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The subcellular localization of AlSQS1 and AlSQS2. a The subcellular localization of 35S:: GFP,35S:: AlSQS1-GFP and 35S:: AlSQS2-GFP in N. benthamiana leaf epidermal cells after 48 h infiltration; the epidermal cells of Nicotiana benthamiana were used for taking images of green fluorescence, chloroplast autofluorescence, visible light and merged visible light. b Green fluorescence show the expression and distribution of GFP protein; red fluorescence was from the expression of endoplasmic reticulum marker plasmid pBWA(V)HS-SPER-MAKTE; pink fluorescence was from chloroplasts; Light field pictures showed the overall morphology of cells under normal light conditions; Overlayed pictures showed if the target protein AlSQS2 colocalized with endoplasmic reticulum or chloroplasts. Scale bars = 20 μm
Source publication
Main conclusion
Two squalene synthase genes AlSQS1 and AlSQS2 were isolated from
Atractylodes lancea
and functionally characterized using in vitro enzymatic reactions.
AbstractAtractylodes lancea is a traditional herb used for the treatment of rheumatic diseases, gastric disorders, and influenza. Its major active ingredients include sesquiterpenoid...
Citations
... The mixture was reacted at 30 °C for 12 h, and the squalene component was then extracted using n-hexane for GC-MS analysis. GC-MS analysis was performed on an Agilent 7890B-7000B platform equipped with a DB-5 MS column (60 m × 0.25 mm × 0.25 μm) (Agilent Technologies, Palo Alto, CA, USA) as previously described (Zhao et al. 2015;Wu et al. 2021;Yang et al. 2022). ...
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.
... In addition, an N-terminal transit peptide with the characteristic features of chloroplast targeting peptides was detected by TargetP software in the OepSQS4 sequence (Figure 2), with a predicted cleavage site after Glu at residue 50. Experimental localization for plant SQS protein not only has been mainly reported in the ER 15,37−39 but also has been located in the cytoplasm, 39,40 nucleus, 39 and plasma membrane. 35 An unrooted phylogenetic tree based on deduced amino acid sequences of known and characterized plant SQS ( Figure S3) was generated to investigate the phylogenetic relationship of olive SQS. ...
The release of new olive cultivars with an increased squalene content in their virgin olive oil is considered an important target in olive breeding programs. In this work, the variability of the squalene content in a core collection of 36 olive cultivars was first studied, revealing two olive cultivars, 'Dokkar' and 'Klon-14', with extremely low and high squalene contents in their oils, respectively. Next, four cDNA sequences encoding squalene synthases (SQS) were cloned from olive. Sequence analysis and functional expression in bacteria confirmed that they encode squalene synthases. Transcriptional analysis in distinct olive tissues and cultivars indicated that expression levels of these four SQS genes are spatially and temporally regulated in a cultivar-dependent manner and pointed to OeSQS2 as the gene mainly involved in squalene biosynthesis in olive mesocarp and, therefore, in the olive oil. In addition, the biosynthesis of squalene appears to be transcriptionally regulated in water-stressed olive mesocarp.
... Squalene synthase (EC 2.5.1.21) in the steroid biosynthesis pathway (ko00100) is the rate-limiting enzyme for squalene synthesis (Wu et al. 2022), which can be annotated in the genome of Muc X . According to the KEGG pathway analysis results, it was predicted that both Muc X and Muc T could synthesize GABA and squalene, which may be one of the reasons for the neuroprotection, bile acid resistance, and anti-inflammatory effects of A. muciniphila. ...
