CRISPR interference‐mediated gene regulation in Pseudomonas putida KT2440

Abstract

SummaryTargeted 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 expected to become a robust tool to reprogram P. putida KT2440 for the development of microbial cell factories producing industrially valuable products.

CRISPR interference-based gene repression in the plant growth promoter Paenibacillus sonchi genomovar Riograndensis SBR5

“…In both cases, CRISPRi was highly efficient, with less than 1% remaining reporter protein signal. Similarly, targeting plasmid GFP reporter gene in Pseudomonas putida decreased fluorescence intensity to background levels (Kim et al 2020). In this study, flow cytometry analysis revealed substantial drop of plasmid-borne GFPUV fluorescence (~70%) in P. sonchi SBR5 expressing dCas9 and gfpUV targeting sgRNA, but about 30% of the reporter signal remained (Table 2).…”

Exploring the synthetic biology potential of bacteriophages for engineering non-model bacteria

“… TF Promoter Inducer(s) Reported pros, cons and main applications in P. putida Ref. (s) Positively regulated induction systems XylS Pm m -toluate and derivatives + High expression levels + Extensively characterized + Cheap inducers − Catabolite repression − Dose-dependent response in absence of inducer metabolization pathways − Bimodal response − Leaky expression 28 , 49 , 63 , 130 – 139 Applications: Expression of I- sceI , dCas9 , trfA , the λ red operon, and recombinases in genome engineering tools; expression of toxic genes for controlled autolysis; production of recombinant antibody fragments and p -coumaric acid RhaRS P rhaB l -rhamnose + No catabolite repression + No metabolization of rhamnose: dose-dependent response + Tight regulation + Non-toxic inducer − Inhomogeneous response at intermediate inducer levels − Expensive inducers 132 , 138 , 140 , 141 Applications: Expression of dCas9 for CRISPRi and Cre for genomic deletions; production of p -coumaric acid AraC P BAD l -arabinose + Characterization + No catabolite repression + No metabolization of arabinose: dose-dependent response + Tight regulation + Non-toxic inducer − Inhomogeneous response at intermediate inducer levels − Poor arabinose uptake without AraE transporter − Expensive inducers 132 , 142 , 143 Applications: Production of p -coumaric acid Negatively regulated induction systems LacI Plac, PlacUV5, Ptac, Ptrc Lactose, isopropyl β...…”

An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida

“…A linear response of downregulation levels of a chromosomally expressed msf·gfp was observed as a function of the inducer (3‐ m Bz) concentration. This feature represents a substantial improvement to previously CRISPRi‐based approaches, which did not allow for titratable gene downregulation (Kim et al , ), sometimes exhibiting high leakiness (> 50%) in the absence of the corresponding inducers (Tan et al , ). Importantly, we also demonstrated the ability to simultaneously downregulate three genes in P. putida with this system.…”

“…Recently, two alternative SpdCas9-based CRISPRi systems were developed and explored in Pseudomonas species (Sun et al, 2018;Kim et al, 2020). Both systems were demonstrated to be functional for repression of genes encoding fluorescent proteins, and Kim et al (2020) also employed CRISPRi for metabolic engineering via gene repression by depleting the GlpR regulator to enhance the glycerol-dependent synthesis of mevalonate. Other examples on the development of CRISPRi systems have been reported for P. aeruginosa (Peters et al, 2019;Qu et al, 2019) and P. fluorescens (Noirot-Gros et al, 2019).…”