Oxidosqualene Cyclase Research Articles

Oxidosqualene cyclases and cytochrome P450s involved in the biosynthesis of diverse triterpenes in Centipeda minima.

PpbZIP23 from a native Kentucky bluegrass (Poa pratensis L.) regulates osmotic stress tolerance in transgenic rice.

The differential cyclization and rearrangement of 2,3-oxidosqualene controlled by oxidosqualene cyclases (OSCs) represents one of the most complex single enzyme transformations in nature and gives rise to a vast array of triterpenoid diversity in the plant kingdom. Here we systematically mine 599 plant genomes representing 387 species and investigate OSC diversity across different plant lineages. From the OSC sequences identified, 20 were selected for functional evaluation. Through analysis of these enzymes, we discover product profiles within clades previously believed to be functionally conserved and OSCs producing triterpenes for which no enzymatic source was known. We also discover OSCs with product profiles that yield mechanistic insights into the control of specific reaction pathways. Our study reveals lineage-specific blooms of OSC subgroups suggestive of adaptation to different environmental niches, opens up previously inaccessible chemistry and provides a framework for systematic investigations of metabolic diversification and underlying enzymatic mechanisms in the plant kingdom.

Boswellia trees produce bioactive boswellic acids (BAs) in response to wounding, which are a rare class of C3-epimeric triterpenoids. Understanding BA biosynthesis, particularly knowing whether C3-epimerization occurs at the triterpenoid epoxidation/cyclization steps catalyzed by squalene epoxidase (SQE)/2,3-oxidosqualene cyclase (OSC) or at the later stage of scaffold modification, remains largely elusive. We identified four BsSQEs (BsSQE1-4) and six BsOSCs (BsOSC1-6) from the Boswellia serrata transcriptome, and functionally characterized them in vitro, in planta assays and using yeast SQE/OSC mutants to know the involvement of BsSQEs/BsOSCs in BA biosynthesis. Wound-inducible BsSQE1 and BsSQE3 formed (3S)-2,3-oxidosqualene, and a wound-inducible BsOSC3 produced α-amyrin and β-amyrin. BsSQEs did not make (3R)-2,3-oxidosqualene, a potential source of C3 epimers, and none of the BsOSCs formed C3 epimers when assayed using racemic (3R,S)-2,3-oxidosqualene. These results excluded the possibility of C3-epimerization at the squalene epoxidation/2,3-oxidosqualene cyclization steps. Remarkably, BsOSC3 overexpression in B. serrata leaves increased the contents of 3-epi-α-amyrin, 3-epi-β-amyrin and BAs, indicating that α/β-amyrin produced by BsOSC3 might get epimerized and subsequently fed into the BA biosynthetic pathway. Overall, the results suggested that 3-epi-α/β-amyrin is not the direct product of squalene epoxidation/2,3-oxidosqualene cyclization reactions; rather, 3-epi-α/β-amyrin might form via epimerization of α/β-amyrin, thus establishing a critical step in the BA biosynthetic pathway.

Exploring oxidosqualene cyclases and cytochrome P450s from Aralia elata for the synthesis of diverse pentacyclic triterpene sapogenins in Nicotiana benthamiana.

CaOSC5-CaOSC7 module governs metabolic partitioning between asiaticoside and asiaticoside B in Centella asiatica.

Plant triterpenoids are structurally diverse specialized metabolites with significant ecological, medicinal, and agricultural importance. Oxidosqualene cyclases (OSCs) catalyze the crucial cyclization step in triterpenoid biosynthesis, generating the fundamental carbon skeletons that determine their structural diversity and biological functions. Genome-wide identification of OSC genes was performed using bioinformatics tools, including HMMER and BLASTP, followed by phylogenetic analysis, gene structure analysis, conserved domain and motifs identification, cis-regulatory element prediction, protein–protein interaction analysis, and expression profiling using publicly available transcriptome data from UV-B treated A. annua six-week-old seedlings. We identified 24 AaOSC genes, classified into CAS, LAS, LUS, and unknown subfamilies. Phylogenetic analysis revealed evolutionary relationships with OSCs from other plant species. Gene structure analysis showed variations in exon–intron organization. Promoter analysis identified cis-regulatory elements related to light responsiveness, plant growth and development, hormone signaling, and stress response. Expression profiling revealed differential expression patterns of AaOSC genes under UV-B irradiation. This genome-wide characterization provides insights into the evolution and functional diversification of the OSC gene family in A. annua. The identified AaOSC genes and their regulatory elements lay the foundation for future studies aimed at manipulating triterpenoid biosynthesis for medicinal and biotechnological applications, particularly focusing on enhancing stress tolerance and artemisinin production.

