Genome Engineering of Pseudomonas putida

Genome Engineering of Pseudomonas putida

1. Research Background

Pseudomonas putida is an environmental and industrial microorganism with tremendous application potential, and is recognized as a Generally Recognized as Safe (GRAS) strain. Its outstanding metabolic diversity, strong environmental adaptability and excellent stress tolerance render it a core organism in the fields of bioremediation, biocatalysis and synthetic biology. Precise and efficient genome editing technology is a key tool to explore its metabolic potential and construct high-performance engineering strains.

Against this backdrop, GeneRulor has developed a CRISPR/Cas9-based method for genomic modification of Pseudomonas putida, which can customarily achieve target gene knockout, large-fragment deletion, site-directed mutagenesis, gene integration (overexpression) and other services in different strains, with positive mutant strains as the final deliverables. We are committed to providing professional and reliable customized genome editing solutions for Pseudomonas putida to scientific research and industrial clients worldwide.

2. Strain Characteristics and Biological Background

(1) Gram-staining property: Pseudomonas putida is a Gram-negative bacterium.

(2) Physical characteristics: It possesses a typical outer membrane structure and a thin peptidoglycan cell wall. The outer membrane acts as an effective permeability barrier, endowing the strain with innate tolerance to a variety of antibiotics and organic solvents. As a model species of the genus Pseudomonas, it has diverse metabolic pathways.

(3) Industrial significance: As a model Gram-negative bacterium, it is an ideal model for studying aromatic hydrocarbon degradation, organic solvent tolerance and biofilm formation. It is widely used in industry for bioremediation, chiral compound synthesis and polymer material production.

(4) Genetic transformation: It has a high capacity for exogenous DNA uptake. The introduction of genome editing tools can be achieved via electroporation or conjugal transfer with relatively high transformation efficiency.

Figure 1. Growth of Pseudomonas putida on culture medium plates

3. Reported Editing Strategies in Literatures

Aiming at the transformation challenges of Pseudomonas putida, the mainstream editing strategies adopted in current literatures and scientific research practices include the following:

3.1 CRISPR/Cas9 System (Mainstream Protocol)：

(1) Principle: The Cas9 protein (containing RuvC and HNH domains) mediates site-specific cleavage of the target genomic DNA under the guidance of sgRNA.

(2) Advantages: Inducing DNA repair via double-strand breaks (DSB) enables efficient gene knockout, large-fragment deletion or gene integration (overexpression).

3.2 Homologous Recombination：

Suicide plasmids incapable of autonomous replication are used to achieve scarless gene knockout or site-directed mutagenesis through two rounds of homologous recombination.

3.3 Transformation Efficiency Optimization：

For specific industrial strains, the barriers of the host restriction-modification system are overcome by optimizing electroporation parameters, using methylation-deficient strains or introducing specific DNA methylation modifications.

4. Core Application Fields

(1) Environmental bioremediation: Enhance the strain's degradation capacity for aromatic hydrocarbons, pesticides and heavy metal pollutants through gene knockout or integration.

(2) Cell factory construction: Improve the yield of target compounds (e.g., polyhydroxyalkanoates, terpenoids) via metabolic pathway optimization and byproduct pathway blocking.

(3) Synthetic biology modification: Integrate biosensors or regulatory elements into the genome to construct environment-responsive intelligent strains.

(4) Stress tolerance improvement: Enhance the strain's tolerance to organic solvents, high salt or extreme pH through site-directed mutagenesis or gene integration, making it suitable for harsh industrial environments.

5. Project Process and Validation

We provide one-stop services from design to delivery to ensure the accuracy of editing results:

(1) Protocol design and vector construction: Design knockout vectors for target loci.

(2) Bacterial transformation and screening: Introduce the editing system via natural transformation or electroporation technology.

(3) Multiplex validation: Confirm positive mutants through PCR identification and Sanger sequencing.

Figure 2. Schematic diagram of the project process

6. Introduction to Genome Editing Projects

6.1 Core Services Include：

(1) Gene knockout/inactivation: Precisely delete protease genes or metabolic byproduct genes to optimize chassis performance.

(2) Gene knock-in/overexpression: Integrate exogenous expression cassettes at specific loci or enhance the expression of endogenous rate-limiting enzymes.

(3) Site-directed mutagenesis/modification: Perform site-directed mutagenesis on key enzymes to improve their thermal stability or catalytic activity.

(4) Multigene editing: Continuously edit multiple genes to construct strains with modified complex metabolic networks.

6.2 Technical Advantages：

(1) High success rate: Rich experience in editing industrial strains and laboratory model strains.

(2) Customized design: Devise the optimal editing strategy according to production objectives (e.g., enzyme activity enhancement, growth improvement).

(3) Full-process validation: Provide a complete closed-loop report from protocol design to final genotype confirmation.

7. Case Introduction

We have successfully provided services for many top universities, research institutions and biotechnology companies at home and abroad. Partial examples are as follows:

Case: Construction of Pseudomonas putida KT2440 mutant strain

Project content: Successful integration of exogenous gene fragments.

Figure 3. Colony picking validation of transposon strains of Pseudomonas putida

(Upper panel: Validation of resistance gene insertion; Lower panel: Validation of plasmid existence)

8. References

[1] Wen Q, et al. A single-plasmid-based, easily curable CRISPR/Cas9 system forrapid, iterative genome editing inPseudomonas putida KT2440. Microb Cell Fact.2024 Dec;23(1):349.

[2] Yunus IS, et al.Predictive CRISPR-mediated gene downregulation for enhancedproduction ofsustainable aviation fuel precursor in Pseudomonas putida. MetabEng. 2026Mar;94:67-76.

[3] Köbbing S, et al.Reliable Genomic Integration Sites in Pseudomonasputida Identifiedby Two-Dimensional Transcriptome Analysis. ACS SynthBiol. 2024 Jul19;13(7):2060-2072.

[4] Nong LK, et al.Redefining HexR regulatory landscape in Pseudomonas putidaKT2440 throughintegrative systems biology. Metab Eng. 2026 Mar;94:77-89.

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