Induction of point and structural mutations in engineered yeast Saccharomyces cerevisiae improve carotenoid production.

ß-Carotene is an attractive compound and that its biotechnological production can be achieved by using engineered Saccharomyces cerevisiae. In a previous study, we developed a technique for the efficient establishment of diverse mutants through the introduction of point and structural mutations into the yeast genome. In this study, we aimed to improve ß-carotene production by applying this mutagenesis technique to S. cerevisiae strain that had been genetically engineered for ß-carotene production. Point and structural mutations were introduced into ß-carotene-producing engineered yeast. The resulting mutants showed higher ß-carotene production capacity than the parental strain. The top-performing mutant, HP100_74, produced 37.6 mg/L of ß-carotene, a value 1.9 times higher than that of the parental strain (20.1 mg/L). Gene expression analysis confirmed an increased expression of multiple genes in the glycolysis, mevalonate, and ß-carotene synthesis pathways. In contrast, expression of ERG9, which functions in the ergosterol pathway competing with ß-carotene production, was decreased in the mutant strain. The introduction of point and structural mutations represents a simple yet effective method for achieving mutagenesis in yeasts. This technique is expected to be widely applied in the future to produce chemicals via metabolic engineering of S. cerevisiae.

The mechanism and clinical application of farnesyl diphosphate farnesyltransferase 1 in cancer metabolism.

Cancer poses a significant risk to human well-being. Among the crucial characteristics of cancer is metabolic reprogramming. To meet the relentless metabolic needs, cancer cells enhance cholesterol metabolism within the adverse tumor microenvironment. Reprograming cholesterol metabolism includes a series of modifications in the synthesis, absorption, esterification, and metabolites associated with cholesterol. These adjustments have a strong correlation with the proliferation, invasion, metastasis, and other characteristics of malignant tumors. FDFT1, also known as farnesyl diphosphate farnesyltransferase 1, is an enzyme crucial in the process of cholesterol biosynthesis. Its significant involvement in tumor metabolism has garnered considerable interest. The significance of FDFT1 in cancer metabolism cannot be overstated, as it actively interacts with cancer cells. This paper aims to analyze and consolidate the mechanism of FDFT1 in cancer metabolism and explore its clinical application. The goal is to contribute new strategies and targets for the prevention and treatment of cancer metabolism.

Spectroscopic diagnosis and metabolite characterization of cisplatin resistance regulated by FDFT1 in bladder cancer tissue.

As is the case for most solid tumours, chemotherapy remains the backbone in the management of metastatic disease. However, the occurrence of chemotherapy resistance is a cause to worry, especially in bladder cancer. Extensive evidence indicates molecular changes in bladder cancer cells to be the underlying cause of chemotherapy resistance, including the reduced expression of farnesyl-diphosphate farnesyltransferase 1 (FDFT1) - a gene involved in cholesterol biosynthesis. This can likely be a hallmark in examining the resistance and sensitivity of chemotherapy drugs. This work performs spectroscopic analysis and metabolite characterization on resistant, sensitive, stable-disease and healthy bladder tissues. Raman spectroscopy has detected peaks at around 1003 cm-1 (squalene), 1178 cm-1 (cholesterol), 1258 cm-1 (cholesteryl ester), 1343 cm-1 (collagen), 1525 cm-1 (carotenoid), 1575 cm-1 (DNA bases) and 1608 cm-1 (cytosine). The peak parameters were examined, and statistical analysis was performed on the peak features, attaining significant differences between the sample groups. Small-angle x-ray scattering (SAXS) measurements observed the triglyceride peak together with 6th, 7th and 8th - order collagen peaks; peak parameters were also determined. Neutron activation analysis (NAA) detected seven trace elements. Carbon (Ca), magnesium (Mg), chlorine (Cl) and sodium (Na) have been found to have the greatest concentration in the sample groups, suggestive of a role as a biomarker for cisplatin resistance studies. Results from the present research are suggested to provide an important insight into understanding the development of drug resistance in bladder cancer, opening up the possibility of novel avenues for treatment through personalised interventions.

