CRISPR interference-mediated gene regulation in Pseudomonas putida KT2440.

Microbial Biotechnology
Seong Keun KimSeung-Goo Lee

Abstract

Targeted gene regulation is indispensable for reprogramming a cellular network to modulate a microbial phenotype. Here, we adopted the type II CRISPR interference (CRISPRi) system for simple and efficient regulation of target genes in Pseudomonas putida KT2440. A single CRISPRi plasmid was generated to express a nuclease-deficient Cas9 gene and a designed single guide RNA, under control of l-rhamnose-inducible Prha BAD and the constitutive Biobrick J23119 promoter respectively. Two target genes were selected to probe the CRISPRi-mediated gene regulation: exogenous green fluorescent protein on the multicopy plasmid and endogenous glpR on the P. putida KT2440 chromosome, encoding GlpR, a transcriptional regulator that represses expression of the glpFKRD gene cluster for glycerol utilization. The CRISPRi system successfully repressed the two target genes, as evidenced by a reduction in the fluorescence intensity and the lag phase of P. putida KT2440 cell growth on glycerol. Furthermore, CRISPRi-mediated repression of glpR improved both the cell growth and glycerol utilization, resulting in the enhanced production of mevalonate in an engineered P. putida KT2440 harbouring heterologous genes for the mevalonate pathway. CRISPRi is ex...Continue Reading

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Citations

Feb 12, 2020·Microbial Biotechnology·Christos BatianisVitor A P Martins Dos Santos
Feb 18, 2020·Biotechnology and Applied Biochemistry·Kerstin SchultenkämperVolker F Wendisch
Aug 14, 2020·Applied Microbiology and Biotechnology·Anna WeimerChristoph Wittmann
Apr 11, 2020·Applied Microbiology and Biotechnology·Luciana F BritoVolker F Wendisch
Jun 15, 2020·Applied Microbiology and Biotechnology·Jun XuXinle Liang
Jul 28, 2020·Microbial Biotechnology·Stamatios G DamalasVitor A P Martins Dos Santos
Oct 22, 2020·Nature Communications·Eveline-Marie LammensRob Lavigne
Jun 5, 2020·Current Opinion in Biotechnology·Anita Loeschcke, Stephan Thies
May 11, 2020·Current Opinion in Chemical Biology·Kirsten Davis, Tae Seok Moon
May 1, 2021·Metabolic Engineering·Cholpisit KiattiseweeJesse G Zalatan
Jul 6, 2021·Essays in Biochemistry·Nora Lisa BitzenhoferAnita Loeschcke

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Methods Mentioned

BETA
PCR
flow cytometry

Software Mentioned

flowjo
artemis Software
bd cellquest [UNK] pro
artemis

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