Gan Lu, Zhang Zhengyu, Chen Jingtao, Shen Zhichun, Chen Wujie, Chen Shaoxin, Li Jiyang
Department of Biological Medicines & Shanghai Engineering Research Center of Immunotherapeutics, School of Pharmacy, Fudan University, Shanghai, 201203, China.
National Key Laboratory of Lead Druggability Research, China State Institute of Pharmaceutical Industry, Shanghai Institute of Pharmaceutical Industry, Shanghai, 201203, China.
Microb Cell Fact. 2025 May 8;24(1):102. doi: 10.1186/s12934-025-02724-x.
Spinosad, a secondary metabolite produced by Saccharopolyspora spinosa, is a polyketide macrolide insecticide with low toxicity and environmental friendliness. Owing to the high level of DNA methylation and unclear regulatory mechanisms, gene engineering to increase spinosad production is challenging. Limited improvements in yield have been observed with heterologous expression or partial overexpression of the 74-kb spinosyn gene cluster (spn), and research on the overexpression of the complete spinosyn gene cluster is lacking.
The plasmid pCM265-spn was constructed using CRISPR/Cas9-mediated Transformation-Associated Recombination cloning to enable the overexpression of the complete spn gene cluster in Sa. spinosa. The engineered strain Sa. spinosa-spn achieved a 124% increase in spinosad yield (693 mg/L) compared to the wild type (309 mg/L). The overexpression of the spn gene cluster also delayed spore formation and reduced hyphal compartmentalization by influencing the transcription of related genes (bldD, ssgA, whiA, whiB, and fstZ). Transcriptional analysis revealed significant upregulation of genes in the spn gene cluster, thereby enhancing secondary metabolism. Additionally, optimization of the fermentation medium through response surface methodology further increased spinosad production to 920 mg/L.
This study is the first to successfully overexpress the complete spn gene cluster in Sa. spinosa, significantly enhancing spinosad production. These findings have significance for further optimization of spinosad biosynthesis.
多杀菌素是由刺糖多孢菌产生的一种次生代谢产物，是一种低毒且环境友好的聚酮类大环内酯杀虫剂。由于DNA甲基化水平高且调控机制不明，通过基因工程提高多杀菌素产量具有挑战性。通过异源表达或部分过表达74 kb的多杀菌素基因簇（spn），产量提升有限，且缺乏对完整多杀菌素基因簇过表达的研究。
利用CRISPR/Cas9介导的转化相关重组克隆构建了质粒pCM265-spn，以实现完整spn基因簇在刺糖多孢菌中的过表达。工程菌株刺糖多孢菌-spn与野生型（309 mg/L）相比，多杀菌素产量提高了124%（693 mg/L）。spn基因簇的过表达还通过影响相关基因（bldD、ssgA、whiA、whiB和fstZ）的转录延迟了孢子形成并减少了菌丝分隔。转录分析显示spn基因簇中的基因显著上调，从而增强了次生代谢。此外，通过响应面法优化发酵培养基进一步将多杀菌素产量提高到920 mg/L。
本研究首次成功在刺糖多孢菌中过表达完整的spn基因簇，显著提高了多杀菌素产量。这些发现对进一步优化多杀菌素生物合成具有重要意义。
