Abstract
BackgroundThe soil bacterium Pseudomonas putida KT2440 is a “generally recognized as safe”-certified strain with robust property and versatile metabolism. Thus, it is an ideal candidate for synthetic biology, biodegradation, and other biotechnology applications. The known genome editing approaches of Pseudomonas are suboptimal; thus, it is necessary to develop a high efficiency genome editing tool.ResultsIn this study, we established a fast and convenient CRISPR–Cas9 method in P. putida KT2440. Gene deletion, gene insertion and gene replacement could be achieved within 5 days, and the mutation efficiency reached > 70%. Single nucleotide replacement could be realized, overcoming the limitations of protospacer adjacent motif sequences. We also applied nuclease-deficient Cas9 binding at three locations upstream of enhanced green fluorescent protein (eGFP) for transcriptional inhibition, and the expression intensity of eGFP reduced to 28.5, 29.4, and 72.1% of the control level, respectively. Furthermore, based on this CRISPR–Cas9 system, we also constructed a CRISPR–Cpf1 system, which we validated for genome editing in P. putida KT2440.ConclusionsIn this research, we established CRISPR based genome editing and regulation control systems in P. putida KT2440. These fast and efficient approaches will greatly facilitate the application of P. putida KT2440.
Highlights
The soil bacterium Pseudomonas putida KT2440 is a “generally recognized as safe”-certified strain with robust property and versatile metabolism
Establishment of a two‐plasmid Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–Cas9 system in P. putida KT2440 Various CRISPR/Cas9 systems have been established in several common microorganisms
The E. coli CRISPR/Cas9 version 2.0 system developed by Jiang is a typical two-plasmid system that consists of several common inducible expression systems and antibiotics markers used in Gram-negative bacteria
Summary
The soil bacterium Pseudomonas putida KT2440 is a “generally recognized as safe”-certified strain with robust property and versatile metabolism. It is an ideal candidate for synthetic biology, biodegradation, and other biotechnology applications. Based on homologous recombination in double-stranded breaks (DSB), the I-SceI homing endonuclease has been developed as a seamless genome editing tool in P. putida KT2440 [14]. In another chromosomal engineering approach used in Pseudomonas, transposon vectors-based methods [15,16,17] have excellent transposition frequencies. These various strategies have been applied into genome engineering of Pseudomonas, they still have many drawbacks, such as time-consuming manipulation, scars left
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