Pseudomonas putida KT2440 markerless gene deletion using a combination of λ Red recombineering and Cre/loxP site-specific recombination

FEMS Microbiology Letters
Xi LuoGuangdong Shang

Abstract

Pseudomonas putida KT2440 is a saprophytic, environmental microorganism that plays important roles in the biodegradation of environmental toxic compounds and production of polymers, chemicals and secondary metabolites. Gene deletion of KT2440 usually involves cloning of the flanking homologous fragments of the gene of interest into a suicide vector followed by transferring into KT2440 via triparental conjugation. Selection and counterselection steps are then employed to generate gene deletion mutant. However, these methods are tedious and are not suitable for the manipulation of multiple genes simultaneously. Herein, a two-step, markerless gene deletion method is presented. First, homologous armsflanked loxP-neo-loxP was knocked-in to replace the gene of interest, then the kanamycin resistance marker is removed by Cre recombinase catalyzed site-specific recombination. Both two-plasmid and one-plasmid gene systems were established. MekR/PmekA regulated gene expression system was found to be suitable for tight Cre expression in one-plasmid deletion system. The straightforward, time saving and highly efficient markerless gene deletion strategy has the potential to facilitate the genetics and functional genomics study of P. putida ...Continue Reading

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Citations

Oct 22, 2016·FEMS Microbiology Letters·Zhongqiu ChenGuangdong Shang
Jan 5, 2018·Journal of Industrial Microbiology & Biotechnology·Taylor B CookBrian F Pfleger
Feb 15, 2019·Applied Microbiology and Biotechnology·Zhixin WuGuangdong Shang
Feb 23, 2019·Microbial Biotechnology·Seong Keun KimSeung-Goo Lee
Nov 3, 2017·Microbial Biotechnology·Deirdre E RicaurteTomás Aparicio
Aug 28, 2020·Frontiers in Bioengineering and Biotechnology·Jun SunJian-Ping Wu
Oct 22, 2020·Nature Communications·Eveline-Marie LammensRob Lavigne
Nov 17, 2019·Enzyme and Microbial Technology·Jeongmo YangByung-Keun Oh
Jan 27, 2021·Biotechnology for Biofuels·Lauren A Riley, Adam M Guss
Jun 5, 2020·Current Opinion in Biotechnology·Anita Loeschcke, Stephan Thies
Apr 1, 2021·Biotechnology Advances·Maria Martin-PascualVitor A P Martins Dos Santos

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