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Made available in DSpace on 2020-12-12T02:09:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-01 Plants produce a vast array of defense compounds to protect themselves from pathogen attack or herbivore predation. Saponins are a specific class of defense compounds comprising bioactive glycosides with a steroidal or triterpenoid aglycone backbone. The model legume Medicago truncatula synthesizes two types of saponins, hemolytic saponins and nonhemolytic soyasaponins, which accumulate as specific blends in different plant organs. Here, we report the identification of the seed-specific transcription factor TRITERPENE SAPONIN ACTIVATION REGULATOR3 (TSAR3), which controls hemolytic saponin biosynthesis in developing M. truncatula seeds. Analysis of genes that are coexpressed with TSAR3 in transcriptome data sets from developing M. truncatula seeds led to the identification of CYP88A13, a cytochrome P450 that catalyzes the C-16a hydroxylation of medicagenic acid toward zanhic acid, the final oxidation step of the hemolytic saponin biosynthesis branch in M. truncatula. In addition, two uridine diphosphate glycosyltransferases, UGT73F18 and UGT73F19, which glucosylate hemolytic sapogenins at the C-3 position, were identified. The genes encoding the identified biosynthetic enzymes are present in clusters of duplicated genes in the M. truncatula genome. This appears to be a common theme among saponin biosynthesis genes, especially glycosyltransferases, and may be the driving force of the metabolic evolution of saponins. Ghent University Department of Plant Biotechnology and Bioinformatics VIB Center for Plant Systems Biology Department of Organic Chemistry Institute of Chemistry São Paulo State University (UNESP) Institut de Recherche en Horticulture et Semences-Unités Mixtes de Recherche Université d’Angers INRAE Institut Agro, SFR 4207 QuaSaV VIB Metabolomics Core Department of Organic Chemistry Institute of Chemistry São Paulo State University (UNESP)

Triterpenoids are widespread bioactive plant defence compounds with potential use as pharmaceuticals, pesticides and other high-value products. Enzymes belonging to the cytochrome P450 family have an essential role in creating the immense structural diversity of triterpenoids across the plant kingdom. However, for many triterpenoid oxidation reactions, the corresponding enzyme remains unknown. Here we characterize CYP716 enzymes from different medicinal plant species by heterologous expression in engineered yeasts and report ten hitherto unreported triterpenoid oxidation activities, including a cyclization reaction, leading to a triterpenoid lactone. Kingdom-wide phylogenetic analysis of over 400 CYP716s from over 200 plant species reveals details of their evolution and suggests that in eudicots the CYP716s evolved specifically towards triterpenoid biosynthesis. Our findings underscore the great potential of CYP716s as a source for generating triterpenoid structural diversity and expand the toolbox available for synthetic biology programmes for sustainable production of bioactive plant triterpenoids., Cytochrome P450 family enzymes have an essential role in the creation of triterpenoid diversity in plants. Here, the authors describe triterpenoid synthesis as mediated by CYP716 enzymes in medicinal plant species, and perform phylogenetic analysis to describe CYP716 molecular evolution in plants.

Made available in DSpace on 2020-12-12T01:45:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-11-01 The native Brazilian plant Maytenus ilicifolia accumulates a set of quinone methide triterpenoids with important pharmacological properties, of which maytenin, pristimerin and celastrol accumulate exclusively in the root bark of this medicinal plant. The first committed step in the quinone methide triterpenoid biosynthesis is the cyclization of 2,3-oxidosqualene to friedelin, catalyzed by the oxidosqualene cyclase friedelin synthase (FRS). In this study, we produced heterologous friedelin by the expression of M. ilicifolia FRS in Nicotiana benthamiana leaves and in a Saccharomyces cerevisiae strain engineered using CRISPR/Cas9. Furthermore, friedelin-producing N. benthamiana leaves and S. cerevisiae cells were used for the characterization of CYP712K4, a cytochrome P450 from M. ilicifolia that catalyzes the oxidation of friedelin at the C-29 position, leading to maytenoic acid, an intermediate of the quinone methide triterpenoid biosynthesis pathway. Maytenoic acid produced in N. benthamiana leaves was purified and its structure was confirmed using high-resolution mass spectrometry and nuclear magnetic resonance analysis. The three-step oxidation of friedelin to maytenoic acid by CYP712K4 can be considered as the second step of the quinone methide triterpenoid biosynthesis pathway, and may form the basis for further discovery of the pathway and heterologous production of friedelanes and ultimately quinone methide triterpenoids. Department of Plant Biotechnology and Bioinformatics Ghent University VIB Center for Plant Systems Biology Department of Organic Chemistry Institute of Chemistry so Paulo State University (UNESP) Department of Biological Sciences School of Pharmaceutical Sciences so Paulo State University (UNESP) VIB Metabolomics Core Department of Organic Chemistry Institute of Chemistry so Paulo State University (UNESP) Department of Biological Sciences School of Pharmaceutical Sciences so Paulo State University (UNESP)

