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Gas chromatography analysis of the products of IaAS1 and IaAS2 over-expressed in yeast. pYES-DEST52 was served as a negative control and α-amyrin and β-amyrin as standards.
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Ilex asprella, a plant widely used as a folk herbal drug in southern China, produces and stores a large amount of triterpenoid saponins, most of which are of the α-amyrin type. In this study, two oxidosqualene cyclase (OSC) cDNAs, IaAS1 and IaAS2, were cloned from the I. asprella root. Functional characterisation was performed by heterologous expre...
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... products were extracted after 72 h of induction and analysed using gas chromatography (GC). Both IaAS1 and IaAS2 extracts contain two compounds that were not present in the control cells carrying empty vector (see Figure 4). To identify the products of IaAS1 and IaAS2, the cell extracts were submitted to gas chromatography-mass spectrometry (GC-MS) analysis. Two compounds found in IaAS1 and IaAS2 extracts were identified as α-amyrin and β-amyrin, respectively, by comparing their retention times and mass fragment patterns with an authentic standard, indicating that both enzymes are mixed ASs (see Figure 5). α-Amyrin was the major product of IaAS1, with a 4:1 ratio to β-amyrin, whereas β-amyrin was the main product of IaAS2, with a ratio of 19:1 to α-amyrin. Compared with other mixed ASs, IaAS1 exhibits a unique product specificity, with α-amyrin accounting for up to 80% of the enzyme product, second only to MdOSC1 in Malus × domestica [23]. ...
Citations
... Illex aspera a folklore medicinal plant from China had been the store house of large amounts of triterpenoid saponins. Two OSC cDNAs, IaAS1 and IaAS2 were cloned and expressed in Saccharomyces cerevisiae; which encoded a mixture of α-amyrin and β-amyrin respectively (Zheng et al. 2015). Adiantum capillus veneris was a widely distributed fern and its molecular basis for the biosynthesis of triterpenes and sterols in ferns were studied by cDNA cloning of squalene hopene cyclase and oxidosqualene cyclase in a yeast mutant GIL17. ...
Squalene hopene cyclases (SHC) convert squalene, the linear triterpene to fused ring product hopanoid by the cationic cyclization mechanism. The main function of hopanoids, a class of pentacyclic triterpenoids in bacteria involves the maintenance of membrane fluidity and stability. 2, 3-oxido squalene cyclases are functional analogues of SHC in eukaryotes and both these enzymes have fascinated researchers for the high stereo selectivity, complexity, and efficiency they possess. The peculiar property of the enzyme squalene hopene cyclase to accommodate substrates other than its natural substrate can be exploited for the use of these enzymes in an industrial perspective. Here, we present an extensive overview of the enzyme squalene hopene cyclase with emphasis on the cloning and overexpression strategies. An attempt has been made to explore recent research trends around squalene cyclase mediated cyclization reactions of flavour and pharmaceutical significance by using non-natural molecules as substrates.
... The expression differences may be attributed to the tissue specificity of BcBAS1. The phenomenon that the β-AS genes were specifically expressed in some specific tissues was also reported in other plants (Dhar et al. 2014;Zheng et al. 2015), which might also be one of the reasons for the inequitable distribution of SSs or other triterpenoid saponins. The relationship between the expression levels of BcBAS1 and the content of SSs was also investigated in this study. ...
Bupleurum chinense DC. is a commonly used plant in traditional Chinese medicine, and saikosaponins(SSs) are the main active oleanane-typetriterpene saponins in B. chinense. β-Amyrin synthase (β-AS) is an important enzyme in oleanane-type triterpenoid saponin synthesis, but its role in saikosaponin synthesis has rarely been studied. Here, the putative β-AS gene BcBAS1(Accession No.ON890382) selected according to metabolomic and transcriptomic analyses was cloned and functionally characterized by heterologous expression in Escherichia coli and Pichia pastoris, and its subcellular localization and expression patterns were examined. The molecular weight of the BcBAS1 recombinant protein was approximately 87 kDa, and this protein could catalyse the production of β-amyrin, the precursor of SSs. Furthermore, BcBAS1 was located in the cytosol, and relative expression in four tissues of the four genotypes was positively correlated with SSa and SSd contents. Our results indicate that BcBAS1 is a β-AS gene and may play an important role in saikosaponin biosynthesis and regulation. This study sheds light on the role of β-AS genes in the synthesis of SSs and provides insights for the metabolic engineering of SSs.
