Lactococcus lactis Gene Editing Service

1. ResearchBackground

Lactococcus lactis is a Gram-positive bacterium of greatindustrial importance and generally recognized as safe (GRAS). As a key chassisorganism for dairy fermentation, flavor compound production, and oralvaccine/drug delivery systems, its genetic engineering is critical foroptimizing industrial performance and developing novel probiotic functions.

Based on theadvanced CRISPR/Cas9 system and classic homologous recombination technology,Shutong Biotech provides highly efficient and precise large-scale genomeediting services to support global research and industrial clients.

2. StrainCharacteristics and Biological Background

(1) Gram StainProperty:

Lactococcus lactis is a Gram-positive bacterium characterizedby a thick peptidoglycan cell wall, non-spore-forming and non-motile.

(2) Safety andIndustrial Significance:

As the firstmicroorganism to obtain GRAS status, it plays an essential role in fermentedfoods such as cheese and yogurt. Meanwhile, its simple metabolic pathways andsecretion machinery make it an ideal host for heterologous protein expression(e.g., nisin, GABA).

(3) MetabolicTraits:

A typicalhomofermentative lactic acid bacterium that rapidly metabolizes sugars intolactic acid with excellent acid tolerance.

3. Reported EditingStrategies in Literature

To overcometransformation barriers caused by the thick cell wall andrestriction-modification (RM) systems in industrial Lactococcus lactisstrains, mainstream editing strategies include:

3.1 CRISPR/Cas9System

(1) Mechanism:The Cas9 protein, guided by sgRNA, performs site-specific cleavage of thetarget locus as a lethal selection tool.

(2) Advantages:Combined with donor DNA, it enables marker-free knockout, point mutation orgene integration with efficiency exceeding 90%.

3.2 HomologousRecombination

Temperature-sensitivesuicide plasmids (e.g., pG+host series) are commonly used to achieve preciseediting via double crossover.

3.3 Recombineering

Red/ET-likerecombination systems are applied to enhance integration efficiency of linearDNA fragments.

3.4 TransformationEfficiency Optimization

Forhard-to-transform strains such as NZ9000 and IL1403, electroporation parametersare optimized or vectors are modified to evade host RM systems.

4. Core ApplicationAreas

(1) MetabolicPathway Optimization: Improve production of lactic acid, nisin or flavorcompounds via gene knockout or overexpression.

(2) SyntheticBiology Applications: Construct oral delivery chassis for secretoryexpression of therapeutic proteins (e.g., IL-10) or vaccine antigens.

(3) StressResistance Engineering: Enhance phage resistance, acid tolerance and salttolerance during fermentation.

(4) FunctionalGenomics: Precisely inactivate uncharacterized genes to reveal theirphysiological roles in complex fermentation processes.

5. Project Workflowand Validation

We provide one-stopservices from design to delivery to ensure accurate editing results:

(1) ProtocolDesign and Vector Construction: Design of optimal sgRNAs and homology armsfor target loci.

(2) BacterialTransformation and Screening: Overcome cell wall barriers using optimizedelectroporation.

(3) Targeted DNACleavage and Repair: Cas9-mediated double-strand break followed by preciseediting via homologous repair templates.

(4) MultilevelValidation: Positive mutants confirmed by PCR identification and Sangersequencing.

6. Gene EditingProject Introduction

6.1 Core Services

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

(2) Gene Knock-in/ Overexpression: Insert heterologous gene clusters (e.g., GABA synthesisgenes) at safe chromosomal loci to construct stable expression strains.

(3) Precise PointMutation: Improve activity or stability of key enzymes through basesubstitution.

(4) MultigeneEditing: Achieve sequential editing of multiple metabolic targets usingCRISPR technology.

6.2 TechnicalAdvantages

(1) High SuccessRate: Extensive experience with various industrial isolates and laboratorymodel strains (MG1363, NZ9000).

(2) Marker-FreeEditing: No antibiotic resistance markers introduced, fully compliant withfood-grade applications.

