Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing, 210023, Jiangsu Province, China.
Appl Microbiol Biotechnol. 2019 Mar;103(6):2783-2795. doi: 10.1007/s00253-019-09654-w. Epub 2019 Feb 14.
Pseudomonas putida KT2440 is a Gram-negative, biosafety strain that plays important roles in environmental and biotechnological applications. Highly efficient genome editing strategy is essential to the elucidation of gene function and construction of metabolic engineered strains. Building on our previously established recombineering-mediated markerless and scarless P. putida KT2440 chromosomal gene deletion methods, herein we combined single-stranded DNA (ssDNA) recombineering and CRISPR-Cas9 technologies for P. putida KT2440 genome editing. Firstly, an inactive kanamycin resistance gene was knocked into the P. putida KT2440 chromosome. Then, based on kanamycin selection, recombinase gene selection, ssDNA recombineering condition optimization, and gRNA expression promoter selection were performed. A two-plasmid genome editing system was established; the first is a broad host range, RK2 replicon-based plasmid cloned with the tightly regulated redβ and cas9 genes; the second is a broad host range, pBBR1 replicon-based, sgRNA expression plasmid. Gene point mutations and gene deletions were carried out; the genome editing efficiency is as high as 100%. The method will expedite the P. putida KT2440 metabolic engineering and synthetic biology studies.
恶臭假单胞菌 KT2440 是一种革兰氏阴性、生物安全菌株，在环境和生物技术应用中发挥着重要作用。高效的基因组编辑策略对于阐明基因功能和构建代谢工程菌株至关重要。基于我们之前建立的重组介导的无痕和无疤恶臭假单胞菌 KT2440 染色体基因缺失方法，本文将单链 DNA（ssDNA）重组和 CRISPR-Cas9 技术结合用于恶臭假单胞菌 KT2440 的基因组编辑。首先，将一个失活的卡那霉素抗性基因敲入恶臭假单胞菌 KT2440 染色体中。然后，基于卡那霉素选择、重组酶基因选择、ssDNA 重组条件优化和 gRNA 表达启动子选择进行操作。建立了一个双质粒基因组编辑系统；第一个是基于 RK2 复制子的广泛宿主范围质粒，克隆了受严格调控的 redβ 和 cas9 基因；第二个是基于 pBBR1 复制子的广泛宿主范围 sgRNA 表达质粒。进行了基因点突变和基因缺失；基因组编辑效率高达 100%。该方法将加速恶臭假单胞菌 KT2440 的代谢工程和合成生物学研究。
