Lactococcus lactis Genome Editing Service

Lactococcus lactis Genome Editing Service

1. Research Background

Lactococcus lactis is a Gram-positive bacterium with immense industrial value and generally recognized as sae(GRAS). As a core chassis organism for dairy fermentation, flavor compound synthesis, and oral vaccine/drug delivery systems, its genetic modification is crucial for optimizing industrial production performance and developing novel probiotic functions. Based on advanced CRISPR/Cas9 systems and classic homologous recombination technologies, GeneRulor provides efficient, precise and large-scale genome editing services to support scientific research and industrial clients worldwide.

2. Bacterial Characteristics and Biological Background

（1）Gram Staining Property: Lactococcus lactis is a Gram-positive bacterium, characterized by a thick peptidoglycan cell wall, non-sporulating and non-motile features.

（2）Safety and Industrial Significance: As the first microorganism to obtain GRAS certification, it plays a key role in fermented foods such as cheese and yogurt. Meanwhile, its simple metabolic pathways and secretion mechanisms make it an ideal host for heterologous protein expression (e.g., Nisin, GABA).

（3）Metabolic Traits: A typical homofermentative lactic acid bacterium that can rapidly utilize carbohydrates to produce lactic acid and exhibits excellent acid tolerance.

Figure 1 Growth of Lactococcus lactis NZ9000 on culture medium plates

3. Reported Editing Strategies

Targeting the transformation barriers caused by the thick cell wall of Lactococcus lactis and the restriction-modification (RM) systems present in industrial strains, the mainstream editing strategies include:

3.1 CRISPR/Cas9 System:

（1）Principle: The Cas9 protein, guided by sgRNA, performs site-specific cleavage of target loci and acts as a lethal screening tool.

（2）Advantages: Combined with donor DNA, it enables scarless knockout, site-directed mutagenesis or gene integration with an efficiency of over 90%.

3.2 Homologous Recombination:

Temperature-sensitive suicide plasmids (e.g., pG+host series) are commonly used to achieve precise editing through double crossover.

3.3 Recombineering Technology:

Red/ET-like recombination systems are utilized to enhance the integration efficiency of linear fragments.

3.4 Transformation Efficiency Optimization:

For hard-to-transform strains (e.g., NZ9000, IL1403), electroporation parameters are optimized or vectors are modified to evade the host RM system.

4. Core Application Fields

（1）Metabolic Pathway Optimization: Improve the yield of lactic acid, Nisin or flavor compounds through gene knockout or overexpression.

（2）Synthetic Biology Applications: Construct oral delivery chassis for secretory expression of therapeutic proteins (e.g., IL-10) or vaccine antigens.

（3）Strain Stress Resistance Modification: Enhance the phage resistance, acid tolerance and salt tolerance of strains during fermentation.

（4) Functional Genomics: Precisely inactivate genes of unknown function to elucidate their physiological mechanisms in complex fermentation processes.

5. Project Process and Validation

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

（1）Protocol Design and Vector Construction: Design optimal sgRNAs and homologous arms for target loci.

（2）Bacterial Transformation and Screening: Overcome cell wall barriers through optimized electroporation technology.

（3）DNA Targeted Cleavage and Repair: The Cas9 system induces double-strand breaks, and precise editing is completed using homologous repair templates.

（4）Multiplex Validation: Confirm positive mutations through PCR identification and Sanger sequencing.

Figure 2 Schematic of the Project Process

6. Introduction to Genome Editing Projects

6.1 Our core services include:

（1）Gene Knockout/Inactivation: Precisely delete metabolic byproduct genes (e.g., nadR) to optimize industrial production phenotypes.

（2）Gene Knock-in/Overexpression: Insert exogenous gene clusters (e.g., GABA synthesis genes) into chromosomal safe harbors to construct stably expressing strains.

（3）Precise Site-Directed Mutagenesis: Improve the activity or stability of key enzymes through base substitution.

（4）Multi-Gene Editing: Realize sequential editing of multiple metabolic targets using CRISPR technology.

6.2 Technical Advantages

（1） High Success Rate: Rich experience in handling various industrial isolates and laboratory model strains (MG1363, NZ9000).

（2）Scarless Editing: No resistance markers are introduced, complying with food-grade application requirements.

（3）Short Cycle: Optimized dual-plasmid or single-plasmid editing systems significantly shorten R&D time.