(3) ShortTimeline: Optimized single- or two-plasmid editing systems significantlyshorten R&D cycles.

7. Case Study

CRISPR-Cas9-MediatedGenome Engineering in Lactococcus lactis NZ9000

Project Summary: An efficient CRISPR-Cas9 gene editingplatform was successfully developed for the food-grade lactic acid bacterium Lactococcuslactis.

(1) ResearchBackground

Lactococcus lactis is an important food-grade lactic acidbacterium widely used in dairy fermentation and the medical industry (e.g.,probiotics and recombinant protein production).

Traditional geneediting methods (e.g., RecA-dependent homologous recombination) aretime-consuming (≈20 days) and inefficient.

Although CRISPR-Cas9is well-established in other microbes, tools for large-fragment editing in Lactococcuslactis remain limited.

This study aimed todevelop a single-plasmid CRISPR-Cas9 system (pLL series) for rapid and precisegene editing, accelerating functional genomic research and industrialoptimization of Lactococcus lactis.

(2) ExperimentalDesign

The research teamconstructed a single-plasmid CRISPR-Cas9 editing system (pLL) with thefollowing core features:

Plasmid Construction:

Based on the pHSP02backbone, the system integrated a Cas9 expression cassette, sgRNA module,homology arm targeting sequences and resistance gene.

Different promoters(P11, P23, P32, P44) were tested to drive sgRNA expression for editingefficiency optimization.

Target Selection:

Deletion assays wereperformed on the lactate dehydrogenase gene (ldh, LLNZ_02045) and uracilphosphoribosyltransferase gene (upp, LLNZ_11240).

sgRNAs targeting the3′, middle and 5′ regions were designed to evaluate positional effects onediting efficiency.

ExperimentalWorkflow:

The pLL plasmid wasintroduced into Lactococcus lactis NZ9000 by electroporation.

Cas9-induceddouble-strand breaks were repaired by homologous recombination to achieve genedeletion.

Transformants wereselected via erythromycin resistance, and editing results were verified by PCRand sequencing.

The P23 promotersignificantly improved sgRNA expression and editing efficiency.

The entire workflowcould be completed within 7 days, greatly reducing the time required bytraditional methods.

(3) ExperimentalConclusions

Editing Efficiency:

The pLL systemachieved 8%–50% deletion efficiency for the ldh gene, with the P23promoter yielding the highest rate (50%).

Sequential deletionof upp and upp1 confirmed the system’s capacity for multigeneediting.

No significantefficiency differences were observed among sgRNAs targeting different regionsof upp (16%–38%), indicating that binding position had minimal impact.

Promoter Activity:

Transcriptionalactivity of P23 and P32 was significantly higher than P11, effectively reducingwild-type background (transformant survival reduced by >99.9%).

FunctionalValidation:

upp-deleted strains gained resistance to5-fluorouracil (5-FU), confirming that upp encodes uracilphosphoribosyltransferase and validating gene function.

sgRNA Efficiency:

sgRNAs targetingdifferent regions of ldh and upp induced strong lethality(99.93%–99.98%), demonstrating high system robustness.

(4) ApplicationSignificance

Technical Advantages:

The pLL systemshortened editing time from 20 days to 7 days with up to 50% efficiency,outperforming traditional homologous recombination.

The single-plasmiddesign simplifies operation without exogenous recombinases, making it suitablefor high-throughput gene function screening.

Industrial Potential:

Enables deletion ofdisadvantageous genes (e.g., acid-producing genes) or introduction ofbeneficial traits to optimize dairy fermentation strains.

Provides a precisionediting tool for developing next-generation probiotics with enhanced intestinalcolonization.

Scientific Value:

Serves as ablueprint for gene editing in other lactic acid bacteria and promotes syntheticbiology applications in food microorganisms.

Acceleratesmetabolic pathway dissection through rapid gene function identification (e.g., upp).

Work With Us

Partner with us, andyou will gain a highly experienced and technically proficient gene editingcollaborator.

We are committed toaccelerating your research and projects with professional expertise, rigorousworkflows, and efficient communication.