Discovery of Novel 11-Membered Templates as Squalene Synthase Inhibitors.

Squalene synthase is one of the most promising pharmaceutical targets to treat hyperlipidemia. Inhibition of the squalene synthase causes a decrease in the hepatic cholesterol concentration. We have already reported the design and synthesis of highly potent benzhydrol-type squalene inhibitors. Although these templates showed unique and potent cyclic active conformations via intramolecular hydrogen bonds, the in vivo cholesterol-lowering efficacy was insufficient. We attempted to improve their potential as an orally active medicine. In our medicinal chemistry effort, cyclized 11-membered ring templates were acquired. The novel series of compounds exhibited potent squalene synthase inhibitory activity, and one of the derivatives, isomer A-(1S, 3R)-14i, showed plasma lipid-lowering efficacy in hamster and marmoset repeated-dose studies. Our findings provide valuable insights into the design and development of novel and unique 11-membered ring-type highly potent squalene synthase inhibitors.

Terpenoid balance in Aspergillus nidulans unveiled by heterologous squalene synthase expression.

Filamentous fungi produce numerous uncharacterized natural products (NPs) that are often challenging to characterize because of cryptic expression in laboratory conditions. Previously, we have successfully isolated novel NPs by expressing fungal artificial chromosomes (FACs) from a variety of fungal species into Aspergillus nidulans. Here, we demonstrate a twist to FAC utility wherein heterologous expression of a Pseudogymnoascus destructans FAC in A. nidulans altered endogenous terpene biosynthetic pathways. In contrast to wild type, the FAC transformant produced increased levels of squalene and aspernidine type compounds, including three new nidulenes (1- 2, and 5), and lost nearly all ability to synthesize the major A. nidulans characteristic terpene, austinol. Deletion of a squalene synthase gene in the FAC restored wild-type chemical profiles. The altered squalene to farnesyl pyrophosphate ratio leading to synthesis of nidulenes and aspernidines at the expense of farnesyl pyrophosphate-derived austinols provides unexpected insight into routes of terpene synthesis in fungi.

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.

Intramembrane protease SPP defines a cholesterol-regulated abundance control of the mevalonate pathway enzyme squalene synthase.

Intramembrane proteolysis regulates important processes such as signaling and transcriptional and posttranslational abundance control of proteins with key functions in metabolic pathways. This includes transcriptional control of mevalonate pathway genes, thereby ensuring balanced biosynthesis of cholesterol and other isoprenoids. Our work shows that, at high cholesterol levels, signal peptide peptidase (SPP) cleaves squalene synthase (SQS), an enzyme that defines the branching point for allocation of isoprenoids to the sterol and nonsterol arms of the mevalonate pathway. This intramembrane cleavage releases SQS from the membrane and targets it for proteasomal degradation. Regulation of this mechanism is achieved by the E3 ubiquitin ligase TRC8 that, in addition to ubiquitinating SQS in response to cholesterol levels, acts as an allosteric activator of SPP-catalyzed intramembrane cleavage of SQS. Cellular cholesterol levels increase in the absence of SPP activity. We infer from these results that, SPP-TRC8 mediated abundance control of SQS acts as a regulation step within the mevalonate pathway.

Identification and functional characterization of two trans-isopentenyl diphosphate synthases and one squalene synthase involved in triterpenoid biosynthesis in Platycodon grandiflorus.

