Improving spinosad production by tuning expressions of the forosamine methyltransferase and the forosaminyl transferase to reduce undesired less active byproducts in the heterologous host Streptomyces albus J1074

Microbial Cell Factories

Research

Jun Fu¹  Xiaochen Li¹  Ruofei Guo¹  Hailong Wang¹  Youming Zhang¹  Ji Luan¹ 

[1]State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Helmholtz International Lab for Anti-infectives, Shandong University–Helmholtz Institute of Biotechnology, Shandong University, Binhai Rd 72, 266237, Qingdao, Shandong, China

关键词: Spinosad;    Forosamine;    N,N;    Forosaminyl transferase;    Biosynthesis;    Heterologous expression;   

DOI  :  10.1186/s12934-023-02023-3

 received in 2022-11-07, accepted in 2023-01-12,  发布年份 2023

来源: Springer

【 摘 要 】

BackgroundSpinosad is a macrolide insecticide with the tetracyclic lactone backbone to which forosamine and tri-o-methylrhamnose are attached. Both the sugar moieties are essential for its insecticidal activity. In biosynthesis of spinosad, the amino group of forosamine is dimethylated by SpnS and then transferred onto the lactone backbone by SpnP. Because the spinosad native producer is difficult to genetically manipulate, we previously changed promoters, ribosome binding sites and start codons of 23 spinosad biosynthetic genes to construct an artificial gene cluster which resulted in a 328-fold yield improvement in the heterologous host Streptomyces albus J1074 compared with the native gene cluster. However, in fermentation of J1074 with the artificial gene cluster, the N-monodesmethyl spinosad with lower insecticidal activity was always produced with the same titer as spinosad.ResultsBy tuning expression of SpnS with an inducible promotor, we found that the undesired less active byproduct N-monodesmethyl spinosad was produced when SpnS was expressed at low level. Although N-monodesmethyl spinosad can be almost fully eliminated with high SpnS expression level, the titer of desired product spinosad was only increased by less than 38%. When the forosaminyl transferase SpnP was further overexpressed together with SpnS, the titer of spinosad was improved by 5.3 folds and the content of N-desmethyl derivatives was decreased by ~ 90%.ConclusionN-monodesmethyl spinosad was produced due to unbalanced expression of spnS and upstream biosynthetic genes in the refactored artificial gene cluster. The accumulated N-desmethyl forosamine was transferred onto the lactone backbone by SpnP. This study suggested that balanced expression of biosynthetic genes should be considered in the refactoring strategy to avoid accumulation of undesired intermediates or analogues which may affect optimal production of desired compounds.

【 授权许可】

CC BY   
© The Author(s) 2023

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