Akkermansia muciniphila is considered to be a next-generation probiotic, and closely related to host metabolism and immune response. Compared with other probiotics, little is known about its genomic analysis. Therefore, further researches about isolating more A. muciniphila strains and exploring functional genes are needed. In the present study, a new strain isolated from mice feces was identified as A. muciniphila (MucX). Whole-genome sequencing and annotation revealed that MucX possesses key genes necessary for human milk oligosaccharides (HMO) utilization, including α-l-fucosidases, β-galactosidases, exo-α-sialidases, and β-acetylhexosaminidases. The complete metabolic pathways for γ-aminobutyric acid and squalene and genes encoding functional proteins, such as the outer membrane protein Amuc_1100, were annotated in the MucX genome. Comparative genome analysis was used to identify functional genes unique to MucX compared to six other A. muciniphila strains. Results showed MucX genome possesses unique genes, including sugar transporters and transferases. Single-strain incubation revealed faster utilization of 2′-fucosyllactose (2′-FL), galacto-oligosaccharides, and lactose by MucX than by A. muciniphila DSM 22959. This study isolated and identified an A. muciniphila strain that can utilize 2′-FL, and expolored the genes related to HMO utilization and special metabolites, which provided a theoretical basis for the further excavation of A. muciniphila function and the compound application with fucosylated oligosaccharides.
Plants are major factories for the biosynthesis of valuable bioactive compounds having pharmaceutical applications. These phytocompounds are synthesised via the diversion of primary metabolites toward secondary metabolic pathways. The most varied class of metabolites are triterpenoids that are biosynthesized through the MVA-MEP pathways, leading to the production of oxidosqualene followed by cyclization, and enzymatic modifications. Triterpenoids play a significant role in growth, development, reproductive behaviour, plant adaptation, and plant-to-plant communication. Various signals, through the signal transduction mechanism, are transmitted through receptors from cell to cell by a series of molecular events to initiate/ activate the cascade of the triterpenoid biosynthesis network. In this review, different naturally occurring classes of triterpenoids with their medicinal potential have been summarized. Further, this review provides insight into the current status of its synthesis through heterologous gene expression, structure elucidation, biosynthetic regulation via transcriptional regulation, and miRNAs. Recently, triterpenoids are in demand due to their variety of pharmacological importance which requires a fast rate of production. Furthermore, to ramp up the synthesis of these vital triterpenoids, the applications of modern bioengineering technologies and green nanoparticle synthesis, have been also highlighted here, offering sustainable alternatives for their large-scale production.
... A previous study reported the transcriptome of A. lancea. Our group also cloned and characterised genes in the MVA pathway of terpenoids and the triterpene synthesis pathway in A. lancea (Wu et al. 2021a(Wu et al. , 2021bXu et al. 2021). Although the DXS and DXR genes have been extensively studied, only few molecular studies have been conducted on these genes in A. lancea. ...
1-Deoxy-d-xylulose-5-phosphate synthase and 1-deoxy-d-xylulose-5-phosphate reductoismerase are considered two key enzymes in the 2-C-methyl-d-erythritol-4-phosphate pathway of terpenoid biosynthesis and are related to the synthesis and accumulation of sesquiterpenoids. We cloned two DXS and DXR genes from Atractylodes lancea and analysed their expression in different tissues and in response to methyl jasmonate (MeJA). Subcellular localisation analysis revealed that the AlDXS and AlDXR1 proteins are located in the chloroplasts and cytoplasm, whereas AlDXR2 is only located in the chloroplasts. pET-AlDXS-28a and pGEX-AlDXR-4T-1 were expressed in Escherichia coli BL21(DE3) and BL21, respectively. Based on the abiotic stress analysis, the growth rate of the recombinant pGEX-AlDXR-4T-1 was higher than that of the control in HCl and NaOH. AlDXS exhibited the highest expression level in rhizomes of A. lancea from Hubei but was highest in leaves from Henan. In contrast, AlDXR showed maximum expression in the leaves of A. lancea from Hubei and Henan. Moreover, DXS and DXR gene expression, enzyme activities, and antioxidant enzyme activities oscillated in response to MeJA, with expression peaks appearing at different time points. Our findings indicated that the characterisation and function of AlDXS and AlDXR could be useful for further elucidating the functions of DXR and DXR genes in A. lancea.
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.
