Promiscuous Oxidosqualene Cyclases from Neoalsomitra integrifoliola Catalyzing the Formation of Tetracyclic, Pentacyclic, and Heterocyclic Triterpenes.

Six oxidosqualene cyclases (NiOSC1-NiOSC6) from Neoalsomitra integrifoliola were characterized for the biosynthesis of diverse triterpene scaffolds, including tetracyclic and pentacyclic triterpenes from the 2,3-oxidosqualene (1) and oxacyclic triterpenes from the 2,3:22,23-dioxidosqualene (2). NiOSC1 showed high efficiency in the production of naturally rare (20R)-epimers of oxacyclic triterpenes. Mutagenesis results revealed that the NiOSC1-F731G mutant significantly increased the yields of (20R)-epimers compared to the wild type. Homology modeling and molecular docking elucidated the origin of the (20R)-configuration in the epoxide addition step.

Differential regulation of key triterpene synthase gene under abiotic stress in Withania somnifera L. Dunal and its co-relation to sterols and withanolides.

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 ß-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.

The Promiscuity of Squalene Synthase-Like Enzyme: Dehydrosqualene Synthase, a Natural Squalene Hyperproducer?

Dehydrosqualene synthase (CrtM), as a squalene synthase-like enzyme from Staphylococcus aureus, can naturally utilize farnesyl diphosphate to produce dehydrosqualene (C30H48). However, no study has documented the natural production of squalene (C30H50) by CrtM. Here, based on an HPLC-Q-Orbitrap-MS/MS study, we report that the expression of crtM in vitro or in Bacillus subtilis 168 both results in the output of squalene, dehydrosqualene, and phytoene (C40H64). Notably, wild-type CrtM exhibits a significantly higher squalene yield compared to squalene synthase (SQS) from Bacillus megaterium with an approximately 2.4-fold increase. Moreover, the examination of presqualene diphosphate's stereostructures in both CrtM and SQS enzymes provides further understanding into the presence of multiple identified terpenoids. In summary, this study not only provides insights into the promiscuity demonstrated by squalene synthase-like enzymes but also highlights a new strategy of utilizing CrtM as a potential replacement for SQS in cell factories, thereby enhancing squalene production.

Functional characterization of genes related to triterpene and flavonoid biosynthesis in Cyclocarya paliurus.

MAIN CONCLUSION: The oxidosqualene cyclases (OSCs) generating triterpenoid skeletons in Cyclocarya paliurus were identified for the first time, and two uridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyzing the glycosylation of flavonoids were characterized. Cyclocarya paliurus, a native rare dicotyledonous plant in China, contains an abundance of triterpenoid saponins and flavonoid glycosides that exhibit valuable pharmaceutical effects in preventing hypertension, hyperlipidemia, and diabetes. However, the molecular mechanism explaining the biosynthesis of triterpenoid saponin and flavonoid glycoside in C. paliurus remains unclear. In this study, the triterpene content in different tissues and the expression pattern of genes encoding the key enzymes associated with triterpenoid saponin and flavonoid glycoside biosynthesis were studied using transcriptome and metabolome analysis. The eight upstream oxidosqualene cyclases (OSCs) involved in triterpenoid saponin biosynthesis were functionally characterized, among them CpalOSC6 catalyzed 2,3;22,23-dioxidosqualene to form 3-epicabraleadiol; CpalOSC8 cyclized 2,3-oxidosqualene to generate dammarenediol-II; CpalOSC2 and CpalOSC3 produced ß-amyrin and CpalOSC4 produced cycloartenol, while CpalOSC2-CpalOSC5, CpalOSC7, and CpalOSC8 all produced lanosterol. However, no catalytic product was detected for CpalOSC1. Moreover, two downstream flavonoid uridine diphosphate (UDP)-glycosyltransferases (UGTs) (CpalUGT015 and CpalUGT100) that catalyze the last step of flavonoid glycoside biosynthesis were functionally elucidated. These results uncovered the key genes involved in the biosynthesis of triterpenoid saponins and flavonoid glycosides in C. paliurus that could be applied to produce flavonoid glycosides and key triterpenoid saponins in the future via a synthetic strategy.

Identification of a cytochrome P450 from Tripterygium hypoglaucum (Levl.) Hutch that catalyzes polpunonic acid formation in celastrol biosynthesis.

