构建基于染色体表达人工合成途径的萜类化合物高产工程菌

Synthetic biology provides an alternative to traditional methods that are concentrated on extraction of bioactive compounds from their natural source or on chemical synthesis. Terpenoids are an example of a class of natural compounds that are of particular interest for microbial production, such as artemisinin, paclitaxel and carotenoids. Escherichia coli is one of the most common chassis host used in the field of synthetic biology. Heterologous biosynthetic pathways are generally brought into the host through one or more engineered plasmids. While proving extraordinarily helpful, problems with plasmid capacity and stability often arise. It is advantageous to incorporate the heterologous synthetic pathways into the chromosome as it is the most stable vector and has more than 1 Mb capacity. In this study, synthetic pathways were integrated into the E. coli chromosome by a markless, sequence-mutationless and locus-targeted integration system based on Red homologous recombination and I-SceI endonuclease. The artificial synthetic pathways were controlled by the orthogonal transcription system for producting amorphadiene, the precursor of artemisinin. When the biosynthetic pathways are integrated into the chromosome by this recombination system the production level of amorphadiene was found to be higher than that of in the plasmid vector. The effect of insertion sites and the direction on the expression of the heterologous pathways will be thoroughly investigated in the study. To further increasing production of amorphadiene, key-genes' expression was regulated by othorgonal T7 promoters and SD sites of different strength. The transcription level of genes in the terpenoids pathway will be measured by quantitative RT-PCR, and their protein levels will be analyzed by targeted proteomics via selected-reaction monitoring mass spectrometry, and the difference of expression levels of host central metabolic pathways between the two conditions will be evaluated by genechips, and the genetic stability of heterologous pathways in two conditions will be checked by replica-plating method and PFGE, and the products will be quantified by GC-MS. The results will be helpful to biosynthesis of other natural compounds.

萜类化合物如青蒿素、紫杉醇、类胡萝卜素等具有重要药用价值，其生物合成途径涉及多个基因。因天然来源稀少，利用合成生物学技术实现其高效生产极具应用前景。大肠杆菌是常用细胞工厂之一，前人研究主要采用质粒载体引入合成途径，由于质粒不稳定性导致合成途径不稳定，难以在商业规模实现目标产物合成。染色体是细胞内最稳定的载体，本课题以青蒿素前体amorphadiene（AD）为目标产物，构建基于正交转录体系的人工合成途径，利用自主建立的无痕-无突变-定点整合技术将整个合成途径移植到染色体上，研究染色体整合表达异源合成途径工程菌的遗传稳定性；同时应用定量PCR技术、基因芯片分析技术、蛋白质组定量分析技术、GC-MS定量分析技术，研究整合位点、整合方向及其表达调控方法等对合成途径基因表达和产物合成的影响，确定最佳整合表达策略，最终实现AD高效稳定合成。本研究结果对其他天然化合物的生物合成研究具有指导意义。

萜类化合物家族包括了超过5万种物质，其中的代表如青蒿素、番茄红素和蒎烯等在药品、食品、保健品和化妆品等行业具有重要应用价值。因天然来源稀少，利用合成生物学技术实现其高效生产极具应用前景。在生物体内，所有的萜类化合物均来源于甲羟戊酸（MVA）途径或5-磷酸脱氧木酮糖（DXP）途径。大肠杆菌是常用细胞工厂之一，前人研究主要采用质粒载体引入异源MVA途径，由于质粒不稳定性导致合成途径不稳定，并且质粒需要抗生素维持，难以在商业规模实现目标产物合成。大肠杆菌染色体是其最稳定的载体，本项目以番茄红素（Lycopene）目标产物，利用自主建立的无痕-无突变-定点整合技术，结合代谢通路在基因组的整合位点及方向，首次构建了染色体整合型高水平稳定合成番茄红素工程菌。利用该工程菌菌株，在试管中番茄红素产量为390 mg/L，在5 L发酵罐将产量提高到1.3 g/L。本研究结果对其他天然化合物的生物合成研究具有指导意义。