Molecular Cloning and Functional Analysis of Squalene Synthase 2(SQS2) in Salvia miltiorrhiza Bunge

Abstract

Salvia miltiorrhiza Bunge, which is also known as a traditional Chinese herbal medicine, is widely studied for its ability to accumulate the diterpene quinone Tanshinones. In addition to producing a variety of diterpene quinone, S. miltiorrhiza Bunge also accumulates sterol, brassinosteroid and triterpenoids. During their biosynthesis, squalene synthase (SQS, EC 2.5.1.21) converts two molecules of the hydrophilic substrate farnesyl diphosphate (FPP) into a hydrophobic product, squalene. In the present study, cloning and characterization of S. miltiorrhiza Bunge squalene synthase 2 (SmSQS2, Genbank Accession Number: KM408605) cDNA was investigated subsequently followed by its recombinant expression and preliminary enzyme activity. The full-length cDNA of SmSQS2 was 1 597 bp in length, with an open reading frame of 1 245 bp encoding 414 amino acids. The deduced amino acid sequence of SmSQS2 shared high similarity with those of SQSs from other plants. To obtain soluble recombinant enzymes, the truncated SmSQS2 in which 28 amino acids were deleted from the carboxy terminus was expressed as GST-Tag fusion protein in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western Blot analysis, and the resultant bacterial crude extract was incubated with FPP and NADPH. Gas chromatograph-mass spectrometer analysis showed that squalene was detected in the in vitro reaction mixture. The gene expression level was analyzed through Quantitative real-time PCR, and was found to be higher in roots as compared to the leaves, and was up-regulated upon YE+ Ag+ treatment. These results could serve as an important to understand the function of the SQS family. In addition, the identification of SmSQS2 is important for further studies of terpenoid and sterol biosynthesis in S. miltiorrhiza Bunge.

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

“…In vitro enzyme reactions demonstrated that TwSQS could catalyze the reaction of FPP form to squalene under the coordination of Mg 2+ and NADPH. Somewhat similar results were also reported in M. officinalis SQS [ 21 ], S. miltiorrhiza SQS2 [ 22 ], P. notoginseng SQS [ 24 ], tobacco SQS [ 31 ], and W. somnifera SQS [ 14 ]. In addition, the recombinant AtSQS2 which was deemed to be a non-functional SQS, was unable to synthesize squalene from FPP in the presence of NADPH and either Mg 2+ or Mn 2+ [ 18 ].…”

“…Some studies found that the tissue expression pattern of SQS varies greatly in different plants. Similar patterns of gene expression were found in S. miltiorrhiza [ 22 ], Euphorbia pekinensis [ 36 ], Taxus cuspidata [ 32 ], while some different expression patterns were found in B. platyphylla [ 37 ], W. somnifera [ 38 ], and Euphorbia tirucalli [ 39 ]. In addition, several studies have reported that the MeJA could upregulates the expression of SQS [ 21 , 36 ].…”

“…Thus, the genes encoding the enzyme have been cloned from several organisms including fungi [ 14 , 15 ], bacteria [ 16 ], animals [ 17 ], and human beings [ 18 , 19 ]. Botanical squalene synthase enzymes have also been identified in Arabidopsis thaliana [ 20 ], Magnolia officinalis [ 21 ], Salvia miltiorrhiza Bunge [ 22 ], Panax ginseng [ 23 ], Panax notoginseng [ 24 ], and Glycine max [ 25 ]. The squalene synthases identified from P. ginseng were able to convert yeast erg9 mutant cells to ergosterol prototrophy in spite of sequence divergence to yeast [ 21 ].…”

“…In addition, many SQSs have been investigated subsequently followed by recombinant expression and preliminary enzyme activity. These recombinant SQSs could synthesize squalene from FPP in the presence of NADPH and Mg 2+ [ 19 , 20 , 22 ]. Many studies have demonstrated that overexpression of SQS genes could enhance the accumulation of triterpenoid and/or phytosterols compounds in Withania somnifera [ 26 ], Panax ginseng [ 27 ], Eleutherococcus senticosus [ 28 ], Bupleurum falcatum [ 29 ] and Solanum chacoense [ 30 ].…”

Recent advances in steroidal saponins biosynthesis and in vitro production

“…It catalyzes the condensation of two FPP molecules into squalene. SQS was characterized in a wide variety of plant species including Panax ginseng , Chlorophytum borivilianum (Kalra et al 2013), Ornithogalum caudatum , Panax notoginseng (Jiang et al 2017), Siraitia grosvenorii (Zhao et al 2017), Salvia miltiorrhiza (Rong et al 2016) and Magnolia officinalis .…”