... It contains ursane-type triterpenoids and triterpenoid saponins with well-known pharmacological activities. The ursane triterpene α-amyrin is catalyzed by the key gene laAS1(AIS39793) [45]. The leaves of loquat (Eriobotrya japonica) possess high medicinal value due to the high amount of ursolic acid, which is one of the most effective active compounds. ...
Triterpenes are natural products of plants that can defend against microorganisms and various stresses. Oxidosqualene cyclase (OSC), the key rate-limiting enzyme of the triterpene biosynthetic pathway, catalyzes 2,3-oxidosqualene into sterols and triterpenes with different skeletons through the chair–boat–chair (CBC) conformation or chair–chair–chair (CCC) conformation. They were expanded in plants mainly by tandem duplication and are distributed in many plant lineages. They have multiple biological activities, including as functional foods and drugs. Here, we summarize the current characterized forest OSCs and their potential functions, especially for pharmacological applications. The study of triterpene-catalyzed enzyme OSC has an important scientific role and potential economic value. This paper summarizes the research advances of the main members of the OSC family in plants, their structure and function, the biosynthesis of triterpenes, and the molecular evolution of OSC.
... IaAS1 mainly produces α-amyrin, accounting to ≥80% of total amyrin production. IaAS2 mainly synthesizes β-amyrin with a yield of 95% (Zheng et al., 2015). And IaAO1 (a CYP716A210 homolog) catalyzes the C-28 carboxylation of amyrin (Ji et al., 2020). ...
... The resulted strain was named as WAT11tfAX-pTIaAO1-pUIaAO4. In addition, strain WAT11tfAX-pTIaAO1 which contained only plasmid pTIaAO1 and strain WAT11tfAX-pTIaAO1-pU which contained plasmid pTIaAO1 and empty vector pESC-URA were constructed and used as negative control. On the other hand, the plasmids pTIaAO5 mentioned above and pYES-DEST52 IaAS2 carrying the amyrin synthase IaAS2 from I. asprella (Zheng et al., 2015) were co-transformed into the engineered strain of S. cerevisiae WAT11tfA (Supplementary Table 2) to give strain WAT11tfA-pDIaAS2-pTIaAO5. For negative control, strain WAT11tfA-pDIaAS2 and strain WAT11tfA-pDIaAS2-pT were used. ...
Ilex asprella is a plant from Aquifoliaceae. Its root is commonly used as folk medicinal materials in southern China. The chemical compositions of I. asprella are rich in pentacyclic triterpenoids, which show various biological activities and demonstrate a good prospect for drug development. The elucidation of biosynthesis mechanism of triterpenoids in I. asprella could lay important foundations for the production of these precious plant secondary metabolites by metabolic engineering. Our previous studies have revealed IaAO1 (a CYP716A210 homolog) responsible for the C-28 oxidation of α- and β-amyrin. Herein, we reported the identification of three more cytochrome P450 monooxygenase genes IaAO2 (a CYP716A212 homolog), IaAO4 (CYP714E88), IaAO5 (CYP93A220), and a cytochrome P450 reductase gene IaCPR by using Saccharomyces cerevisiae eukaryotic expression system and gas chromatography-mass spectrometry. Among them, the protein encoded by IaAO2 can catalyze the C-28 oxidation of α-amyrin and β-amyrin, IaAO4 can catalyze the C-23 oxidation of ursolic acid and oleanolic acid, while IaAO5 is responsible for the C-24 oxidation of β-amyrin. By introducing three genes IaAO1, IaAO4 and IaCPR into S. cerevisiae. We constructed an engineered yeast strain that can produce C-23 hydroxyl ursane-type triterpenoid derivatives. This study contributes to a thorough understanding of triterpenoid biosynthesis of medicinal plants and provides important tools for further metabolic engineering.
