中药抗肿瘤成分薏苡仁甘油三酯在产油酵母代谢途径中的解析及酵母途径重构的研究

Coix seed enjoys the good reputation of "the king of gramineous plants in the world". The intravenous forms Kanglaite (KLT) injection has better clinical application value for malignant tumors, whose main anti-cancer activity components were ciox seed triglyceride. It would be more controllable and sustainable to produce coix seed triglyceride by constructing microbial “cell factories” through synthetic biology and metabolic engineering than plant material. Based on the above background, Rhodosporidium toruloides was chosen as the start strain to synthesize coix seed triglyceride. Comparing to coix seed triglyceride, the unsaturated fatty acids contents in R. toruloides was lower. The aim of this project is to obtain the two key genes Δ9- and Δ12- fatty acid desaturase (Fatty acid desaturase, FADS) of coix seeds, and explore their functions in R. toruloides, then taking this yeast to be “microbial cell factories” of producing coix seed triglyceride. This study will reconstructe the metabolic pathway of a non-model strain, give a new thought of biosynthesis of the active components from plant materials, and provide the theoretical basis for the research of the metabolic pathway of coix seed triglyceride.

薏苡仁享有“世界禾本科植物之王”的美誉。静脉制剂康莱特注射液对多种恶性肿瘤具有较好疗效，主要抗癌活性成分为薏苡仁甘油三酯。采用合成生物学和代谢工程创制微生物“细胞工厂”较植物源生产薏苡仁甘油三酯更为可控且可持续。基于以上背景，选择产油酵母圆红冬孢酵母（Rhodosporidium toruloides）为出发菌株进行薏苡仁甘油三酯的生物合成。与薏苡仁甘油三酯相比，R. toruloides中不饱和脂肪酸比例较低。本项目拟调取薏苡仁中合成不饱和脂肪酸的两个关键基因Δ9-和Δ12-脂肪酸去饱和酶（Fatty acid desaturase, FADS）并探索其在R. toruloides中的功能，进而将R. toruloides重构成薏苡仁甘油三酯生产的“细胞工厂”。以非模式菌株为出发菌株进行代谢途径重构，为植物源活性成分的生物合成提供了新思路，为薏苡仁甘油三酯代谢途径的研究奠定理论基础。

康莱特注射液对多种恶性肿瘤具有较好疗效，其主要抗癌活性成分为薏苡仁甘油三酯。Δ9-和Δ12-脂肪酸去饱和酶 (Fatty acid desaturase, FADS) 是薏苡仁油生物合成途径的两个关键酶。本项目以薏苡（Coix lacryma-jobi L.）作为研究材料，对不同部位不同时期的薏苡样品进行脂肪酸成分分析，然后进行转录组测序，通过数据分析设计引物，从薏苡的cDNA中克隆获得两个基因，并对其进行生物信息学分析。将获得的序列按照产油酵母圆红冬孢酵母（Rhodosporidium toruloides）的密码子进行优化合成，合成后的片段构建到我们改造后的组成型表达载体上，通过农杆菌介导转化的方法获得酵母工程菌株。最后采用药渣作为发酵底物，摸索不同的发酵条件，对产油酵母进行发酵培养，确定最终的发酵条件。发现，工程菌株亚油酸含量是野生型菌株的2倍左右，表明基因在产油酵母菌株中发挥了功能。本项目表明以合适的非模式菌株为出发菌株进行代谢途径改造，既可以为目的化合物生物合成提供充足的底物，也可以进一步完善其遗传操作体系，为进一步扩大利用范围提供坚实基础。