Tripterygium hypoglaucum (Levl.) Hutch, a traditional Chinese medicinal herb with a long history of use, is widely distributed in China. One of its main active components, celastrol, has great potential to be developed into anti-cancer and anti-obesity drugs. Although it exhibits strong pharmacological activities, there is a lack of sustainable sources of celastrol and its derivatives, making it crucial to develop novel sources of these drugs through synthetic biology. The key step in the biosynthesis of celastrol is considered to be the cyclization of 2,3-oxidosqualene into friedelin under the catalysis of 2,3-oxidosqualene cyclases. Friedelin was speculated to be oxidized into celastrol by cytochrome P450 oxidases (CYP450s). Here, we reported a cytochrome P450 ThCYP712K1 from Tripterygium hypoglaucum (Levl.) Hutch that catalyzed the oxidation of friedelin into polpuonic acid when heterologously expressed in yeast. Through substrate supplementation and in vitro enzyme analysis, ThCYP712K1 was further proven to catalyze the oxidation of friedelin at the C-29 position to produce polpunonic acid, which is considered a vital step in the biosynthesis of celastrol, and will lay a foundation for further analysis of its biosynthetic pathway.

Effect of squalane-based emulsion on polyphenols skin penetration: Ex vivo skin study.

Polyphenols have gained attractiveness as ingredients in cosmetic formulations as result of their ability to delay the aging process. However, different factors limit their use, including low solubility and poor skin permeability. In this sense, this study describes the potential of squalane to increase the polyphenols ex vivo skin penetration, incorporated into a water-in-oil emulsion. Polyphenols skin permeation followed the Fick's first law and, p-coumaric acid, vitexin, schaftoside and ferulic acid had the higher permeability coefficients (Kp = 6.0-8.0 × 10-3 cm-2 h-1). Addition of squalane to phenolic compounds decreased the permeability coefficients (Kp = 4.1-5.9 × 10-3 cm-2 h-1), indicating that squalane increased the retention of polyphenols in the skin. Gentisic acid, ferulic acid, and p-coumaric acid were the only compounds permeating from water-in-oil emulsion, in the first 8 h of study and, according Krosmeyer-Peppas model, its n value was > 1 indicating a high transport resistance from the formulation and throughout the skin. Results suggest squalane as an efficient vehicle to increase the dermal availability increasing phenolic compounds physiological functions, by enhancing the skin retention time where they should exert antiaging effect.

Characterization of oxidosqualene cyclases from Trichosanthes cucumerina L. reveals key amino acids responsible for substrate specificity of isomultiflorenol synthase.

MAIN CONCLUSION: Two key amino acids of isomultiflorenol synthase, Y125 and M254, were first proposed. They could be associated with the production of isomultiflorenol. Oxidosqualene cyclases (OSCs) are the first committed enzymes in the triterpenoid biosynthesis by converting 2,3-oxidosqualene to specific triterpenoid backbones. Thus, these enzymes are potential targets for developing plant-active compounds through the study of triterpenoid biosynthesis. We applied transcriptome information and metabolite profiling from Trichosanthes cucumerina L. to define the diversity of triterpenoids in this plant through OSCs. Isomultiflorenol synthase and cucurbitadienol synthase were previously identified in this plant. Here, three new OSCs, TcBAS, TcLAS, and TcCAS, were cloned and functionally characterized as ß-amyrin synthase, lanosterol synthase, and cycloartenol synthase activities, respectively. We also took advantage of the multiple sequence alignment and molecular docking of OSCs exhibiting in this plant and other plant OSCs to identify key residues associated with isomultiflorenol synthase specificity. Two novel key amino acids, referred to the Y125 and M254, were first discovered. These results provide information on a possible catalytic mechanism for plant OSCs that produce specific products.

Oxidosqualene Cyclases Involved in the Biosynthesis of Diverse Triterpenes in Camellia sasanqua.

Camellia sasanqua is an important economic plant that is rich in lipophilic triterpenols with pharmacological activities including antiallergic, anti-inflammatory, and anticancer activities. However, the key enzymes related to triterpene biosynthesis have seldom been studied in C. sasanqua. Oxidosqualene cyclases (OSCs) are the rate-limiting enzymes related to triterpene biosynthesis. In this study, seven putative OSC genes (CsOSC1-7) were mined from the C. sasanqua transcriptome. Six CsOSCs were characterized for the biosynthesis of diverse triterpene skeletons, including α-amyrin, ß-amyrin, δ-amyrin, dammarenediol-II, ψ-taraxasterol, taraxasterol, and cycloartenol by the heterologous expression system. CsOSC3 was a multiple functional α-amyrin synthase. Three key residues, Trp260, Tyr262, and Phe415, are critical to the catalytic performance of CsOSC3 judging from the results of molecular docking and site-directed mutagenesis. These findings provide important insights into the biosynthesis pathway of triterpenes in C. sasanqua.