... b-Amyrin synthase (bAS) is a very popular OSC, which has been discovered in more than 30 plant species. 24 of them have been characterized as solo-product triterpene synthases using a yeast heterogenous expression system (Kushiro et al. 1998;Morita et al. 2000;Haralampidis et al. 2001;Hayashi, Huang, Kirakosyan, et al. 2001;Iturbe-Ormaetxe et al. 2003;Zhang et al. 2003;Kajikawa et al. 2005;Basyuni, Oku, Tsujimoto, Kinjo, et al. 2007;Meesapyodsuk et al. 2007;Kirby et al. 2008;Liu et al. 2009;Scholz et al. 2009;Shibuya et al. 2009;Wang, Guhling, et al. 2011;Dhar et al. 2014;Huang et al. 2015;Zheng et al. 2015;Liu et al. 2016;Han et al. 2018;Putter et al. 2019;Zhou et al. 2019). It appears that bASs are more common in dicots, as only Avena strigosa bAS1 serves as the monocotyledonous single-functional b-amyrin synthase (Haralampidis et al. 2001). ...
... However, mono-functional aAS has not yet been cloned from any plant. Several characterized multi-functional OSCs could yield a-amyrin as a predominant product associated with more or less b-amyrin (Saimaru et al. 2007;Brendolise et al. 2011;Huang et al. 2012;Moses et al. 2015;Zheng et al. 2015;Yu et al. 2018). On the contrary, the percentage of a-amyrin in the total product of OsABAS and a multifunctional triterpene synthase (PsPSM) was almost equal to or lower than that of b-amyrin (Morita et al. 2000;Sun et al. 2013). ...
Triterpenoids are one of the largest groups of secondary metabolites and exhibit diverse structures, which are derived from C30 skeletons that are biosynthesized via the isoprenoid pathway by cyclization of 2,3-oxidosqualene. Triterpenoids have a wide range of biological activities, and are used in functional foods, drugs, and as industrial materials. Due to the low content levels in their native plants and limited feasibility and efficiency of chemical synthesis, heterologous biosynthesis of triterpenoids is the most promising strategy. Herein, we classified 121 triterpene alcohols/ketones according to their conformation and ring numbers, among which 51 skeletons have been experimentally characterized as the products of oxidosqualene cyclases (OSCs). Interestingly, 24 skeletons that have not been reported from nature source were generated by OSCs in heterologous expression. Comprehensive evolutionary analysis of the identified 152 OSCs from 75 species in 25 plant orders show that several pentacyclic triterpene synthases repeatedly originated in multiple plant lineages. Comparative analysis of OSC catalytic reaction revealed that stabilization of intermediate cations, steric hindrance, and conformation of active center amino acid residues are primary factors affecting triterpene formation. Optimization of OSC could be achieved by changing of side-chain orientations of key residues. Recently, methods, such as rationally design of pathways, regulation of metabolic flow, compartmentalization engineering, etc., were introduced in improving chassis for the biosynthesis of triterpenoids. We expect that extensive study of natural variation of large number of OSCs and catalytical mechanism will provide basis for production of high level of triterpenoids by application of synthetic biology strategies.
... In another study, researchers balanced the metabolic pathway and achieved transcriptional regulation of aligned oleanane-type triterpenoids by overexpression of UPC2-1, a global transcription factor for ergosterol synthesis in yeast. [77] In addition, they reconstructed the promoter at the binding site of UPC2 and the galactose regulatory network to promote gene expression, resulting in a 65 and 6.Eightfold increase in β-amyrin and OA, respectively. [78] This also provides a new idea for the synthesis and regulation of both α-amyrin and UA in yeast. ...
Background
Ursolic acid (UA) is a ursane-type pentacyclic triterpenoid compound, naturally produced in plants via specialized metabolism and exhibits vast range of remarkable physiological activities and pharmacological manifestations. Owing to significant safety and efficacy in different medical conditions, UA may serve as a backbone to produce its derivatives with novel therapeutic functions.
Purpose and Scope
This review aims to provide ideas for exploring more diverse structures to improve UA pharmacological activity and increasing its biological yield to meet the industrial requirements by systematically reviewing the current research progress of UA .