MAIN CONCLUSION: Two trans-isopentenyl diphosphate synthase and one squalene synthase genes were identified and proved to be involved in the triterpenoid biosynthesis in Platycodon grandiflorus. Platycodon grandiflorus is a commonly used traditional Chinese medicine. The main bioactive compounds of P. grandiflorus are triterpenoid saponins. The biosynthetic pathway of triterpenoid saponins in P. grandiflorus has been preliminarily explored. However, limited functional information on related genes has been reported. A total of three trans-isopentenyl diphosphate synthases (trans-IDSs) genes (PgFPPS, PgGGPPS1 and PgGGPPS2) and one squalene synthase (SQS) gene (PgSQS) in P. grandiflorus were screened and identified from transcriptome dataset. Subcellular localization of the proteins was defined based on the analysis of GFP-tagged. The activity of genes was verified in Escherichia coli, demonstrating that recombinant PgFPPS catalysed the production of farnesyl diphosphate. PgGGPPS1 produced geranylgeranyl diphosphate, whereas PgGGPPS2 did not exhibit catalytic activity. By structural identification of encoding genes, a transmembrane region was found at the C-terminus of the PgSQS gene, which produced an insoluble protein when expressed in E. coli but showed no apparent effect on the enzyme function. Furthermore, some triterpenoid saponin synthesis-related genes were discovered by combining the component content and the gene expression assays at the five growth stages of P. grandiflorus seedlings. The accumulation of active components in P. grandiflorus was closely associated with the expression level of genes related to the synthesis pathway.

Effect of a new squalene synthase inhibitor on an ApoE-/- mouse model of atherosclerosis.

Ηypercholesterolemia/hyperlipidemia in conjunction with oxidative stress and inflammatory processes contribute synergistically to the pathogenesis of atherosclerosis. We hereby evaluated the antiatherosclerotic effect of the multi-target derivative 4-methyl-2-(10H-phenothiazin-3-yl)morpholin-2-ol hydrobromide 1 in apoE-/- mice; compound 1 is a potent antihyperlipidemic agent acting through Squalene Synthase inhibition, while it has exhibited an outstanding antioxidant and anti-inflammatory activity in various experimental animal models. The new analogue was evaluated in terms of its antiatherosclerotic/antioxidant effect in the ApoE-/- transgenic mouse model. Its toxicity profile was also assessed by measuring the levels of four sensitive indicators of liver toxicity. Prolonged administration of 1 in ApoE-/- mice fed with a western-type (wt) diet efficiently reduced the aortic atheromatic lesions, an effect that took place through a cholesterol lowering independent manner. In addition, 1 displayed a significant reduction not only of glucose but also of oxidative stress levels, while it did not cause any toxicity. To the best of our knowledge this is the first time that the antiatherosclerotic effect of a Squalene Synthase inhibitor is studied in this specific atherosclerosis mouse model. As a result, compound 1 may serve as a promising starting point towards developing new bioactive analogues against the onset and subsequent development of atherosclerosis.

Expression of PnSS Promotes Squalene and Oleanolic Acid (OA) Accumulation in Aralia elata via Methyl Jasmonate (MeJA) Induction.

Aralia elata is an important herb due to the abundance of pentacyclic triterpenoid saponins whose important precursors are squalene and OA. Here, we found that MeJA treatment promoted both precursors accumulation, especially the latter, in transgenic A. elata, overexpressing a squalene synthase gene from Panax notoginseng(PnSS). In this study, Rhizobium-mediated transformation was used to express the PnSS gene. Gene expression analysis and high-performance liquid chromatography (HPLC) were used to identify the effect of MeJA on squalene and OA accumulation. The PnSS gene was isolated and expressed in A. elata. Transgenic lines showed a very high expression of the PnSS gene and farnesyl diphosphate synthase gene (AeFPS) and a slightly higher squalene content than the wild-type, but endogenous squalene synthase (AeSS), squalene epoxidase (AeSE), and ß-amyrin synthase (Aeß-AS) gene were decreased as well as OA content. Following one day of MeJA treatment, the expression levels of PeSS, AeSS, and AeSE genes increased significantly. On day 3, the maximum content of both products reached 17.34 and 0.70 mg·g-1, which increased 1.39- and 4.90-fold than in the same lines without treatment. Transgenic lines expressing PnSS gene had a limited capability to promote squalene and OA accumulation. MeJA strongly activated their biosynthesis pathways, leading to enhance yield.