[Oxidosqualene cyclases in triterpenoids biosynthesis: a review].

Triterpenoids are one of the most diverse compounds in plant metabolites, and they have a wide variety of physiological activities and are of important economic value. Oxidosqualene cyclases catalyze the cyclization of 2, 3-oxidosqualene to generate different types of sterols and plant triterpenoids, which is of great significance to the structural diversity of natural products. However, the mechanism of the diversified cyclization of 2, 3-oxidosqualene catalyzed by oxidosqualene cyclases remains unclear. This review summarized the research progress of oxidosqualene cyclases from the aspects of catalytic function, molecular evolutionary relationship between genes and proteins, protein structure, molecular simulation and molecular calculations, which may provide a reference for protein engineering and metabolic engineering of triterpene cyclase.

A novel carotenoid biosynthetic route via oxidosqualene.

Over 800 known carotenoids are synthesized from phytoene or 4,4'-diapophytoene (dehydrosqualene) characterized by three conjugated double bonds. In this paper, we report that carotenoid desaturase CrtN from Staphylococcus aureus and Methylomonas can accept oxidosqualene, which is the precursor for plant- or animal-type triterpenoids, yielding the yellow carotenoid pigments with 8, 9, or 10 conjugated double bonds. The resulting pathway is the second nonnatural route for carotenoid pigments and the first pathway for carotenoid pigments not biosynthesized via (diapo)phytoene.

Diverse triterpene skeletons are derived from the expansion and divergent evolution of 2,3-oxidosqualene cyclases in plants.

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.

Corneal protective effects of novel tear substitutes containing sodium hyaluronate and dodecahydrosqualene, squalane, in a porcine dry eye model.

To develop a novel tear substitute (TS) containing sodium hyaluronate (SH) and dodecahydrosqualene (DHS, squalane), we improved the prescription of a previously developed TS containing saline, 0.5% SH and 1% castor oil (CO), which had corneal protective effects against 60-min desiccation in a porcine dry eye model and viscosity of 106.8 mPa·S. Fresh porcine eyes were treated with a TS containing saline, 0.1%, 0.25%, 0.3% or 0.5% SH, and 1% CO or 1%, 2.5% or 5% DHS, and TS-treated eyes were desiccated for up to 180 min. The corneal damage was evaluated by the staining score of methylene blue (MB), absorbance of MB extracted from the cornea, the staining density of lissamine green (LG) and histopathology. The viscosities of the examined TS were also measured. A saline/0.5% SH/1% DHS solution had corneal protective effects for 90 min under desiccation and a viscosity of 110.0 mPa·s. A TS with saline, 0.1%, 0.25% or 0.3% SH and 1% or 2.5% DHS did not have better protective effects than a saline/0.5% SH/1% DHS solution, although a saline/0.3% SH/5% DHS solution exhibited greater corneal protection against 180-min desiccation on MB and LG staining and histopathological examination, and its viscosity was 34.5 mPa·s, which was similar to the 29.5 mPa·s of 0.3% SH. The saline/0.3% SH/5% DHS solution is available as a novel 3-hr long-lasting TS containing mucinomimetic and liquid oil components to treat and relieve dry eye symptoms in animals and humans.

A gas chromatography-mass spectrometry method for quantifying squalane and squalene in aerosol emissions of electronic cigarette, or vaping, products.

Numerous chemicals of unknown inhalational toxicity have been measured in electronic cigarette, or vaping, products (EVPs). In addition, little is known about the liquid-to-aerosol transmission and deliveries of these chemicals, including oil-like terpenes such as squalene (SQE) and squalane (SQA). To provide information on the aerosol deliveries of these compounds from EVPs, we developed and validated a quantitative method to measure squalene and squalane in EVP aerosol emissions. Validation parameters include measurement repeatability (SQA and SQE %RSD <6%), intermediate precision (SQA: %RSD 11%, SQE: %RSD 17%), accuracy (SQA: 86-107%, SQE: 104-113%), matrix effects, method robustness, and analyte stability. Limits of detection were 6.06 ng/mL puffed air volume for both squalene and squalane. The method was used to measure squalene and squalane in aerosol emissions of 153 EVPs associated with case patients from a recent outbreak of e-cigarette, or vaping, product use associated lung injury (EVALI). The EVPs analyzed were organized into nicotine, cannabidiol, and tetrahydrocannabinol products by the percentage of nicotine, cannabidiol, and tetrahydrocannabinol in total particulate matter after vaping. In case-associated tetrahydrocannabinol products the detection rates and mean concentrations were 82.4% and 33.0 ng/mL puffed air for squalene and 4.41% and 7.80 ng/mL puffed air for squalane.

