Cloning, Expression Analysis and Functional Characterization of Squalene Synthase (SQS) from Tripterygium wilfordii

Abstract

Celastrol is an active triterpenoid compound derived from Tripterygium wilfordii which is well-known as a traditional Chinese medicinal plant. Squalene synthase has a vital role in condensing two molecules of farnesyl diphosphate to form squalene, a key precursor of triterpenoid biosynthesis. In the present study, T. wilfordii squalene synthase (TwSQS) was cloned followed by prokaryotic expression and functional verification. The open reading frame cDNA of TwSQS was 1242 bp encoding 413 amino acids. Bioinformatic and phylogenetic analysis showed that TwSQS had high homology with other plant SQSs. To obtain soluble protein, the truncated TwSQS without the last 28 amino acids of the carboxy terminus was inductively expressed in Escherichia coli Transetta (DE3). The purified protein was detected by SDS-PAGE and Western blot analysis. Squalene was detected in the product of in vitro reactions by gas chromatograph-mass spectrometry, which meant that TwSQS did have catalytic activity. Organ-specific and inducible expression levels of TwSQS were detected by quantitative real-time PCR. The results indicated that TwSQS was highly expressed in roots, followed by the stems and leaves, and was significantly up-regulated upon MeJA treatment. The identification of TwSQS is important for further studies of celastrol biosynthesis in T. wilfordii.

Molecular Cloning and Functional Identification of a Squalene Synthase Encoding Gene from Alfalfa (Medicago sativa L.)

“…Relatively, the expression level in leaves and roots was about 1.5- and 4.8-times of that in stems (Figure 6a), suggesting that MsSQS was preferentially expressed in root tissues. The result is consistent with the observations that SQS was predominantly expressed in roots of soybean and Tripterygium wilfordii [11,37].…”

“…Some plants, such as Withania somnifera [47], Betula platyphylla [15] and Arabidopsis [22], displayed a leaf-predominant pattern, suggesting that the spatial and temporal expression patterns of SQS genes vary greatly in different plants. Consistent with the observations that the SQS transcript was activated by MeJA induction [17,18,37], MsSQS was up-regulated upon exposure to MeJA and the stimulation resulted in an increased amount of the MsSQS enzyme (Figure 6). It has been reported that the hydrophobic amino acid residues at the C-terminal of SQS contribute to the membrane anchoring function [4,5].…”

“…Expression analysis indicated that the ubiquitous MsSQS was expressed preferentially in roots (Figure 6). The root-preferred pattern was observed for GmSQS1 in soybean [11], SgSQS in Siraitia grosvenorii [44], HsSQS1 in Huperzia serrata [46] and TwSQS in traditional Chinese medicinal plant Tripterygium wilfordii [37]. Some plants, such as Withania somnifera [47], Betula platyphylla [15] and Arabidopsis [22], displayed a leaf-predominant pattern, suggesting that the spatial and temporal expression patterns of SQS genes vary greatly in different plants.…”

“…It has been reported that the hydrophobic amino acid residues at the C-terminal of SQS contribute to the membrane anchoring function [4,5]. Deletion of the transmembrane domain enhanced the solubility of MsSQS, as well as the recombinant SQS proteins from several species [37,48], and the truncated SQS was capable of converting FPP to form squalene, indicating the folding capability and the catalytic activity remained unchanged [45,49]. Interestingly, fungal squalene synthases have a unique hinge region (26 amino acid residues) linking the catalytic and membrane-spanning domains, and the hinge domain is essential for functional SQS in yeast but not for animals or plants [4].…”

Biosynthesis, total synthesis, structural modifications, bioactivity, and mechanism of action of the quinone‐methide triterpenoid celastrol

“…The farnesyl pyrophosphate synthase catalyzes DMAPP and IPP to form FPP, which is a key precursor of triterpenoids 58–60 . Squalene synthase (SQS) plays an essential role in isoprenoid biosynthesis since it converts FPP into squalene 61 . The next enzyme, squalene epoxidase (SQE), which is also known as squalene monooxygenase, converts squalene into 2,3‐oxidosqualene with the participation of NADPH‐cytochrome P450 reductase 62 .…”