Transcription Factors ZjMYB39 and ZjMYB4 Regulate Farnesyl Diphosphate Synthase- and Squalene Synthase-Mediated Triterpenoid Biosynthesis in Jujube.

Jujube (Ziziphus jujuba Mill.) is rich in valuable bioactive triterpenoids. However, the regulatory mechanism underlying triterpenoid biosynthesis in jujube remains poorly studied. Here, we characterized the triterpenoid content in wild jujube and cultivated jujube. The triterpenoid content was higher in wild jujube than in cultivated jujube, triterpenoids were most abundant in young leaves, buds, and later stages of development. The transcriptome analysis and correlation analysis showed that differentially expressed genes (DEGs) were enriched in the terpenoid synthesis pathways, and triterpenoids content was strongly correlated with farnesyl diphosphate synthase (ZjFPS), squalene synthase (ZjSQS), and transcription factors ZjMYB39 and ZjMYB4 expression. Gene overexpression and silencing analysis indicated that ZjFPS and ZjSQS were key genes in triterpenoid biosynthesis and transcription factors ZjMYB39 and ZjMYB4 regulated triterpenoid biosynthesis. Subcellular localization experiments showed that ZjFPS and ZjSQS were localized to the nucleus and endoplasmic reticulum and ZjMYB39 and ZjMYB4 were localized to the nucleus. Yeast one-hybrid, glucuronidase activity, and dual-luciferase activity assays suggested that ZjMYB39 and ZjMYB4 regulate triterpenoid biosynthesis by directly binding and activating the promoters of ZjFPS and ZjSQS. These findings provide insights into the underlying regulatory network of triterpenoids metabolism in jujube and lay theoretical and practical foundation for molecular breeding.

The Identification of SQS/SQE/OSC Gene Families in Regulating the Biosynthesis of Triterpenes in Potentilla anserina.

The tuberous roots of Potentilla anserina (Pan) are an edible and medicinal resource in Qinghai-Tibetan Plateau, China. The triterpenoids from tuberous roots have shown promising anti-cancer, hepatoprotective, and anti-inflammatory properties. In this study, we carried out phylogenetic analysis of squalene synthases (SQSs), squalene epoxidases (SQEs), and oxidosqualene cyclases (OSCs) in the pathway of triterpenes. In total, 6, 26, and 20 genes of SQSs, SQEs, and OSCs were retrieved from the genome of Pan, respectively. Moreover, 6 SQSs and 25 SQEs genes expressed in two sub-genomes (A and B) of Pan. SQSs were not expanded after whole-genome duplication (WGD), and the duplicated genes were detected in SQEs. Twenty OSCs were divided into two clades of cycloartenol synthases (CASs) and ß-amyrin synthases (ß-ASs) by a phylogenetic tree, characterized with gene duplication and evolutionary divergence. We speculated that ß-ASs and CASs may participate in triterpenes synthesis. The data presented act as valuable references for future studies on the triterpene synthetic pathway of Pan.

Switching Prenyl Donor Specificities in Squalene Synthase-Like Aromatic Prenyltransferases from Bacterial Carbazole Alkaloid Biosynthesis.

Lavanduquinocin (LDQ) is a potent neuroprotective carbazole alkaloid from Streptomyces species that features a rare cyclic monoterpene/cyclolavandulyl moiety attached to the tricyclic carbazole nucleus. We elucidated the biosynthetic logic of LDQ by enzymatically reconstituting the total biosynthetic pathway and identified the genes required for generating the cyclolavandulyl moiety in LDQ based on mutagenetic analysis, including a cyclolavandulyl diphosphate synthase gene ldqA and a squalene synthase-like aromatic prenyltransferase gene ldqG. LdqG is homologous to carbazole prenyltransferases, NzsG and CqsB4, discovered from the biosynthetic pathways of two bacterial carbazoles, neocarazostatin and carquinostatin. Based on analysis of the sequences and modeled protein structures, further in vitro and in vivo site-directed mutagenetic analyses led to identification of two residue sites, F53 and C57 in NzsG vs I54 and A58 in LdqG, which play crucial roles in governing the prenyl donor specificities toward cyclolavandulyl, dimethylallyl, and geranyl diphosphates. By applying this knowledge in strain engineering, prenyl donor delivery was rationally switched to produce the desired prenylated carbazoles. The study provides an opportunity to rationally manipulate the prenylation modification to carbazole alkaloids, which could influence the biological activities by increasing the affinity for membranes as well as the interactions with cellular targets.