Three cytochrome P450 88A subfamily enzymes, CYP88A108, CYP88A164, and CYP88A222, act as β-amyrin 11-oxidases involved in triterpenoid biosynthesis in Melia azedarach L.

Functional characterization of oxidosqualene cyclases and CYP716As associated with triterpene biosynthesis from Corydalis yanhusuo.

Neopicrorhiza scrophulariiflora (Pennell) D.Y.Hong, an endangered perennial herb, is rich in triterpenes, iridoids, and phenolic compounds, which exhibit significant pharmacological effects. However, the molecular mechanisms of triterpenoid biosynthesis in N. scrophulariiflora remain unclear. Here, transcriptomic and metabolomic analyses were performed to investigate the triterpene content in different tissues and the expression patterns of key enzyme-encoding genes related to triterpenoid biosynthesis. We functionally characterized eight upstream oxidosqualene cyclases (OSCs) involved in triterpenoid biosynthesis, among which NsOSC2 is a bifunctional enzyme capable of catalyzing the conversion of 2,3-oxidosqualene to β-amyrin and α-amyrin. Additionally, an efficient regeneration system and a stable genetic transformation system were established for N. scrophulariiflora. These findings reveal key genes in triterpenoid biosynthesis, providing a theoretical foundation for the future production of key triterpenoids in N. scrophulariiflora through synthetic biology approaches.

Plant cell suspension cultures offer a sustainable method for producing valuable secondary metabolites, such as bioactive pentacyclic triterpenes. This study established a high-triterpene-yielding cell suspension culture from the apple cultivar "Cox Orange Pippin". Through transcriptomic analysis and triterpene profiling across growth phases, we uncovered complex regulatory networks that govern biomass production and triterpene biosynthesis. Key biological processes, including cell cycle regulation, cell wall biosynthesis, lipid metabolism, and stress response mechanisms, play pivotal roles in culture dynamics. Differential gene expression linked to these processes revealed how the culture adapts to growth conditions and nutrient availability at each growth phase. Methyl jasmonate elicitation enhanced phenylpropanoid and flavonoid biosynthesis, along with specific triterpene production pathways, highlighting its potential for optimizing secondary metabolite production. Key enzymes, such as oxidosqualene cyclase 4 and a putative C-2α hydroxylase, were identified as promising targets for future metabolic engineering efforts. This study represents the first in-depth report on the molecular mechanisms underlying plant cell growth in bioreactors, specially focusing on a cell suspension culture derived from a semi-russeted apple cultivar. The findings reveal key regulatory pathways in biomass accumulation and triterpene production, offering valuable insights for optimizing bioreactor cultures for industrial applications.

Triterpenoids and steroids are structurally complex polycyclic natural products with potent biological functions, for example, as hormones. In all eukaryotes, the carbon skeletons of these compounds are generated by oxidosqualene cyclases, which carry out a polycyclization cascade to generate four or five rings with up to nine stereogenic centers in a targeted manner. The tight stereochemical control of this cascade reaction severely limits the stereochemical space accessible by known oxidosqualene cyclases. Considering that naturally occurring hormone stereoisomers have markedly different biological activities, finding ways to produce stereoisomers of triterpenes would be highly desirable to open new avenues for developing triterpenoid and steroid drugs. Here, we present a plant kingdom-wide sequence mining approach based on sequence similarity networks to search for noncanonical oxidosqualene cyclases that might produce triterpene stereoisomers. From 1,891 oxidosqualene cyclase sequences representing the diversity of green plants, six candidates were selected for functional evaluation by heterologous production in Nicotiana benthamiana. Of these six candidates, three produced rare or previously inaccessible triterpene stereoisomers, namely, (3S,13S)-malabarica-17,21-diene-3β,14-diol, 19-epi-lupeol, and a previously unknown hopanoid stereoisomer that we call protostahopenol. Site-directed mutagenesis revealed key residues important for catalytic activity. The sequence similarity network mining strategy employed here will facilitate the targeted discovery of enzymes with unusual activity in higher organisms, which are not amenable to common genome mining approaches. More importantly, our work expands the accessible stereochemical space of triterpenes and represents the first step to the development of new triterpenoid-derived drugs.

