Zhu Fayin, Zhong Xiaofang, Hu Mengzhu, Lu Lei, Deng Zixin, Liu Tiangang
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, 430071, PR China.
Biotechnol Bioeng. 2014 Jul;111(7):1396-405. doi: 10.1002/bit.25198. Epub 2014 Feb 17.
Approaches using metabolic engineering and synthetic biology to overproduce terpenoids, such as the precursors of taxol and artemisinin, in microbial systems have achieved initial success. However, due to the lack of steady-state kinetic information and incomplete understanding of the terpenoid biosynthetic pathway, it has been difficult to build a highly efficient, universal system. Here, we reconstituted the mevalonate pathway to produce farnesene (a precursor of new jet fuel) in vitro using purified protein components. The information from this in vitro reconstituted system guided us to rationally optimize farnesene production in E. coli by quantitatively overexpressing each component. Targeted proteomic assays and intermediate assays were used to determine the metabolic status of each mutant. Through targeted engineering, farnesene production could be increased predictably step by step, up to 1.1 g/L (∼ 2,000 fold) 96 h after induction at the shake-flask scale. The strategy developed to release the potential of the mevalonate pathway for terpenoid overproduction should also work in other multistep synthetic pathways.
利用代谢工程和合成生物学方法在微生物系统中过量生产萜类化合物（如紫杉醇和青蒿素的前体）已取得初步成功。然而，由于缺乏稳态动力学信息以及对萜类生物合成途径的理解不完整，构建一个高效、通用的系统一直很困难。在此，我们使用纯化的蛋白质组分在体外重建了甲羟戊酸途径以生产法尼烯（一种新型喷气燃料的前体）。来自这个体外重建系统的信息指导我们通过定量过表达每个组分来合理优化大肠杆菌中法尼烯的生产。使用靶向蛋白质组学分析和中间产物分析来确定每个突变体的代谢状态。通过靶向工程，法尼烯产量可逐步可预测地增加，在摇瓶规模下诱导96小时后可达1.1 g/L（约2000倍）。为释放甲羟戊酸途径在萜类化合物过量生产方面的潜力而开发的策略也应适用于其他多步合成途径。
