罗汉果甜苷，是一种葫芦烷型三萜皂苷，不仅是传统中药罗汉果的主要活性成分，还是非糖类纯天然甜味剂。药理学研究表明罗汉果甜苷具有镇咳祛痰、润肺保肝，抗炎、抗癌、降血糖等作用，已成为为数不多的从中药中发掘出来的具有治疗功能的新型甜味剂。在罗汉果甜苷的生物合成途径中，葫芦二烯醇合酶是葫芦烷型三萜骨架合成的第一个限速酶，也是此条途径唯一的环化酶，可催化底物2,3-氧化鲨烯生成葫芦二烯醇。随后葫芦二烯醇在P450酶和和葡萄糖基转移酶的作用下，依次形成罗汉果醇和罗汉果苷。葫芦二烯醇是葫芦烷型三萜化合物罗汉果苷和葫芦素的基本母核，具有抗炎、癌细胞防治功效，与很多萜类相似，葫芦二烯醇在植物中的含量很低，因而限制了其规模化生产和广泛应用。本文利用合成生物学方法在酿酒酵母中构建葫芦二烯醇合成途径，经途径和发酵过程优化，为实现葫芦二烯醇及其下游产物的生物合成奠定了有利基础。此外，还以罗汉果甜苷V及其前体罗汉果醇和葫芦二烯醇为研究对象，进行体外活性及大鼠血浆中药代动力学研究，为葫芦烷型化合物的开发利用提供了理论依据。现将主要研究结果叙述如下：

（1）构建了葫芦二烯醇酵母表达体系  在对比酵母菌株生长速率、鲨烯底物的前提下，初步选择改造实例丰富，代谢旺盛的优势酵母菌株BY4742作为底盘菌株。同源CS基因进行密码子优化后导入酿酒酵母，实现了葫芦二烯醇在酿酒酵母中的合成，而且表达SgCS基因获得葫芦二烯醇产量可达7.30 mg/L。

（2）建立了多种代谢调控策略  克隆到酵母转录因子UPC2基因，转化到重组酵母菌株后，GC-MS分析产物发现，过表达UPC2基因可提高葫芦二烯醇产量，是出发菌株产量的3倍，为21.47 mg/L。此外，外源物质的添加对产物葫芦二烯醇的合成具有一定的促进作用，茉莉酸甲酯添加浓度为0.5 mg/L时，产物葫芦二烯醇与对照相比约增加了50%；水杨酸添加浓度为0.5 mg/L时，产物葫芦二烯醇与对照相比约增加了35%。

（3）发酵工艺优化  正交试验优化了重组酵母发酵生产葫芦二烯醇的培养条件，发现以200 rpm，pH 5.0，空气流量50 L/h在30℃条件下进行发酵培养有利于酵母菌株生长和产物葫芦二烯醇的积累。并在此基础上进行分批补料发酵，结果发现葡萄糖补料发酵可显著提高目标产物单位体积产量和菌株BY4742-SgCS的生物量，最终产物滴度为16 mg/L，约为对照组的2.8倍。此外，在补加葡萄糖的同时添加无机盐溶液，可进一步提高产物约25%，最终得到产物滴度为20 mg/L。

（4）体外药理活性  肿瘤细胞增殖抑制活性研究结果表明，罗汉果醇（MO）对A-549、BEL-7402和HL-60细胞均具有一定抑制作用；罗汉果苷V（MV）和葫芦二烯醇（CU）对A-549细胞具有一定抑制作用，可作为候选药物进行后续研究。AMPK激活试验结果表明，受试物MO和MV在分子水平可以激活AMPK，MO的最大激活倍数和EC₅₀分别为2.3倍和4.2 μM；MV的最大激活倍数和EC₅₀分别为2.4倍和20.4 μM。这一发现为我们深入了解罗汉果甜苷的降糖机制提供了线索，并为天然AMPK激动剂类新型抗糖尿病药物的发现提供参考。

（5）药代动力学研究  建立了同时测定大鼠血浆中MV及其苷元MO的液相色谱-串联质谱（LC-MS/MS）法，并进行了完整的方法确证。大鼠静脉注射给药MV后，可在大鼠血浆中同时检测到MV和MO的存在，2 min时MO与MV的浓度比仅为4.61%；但随着时间的推移，MO与MV的浓度比越来越大，直至6 h时二者的比值为132%，表明MV在大鼠体内可迅速脱糖，生成代谢产物MO。大鼠灌胃给药MV后，在大鼠血浆中未检出MV的存在，但可检出少量的MO，并经浓度和剂量归一化处理后测得生物利用度值为8.73±1.46%。大鼠静脉注射给药MO后，MO在大鼠体内的消除半衰期为1.40±0.09 h。大鼠灌胃给药MO后，MO在大鼠体内的消除半衰期为2.41±0.11 h，达峰时间t_max为0.39±0.10 h，并经剂量归一化处理后获得的绝对生物利用度值为10.3±2.15%。结果表明，口服给药MV及其苷元MO后，MO可迅速吸收进入循环系统，约在0.38 h达峰浓度。然而，生物利用度相对偏低，可能与首过效应和给药后在体内发生强烈代谢相关。

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 (t_1/2) 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 low with a value being 10.3 ± 2.15% and t_1/2 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.