Functional diversity of isoprenoid lipids in Methylobacterium extorquens PA1.

Hopanoids and carotenoids are two of the major isoprenoid-derived lipid classes in prokaryotes that have been proposed to have similar membrane ordering properties as sterols. Methylobacterium extorquens contains hopanoids and carotenoids in their outer membrane, making them an ideal system to investigate the role of isoprenoid lipids in surface membrane function and cellular fitness. By genetically knocking out hpnE and crtB we disrupted the production of squalene and phytoene in M. extorquens PA1, which are the presumed precursors for hopanoids and carotenoids respectively. Deletion of hpnE revealed that carotenoid biosynthesis utilizes squalene as a precursor resulting in pigmentation with a C30 backbone, rather than the previously predicted canonical C40 phytoene-derived pathway. Phylogenetic analysis suggested that M. extorquens may have acquired the C30 pathway through lateral gene transfer from Planctomycetes. Surprisingly, disruption of carotenoid synthesis did not generate any major growth or membrane biophysical phenotypes, but slightly increased sensitivity to oxidative stress. We further demonstrated that hopanoids but not carotenoids are essential for growth at higher temperatures, membrane permeability and tolerance of low divalent cation concentrations. These observations show that hopanoids and carotenoids serve diverse roles in the outer membrane of M. extorquens PA1.

Non-Polar Lipids as Regulators of Membrane Properties in Archaeal Lipid Bilayer Mimics.

The modification of archaeal lipid bilayer properties by the insertion of apolar molecules in the lipid bilayer midplane has been proposed to support cell membrane adaptation to extreme environmental conditions of temperature and hydrostatic pressure. In this work, we characterize the insertion effects of the apolar polyisoprenoid squalane on the permeability and fluidity of archaeal model membrane bilayers, composed of lipid analogues. We have monitored large molecule and proton permeability and Laurdan generalized polarization from lipid vesicles as a function of temperature and hydrostatic pressure. Even at low concentration, squalane (1 mol%) is able to enhance solute permeation by increasing membrane fluidity, but at the same time, to decrease proton permeability of the lipid bilayer. The squalane physicochemical impact on membrane properties are congruent with a possible role of apolar intercalants on the adaptation of Archaea to extreme conditions. In addition, such intercalant might be used to cheaply create or modify chemically resistant liposomes (archeaosomes) for drug delivery.

Characterisation of a synthetic Archeal membrane reveals a possible new adaptation route to extreme conditions.

It has been proposed that adaptation to high temperature involved the synthesis of monolayer-forming ether phospholipids. Recently, a novel membrane architecture was proposed to explain the membrane stability in polyextremophiles unable to synthesize such lipids, in which apolar polyisoprenoids populate the bilayer midplane and modify its physico-chemistry, extending its stability domain. Here, we have studied the effect of the apolar polyisoprenoid squalane on a model membrane analogue using neutron diffraction, SAXS and fluorescence spectroscopy. We show that squalane resides inside the bilayer midplane, extends its stability domain, reduces its permeability to protons but increases that of water, and induces a negative curvature in the membrane, allowing the transition to novel non-lamellar phases. This membrane architecture can be transposed to early membranes and could help explain their emergence and temperature tolerance if life originated near hydrothermal vents. Transposed to the archaeal bilayer, this membrane architecture could explain the tolerance to high temperature in hyperthermophiles which grow at temperatures over 100 °C while having a membrane bilayer. The induction of a negative curvature to the membrane could also facilitate crucial cell functions that require high bending membranes.

Effectiveness and tolerability of a squalane and dimethicone-based treatment for head lice.

Head lice (Pediculus humanus capitis) are worldwide obligate human ectoparasites, with high implications in pediatrics. In Europe, first-line topical neurotoxic insecticidal therapeutic strategies are being replaced by topical physically acting agents as the first-choice treatment. Safety of the active ingredients and high efficacy in a one-time single-dose treatment with a brief treatment application time are key issues for consumer use and effective compliance. The aim of this study was to assess the in vitro efficacy of a newly developed squalane and dimethicone-based pediculicidal formula, against motile head lice and eggs after 2 and 5 min immersion in the product, as well as its skin tolerance and acceptability under dermatological and pediatric expert control in children with atopic skin. The results indicate that at both time points, 100% mortality rate of head lice crawling stages and late-stage eggs was achieved. The formula was well tolerated and suitable for children with atopic skin from 12 months of age. Showing high in vitro efficacy and good skin acceptability, this solution is presented as a new safe alternative therapy for treatment of head lice infestations.