Introduction: Triterpene saponins are important bioactive plant natural products found in many plant families including the Leguminosae. Objectives: We characterize two Medicago truncatula cytochrome P450 enzymes, MtCYP72A67 and MtCYP72A68, involved in saponin biosynthesis including both in vitro and in planta evidence. Methods: UHPLC-(-)ESI-QToF-MS was used to profile saponin accumulation across a collection of 106 M. truncatula ecotypes. The profiling results identified numerous ecotypes with high and low saponin accumulation in root and aerial tissues. Four ecotypes with significant differential saponin content in the root and/or aerial tissues were selected, and correlated gene expression profiling was performed. Results: Correlation analyses between gene expression and saponin accumulation revealed high correlations between saponin content with gene expression of -amyrin synthase, MtCYP716A12, and two cytochromes P450 genes, MtCYP72A67 and MtCYP72A68. In vivo and in vitro biochemical assays using yeast microsomes containing MtCYP72A67 revealed hydroxylase activity for carbon 2 of oleanolic acid and hederagenin. This finding was supported by functional characterization of MtCYP72A67 using RNAi-mediated gene silencing in M. truncatula hairy roots, which revealed a significant reduction of 2-hydroxylated sapogenins. In vivo and in vitro assays with MtCYP72A68 produced in yeast showed multifunctional oxidase activity for carbon 23 of oleanolic acid and hederagenin. These findings were supported by overexpression of MtCYP72A68 in M. truncatula hairy roots, which revealed significant increases of oleanolic acid, 2-hydroxyoleanolic acid, hederagenin and total saponin levels. Conclusions: The cumulative data support that MtCYP72A68 is a multisubstrate, multifunctional oxidase and MtCYP72A67 is a 2-hydroxylase, both of which function during the early steps of triterpene-oleanate sapogenin biosynthesis.

Nature Plants 3, 16205 (2016); published 22 December 2016; corrected 12 June 2017. Two of the Supplementary Information files originally published contained errors. In Supplementary Table 1a, the SolycIDs for SMO1 and SMO2 were interchanged. In Supplementary Data 1, the SolycIDs for SMO3 and SMO4 were interchanged.

The red seaweed Laurencia dendroidea belongs to the Rhodophyta, a phylum of eukaryotic algae that is widely distributed across the oceans and that constitute an important source of bioactive specialized metabolites. Laurencia species have been studied since 1950 and were found to contain a plethora of specialized metabolites, mainly halogenated sesquiterpenes, diterpenes and triterpenes that possess a broad spectrum of pharmacological and ecological activities. The first committed step in the biosynthesis of triterpenes is the cyclization of 2,3-oxidosqualene, an enzymatic reaction carried out by oxidosqualene cyclases (OSCs), giving rise to a broad range of different compounds, such as the sterol precursors cycloartenol and lanosterol, or triterpene precursors such as cucurbitadienol and β-amyrin. Here, we cloned and characterized the first OSC from a red seaweed. The OSC gene was identified through mining of a L. dendroidea transcriptome dataset and subsequently cloned and heterologously expressed in yeast for functional characterization, which indicated that the corresponding enzyme cyclizes 2,3-oxidosqualene to the sterol precursor cycloartenol. Accordingly, the gene was named L. dendroidea cycloartenol synthase (LdCAS). A phylogenetic analysis using OSCs genes from plants, fungi and algae revealed that LdCAS grouped together with OSCs from other red algae, suggesting that cycloartenol could be the common product of the OSC in red seaweeds. Furthermore, profiling of L. dendroidea revealed cholesterol as the major sterol accumulating in this species, implicating red seaweeds contain a ‘hybrid’ sterol synthesis pathway in which the phytosterol precursor cycloartenol is converted into the major animal sterol cholesterol.

With tens of thousands of characterized members, terpenoids constitute the largest class of natural compounds that are synthesized by all living organisms. Several terpenoids play primary roles in the maintenance of cell membrane fluidity, as pigments or as phytohormones, but most of them function as specialized metabolites that are involved in plant resistance to herbivores or plant-environment interactions. Terpenoids are an essential component of human nutrition, and many are economically important pharmaceuticals, aromatics and potential next-generation biofuels. Because of the often low abundance in their natural source, as well as the demand for novel terpenoid structures with new or improved bioactivities, terpenoid biosynthesis has become a prime target for metabolic engineering and synthetic biology projects. In this review we focus on the creation of new-to-nature or tailor-made plant-derived terpenoids in photosynthetic organisms, in particular by means of combinatorial biosynthesis and the activation of silent metabolism. We reflect on the characteristics of different potential photosynthetic host organisms and recent advances in synthetic biology and discuss their utility for the (heterologous) production of (novel) terpenoids.