功能模块的集成与测试研究

摘 要：谷氨酸棒杆菌作为棒杆菌中的模式生物，拥有多条完整的芳香化合物代谢途径，全基因组测序的完成为在谷氨酸棒杆菌中进行代谢调控研究提供了良好的生物信息学平台；该菌包括基因敲除及互补在内的遗传操作系统非常成熟，是研究芳香化合物代谢调控机制的良好模型。该研究旨在利用棒杆菌等模式生物中的莽草酸合成及芳香化合物代谢相关元件进行元件适配性研究，同时结合生物信息学、分子生物学及生物化学方法发掘其他微生物中这两类元件，并对元件进行表征、改造及标准化并建立元件库；选取高效能的功能元件拼接组装为功能模块，并在棒杆菌等底盘细胞中进行检测适配性，从而构建出高效合成以莽草酸为代表性芳香化合物的人工细胞。到目前为止，研究工作完成了谷氨酸棒杆菌莽草酸合成途径酶元件的鉴定，重点完成了谷棒DAHP合酶和分支酸异构酶功能表征及元件间适配性关系，获得大量潜在的莽草酸合成相关代谢元件；并对部分代谢元件进行功能验证和表征；同时建立高效蛋白表达及酶活测定体系。鉴定了谷棒莽草酸途径的7个酶以及分支酸异构酶，完成了谷棒DAHP合酶、分支酸异构酶、脱氢奎尼酸脱水酶以及莽草酸激酶功能表征，揭示了DAHP合酶和分支酸异构酶相互作用机理和调控关系。获得了3 549个莽草酸途径相关基因序列，确定了516个合成目标基因，完成了这些基因密码子优化和基因序列重新设计，选取了37个脱氢奎尼酸脱水酶基因人工合成，构建标准元件库，并表征了它们的酶促动力学参数。构建并验证了快速高通量的筛选—优化—合成—表征莽草酸途径元件库的Pipeline。另外在调控元件库构建方面，构建了包括RBS、Promoter、Terminator以及Insulator等4類共226个元件的调控元件库，为莽草酸通路模块的优化和精细调控的奠定了基础。 模块的组装和优化方面，构建了基于RiboJ Insulator的基因表达定量调控模型，合成了莽草酸本地途径酶元件和调控元件元件进行模块组装，并在底盘细胞谷氨酸棒杆菌中实现了表达，对最优配比进行了初步筛选，将莽草酸产量提高了7倍。

关键词：莽草酸 莽草酸途径 谷氨酸棒杆菌 合成生物学 元件库

Abstract： Corynebacterium glutamicum as a type strain has a number of complete metabolic pathways of aromatic compounds. The completion of whole genome sequencing provides a good bioinformatic platform for metabolic and regulatory study of cells. Besides， the genetic manipulation systems （including knockout and complementary systems） are very mature， which makes this strain a perfect model to study the metabolic and regulatory mechanisms of aromatic compounds.With techniques of bioinformatics， molecular biology and biochemistry， more devices with similar functions are explored from all other microorganisms. And device libraries are subsequently established after characterization， modification and standardization of these devices. High-performance functional devices are selected to assemble modules which are then transferred into chassis cells for suitability test. After optimization of the suitability， artificially synthesized cell can provide a much more efficient synthesis of shikimic acid -a representation of aromatic compounds- than the wild type strain do. So for， all enzymatic devices involved in shikimic acid synthetic pathway have been identified， and a lot of potential function devices that may related to shikimic acid synthesis have been achieved. In total， 3549 gene sequences that are relative to shikimate pathway are identified and 516 of them are verified to be target genes for shikimic acid synthesis. After codon optimization and sequence redesign， 37 dehydrogenation quinic acid dehydratase genes are selected to be synthesis chemically， and these synthetic devices are used for characterization of their enzymatic kinetic parameters. By then， a highly efficient pipeline for construction of device library with a high-throughput “Screening—Optimization—Synthesis—Characterization” process is built. In terms of regulatory devices， a library is constructed with 226 regulatory devices， including RBS， Promoter， Terminator and Insulator.， which provide a steady foundation for optimization and accurate regulation of shikimic acid pathway modules. Based on a quantitative model named RiboJ Insulator for regulation of gene expression， the local enzymes in shikimic acid pathway are assembled with regulatory devices from the previous library in chassis cells Corynebacterium glutamicum. And the production of shikimic acid is increased by 7 times comparing with the wild type ones.

Key Words： Shikimic acid； Shikimic acid pathway； Corynebacterium glutamicum； Synthetic biology； Device library

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