Identification of triterpenes and functional characterization of oxidosqualene cyclases involved in triterpene biosynthesis in lettuce (Lactuca sativa).

Lettuce (Lactuca sativa) is a member of the family Asteraceae and is most often used for green salads. Triterpenes are the largest class of natural compounds in plants and have beneficial health effects. Here, we identified various triterpene esters (taraxasterol acetates, ψ-taraxasterol acetates, taraxerol acetates, lupeol acetates, α-amyrin acetates, ß-amyrin acetates, and germanicol acetate) and free triterpenes (α-amyrin, ß-amyrin, taraxerol, and taraxasterol) in both the leaves and roots of lettuce. Triterpene biosynthesis occurs through the action of oxidosqualene cyclase (OSC), which generates various types of triterpenes from 2,3-oxidosqualene. None of the OSC genes involved in triterpene biosynthesis in lettuce have been characterized. Five putative lettuce OSC genes (LsOSC1, LsOSC2, LsOSC3, LsOSC4, and LsOSC5) were selected from a transcriptome database. These five genes were functionally characterized via heterologous expression in yeast. The first two enzymes were multifunctional triterpene synthase and the last three genes were monofunctional. Transgenic yeast expressing LsOSC1 produced five triterpenes, namely, taraxasterol, Ψ-taraxasterol, α-amyrin, ß-amyrin, and dammarenediol-II. Yeast expressing LsOSC2 produced baurenol and Ψ-taraxasterol. LsOSC3, LsOSC4, and LsOSC5 expression led to ß-amyrin, taraxerol, and lupeol production, respectively. Transcriptional activity assessment of the five genes revealed that all the OSC genes were more actively transcribed in roots than in leaves, and LsOSC5 among the five OSC genes showed the highest expression in both the leaves and the roots. In conclusion, we identified structurally diverse free triterpenes and triterpene esters in lettuce plants and characterized five OSC genes, which are key enzymes involved in the biosynthesis of diverse triterpenes in lettuce.

Molecular cloning and functional characterization of multiple ApOSCs from Andrographis paniculata.

Triterpenoids have been described in Andrographis paniculata. Oleanolic acid exhibits high biological activity and is widely used in the clinic, and ß-sitosterol not only has good biological activity but also plays an important physiological role in plants. However, analysis of the biosynthetic pathway of triterpenoids in Andrographis paniculata has not been reported. Here, we provide the first report of the isolation and identification of nine 2, 3-oxidosqualene cyclases (ApOSC3 to ApOSC11) from A. paniculata. The results showed that ApOSC4 represented a monofunctional synthase that could convert 2, 3-oxidosqualene to ß-amyrin. ApOSC5 as a bifunctional 2, 3-oxidosqualene cyclases, could transfer 2, 3-oxidosqualene to ß-amyrin and α-amyrin. ApOSC6 to ApOSC8 composed the multifunctional 2, 3-oxidosqualene cyclases that could convert 2, 3-oxidosqualene to ß-amyrin, α-amyrin and one or two undetermined triterpenoids. This study provides a better understanding of the biosynthetic pathway of triterpenoids in A. paniculata, and the discovery of multifunctional 2, 3-oxidosqualene cyclases ApOSC5 to ApOSC8 of the facilitates knowledge of the compounds diversity in A. paniculata.

Synthesis, Computational Studies and Assessment of in Vitro Activity of Squalene Derivatives as Carbonic Anhydrase Inhibitors.

We report novel molecules incorporating the nontoxic squalene scaffold and different carbonic anhydrase inhibitors (CAIs). Potent inhibitory action, in the low-nanomolar range, was detected against isoforms hCA II for sulfonamide derivatives, which proved to be selective against this isoform over the tumor-associate hCA IX and XII isoforms. On the other hand, coumarin derivatives showed weak potency but high selectivity against the tumor-associated isoform CA IX. These compounds are interesting candidates for preclinical evaluation in glaucoma or various tumors in which the two enzymes are involved. In addition, an in silico study of inhibitor-bound hCA II revealed extensive interactions with the hydrophobic pocket of the active site and provided molecular insights into the binding properties of these new inhibitors.