带基因检测电路的大肠杆菌异源合成5-羟基色氨酸的组合转录工程研究

项目名称： 带基因检测电路的大肠杆菌异源合成5-羟基色氨酸的组合转录工程研究

项目编号： No.21506185

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 有机化学

项目作者： 瞿亮

作者单位： 浙江大学

项目金额： 21万元

中文摘要： 在大肠杆菌中构建一条由人色氨酸羟化酶、多个辅酶BH4合成和再生途径关键酶组成的异源合成5-羟基色氨酸的新代谢途径。引入非天然氨基酸密码子拓展策略，利用正交5-OH-TrpRS/tRNA对能够特异性识别5-羟基色氨酸的特性，构建基因电路将5-羟基色氨酸含量与抗性报告基因（氯霉素抗性基因）表达相关联，建立高通量检测新方法。从组合转录突变优化角度，构建大肠杆菌中多分支合成途径不同表达强度的组合突变文库，结合高通量筛选，获得5-羟基色氨酸的高产菌株。最后利用荧光定量PCR等技术对多株高产菌进行系统分析，阐明多途径表达强度的组合优化对5-羟基色氨酸合成的影响规律。这一研究不仅能够进化获得高产5-羟基色氨酸的菌株，而且有可能建立一个具有普遍意义的优化平衡微生物多分支代谢流的组合创新策略。

中文关键词： 5-羟基色氨酸；组合转录工程；高通量筛选；基因检测电路；密码子拓展

英文摘要： A new metabolic pathway comprised with tryptophan hydroxylase and key enzymes associated with Tetrahydrobiopterin (BH4) synthesis and recycle pathways will be construted in E coli toenable the heterologous biosynthesis of 5-OH-Trp(5HTP). The unnatural amino acid 5HTP could be genetically encoded into protein at the rare stop codon by orthogonal 5HTP-aminoacyl synthetase/tRNA pair. Thus a genetic detection circuit could be constructed to correlate the content of 5HTP with the expression of resistance gene, and finally establish a novel high-throughput screening approach for live cells. The expression levels of multiple pathways in E coli will be regulated by PCR-generated promoters to obtain the transcriptional mutation library, and the 5HTP high producing strains will be screened from such library via the established high-throughput approach. The systematic analyses of these strains via Real-time PCR could illustrate the possible effects of combinatorial optimization of multiple pathway-expression levels on the production of 5HTP. Our reseach will generate the 5HTP high producing strains via pathway evolution, and the established combinatorial optimization strategy could be generally applied to achieve the flux balance of multiple pathways in microorganisms.

英文关键词： 5-hydroxytryptophan;combinatorial transcriptional engineering;high through screening; genetic detection circuit;5HTP-aminoacyl synthetase/tRNA pair