Abstract：
The isopentenol utilization pathway (IUP) is potential in terpenoids synthesis. This study aimed to construct IUP-employed Escherichia coli chassis for stably synthesizing terpenoids. As to effectiveness, promotor engineering strategy was employed to regulate IUP expression level, while ribosome-binding site (RBS) library of the key enzyme was constructed for screening the optimal RBS, followed by optimization of concentration of inducer and substrates, the titer of reporting production, lycopene, from 0.087 to 8.67 mg OD600 -1 . As about stability, the IUP expression cassette was integrated into the genome through transposition tool based on CRISPR-associated transposases. Results showed that the strain with 13 copies produced 1.78-fold lycopene titer that of the controlled strain with IUP-harbored plasmid, and it exhibited stable expression after ten successions while the plasmid loss was observed in the controlled strain in the 3rd succession. This strategy provides valuable information for rapid construction of highly effective and stable chassis employing IUP for terpenoids production.
摘要：
异戊烯醇利用途径(IUP)在萜类化合物合成中具有潜力。本研究旨在构建IUP应用的大肠杆菌底盘，以稳定合成萜类化合物。至于有效性，采用启动子工程策略调节IUP表达水平，在构建关键酶核糖体结合位点(RBS)文库的同时,其次是诱导物和底物浓度的优化，报告产量的滴度，番茄红素,从0.087到8.67mg/OD600。至于稳定性，IUP表达盒通过基于CRISPR相关转座酶的转座工具整合到基因组中.结果表明，具有13个拷贝的菌株产生的番茄红素滴度是具有IUP携带质粒的对照菌株的1.78倍，在10次连续后表现出稳定的表达，而在第3次连续的对照菌株中观察到质粒丢失。该策略为使用IUP进行萜类化合物生产的高效和稳定的底盘的快速构建提供了有价值的信息。本文受版权保护。保留所有权利。