Summary
We first provides an overview of the pharmacological activities, acquisition methods and structural modifications of UA. Among them, we focused on the synthetic modifications of UA to yield valuable derivatives with enhanced therapeutic potential. Furthermore, harnessing the essential advances for green synthesis of UA and its derivatives by advent of metabolic engineering and synthetic biology are of great concern. In this regard, all pivotal advances for enhancing the production of UA have been discussed.
Conclusion
: In combination with the advantages of UA biosynthesis and transformation strategy, large-scale microbial production of UA is a promising platform for further exploration.
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... Recent transcriptomic studies on I. paraguariensis have focused on elucidating abiotic stress responses, depicting global female gene expression panels, and characterizing the phenylpropanoid pathway (Acevedo et al. 2013(Acevedo et al. , 2016(Acevedo et al. , 2019Debat et al. 2014;Fay et al. 2018). Gene expression studies on other Ilex species, I. asprella (Zheng et al. 2014(Zheng et al. , 2015 and I. pubescens (Wen et al. 2017), characterized the triterpenoid saponin biosynthetic pathway. ...
Differential epigenetic (DNA cytosine methylation) and gene expression patterns were investigated in reproductive and veg-etative organs from Ilex paraguariensis and I. dumosa, at distinct developmental stages. We aimed at contributing towards elucidating major molecular changes underlying the sexual differentiation processes which, in these dioecious species, are completely unknown. Simultaneously, as a first step towards the development of an early sexing system, we searched for promising molecular markers. This was assessed through Methylation Sensitive Amplified Polymorphism (MSAP) and Amplified Fragment Length Polymorphism on cDNA (cDNA-AFLP) techniques, applying discriminant multivariate analyses, and bioinformatic characterization of differential fragments. A significant positive correlation was found between epigenetic and indirect 'genetic' information for both species, indicating influence of the genetic background on the epigenetic variation. Higher epigenetic than genetic diversities were estimated. Our outcomes showed up to 1.86 times more representation of mCG subepiloci than mCCG in all organs sampled. Along the maturing stages of floral buds, the frequency of mCG evidenced an incremental trend, whereas mCCG and unmethylated conditions showed opposite tendencies. Reproductive and vegetative samples tended to cluster apart based on epigenetic patterns; at gene expression level, organs exhibited clear-cut distinctive patterns, nonetheless profiles of young leaves and floral primordia resemble. Epigenetic and expression data allowed discrimination of I. dumosa´s samples according to the gender of the donor; more elusive patterns were observed for I. paraguariensis. In total, 102 differentially methylated and expressed fragments were characterized bioinformatically. Forty-three were annotated in various functional categories; four candidate markers were validated through qPCR, finding statistical differences among organs but not among sexes. The methylation condition of epilocus C13m33 appears as indicative of gender in both species. Thirty-three organ-specific and 34 gender-specific methylated markers were discriminated and deserve further research, particularly those expressed in leaves. Our study contributes concrete candidate markers with potential for practical application.
... Enhancement of precursor supply is also achieved by adopting the traditional metabolic engineering approaches to overexpress the key genes, utilization of balanced pathways and downregulation of competitive pathways 80 . In another study, researchers balanced the metabolic pathway and achieved transcriptional regulation of aligned oleanane-type triterpenoids by overexpression of UPC2-1, a global transcription factor for ergosterol synthesis in yeast 81 . In addition, they reconstructed the promoter at the binding site of UPC2 and the galactose regulatory network to promote gene expression, resulting in a 65 and 6.8-fold increase in β-amyrin and oleanolic acid, respectively 82 . ...
Ursolic acid (UA) is a ursane-type pentacyclic triterpenoid compound, naturally produced in plants via specialized metabolism and exhibits vast range of remarkable physiological activities and pharmacological manifestations. Owing to significant safety and efficacy in different medical conditions, UA may serve as a backbone to produce its derivatives with novel therapeutic functions. This review systematically provides an overview of the pharmacological activities, acquisition methods and structural modification methods of UA. In addition, we focused on the synthetic modifications of UA to yield its valuable derivatives with enhanced therapeutic potential. Furthermore, harnessing the essential advances for green synthesis of UA and its derivatives by advent of metabolic engineering and synthetic biology are highlighted. In combination with the advantages of UA biosynthesis and transformation strategy, large-scale production and applications of UA is a promising platform for further exploration.