Genome-wide profiling of dysregulated piRNAs and their target genes implicated in oncogenicity of tongue squamous cell carcinoma.

PIWI-interacting RNAs (piRNAs) are single-stranded, 23-36 nucleotide long RNAs that regulate gene expression in the germline but are also detected in some cancers. However, there are no reports yet on piRNA expression in tongue squamous cell carcinoma (TSCC), the most common oral cancer (80-90% percent of all oral cancers). We performed small RNA and whole transcriptome sequencing in H357 tongue cancer and HOK cells (GEO database accession numbers: GSE196674 and GSE196688). We also examined nine published sets of gene expression array data of TSCC tissues from the GEO database to decode piRNAs and their putative targets that may be involved in tumorigenesis. We identified a pool of 16,058 and 25,677 piRNAs in H357 and HOK, respectively, among which 406 are differentially expressed. We also found that 2094 protein-coding genes are differentially expressed in either TSCC tissues or cell lines. We performed target predictions for these piRNAs, pathway and disease function (DF) analyses, as well as qRT-PCR validation of piRNA-target pairs. These experiments revealed one up-regulated (FDFT1) and four down-regulated (OGA, BDH1, TAT, HYAL4) target genes that are enriched in 11 canonical pathways (CPs), with postulated roles in the initiation and progression of TSCC. Downregulation of piR-33422 is predicted to upregulate the FDFT1 gene, which encodes a mevalonate/cholesterol-pathway related farnesyl-diphosphate farnesyltransferase. The FDFT1 appears to be involved in the largest number of oncogenesis-related processes and is interacting with statins, which is a classical cancer drug. This study provides the first evidence of the piRNome of TSCC, which could be investigated further to decode piRNA-mediated gene regulations in malignancy and potential drug targets, such as FDFT1.

[Effect of light intensity on growth, accumulation of ginsenosides, and expression of related enzyme genes of Panax quinquefolius].

Appropriate light intensity is favorable for the photosynthesis, biomass accumulation, key enzyme activity, and secondary metabolite synthesis of medicinal plants. This study aims to explore the influence of light intensity on growth and quality of Panax quinquefolius. To be specific, sand culture experiment was carried out in a greenhouse under the light intensity of 40, 80, 120, and 160 µmol·m~(-2)·s~(-1), respectively. The growth indexes, photosynthetic characteristics, content of 6 ginsenosides of the 3-year-old P. quinquefolius were determined, and the expression of ginsenoside synthesis-related enzyme genes in leaves, main roots, and fibrous roots was determined. The results showed that the P. quinquefolius growing at 80 µmol·m~(-2)·s~(-1) light intensity had the most biomass and the highest net photosynthetic rate. The total biomass of P. quinquefolius treated with 120 µmol·m~(-2)·s~(-1) light intensity was slightly lower than that with 80 µmol·m~(-2)·s~(-1). The root-to-shoot ratio in the treatment with 120 µmol·m~(-2)·s~(-1) light intensity was up to 6.86, higher than those in other treatments(P<0.05),and the ginsenoside content in both aboveground and underground parts of P. quinquefolius in this treatment was the highest, which was possibly associated with the high expression of farnesylpyrophosphate synthase(FPS), squalene synthase(SQS), squalene epoxidase(SQE), oxidosqualene cyclase(OSC), dammarenediol-Ⅱ synthase(DS), and P450 genes in leaves and SQE and DS genes in main roots. In addition, light intensities of 120 and 160 µmol·m~(-2)·s~(-1) could promote PPD-type ginsenoside synthesis in leaves by triggering up-regulation of the expression of upstream ginsenoside synthesis genes. The decrease in underground biomass accumulation of the P. quinquefolius grown under weak light(40 µmol·m~(-2)·s~(-1)) and strong light(160 µmol·m~(-2)·s~(-1)) was possibly attributed to the low net photosynthetic rate, stomatal conductance, and transpiration rate in leaves. In the meantime, the low expression of SQS, SQE, OSC, and DS genes in the main roots might led to the decrease in ginsenoside content. However, there was no significant correlation between the ginsenoside content and the expression of synthesis-related genes in the fibrous roots of P. quinquefolius. Therefore, the light intensity of 80 and 120 µmol·m~(-2)·s~(-1) is beneficial to improving yield and quality of P. quinquefolius. The above findings contributed to a theoretical basis for reasonable shading in P. quinquefolius cultivation, which is of great significance for improving the yield and quality of P. quinquefolius through light regulation.

