Song Chaoyi, Luan Ji, Cui Qingwen, Duan Qiuyue, Li Zhen, Gao Yunsheng, Li Ruijuan, Li Aiying, Shen Yuemao, Li Yuezhong, Stewart A Francis, Zhang Youming, Fu Jun, Wang Hailong
Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science , Shandong University , Binhai Road 72 , 266237 Qingdao , People's Republic of China.
Genomics, Biotechnology Center , Technische Universität Dresden , Tatzberg 47-51 , Dresden 01307 , Germany.
ACS Synth Biol. 2019 Jan 18;8(1):137-147. doi: 10.1021/acssynbio.8b00402. Epub 2019 Jan 9.
Refactoring biosynthetic pathways for enhanced secondary metabolite production is a central challenge for synthetic biology. Here we applied advanced DNA assembly methods and a uniform overexpression logic using constitutive promoters to achieve efficient heterologous production of the complex insecticidal macrolide spinosad. We constructed a 79-kb artificial gene cluster in which 23 biosynthetic genes were grouped into 7 operons, each with a strong constitutive promoter. Compared with the original gene cluster, the artificial gene cluster resulted in a 328-fold enhanced spinosad production in Streptomyces albus J1074. To achieve this goal, we applied the ExoCET DNA assembly method to build a plasmid from 13 GC-rich fragments with high efficiency in one step. Together with our previous direct cloning and recombineering tools, we present new synthetic biology options for refactoring large gene clusters for diverse applications.
重构生物合成途径以提高次级代谢产物产量是合成生物学面临的核心挑战。在此，我们应用先进的DNA组装方法和使用组成型启动子的统一过表达逻辑，实现了复杂杀虫大环内酯多杀菌素的高效异源生产。我们构建了一个79 kb的人工基因簇，其中23个生物合成基因被分成7个操纵子，每个操纵子都有一个强组成型启动子。与原始基因簇相比，该人工基因簇使白色链霉菌J1074中的多杀菌素产量提高了328倍。为实现这一目标，我们应用ExoCET DNA组装方法，一步高效地从13个富含GC的片段构建了一个质粒。结合我们之前的直接克隆和重组工程工具，我们为重构大型基因簇以用于各种应用提供了新的合成生物学选择。