Triterpenoids widely exist in nature with diverse structures and possess various functional properties and biological effects. However, research on triterpenoids biosynthesis in Corynebacterium glutamicum is still limited to squalene, which restricts the development of C. glutamicum to produce high-value triterpenoids. In this study, C. glutamicum was developed as an efficient and flexible platform for the biosynthesis of different types of triterpenoids. Squalene was synthesized and the titer was improved to 400.1 mg/L in flask combining strategies of metabolic engineering and fermentation optimization. Particularly, intracellular squalene accounted for more than 97%, addressing the problem of leaking squalene in C. glutamicum, which may restrict the subsequent synthesis of other triterpenoids derived from squalene. Furthermore, 201.9 mg/L (3S)-2,3-oxidosqualene (SQO) and 264.9 mg/L (3S,22S)-2,3,22,23-dioxidosqualene (SDO) were successfully synthesized in strains harboring heterogeneous squalene epoxidase from Arabidopsis thaliana with different expression strengths. Therefore, a platform for de novo triterpenoids synthesis based on SQO or SDO was constructed in C. glutamicum. For instance, biosynthesis of α-amyrin and α-onocerin was achieved for the first time by introducing oxidosqualene cyclases in SQO- and SDO-producing C. glutamicum strains, respectively. After optimization, the titer of α-amyrin and α-onocerin was improved to 65.3 and 136.85 mg/L, respectively. Furthermore, ursolic acid, derived from α-amyrin, was synthesized after expressing cytochrome P450 enzyme and its compatible cytochrome P450 reductases with a titer of 486 μg/L. For the first time, reactions of epoxidation, cyclization, and oxidation from squalene were achieved in C. glutamicum, leading to the production of different types of triterpenoids. Our study provides a new platform for the production of triterpenoids, which will be helpful for the large-scale production of triterpenoids employing C. glutamicum as a chassis strain.

Dipsaci Radix is a commonly used Chinese herbal medicine in China, with triterpenoid saponins as the main active components. β-Amyrin synthase, a member of the oxidosqualene cyclase superfamily, plays a crucial role in the biosynthesis of oleanane-type triterpenoid saponins. Asperosaponin Ⅵ is an oleanane-type triterpenoid saponin. To explore the β-amyrin synthase genes involved in the biosynthesis of asperosaponin Ⅵ in Dipsacus asper, this study screened the candidate genes from the transcriptome data of D. asper. Two β-amyrin synthase genes, Da OSC1 and Da OSC2, were identified by phylogenetic analysis and correlation analysis. The coding sequences of Da OSC1 and Da OSC2 were 2 286 bp and 2 295 bp in length, encoding 761 and 764 amino acids,respectively. Multiple sequence alignments showed that Da OSC1 and Da OSC2 had three conserved motifs( DCTAE, QW, and MWCYCR) unique to the oxidosqualene cyclase family. Real-time quantitative PCR results showed that Da OSC1 and Da OSC2 had the highest expression levels in the roots. Compared with normal growth conditions, the low-temperature treatment significantly upregulated the expression of Da OSC1 and Da OSC2. Agrobacterium-mediated transient expression of Da OSC1 and Da OSC2 in Nicotiana benthamiana resulted in the production of β-amyrin, which suggested that Da OSC1 and Da OSC2 were able to catalyze the synthesis of β-amyrin. This study clarified the catalytic functions of two β-amyrin synthases in D. asper, analyzed their expression patterns in different tissue and at low temperatures. The findings provide a foundation for further studying the biosynthetic pathway and regulatory mechanism of asperosaponin Ⅵ in D. asper.

The clustered distribution of genes involved in metabolic pathways within the plant genome has garnered significant attention from researchers. By comparing and analyzing changes in the flanking regions of metabolic genes across a diverse array of species, we can enhance our understanding of the formation and distribution of biosynthetic gene clusters (BGCs). In this study, we have designed a workflow that uncovers and assesses conserved positional relationships between genes in various species by using synteny neighborhood networks (SNN). This workflow is then applied to the analysis of flanking genes associated with oxidosqualene cyclases (OSCs). The method allows for the recognition and comparison of homologous blocks with unique flanking genes accompanying different subfamilies of OSCs. The examination of the flanking genes of OSCs in 122 plant species revealed multiple genes with conserved positional relationships with OSCs in angiosperms. Specifically, the earliest adjacency of OSC genes and CYP716 genes first appeared in basal eudicots, and the nonrandom occurrence of CYP716 genes in the flanking region of OSC persists across different lineages of eudicots. Our study showed the substitution of genes in the flanking region of the OSC varies across different plant lineages, and our approach facilitates the investigation of flanking gene rearrangements in the formation of OSC-related BGCs.

