功能模塊的集成與測試研究

劉雙江　劉一鳴　劉暢　張博

摘 要：谷氨酸棒桿菌作為棒桿菌中的模式生物，擁有多條完整的芳香化合物代謝途徑，全基因組測序的完成為在谷氨酸棒桿菌中進行代謝調控研究提供了良好的生物信息學平臺；該菌包括基因敲除及互補在內的遺傳操作系統非常成熟，是研究芳香化合物代謝調控機制的良好模型。該研究旨在利用棒桿菌等模式生物中的莽草酸合成及芳香化合物代謝相關元件進行元件適配性研究，同時結合生物信息學、分子生物學及生物化學方法發掘其他微生物中這兩類元件，并對元件進行表征、改造及標準化并建立元件庫；選取高效能的功能元件拼接組裝為功能模塊，并在棒桿菌等底盤細胞中進行檢測適配性，從而構建出高效合成以莽草酸為代表性芳香化合物的人工細胞。到目前為止，研究工作完成了谷氨酸棒桿菌莽草酸合成途徑酶元件的鑒定，重點完成了谷棒DAHP合酶和分支酸異構酶功能表征及元件間適配性關系，獲得大量潛在的莽草酸合成相關代謝元件；并對部分代謝元件進行功能驗證和表征；同時建立高效蛋白表達及酶活測定體系。鑒定了谷棒莽草酸途徑的7個酶以及分支酸異構酶，完成了谷棒DAHP合酶、分支酸異構酶、脫氫奎尼酸脫水酶以及莽草酸激酶功能表征，揭示了DAHP合酶和分支酸異構酶相互作用機理和調控關系。獲得了3 549個莽草酸途徑相關基因序列，確定了516個合成目標基因，完成了這些基因密碼子優化和基因序列重新設計，選取了37個脫氫奎尼酸脫水酶基因人工合成，構建標準元件庫，并表征了它們的酶促動力學參數。構建并驗證了快速高通量的篩選—優化—合成—表征莽草酸途徑元件庫的Pipeline。另外在調控元件庫構建方面，構建了包括RBS、Promoter、Terminator以及Insulator等4類共226個元件的調控元件庫，為莽草酸通路模塊的優化和精細調控的奠定了基礎。 模塊的組裝和優化方面，構建了基于RiboJ Insulator的基因表達定量調控模型，合成了莽草酸本地途徑酶元件和調控元件元件進行模塊組裝，并在底盤細胞谷氨酸棒桿菌中實現了表達，對最優配比進行了初步篩選，將莽草酸產量提高了7倍。

關鍵詞：莽草酸 莽草酸途徑 谷氨酸棒桿菌 合成生物學 元件庫

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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