Benzylamines as highly potent inhibitors of the sterol biosynthesis pathway in Leishmania amazonensis leading to oxidative stress and ultrastructural alterations.

Leishmaniasis is a neglected disease caused by protozoan parasites of the Leishmania genus. Benzylamines are a class of compounds selectively designed to inhibit the squalene synthase (SQS) that catalyzes the first committed reaction on the sterol biosynthesis pathway. Herein, we studied seven new benzylamines (SBC 37-43) against Leishmania amazonensis. After the first screening of cell viability, two inhibitors (SBC 39 and SBC 40) were selected. Against intracellular amastigotes, SBC 39 and SBC 40 presented selectivity indexes of 117.7 and 180, respectively, indicating high selectivity. Analysis of the sterol composition revealed a depletion of endogenous 24-alkylated sterols such as episterol and 5-dehydroepisterol, with a concomitant accumulation of fecosterol, implying a disturbance in cellular lipid content. This result suggests a blockade of de novo sterol synthesis at the level of SQS and C-5 desaturase. Furthermore, physiological analysis and electron microscopy revealed three main alterations: (1) in the mitochondrion; (2) the presence of lipid bodies and autophagosomes; and (3) the appearance of projections in the plasma membrane. In conclusion, our results support the notion that benzylamines have a potent effect against Leishmania amazonensis and should be an exciting novel pharmaceutical lead for developing new chemotherapeutic alternatives to treat leishmaniasis.

Pharmacological effects of Artocarpus lakoocha methanol extract on inhibition of squalene synthase and other downstream enzymes of the cholesterol synthesis pathway.

CONTEXT: Artocarpus lakoocha Roxb. (Moraceae) is reported to possess antioxidant, anti-inflammatory, and anti-skin ageing agents. OBJECTIVE: This study evaluates the pharmacological effects of A. lakoocha leaves methanol extract on enzymes involved in the cholesterol synthesis pathway in high-fat diet-induced hyperlipidemic rats. MATERIALS AND METHODS: Twenty-four male Wistar rats, weighing approximately 180-220 g, were divided into four groups: control, diseased (hyperlipidemic), A. lakoocha leaves extract treated, and simvastatin treated. The rats were fed with high-fat diet for 2 months to induce hyperlipidaemia, afterward, experimental groups received A. lakoocha leaves methanol extract (250 mg/kg) and simvastatin (10 mg/kg) orally until the 89th day of the experiment, while the diseased group continued to receive high-fat diet along with normal saline. RESULTS: It was found that A. lakoocha extract significantly lowered the serum total cholesterol, triglycerides, and low-density lipoprotein (LDL) levels, while effectively increasing serum high-density lipoprotein (HDL) levels as compared to the diseased group (p ≤ 0.05). The mRNA expression levels of squalene synthase and HMG-CoA reductase were found to be effectively down-regulated after the treatment with A. lakoocha leaves extract (17.45 ± 2.48 vs. 31.91 ± 5.292 and 5.85 ± 3.164 vs. 37.37 ± 6.492) and simvastatin (7.148 ± 0.76 vs. 31.91 ± 5.292, and 3.098 ± 2.09 vs. 37.37 ± 6.492) as compared to the diseased group. DISCUSSION AND CONCLUSIONS: The results suggested that A. lakoocha leaves extract have observable beneficial effects on inhibition of enzymes involved in cholesterol synthesis pathway and improve lipid profile analogous to simvastatin.

cDNA cloning, prokaryotic expression, and functional analysis of squalene synthase (SQS) in Camellia vietnamensis Huang.