Gynostemma pentaphyllum is a traditional Chinese medicinal plant of considerable application value and commercial potential, primarily due to its production of various bioactive compounds, particularly dammarane-type triterpenoid saponins that are structurally analogous to ginsenosides. Oxidosqualene cyclase (OSC), a pivotal enzyme in the biosynthesis of triterpenoid metabolites in plants, catalyzes the conversion of oxidosqualene into triterpenoid precursors, which are essential components of the secondary metabolites found in G. pentaphyllum. To elucidate the role of OSC gene family members in the synthesis of gypenosides within G. pentaphyllum, this study undertook a comprehensive genome-wide identification and characterization of OSC genes within G. pentaphyllum and compared their expression levels across populations distributed over different geographical regions by both transcriptome sequencing and qRT-PCR experimental validation. The results identified a total of 11 members of the OSC gene family within the genome of G. pentaphyllum. These genes encode proteins ranging from 356 to 767 amino acids, exhibiting minor variations in their physicochemical properties, and are localized in peroxisomes, cytoplasm, plasma membranes, and lysosomes. All GpOSCs contain highly conserved DCTAE and QW sequences that are characteristic of the OSC gene family. A phylogenetic analysis categorized the GpOSCs into four distinct subfamilies. A cis-element analysis of the GpOSC promoters revealed a substantial number of abiotic stress-related elements, indicating that these genes may respond to drought conditions, low temperatures, and anaerobic environments, thus potentially contributing to the stress resistance observed in G. pentaphyllum. Expression analyses across different G. pentaphyllum populations demonstrated significant variability in OSC gene expression among geographically diverse samples of G. pentaphyllum, likely attributable to genetic variation or external factors such as environmental conditions and soil composition. These differences may lead to the synthesis of various types of gypenosides within geographically distinct G. pentaphyllum populations. The findings from this study enhance our understanding of both the evolutionary history of the OSC gene family in G. pentaphyllum and the biosynthetic mechanisms underlying triterpenoid compounds. This knowledge is essential for investigating molecular mechanisms involved in forming dammarane-type triterpenoid saponins as well as comprehending geographical variations within G. pentaphyllum populations. Furthermore, this research lays a foundation for employing plant genetic engineering techniques aimed at increasing gypenoside content.

Oxidosqualene cyclases (OSCs) are enzymes responsible for converting linear triterpenes into tetracyclic ones, which are known as precursors of other important and bioactive metabolites. Two OSCs genes encoding parkeol synthase and lanostadienol synthase have been found in representatives of the genera Apostichopus and Stichopus (family Stichopodidae, order Synallactida). As a limited number of sea cucumber OSCs have been studied thus far, OSCs encoding gene(s) of the sea cucumber Eupentacta fraudatrix (family Sclerodactylidae, order Dendrochirotida) were investigated to fill this gap. Here, we employed RACEs, molecular cloning, and Oxford Nanopore Technologies to identify candidate OSC mRNAs and genes. The assembled cDNAs were 2409 bp (OSC1) and 3263 bp (OSC2), which shared the same CDS size of 2163 bp encoding a 721-amino-acid protein. The E. fraudatrix OSC1 and OSC2 had higher sequence identity similarity to each other (77.5%) than to other holothurian OSCs (64.7-71.0%). According to the sequence and molecular docking analyses, OSC1 with L436 is predicted to be parkeol synthase, while OSC2 with Q439 is predicted to be lanostadienol synthase. Based on the phylogenetic analysis, E. fraudatrix OSCs cDNAs clustered with other holothurian OSCs, forming the isolated branch. As a result of gene analysis, the high polymorphism and larger size of the OSC1 gene suggest that this gene may be an ancestor of the OSC2 gene. These results imply that the E. fraudatrix genome contains two OSC genes whose evolutionary pathways are different from those of the OSC genes in Stichopodidae.