... 65 Therefore, the cyclization process of 2,3-oxidosqualene to form triterpenoids or sterols catalyzed by OSCs is regarded as a branch point of the biosynthetic pathway. 66 The cyclization process is primarily divided into four steps: (i) the initial protonation of the substrate, (ii) cyclization via a polyene addition cascade, (iii) rearrangement process via 1,2-hydride shifts or methyl shifts, and (iv) deprotonation to yield diverse products in the last step. 67 OSCs exhibit product specificity, which may depend on the numbers of high-energy intermediates stabilized by the enzyme. ...
Celastrol, a quinone‐methide triterpenoid, was extracted from Tripterygium wilfordii Hook. F. in 1936 for the first time. Almost 70 years later, it is considered one of the molecules most likely to be developed into modern drugs, as it exhibits notable bioactivity, including anticancer and anti‐inflammatory activity, and exerts antiobesity effects. In addition, the molecular mechanisms underlying its bioactivity are being widely studied, which offers new avenues for its development as a pharmaceutical reagent. Owing to its potential therapeutic effects and unique chemical structure, celastrol has attracted considerable interest in the fields of organic, biosynthesis, and medicinal chemistry. As several steps in the biosynthesis of celastrol have been revealed, the mechanisms of key enzymes catalyzing the formation and postmodifications of the celastrol scaffold have been gradually elucidated, which lays a good foundation for the future heterogeneous biosynthesis of celastrol. Chemical synthesis is also an effective approach to obtain celastrol. The total synthesis of celastrol was realized for the first time in 2015, which established a new strategy to obtain celastroid natural products. However, owing to the toxic effects and suboptimal pharmacological properties of celastrol, its clinical applications remain limited. To search for drug‐like derivatives, several structurally modified compounds were synthesized and tested. This review focuses primarily on the latest research progress in the biosynthesis, total synthesis, structural modifications, bioactivity, and mechanism of action of celastrol. We anticipate that this paper will facilitate a more comprehensive understanding of this promising compound and provide constructive references for future research in this field.
... SQE and OSC are the key ratelimiting enzymes in triterpenoid biosynthesis, catalysing the first oxygenation and cyclization steps, respectively [20,21]. Strategies for altering triterpenoid production by manipulating genes that encode triterpenoid pathway enzymes have been reported [22][23][24][25]. Transcription factors (TFs) present great potential for improving the production of secondary metabolites by activating or repressing structural genes in metabolic pathways by binding to their promoter regions [26]. ...
Background:
Limonoids are major bioactive compounds that are produced by the triterpenoid metabolic pathway. The detailed biochemical process of limonoid biosynthesis and the mechanism of its molecular regulation remain elusive. The identification of transcription factors that regulate limonoid biosynthetic pathways is very important for understanding the underlying regulatory mechanisms. This information could also provide tools for manipulating biosynthesis genes to modulate limonoid production.
Results:
In this study, the CiMYB42 transcription factor was isolated to identify its role in limonoid biosynthesis. Multiple alignment analysis and phylogenetic analysis demonstrated that CiMYB42 is a typical R2R3MYB transcription factor that shares high similarity of its amino acid sequence with AtMYB42. Limonoids contents were higher in Citrus sinensis and Citrus grandis than in other species. Limonoid accumulation during leaf development also showed diverse trends in different genotypes. The expression of CiMYB42 was significantly related to the limonoid content and the expression of CiOSC in some citrus accessions. The overexpression of CiMYB42 in sweet orange resulted in significant accumulation of limonin, whereas the downregulation of CiMYB42 by RNAi resulted in a dwarf phenotype and less nomilin accumulation. Furthermore, the results of a yeast one-hybrid assay and EMSA indicated that CiMYB42 binds exclusively to the TTGTTG sequence (type II MYB core) in the promoter of CiOSC. Together, these results suggest that CiMYB42 positively regulates limonoid biosynthesis by regulating the expression of CiOSC by binding to the TTGTTG sequence (type II MYB core) of its promoter.
Conclusions:
CiMYB42 is an important transcription activator involved in limonoid biosynthesis that regulates the expression of CiOSC by binding to the TTGTTG sequence (type II MYB core).
