抗癌药物榄香烯生物合成关键倍半萜合酶基因的克隆、改造和功能研究

Elemene is an important anti-tumor drug with proprietary intellectual property rights in China. It has advantages of strong anti-tumor activity, broad anti-tumor spectrum, slightly side effect and low drug resistance. However, It`s different, costly and low to extract of elemene from stems of Curcuma wenyujin. To study on gene function involved in elemene biosynthetic pathway is a prerequisite for its biosynthesis. Based on the results of the differential expressions at the level of transcriptome in Curcuma wenyujin, the ten unigenes were preliminarily presumed to be positive sesterpene synthases genes. The sts genes was amplified by PCR and RACE technology and then expressed in E. coli. The aim is to obtain the STS with high efficiency and fewer by-products by analysis of the kinds and rates of products. Further, the conservative structure motif in STS were knocked out and replaced by site-directed mutagenesis. Plastic amino acids around the active pocket were analyzed and designed based on mathematical model. Mutants were constructed and the catalytic properties were analyzed. Finally, the catalytic efficiency and product specificity of the STS will be improved, and the synthesized efficiency and purity of elemene catalyzed by STS mutants will be promoted. The results will illuminate the mechanism of the key step for elemene biosynthesis, reveal the effects of the conservative structure motif and plastic amino acids around the active pocket on catalytic properties of STS, and build a foundation for elemene biosynthesis.

榄香烯是我国拥有自主知识产权的抗肿瘤药物，具有抗肿瘤活性强、作用范围广、毒副作用轻微、不易产生耐药性的优点，其主要来源于药用植物温郁金块根，含量较低，使得目前提取生产方式难度大、得率低、成本高。开展榄香烯生物合成途径的基因功能研究是进行榄香烯生物合成制备的必要前提。本项目提出基于温郁金转录组组织差异表达的分析结果，针对筛选到的十个在块根中表达水平较高的倍半萜合酶基因，分析其催化性质和酶学性质，筛选催化效率高、副产物少的榄香烯合成酶基因。进而，通过定点突变技术对榄香烯合成酶的保守结构模块进行敲除和替换，利用数学模型对活性口袋周围的可变氨基酸进行理性设计，实验筛选得到催化效率和产物专一性均提高的突变酶，从而提高榄香烯的生物合成产率和纯度。本项目将从分子水平上阐明榄香烯关键合成步骤的机理，揭示保守模块和活性口袋周围的可变氨基酸对催化性质的影响规律，为榄香烯的生物合成制备奠定基础。

榄香烯是我国拥有自主知识产权的抗肿瘤药物，具有抗肿瘤活性强、作用范围广、毒副作用轻微、不易产生抗药性的优点，本项目针对榄香烯天然提取生产方式成本高、得率低、产品纯度不够高的缺点，采用分子生物学，合成生物学等技术，获得榄香烯生物合成途径上的关键酶，并对其进行理性设计和改造，获得催化性质改进的突变酶，并阐明酶催化机制。本研究基于榄香烯来源植物温郁金转录组差异表达的分析结果，筛选到一条萜类合酶（CwTPS），在大肠杆菌中重组表达鉴定其可催化底物FPP生成榄香烯。并构建了温郁金萜类合酶突变株D364A、K332R、K332G、R327K，确认K332为活性位点，R327的突变会导致酶活性降低。基于Genbank和发表的文献，克隆并鉴定了莴苣来源的LsGAS1（LTC1），并设计了四种突变位点，构建突变酶，获得活性增加了13倍的突变酶LsGAS1-R273G，以及增加了3倍的突变酶LsGAS1-T412S。构建的LsGAS1-Y530F突变株，以及删除第二个天冬氨酸富集的 DDXXD 基序的LsGAS1-DE突变株，活性显著降低，确定了影响LsGAS催化活性的关键位点。进一步，克隆了莴苣来源的LsGAS2（LTC2），针对酶与底物结合的阶段起关键作用的是第273位氨基酸R，而在产物释放阶段起关键作用的是第410位氨基酸T，设计突变位点，并进行重组表达和酶活分析，筛选到LsGAS2-R273G和LsGAS2-T410S两种突变酶，野生型及突变后两个酶基因针对底物FPP的表观Kcat／Km分别为2.14s-1mM-1、8.45s-1mM-1和9.93 s-1mM-1，表明突变酶获得了更高的催化效率，催化底物FPP生成β-榄香烯的产量与野生株相比，分别提高了2.15和 2.9倍。进而构建了双突变株LsGAS2-R273G-T410S，催化底物FPP生成β-榄香烯的产量与野生株相比，提高了5.9倍。本项目从分子水平上阐明榄香烯生物合成途径上的相关酶，并利用计算机辅助分子建模技术能够以提高酶的催化活性为目的进行定向改造，在定向改造后有效的提高了目标产物的产量，为榄香烯的生物合成制备奠定基础。