Camellia vietnamensis Huang, which belongs to Camellia oleifera, is a traditional Chinese medicinal plant widely planted on Hainan Island. Tea saponin is an important functional component of C. vietnamensis, and squalene is the precursor substance that controls its formation. Squalene synthase (SQS: EC 2.5.1.21) synthesizes squalene from 2 molecules of farnesyl pyrophosphate (FPP). In this study, 1683 bp of the C. vietnamensis SQS gene, designated as CvSQS, was cloned and encoded 414 amino acids. Bioinformatics and phylogenetic tree analysis revealed the high homology of CvSQS with squalene synthases from other plants. For soluble proteins, the carboxy-terminal deleted CvSQS was obtained for expression in Escherichia coli Transetta (DE3), and the recombinant protein with a weight of 42.5 kDa was detected using SDS-PAGE and Western blot. After an enzymatic reaction, the presence of squalene in the product was analyzed using GC-MS detection, which indicated that CvSQS had catalytic activity. The tissue specificity of CvSQS and its presence in seeds at various ripening stages was detected by q-RT PCR. CvSQS had the highest transcriptional level in leaves, followed by seeds, roots, and flowers; the amount of CvSQS in the seeds was highest in September. The identification and functional characterization of CvSQS is essential for further studies on the regulation mechanism of tea saponin in C. vietnamensis.

Structural Basis for the Prenylation Reaction of Carbazole-Containing Natural Products Catalyzed by Squalene Synthase-Like Enzymes.

Some enzymes annotated as squalene synthase catalyze the prenylation of carbazole-3,4-quinone-containing substrates in bacterial secondary metabolism. Their reaction mechanisms remain unclear because of their low sequence similarity to well-characterized aromatic substrate prenyltransferases (PTs). We determined the crystal structures of the carbazole PTs, and these revealed that the overall structure is well superposed on those of squalene synthases. In contrast, the stacking interaction between the prenyl donor and acceptor substrates resembles those observed in aromatic substrate PTs. Structural and mutational analyses suggest that the Ile and Asp residues are essential for the hydrophobic and hydrophilic interactions with the carbazole-3,4-quinone moiety of the prenyl acceptor, respectively, and a deprotonation mechanism of an intermediary σ-complex involving a catalytic triad is proposed. Our results provide a structural basis for a new subclass of aromatic substrate PTs.

Structural and Molecular Characterization of Squalene Synthase Belonging to the Marine Thraustochytrid Species Aurantiochytrium limacinum Using Bioinformatics Approach.

The marine microorganisms thraustochytrids have been explored for their potential in the production of various bioactive compounds, such as DHA, carotenoids, and squalene. Squalene is a secondary metabolite of the triterpenoid class and is known for its importance in various industrial applications. The bioinformatic analysis for squalene synthase (SQS) gene (the first key enzyme in the tri-terpenoid synthesis pathway), that is prevailing among thraustochytrids, is poorly investigated. In-silico studies combining sequence alignments and bioinformatic tools helped in the preliminary characterization of squalene synthases found in Aurantiochytrium limacinum. The sequence contained highly conserved regions for SQS found among different species indicated the enzyme had all the regions for its functionality. The signal peptide sequence and transmembrane regions were absent, indicating an important aspect of the subcellular localization. Secondary and 3-D models generated using appropriate templates demonstrated the similarities with SQS of the other species. The 3-D model also provided important insights into possible active, binding, phosphorylation, and glycosylation sites.