7. Case Introduction

Case: Genome Modification of Lactococcus lactis NZ9000 Using the CRISPR-Cas9 System

Project Content: Successfully developed a highly efficient CRISPR-Cas9 gene editing platform for food-grade lactic acid bacterium Lactococcus lactis.

（1）Research Background

Lactococcus lactis is an important food-grade lactic acid bacterium widely used in dairy fermentation and the medical industry (e.g., probiotic and recombinant protein production). Traditional gene editing methods (e.g., RecA-dependent homologous recombination) are time-consuming (taking 20 days) and low in efficiency. Although CRISPR-Cas9 technology is maturely applied in other microorganisms, tools for large-fragment gene editing of Lactococcus lactis are still lacking. This study aimed to develop a single-plasmid CRISPR-Cas9 system (pLL series) to achieve rapid and precise gene editing, thereby accelerating functional gene research and industrial application optimization of Lactococcus lactis.

（2）Protocol Design

The research team constructed a CRISPR-Cas9-based single-plasmid editing system pLL, with the core design including:

Plasmid Construction: Using pHSP02 as the backbone, integrate the Cas9 expression cassette, sgRNA module, homologous arm targeting sequences and resistance genes. Test different promoters (P11, P23, P32, P44) to drive sgRNA expression for optimizing editing efficiency.

Target Selection: Conduct deletion experiments on the lactate dehydrogenase gene (ldh, LLNZ_02045) and uracil phosphoribosyltransferase gene (upp, LLNZ_11240). Design sgRNAs targeting different regions (3', middle, 5') of the genes to evaluate the impact of position on editing efficiency.

Experimental Process: Introduce the pLL plasmid into Lactococcus lactis NZ9000 via electroporation, induce double-strand breaks with Cas9, and achieve gene deletion through homologous recombination repair. Screen transformants via erythromycin resistance, and verify editing results through PCR and sequencing. By comparing the activity of different promoters, the study found that the P23 promoter could significantly improve sgRNA expression efficiency, thus optimizing the editing effect. The entire editing process can be completed within 7 days, greatly shortening the time required for traditional methods.

（3） Experimental Conclusions

Gene Editing Efficiency: The deletion efficiency of the pLL system for the ldh gene reached 8%-50%, with the P23 promoter showing the best effect (50% efficiency). Successful sequential deletion of upp and upp1 genes verified that the system supports multi-gene editing. There was no significant difference in editing efficiency when sgRNAs targeted different regions of the gene (e.g., 16%-38% deletion efficiency for the upp gene), indicating that the sgRNA binding position does not affect the results.

Promoter Activity Verification: The transcriptional activity of P23 and P32 promoters was significantly higher than that of P11, which could effectively reduce the wild-type background (transformant survival rate reduced by more than 99.9%).

Functional Verification: Strains with the upp gene deleted developed resistance to 5-fluorouracil (5-FU), confirming that upp encodes uracil phosphoribosyltransferase and verifying the gene function.

sgRNA Targeting Efficiency: sgRNAs targeting different regions of ldh and upp genes all efficiently induced editing with a lethal efficiency of 99.93%-99.98%, indicating the strong robustness of the system.

（4） Application Significance

Technical Advantages: The pLL system shortens the editing time from 20 days to 7 days and increases the efficiency to 50%, which is superior to traditional homologous recombination methods. The single-plasmid design simplifies operations, does not require exogenous recombinases, and is suitable for high-throughput gene function screening.

Industrial Potential: It can be used to knock out undesirable genes (e.g., acid-producing genes) or introduce beneficial traits to optimize dairy fermentation strains, and provide a precise editing tool for developing novel probiotics (e.g., enhancing intestinal colonization ability).

Scientific Value: It provides a template for gene editing of other lactic acid bacteria and promotes the application of synthetic biology in food microorganisms. Accelerate metabolic pathway analysis through rapid gene function identification (e.g., upp gene).

8. References

[1] Datsenko, K. A., & Wanner, B. L. (2000). One-step inactivation of chromosomal genes. PNAS.

[2] Guo, T., et al. (2019). Single-plasmid systems based on CRISPR-Cas9 for gene editing in Lactococcus lactis. Microbial Cell Factories.

[3] Jiang, W., et al. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology.

[4] Van Pijkeren, J. P., & Britton, R. A. (2012). High efficiency CRISPR-Cas9 genome editing in Lactic Acid Bacteria. Applied and Environmental Microbiology.

[5] Song X, et al. (2021). Single-plasmid systems based on CRISPR-Cas9 for gene editing in Lactococcus lactis. J Dairy Sci.

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