Precision Metabolic Engineering for Advanced Biomanufacturing and Circular Bioeconomy. Pseudomonas putida, particularly the KT2440 strain, is a premier industrial "chassis" known for its diverse metabolic repertoire, high tolerance to organic solvents, and robust growth on non-conventional substrates. CD Biosynsis offers professional Pseudomonas putida Genome Editing Services, utilizing cutting-edge CRISPR/Cas9, Cas12a, and I-SceI counter-selection systems. We empower our clients to transform this versatile host into high-performance microbial cell factories for the production of bioplastic precursors, biosurfactants, and high-value aromatic chemicals.
Get a Technical QuoteOur platform provides a comprehensive suite of genomic tools designed to rewire the complex metabolism of P. putida for industrial-scale efficiency. We specialize in optimizing strains for robust performance on alternative carbon sources.
| Service Tier | Technical Strategy | Primary Application | Standard Deliverables |
|---|---|---|---|
| High-Efficiency Knockout | CRISPR/Cas12a & Suicide Plasmids | Bio-based chemicals (e.g., Malic Acid) | Verified null mutants + Sequencing data |
| Large-Fragment Knock-in | CRISPR/Cas9 λ-Red Recombination | Bioplastic production (e.g., PHA) | Integrated strains + Stability validation |
| Scarless Multi-site Editing | CRISPR/Cas9 + I-SceI Selection | Waste-to-value biosurfactants | Marker-free stable strains + Titer report |
| Dynamic Pathway Tuning | CRISPR/Cas9 + CRISPRi | High-value aromatics (e.g., pHBA) | Regulated strains + Flux prediction data |
1. Target Design & Simulation
2. Editing System Selection
3. Genome Execution
4. Phenotypic Screening
Identifying metabolic bottlenecks and designing high-specificity gRNAs to redirect carbon flux.
Formal project proposal and Mutual NDA signing.
Choosing between Cas9 for high-resolution edits or Cas12a for high-efficiency multiplexed knockouts.
Optimization of λ-Red or suicide plasmid components for specific strain backgrounds.
Simultaneous transformation and selection of genomic edits, including large-fragment pathway integrations.
Application of I-SceI counter-selection to achieve scarless, marker-free engineering.
High-throughput characterization of growth kinetics and product yields under industrial fermentation conditions.
Final genetic verification via Sanger/WGS and delivery of optimized production strains.
To deliver world-class results, our technical team continuously monitors and benchmarks our protocols against landmark research in the field. These studies demonstrate the diverse potential of engineered P. putida.
Malic acid is a critical building block for polyesters. Utilizing a highly efficient CRISPR/Cas12a system combined with suicide plasmids, research has achieved the dual knockout of aceA and icl. This redirection forced carbon flux into the malic acid pathway, resulting in a yield of 26.2 g/L using glycerol—a 3.5-fold increase over wild-type strains.
(Reference: Mikaili et al., 2021)
Polyhydroxyalkanoates (PHAs) are vital biodegradable plastics. Through a CRISPR/Cas9-assisted recombination system, technical benchmarks achieved the knockout of phaZ (degradase) and glpR (repressor), coupled with the knock-in of phaC1 synthase. This allowed for high-efficiency PHA production from glycerol, with PHA content reaching 70% of cell dry weight.
(Reference: Liu et al., 2023)
Rhamnolipids are essential surfactants for cosmetics and oil recovery. Using CRISPR/Cas9 and I-SceI counter-selection, researchers integrated the rhlAB cluster into a safe-harbor site while knocking out gcd and pykA. The engineered strains produced 5 g/L of rhamnolipids using substrates like oil residues, drastically reducing production costs.
(Reference: Bator et al., 2025)
pHBA is a high-value precursor for liquid crystal polymers. Utilizing a combined CRISPR/Cas9 and CRISPRi strategy, researchers knocked out competitive enzymes (catA and pobA) while upregulating the shikimate pathway. This achieved a high titer of 12.5 g/L, demonstrating that engineered P. putida is more cost-competitive than traditional systems for aromatic synthesis.
(Reference: Cui et al., 2024)
1. Why choose P. putida over E. coli for my project?
P. putida possesses a much higher natural tolerance to toxic products and organic solvents. It also features a more versatile native metabolism, making it the preferred host for producing hydrophobic molecules like PHA and aromatics.
2. Can you perform scarless and marker-free edits in P. putida?
Yes. We utilize I-SceI mediated counter-selection and CRISPR-based systems to ensure the final industrial strain is "clean," containing no antibiotic resistance markers or genomic scars.
3. What is the typical turnaround time for a complex pathway integration?
A standard multi-gene project—including target design, genomic execution, and metabolic characterization—typically takes 8 to 12 weeks, depending on host growth rate.
4. How do you handle genes that are essential for P. putida growth?
For essential genes that compete with your target pathway, we use CRISPRi to "knock down" their expression to a low level. This redirects metabolic flux without compromising cell viability.
5. Is the resulting strain stable for large-scale industrial fermentation?
Absolutely. We perform long-term genetic stability tests (>40 generations) and verify the genotype via Whole Genome Sequencing (WGS) to ensure the engineered traits are permanent and robust.
CRISPR-Cas9 technology represents a transformative advancement in gene editing techniques. The main function of the system is to precisely cut DNA sequences by combining guide RNA (gRNA) with the Cas9 protein. This technology became a mainstream genome editing tool quickly after its 2012 introduction because of its efficient, simple and low-cost nature.
The CRISPR gene editing system with its Cas9 version stands as a vital instrument for current biological research. CRISPR technology enables gene knockout (KO) through permanent gene expression blockage achieved by sequence disruption. Various scientific domains including disease modeling and drug screening employ this technology to study gene functions. CRISPR KO technology demonstrates high efficiency and precision but requires confirmation and verification post-implementation because unsatisfactory editing may produce off-target effects or incomplete gene knockouts which impact experimental result reliability. For precise and efficient Gene Editing Services - CD Biosynsis, Biosynsis offers comprehensive solutions tailored to your research needs.
The CRISPR-Cas9 knockout cell line was developed using CRISPR/Cas9 gene editing to allow scientists to remove genes accurately for research on gene function and disease models and pharmaceutical discovery. Genetic research considers this technology essential due to its high efficiency together with simple operation and broad usability.
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CD Biosynsis is a leading customer-focused biotechnology company dedicated to providing high-quality products, comprehensive service packages, and tailored solutions to support and facilitate the applications of synthetic biology in a wide range of areas.