Identification, functional characterization and expression profiling of three triterpene synthases from the legume plant Vigna unguiculata

Platycodon grandiflorus stands as one of the most extensively utilized traditional Chinese medicinal herbs, with triterpenoids and their derivatives serving as its primary medicinal components. Oxido squalene cyclase (OSC), serving as a crucial enzyme in the triterpenoid synthesis pathway, has the capability to enzymatically generate significant quantities of sterols and triterpenoid intermediates. While the OSC gene family has been identified in numerous species, bioinformatics research on this family remains scant. Presently, the specific members of this gene family in Platycodon grandiflorus have yet to be definitively determined. In this study, we successfully identified a total of 15 PgOSC genes within the genome of Platycodon grandiflorus by conducting homology comparisons. These genes were discovered to be unevenly distributed across the five chromosomes of the species, organized in the form of gene clusters. Subsequently, we conducted a thorough analysis of the OSC gene family's evolutionary relationship by constructing a phylogenetic tree. Other characteristics of PgOSC family members, including gene structure, conserved motifs, protein three-dimensional structure, subcellular localization, and cis-acting elements were thoroughly characterized. Furthermore, We analyzed the expression of PgOSC gene in different tissues of Platycodon grandiflorus by qRT-PCR, and found that the expression of PgOSC genes in root was higher than that in stem and leaf. Upon comparing the effects of salt, heat, and drought treatments, we observed a significant induction of PgOSC gene expression in Platycodon grandiflorus specifically under salt stress conditions. In summary, this study comprehensively identified and analyzed the OSC gene family, aiming to provide basic biological information for exploring the members of PgOSC gene family.

Abstract Disclosure: Y. Liang: None. S.M. Hyder: None. Triple-negative breast cancers (TNBC) lack ER, PR and Her2neu proteins that are commonly targeted in breast cancer therapies. As a consequence, women who suffer from TNBC have poor prognosis and limited treatment options, and are usually administered toxic, non-specific chemotherapeutic agents. Drug-resistance almost always occurs, leading to tumor metastasis, the main cause of patient death. New non-toxic therapeutic strategies to control metastasis of TNBC are urgently needed. Cholesterol is an essential structural and functional component of cell membranes that is necessary for the growth of both primary tumors and metastatic colonies that reside in distant organs. Therefore, inhibiting cholesterol production is an attractive therapeutic strategy. Treatment with statins, a class of cholesterol biosynthesis inhibitors that target HMGCoA-reductase, is associated with certain undesirable side effects; consequently, we targeted oxidosqualene cyclase (OSC), an enzyme that occurs downstream of HMGCoA-reductase in the cholesterol biosynthetic pathway, to disrupt tumor metastasis. Potent small molecule inhibitors of OSC have been identified. RO 48-8071 (4′-[6-(allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate) (RO), has emerged as a useful chemotherapeutic agent for treating several forms of primary tumors. We developed a mouse model for studying lung metastasis using a MDA-MB-231 variant (LM2) TNBC cell line obtained from Dr. Massagué (Memorial Sloan-Kettering Cancer Center). These are aggressive cells, and injection of only 2 X 105 cells produces detectable lung metastatic colonies in approximately 4-6 weeks. We inoculated female nu/nu mice, 5-6 weeks old, with 2 X 105 MDA-MB-231/LM2 cells in 0.1 ml DMEM/F12 medium without serum via tail vein. Five days after tumor cell inoculation animals received 20 mg/kg (n=6), 40 mg/kg (n=7) RO (ip) or vehicle alone (n=7) every other day for two weeks. Animals were then sacrificed, and both lungs harvested and immediately fixed in Bouin’s fixative. Metastatic colonies on the lung surface were identified and counted under a dissecting microscope. Our study showed that RO effectively reduced lung metastasis without affecting animal weight. Compared with 56 + 7 colonies (Mean ± SEM) in controls given vehicle alone, RO significantly reduced lung metastatic colonies to 28 ± 5 in animals exposed to 20 mg/kg and 6 ± 1 in 40 mg/kg treated animals (p<0.05, ANOVA). Reduction in number of colonies in 40 mg/kg group was also significantly different from colonies counted in 20 mg/kg treated animals. Our study is the first to demonstrate that disruption of cholesterol biosynthesis in a model of human TNBC leads to reduced metastasis in lungs. Further studies will determine mechanisms through which RO suppresses TNBC metastasis to the lungs. Supported by Zalk Missouri Professor Endowment Funds. Presentation: 6/3/2024