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Oxidosqualene cyclases (OSCs) are the key enzymes accountable for the cyclization of 2,3-oxidosqualene to varied triterpenoids and phytosterols. Hoodia gordonii (from the family Apocynaceae), a native of the Kalahari deserts of South Africa, Namibia, and Botswana, is being sold as a prevalent herbal supplement for weight loss. The appetite suppressant properties are attributed to P57AS3, an oxypregnane steroidal glycoside. At the molecular level, the enzymes involved in the biosynthesis of triterpenes and phytosterols from H. gordonii have not been previously reported. In the current study, predicted transcripts potentially encoding oxidosqualene cyclases were recognized first by searching publicly available H. gordonii RNA-seq datasets. Two OSC-like sequences were selected for functional analysis. A monofunctional OSC, designated HgOSC1 which encodes lupeol synthase, and HgOSC2, a multifunctional cycloartenol synthase forming cycloartenol and other products, were observed through recombinant enzyme studies. These studies revealed that distinct OSCs exist for triterpene formation in H. gordonii and provided opportunities for the metabolic engineering of specific precursors in producing phytosterols in this plant species. [ABSTRACT FROM AUTHOR]

Astragalosides are the main active constituents of traditional Chinese medicine Huang-Qi, of which cycloastragenol-type glycosides are the most typical and major bioactive compounds. This kind of compounds exhibit various biological functions including cardiovascular protective, neuroprotective, etc. Owing to the limitations of natural sources and the difficulties encountered in chemical synthesis, re-engineering of biosynthetic machinery will offer an alternative and promising approach to producing astragalosides. However, the biosynthetic pathway for astragalosides remains elusive due to their complex structures and numerous reaction types and steps. Herein, guided by transcriptome and phylogenetic analyses, a cycloartenol synthase and four glycosyltransferases catalyzing the committed steps in the biosynthesis of such bioactive astragalosides were functionally characterized from Astragalus membranaceus. AmCAS1, the first reported cycloartenol synthase from Astragalus genus, is capable of catalyzing the formation of cycloartenol; AmUGT15, AmUGT14, AmUGT13, and AmUGT7 are four glycosyltransferases biochemically characterized to catalyze 3-O-xylosylation, 3-O-glucosylation, 25-O-glucosylation/O-xylosylation and 2ʹ-O-glucosylation of cycloastragenol glycosides, respectively. These findings not only clarified the crucial enzymes for the biosynthesis and the molecular basis for the structural diversity of astragalosides in Astragalus plants, also paved the way for further completely deciphering the biosynthetic pathway and constructing an artificial pathway for their efficient production.

Oxidosqualene cyclases (OSCs) are the key enzymes accountable for the cyclization of 2,3-oxidosqualene to varied triterpenoids and phytosterols. Hoodia gordonii (from the family Apocynaceae), a native of the Kalahari deserts of South Africa, Namibia, and Botswana, is being sold as a prevalent herbal supplement for weight loss. The appetite suppressant properties are attributed to P57AS3, an oxypregnane steroidal glycoside. At the molecular level, the enzymes involved in the biosynthesis of triterpenes and phytosterols from H. gordonii have not been previously reported. In the current study, predicted transcripts potentially encoding oxidosqualene cyclases were recognized first by searching publicly available H. gordonii RNA-seq datasets. Two OSC-like sequences were selected for functional analysis. A monofunctional OSC, designated HgOSC1 which encodes lupeol synthase, and HgOSC2, a multifunctional cycloartenol synthase forming cycloartenol and other products, were observed through recombinant enzyme studies. These studies revealed that distinct OSCs exist for triterpene formation in H. gordonii and provided opportunities for the metabolic engineering of specific precursors in producing phytosterols in this plant species.

Astragalosides are the main active constituents of traditional Chinese medicine Huang-Qi, of which cycloastragenol-type glycosides are the most typical and major bioactive compounds. This kind of compounds exhibit various biological functions including cardiovascular protective, neuroprotective, etc. Owing to the limitations of natural sources and the difficulties encountered in chemical synthesis, re-engineering of biosynthetic machinery will offer an alternative and promising approach to producing astragalosides. However, the biosynthetic pathway for astragalosides remains elusive due to their complex structures and numerous reaction types and steps. Herein, guided by transcriptome and phylogenetic analyses, a cycloartenol synthase and four glycosyltransferases catalyzing the committed steps in the biosynthesis of such bioactive astragalosides were functionally characterized from Astragalus membranaceus. AmCAS1, the first reported cycloartenol synthase from Astragalus genus, is capable of catalyzing the formation of cycloartenol; AmUGT15, AmUGT14, AmUGT13, and AmUGT7 are four glycosyltransferases biochemically characterized to catalyze 3- O -xylosylation, 3- O -glucosylation, 25- O -glucosylation/ O -xylosylation and 2 ʹ - O -glucosylation of cycloastragenol glycosides, respectively. These findings not only clarified the crucial enzymes for the biosynthesis and the molecular basis for the structural diversity of astragalosides in Astragalus plants, also paved the way for further completely deciphering the biosynthetic pathway and constructing an artificial pathway for their efficient production. A cycloartenol synthase and four glycosyltransferases with different glycosylation patterns in the biosynthesis of cycloastragenol-type astragalosides were identified from Astragalus membranaceus. [Display omitted] [ABSTRACT FROM AUTHOR]

Withania somnifera (Ashwagandha), is one of the most reputed Indian medicinal plants, having immense pharmacological activities due to the occurrence of withanolides. The withanolides are biosynthesized through triterpenoid biosynthetic pathway with the involvement of WsCAS leading to cyclization of 2, 3 oxidosqualene, which is a key metabolite to further diversify to a myriad of phytochemicals. In contrast to the available reports on the studies of WsCAS in withanolide biosynthesis, its involvement in phytosterol biosynthesis needs investigation. Present work deals with the understanding of role of WsCAS triterpenoid synthase gene in the regulation of biosynthesis of phytosterols & withanolides. Docking studies of WsCAS protein revealed Conserved amino acids, DCATE motif, and QW motif which are involved in efficient substrate binding, structure stabilization, and catalytic activity. Overexpression/silencing of WsCAS leading to increment/decline of phytosterols confers its stringent regulation in phytosterols biosynthesis. Differential regulation of WsCAS on the metabolic flux towards phytosterols and withanolide biosynthesis was observed under abiotic stress conditions. The preferential channelization of 2, 3 oxidosqualene towards withanolides and/or phytosterols occurred under heat/salt stress and cold/water stress, respectively. Stigmasterol and β-sitosterol showed major contribution in high/low temperature and salt stress, and campesterol in water stress management. Overexpression of WsCAS in Arabidopsis thaliana led to the increment in phytosterols in general. Thus, the WsCAS plays important regulatory role in the biosynthetic pathway of phytosterols and withanolides under abiotic stress conditions. [Display omitted] • Pharmacologically active withanolides are putatively biosynthesized through triterpenoid biosynthetic pathway. • Cyclization of 2, 3 oxidosqualene to cycloartenol leads to the way towards the diversification into withanolide and sterol biosynthetic pathways. • WsCAS expressed differential regulation for the biosynthesis of phytosterols and withanolides under different abiotic stress conditions. [ABSTRACT FROM AUTHOR]