The Biosynthesis Of Cucubitadienol And The Biological Activities Of Cucurbitane-type Triterpenes In Siraitia Grosvenorii

Mogrosides,the major bioactive components isolated from the fruits of S.grosvenorii,are a family of cucurbitane-type tetracyclic triterpenoid saponins that are used worldwide as natural,zero-calorie,high-potency sweeteners and possess a variety of notable pharmacological activities.To date,S.grosvenorii fruits has been shown to have antitussive,anti-asthmatic,lung congestion,liver-protection,anti-oxidation,anti-inflammatory,anti-cancer and anti-diabetic effects.It has become one of the novel sweeteners with therapeutic function that have been found in traditional Chinese medicine(TCM).Mogrosides are synthesized by the isoprenoid pathway and share the same precursor,2,3-oxidosqualene,with sterol.Cucubitadienol Synthase(CS)is the first rate-limiting enzyme for synthesizing cucurbitane-type triterpene skeleton,which catalysis 2,3-oxidosqualene formating cucurbitane skeleton cucubitadienol.The cucubitadienol then undergoes various modifications(oxidation and glycosylation)mediated by cytochrome P450-dependent monooxygenases,glycosyltransferases and other enzymes,eventually forming mogrosides.Cucubitadienol,the skeleton of cucurbitane-type triterpenoids such as mogrosides and cucurbitacins,has anti-inflammatory and cancer prevention effects.However,similar to a lot of terpenoids,the very low content of cucubitadienol in plants and led to greatly limited its large-scale production and wide application.In present paper,synthetic biology is employed to synergistically express multi genes from various species for construction of cucubitadienol biosynthetic pathway in Saccharomyces cerevisiae.Through the optimization of pathway and fermentation process,cucubitadienol is expected to efficiently be produced.In addition,In vitro pharmacological activities and phannacokineties of cucurbitane-type triterpenes in S.grosvenorii were systematically investigated.The main results are described as follows:(1)The yeast expression system of cucurbitadienol was constructed:We compared the growth rate and the accumulation of squalene in 4 different yeast strains,and finally chose the popular experimental strain BY4742 as original strain.The genes of cucurbitadienol synthase from Siraitia grosvenorii(SgCS),Cucurbita pepo(CpCS),Cucumis sativus(CsCS)and Citrullus colocynthis(CcCS)were firstly chosen and codon-optimized.After expressed using different plasmids in yeast,GC-MS analysis showed that cucurbitadienol was successfully produced by Saccharomyces cerevisiae,and the titer was 7.3 mg/L after SgCS gene expression.This study indicated that it is feasible to produce cucurbitadienol by constructing cucurbitadienol biosynthetic pathway in S.cerevisiae.(2)Multiple metabolic regulation strategies were constructed:We cloned yeast transcription factor UPC2,then over-expressed using a plasmid in yeast.GC-MS analysis showed that over-expression of UPC2 gene could increase the yield of cucurbitadienol.Compared with the initial strain,the yield was increased by 3 times and the titer of cucurbitadienol increased to was 21.47 mg/L.Moreover,adding exogenous substances can promote the synthesis of cucurbitadienol.When the concentration of methyl jasmonate was added at 0.5 mg/L,leading to the production of cucurbitadienol increased by 50%;the concentration of salicylic acid was added at 0.5 mg/L,the production of cucurbitadienol increased by 35%.(3)Optimization of yeast strain fermentation process:The culture conditions of engineered S.cerevisiae were optimized to produce cucurbitadienol,and the optimal conditions were obtained as the rotation rate was 200 rpm,the cultivation temperature was 30 ℃,the air flow was 50 L/h and the pH was 5.0,respectively.Fed-batch fermentation using glucose as substrate resulted in the production of cucurbitadienol increased by 2.8 times.Furthermore,Fed-batch fermentation using glucose and inorganic salt as substrate,cucurbitadienol titer was up to 20 mg/L.(4)In vitro pharmacological activity:The results showed that mogroside V(MV)and cucubitadienol(CU)have a potent growth inhibitory effect on A-549 cancer cell line(73%and 67%inhibition at 100 μM,respectively).Mogrol(MO)showed significantly inhibit the proliferation of A-549,HL-60 and BEL-7402 cells and in a dose-dependent manner.In vitro activities indicated that MV and its aglycone MO were both potent AMPK activators.MV and MO could activate the AMPK by 2.4 and 2.3 fold with an EC50 of 20.4 and 4.2 μM,respectively.This result suggested AMPK activation by MV and MO was proved to contribute at least partially to the anti-diabetic properties of S.grosvenorii fruits.(5)Studies on pharmaeokineties of MV and MO in rat plasma:An LC/MS/MS method was developed and validated for the determination of MV and MO in rat plasma with buspirone as the intimal standard.The Phmaraeokinetics of MV and MO were investigated in rat plasma by the LC/MS/MS method.The pharmacokinetic results showed that MV was rapidly deglycosylated and metabolized into MO,and both of these were determined after intravenous administration of MV in rats.The concentration ratio between MO and MV was only 4.61%at 2 min after intravenous administration,but as time goes by,the ratio was growing up to 132%.MV was not detected in rat plasma after oral administration,whereas a trace amount of MO was found.The oral absolute bioavailability(F)of MV was estimated to be 8.73 ±1.46%and the elimination half-life(t1/2)of metabolite MO in rats was 2.46 ±0.19 h.It was indicated that MV showed poor absorption and/or strong metabolism in vivo.After oral administration MO,we found that MO was rapidly absorbed into the circulation system and reached its peak concentration at 0.38 ± 0.11 h.and the absolute bioavailability(F)was relative lowwith a value being 10.3±2.15%and t1/2 was 2.41 ±0.11 h.A significant first pass effect and poor permeability through the intestinal epithelial membrane after oral administration might be responsible for the low bioavailability of this compound.