Li Ya-Wen, Yang Cai-Ling, Shen Qi, Peng Qian-Qian, Guo Qi, Nie Zhi-Kui, Sun Xiao-Man, Shi Tian-Qiong, Ji Xiao-Jun, Huang He
School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, China.
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, China.
Front Bioeng Biotechnol. 2022 Mar 2;9:816980. doi: 10.3389/fbioe.2021.816980. eCollection 2021.
Non-homologous end-joining (NHEJ)-mediated random integration in has been demonstrated to be an effective strategy for screening hyperproducer strains. However, there was no multigene assembly method applied for NHEJ integration, which made it challenging to construct and integrate metabolic pathways. In this study, a Golden Gate modular cloning system (YALIcloneNHEJ) was established to develop a robust DNA assembly platform in . By optimizing key factors, including the amounts of ligase and the reaction cycles, the assembly efficiency of 4, 7, and 10 fragments reached up to 90, 75, and 50%, respectively. This YALIcloneNHEJ system was subsequently applied for the overproduction of the sesquiterpene (-)-α-bisabolol by constructing a biosynthesis route and enhancing the flux in the mevalonate pathway. The resulting strain produced 4.4 g/L (-)-α-bisabolol, the highest titer reported in yeast to date. Our study expands the toolbox of metabolic engineering and is expected to enable a highly efficient production of various terpenoids.
非同源末端连接（NHEJ）介导的随机整合已被证明是筛选高产菌株的有效策略。然而，尚无适用于NHEJ整合的多基因组装方法，这使得构建和整合代谢途径具有挑战性。在本研究中，建立了一种金门模块化克隆系统（YALIcloneNHEJ），以在[具体物种]中开发一个强大的DNA组装平台。通过优化关键因素，包括连接酶用量和反应循环数，4、7和10个片段的组装效率分别达到了90%、75%和50%。随后，该YALIcloneNHEJ系统通过构建生物合成途径并增强甲羟戊酸途径中的通量，用于倍半萜（-）-α-红没药醇的过量生产。所得菌株产生了4.4 g/L的（-）-α-红没药醇，这是迄今为止酵母中报道的最高滴度。我们的研究扩展了代谢工程的工具箱，有望实现各种萜类化合物的高效生产。
