Cloning, Expression Analysis and Functional Characterization of Squalene Synthase (SQS) from Tripterygium wilfordii
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
... ncoding 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. Molecules 2018, 23, 269 2 of 12 obtained by extraction from the root of T. wilfordii. However, T. wilfordii is not widely cultivated and the content of celastrol in natural plants is far below demand. The chemical synthesis of this compound is not an economical alternative because of its multistep reactions and low overall yield  . Tissue cultivation is a potential choice for the production of natural products, but up to now, only the culture of T. wilfordii in a small-scale 10-L bioreactor has been reported  . Synthesis on an industrial scale is still far away. Therefore, it is of interest to produce the compound through synthetic biology strategies. However, the development of these strategies requires detailed understanding of the celastrol biosynthetic pathway. Squalene synthase (SQS, EC 22.214.171.124) is a membrane-associated enzyme that catalyzes the first enzymatic step in sterols and triterpenoid biosynthesis . It converts two molecules of farnesyl diphosphate (FPP) into squalene, which is a precursor to both sterols and triterpenoid  . The SQS enzyme is considered to be a pivotal enzyme in the regulation of triterpenoid biosynthesis. Thus, the genes encoding the enzyme have been cloned from several organisms including fungi [14, 15] , bacteria , animals , and human beings [18,19]. Botanical squalene synthase enzymes have also been identified in Arabidopsis thaliana , Magnolia officinalis , Salvia miltiorrhiza Bunge , Panax ginseng , Panax notoginseng , and Glycine max  . 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  . Likewise, similar results have been reported in Glycine max. The GmSQS1 was able to convert yeast sterol auxotrophy erg9 mutant to sterol prototrophy and overexpression of GmSQS1 increased end product sterols in Arabidopsis seeds  . 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 , Panax ginseng , Eleutherococcus senticosus , Bupleurum falcatum  and Solanum chacoense . According to these studies, we believe that TwSQS may play a pivotal role in celastrol biosynthesis. However, the function of SQS is still unexplored in T. wilfordii. Here, we describe the prokaryotic expression, functional characterization and expression analysis of a squalene synthase gene from T. wilfordii and establish the foundation to study the celastrol biosynthetic pathway. In addition, the tissue expression of TwSQS in T. wilfordii natural plants and the expression in T. wilfordii hairy root induced by methyl jasmonate (MeJA) were investigated.