基于恶臭假单胞菌F1的条件性自杀底盘细胞构建

Construction of a chassis cell with conditional suicide from Pseudomonas putida F1

摘要:

【背景】恶臭假单胞菌(Pseudomonas putida) F1营养需求简单、生长迅速以及多样的代谢途径，被认为是合成生物学领域中最有潜力的底盘细胞之一。然而，将P.putidaF1的基因组进行精简构建生物降解的底盘细胞的研究鲜有报道。【目的】构建生物安全性较高的底盘细胞。【方法】利用基于同源重组原理的无痕基因编辑技术对P.putida F1基因组进行精简，并将条件性自杀系统整合到基因组中。【结果】成功敲除了占基因组大小1.83%的2个溶源噬菌体基因组岛PP1 (50.7 kb)和PP2 (58.4 kb)，构建了敲除菌株P.putida F1ΔPP12，进一步将由阿拉伯糖启动子控制的条件性自杀系统整合到P. putida F1ΔPP12基因组中的upp基因位点，构建了生物安全性更高的底盘细胞P.putida F1ΔPP12-P_ara-nuc。【结论】提高了P.putida F1的现实应用价值，并为使用其或以其为底盘细胞进一步改造的基因工程菌进行环境原位修复的可能性提供了理论依据。

Abstract:

[Background] Pseudomonas putidaF1 with simple nutritional requirements, rapid growth, and diverse metabolic pathways is regarded as one of the most promising chassis cells in synthetic biology. However, few studies have reported the application of P. putida F1 as a biodegradable chassis cell by streamlining the genome. [Objective] To construct a chassis cell with high biosafety. [Methods] We employed scarless genome editing based on homologous recombination principle to streamline the genome of P. putida F1 and integrated the conditional suicide system into the genome. [Results] Two lysogenic phage genomic islands PP1 (50.7 kb) and PP2 (58.4 kb), which account for 1.83% of the genome size, were successfully knocked out, and a knockout strain P. putida F1ΔPP12 was constructed. Furthermore, the conditional suicide system controlled by the arabinose promoter was integrated into the site of upp in the genome of P. putida F1ΔPP12, and thus a chassis cell P.putida F1ΔPP12-P_ara-nuc with high biosafety was successfully constructed. [Conclusion] This study increases the application value of P.putida F1 and provides a theoretical basis for the application of this strain or genetically engineered bacteria from this strain in environment remediation in situ.