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Enhanced Heterologous Spinosad Production from a 79-kb Synthetic Multioperon Assembly.

ACS synthetic biology (2018-12-29)
Chaoyi Song, Ji Luan, Qingwen Cui, Qiuyue Duan, Zhen Li, Yunsheng Gao, Ruijuan Li, Aiying Li, Yuemao Shen, Yuezhong Li, A Francis Stewart, Youming Zhang, Jun Fu, Hailong Wang
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
L-Rhamnose, natural sourced, 99%, FG
Supelco
Spinosad, PESTANAL®, analytical standard

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