalization of phosphatidylserine, indicating the induction of yeast apoptosis by β-amyrin in C. albicans.     

Triterpenoids of Cnidosculus urens

The ethanol extract of Cnidosculus urens yielded β-amyrin cinnamate and germanicol acetate. Also, using ¹³C NMR spectroscopy, mixtures of β-amyrin, germanicol and lupeol as well as their acetates were identified.     

Triterpene functional genomics in licorice for identification of CYP72A154 involved in the biosynthesis of glycyrrhizin

Glycyrrhizin, a triterpenoid saponin derived from the underground parts of Glycyrrhiza plants (licorice), has several pharmacological activities and is also used worldwide as a natural sweetener. The biosynthesis of glycyrrhizin involves the initial cyclization of 2,3-oxidosqualene to the triterpene skeleton β-amyrin, followed by a series of oxidative reactions at positions C-11 and C-30, and glycosyl transfers to the C-3 hydroxyl group. We previously reported the identification of a cytochrome P450 monooxygenase (P450) gene encoding β-amyrin 11-oxidase (CYP88D6) as the initial P450 gene in glycyrrhizin biosynthesis. In this study, a second relevant P450 (CYP72A154) was identified and shown to be responsible for C-30 oxidation in the glycyrrhizin pathway. CYP72A154 expressed in an engineered yeast strain that endogenously produces 11-oxo-β-amyrin (a possible biosynthetic intermediate between β-amyrin and glycyrrhizin) catalyzed three sequential oxidation steps at C-30 of 11-oxo-β-amyrin supplied in situ to produce glycyrrhetinic acid, a glycyrrhizin aglycone. Furthermore, CYP72A63 of Medicago truncatula, which has high sequence similarity to CYP72A154, was able to catalyze C-30 oxidation of β-amyrin. These results reveal a function of CYP72A subfamily proteins as triterpene-oxidizing enzymes and provide a genetic tool for engineering the production of glycyrrhizin.     

CYP716A subfamily members are multifunctional oxidases in triterpenoid biosynthesis

Triterpenoids are a diverse group of secondary metabolites that are associated with a variety of biological activities. Oleanolic acid, ursolic acid and betulinic acid are common triterpenoids in plants with diverse biological activities, including antifungal, antibacterial, anti-human immunodeficiency virus (HIV) and/or antitumor activities. In the present study, using the gene co-expression analysis tool of Medicago truncatula, we found a strong correlation between CYP716A12 and β-amyrin synthase (bAS), which encodes the enzyme responsible for the initial cyclization of 2,3-oxidosqualene to β-amyrin (the basic structural backbone of most triterpenoid saponins). Through an in vitro assay, we identified CYP716A12 as a β-amyrin 28-oxidase able to modify β-amyrin to oleanolic acid (through erythrodiol and, possibly, oleanolic aldehyde). We also confirmed its activity in vivo, by expressing CYP716A12 in transgenic yeast that endogenously produce β-amyrin. In addition, CYP716A12 was evaluated for its potential α-amyrin- and lupeol-oxidizing activities. Interestingly, CYP716A12 was able to generate ursolic acid (through uvaol and, possibly, ursolic aldehyde) and betulinic acid (through betulin). Hence, CYP716A12 was characterized as a multifunctional enzyme with β-amyrin 28-oxidase, α-amyrin 28-oxidase and lupeol 28-oxidase activities. We also identified homologs of CYP716A12 in grape (CYP716A15 and CYP716A17) that are involved in triterpenoid biosynthesis, which indicates the highly conserved functionality of the CYP716A subfamily among plants. These findings will be useful in the heterologous production of pharmacologically and industrially important triterpenoids, including oleanolic acid, ursolic acid and betulinic acid.     

构树皮中三萜类化学成分

对构树皮的化学成分进行进一步研究,通过反复硅胶柱层析从构树皮乙醇提取物的石油醚萃取部位中分离得到六个三萜成分,利用波谱技术及理化性质鉴定其化学结构分别为3β-乙酰氧基-甘遂-7-烯-24S,25二醇(1),3β-乙酰氧基-7,23-甘遂二烯-25-醇(2),羽扇豆醇(3),α-香树脂醇乙酸酯(4),3β,25-二羟基-7,23-甘遂二烯(5)和β-香树脂醇(6)。其中化合物1为新的甘遂烷型三萜,化合物2～5为首次从该植物中分离得到。     

Three isoflavonoids from Iris germanica

From the rhizome of Iris germanica one new hexaoxygenated isoflavone, two new polyoxygenated isoflavone glucosides, the known isoflavonoids irisolidone, irigenin and iridin and acetovanillone, sitosterol, α-amyrin and β-amyrin were isolated. The structures of the new compounds were established by chemical and spectroscopic means and by correlation with known constitutents.     

Sterols and triterpenols in latex and cultured tissues of Euphorbia pulcherrima

The sterol and triterpenol constituents of Euphorbia pulcherrima latex and cultured callus tissues were examined by GLC and mass spectrometry. Latex extracts from different varieties contained sitosterol, β-amyrin, germanicol, cycloartenol, β-amyrin acetate, and germanicol acetate. Capillary GC profiles of these varieties indicated that the triterpene content was essentially identical for examined latices. Cultured tissues derived from petioles and stem internodes synthesized only sitosterol in significant quantities, although trace amounts of several sterols that occur in latex were also detected in cultured tissues. This study supports the interpretation that the pattern of triterpene synthesis in the laticifer of the normal plant is a highly controlled and stable phenomenon among varieties of this species.     

Hexane soluble non-volatiles in flowering to fruiting stages of Fatsia japonica

The n-hexane soluble non-volatile fraction of the acetone extracts from the flower buds, the flowers and the immature and the mature fruits of Fatsia japonica were all found to contain fatty acids, fatty acid methyl esters, squalene, β-amyrin and sterols. At all the stages between budding to the mature fruit, the major fatty acids were palmitic and linoleic acids and the major phytosterol was stigmasterol. In addition steryl and β-amyrenyl esters were found in the flowers and the immature and the mature fruits, but these esters were not present in the flower buds. Sitosteryl ester was the major constituent of the steryl ester fraction in the fruiting stages. Phytol was found in only the flowering stage and triglycerides in only the mature fruits. The variations in the lipid constituents is discussed in relation to the stages from budding to the mature fruit.