Lactobacillus Gene Editing Service

1. ResearchBackground

Lactobacillus represents a group of highly important Gram‑positiveprobiotics widely used in food fermentation, pharmaceutical development, andanimal feed production.

Owing to theircentral roles in maintaining intestinal microecological balance, regulating theimmune system, and synthesizing bioactive metabolites, lactobacilli have becomea major focus in microbiological research.

Precise andefficient gene editing technologies provide essential tools for dissecting thefunctional genome of Lactobacillus, optimizing probiotic traits, andconstructing novel mucosal vaccine delivery systems.

Against thisbackground, Shutong Biotech offers genome engineering services for Lactobacillususing the CRISPR/Cas9 system.

We providecustomized solutions for targeted gene knockout, large‑fragment deletion, pointmutation, and gene integration (overexpression) in various Lactobacillusspecies, including L. plantarum, L. casei, L. reuteri, andothers, with verified positive mutant strains delivered.

We are committed toproviding professional and reliable customized Lactobacillus geneediting solutions for global research and industrial clients.

2. StrainCharacteristics and Biological Background

(1) Gram StainProperty:

Lactobacillus are typical Gram‑positive bacteria.

(2) MorphologicalFeatures:

Their most prominentstructural characteristic is a thick peptidoglycan cell wall (approximately20–80 nm), with no outer membrane as found in Gram‑negative bacteria.

This thick cell walloften acts as a physical barrier to genetic transformation.

(3) ApplicationValue:

As generallyrecognized as safe (GRAS) microorganisms, they serve as important models forstudying probiotic mechanisms, organic acid metabolism, and anti‑infectiveimmunity.

(4) Metabolismand Adaptability:

They exhibit strongacid tolerance and can survive in extremely low‑pH environments, making themideal chassis for developing functional foods and biological products.

3. Reported EditingStrategies in Literature

To addresstransformation challenges caused by the thick cell wall and strain-specificcharacteristics of Lactobacillus, widely adopted strategies inliterature and research practice include:

3.1 CRISPR/Cas9System (Mainstream Approach)

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

(2) Advantages:Induces repair via double-strand breaks (DSBs), enabling efficient geneknockout, large-fragment deletion, or gene integration (overexpression).

3.2 HomologousRecombination

Often used withtemperature-sensitive plasmids or suicide plasmids to achieve precise basesubstitution or marker-free editing through double crossover.

3.3 Site-SpecificRecombination

Uses recombinasesystems (e.g., Cre/loxP) for targeted gene deletion, suitable for sequentialmultigene engineering.

3.4 TransformationEfficiency Optimization

For differentstrains (e.g., L. plantarum WCFS1), transformation barriers are overcomeby optimizing electroporation parameters (field strength, buffer composition)or treatment with cell wall-degrading enzymes.

4. Core ApplicationAreas

(1) FunctionalGenomics: Precise deletion of metabolism-related genes (knockout) to studytheir roles in colonization, acid resistance, or carbon source utilization.

(2) ProbioticMechanism Research: Introduction of point mutations to simulate key proteinvariations, revealing interactions between lactobacilli and host intestinalcells.

(3) SyntheticBiology Engineering: Integration of foreign antigen genes or reporter genesinto the genome to construct oral vaccine delivery systems or environmentalmonitoring strains.

5. Project Workflowand Validation

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

(1) ProtocolDesign and Vector Construction: Design of knockout vectors targetingspecific loci.

(2) BacterialTransformation and Screening: Overcome transformation difficulties inlactobacilli using optimized electroporation.

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

6. Gene EditingProject Introduction

6.1 Core Services

(1) Gene Knockout/ Inactivation: Precise deletion of key metabolic genes for mutant strainconstruction or gene function research.

(2) Gene Knock-in/ Overexpression: Insertion of foreign sequences (fluorescent proteins,antigen sequences) at chromosomal loci to generate genetically stableexpression strains.

(3) PointMutation / Modification: Introduction of specific point mutations to studyacid tolerance, bile salt resistance, or protein function.

(4) MultigeneEditing: Sequential editing of multiple targets for complex metabolicpathway construction or highly attenuated strain development.

6.2 TechnicalAdvantages

(1) High SuccessRate: Optimized transformation and editing protocols for diverselactobacilli, including industrial production strains.

(2) CustomizedDesign: Tailored editing strategies based on research goals (e.g., yieldimprovement, stress resistance enhancement).

(3) Full-ProcessValidation: Closed-loop services covering design to final genotypicvalidation.

7. Case Study

CRISPR/Cas9-MediatedGenomic Insertion of Functional Genes in Lactobacillus plantarum WCFS1

Project Summary: An inducible two-plasmid CRISPR/Cas9system was successfully developed for efficient, marker-free gene knock-in in L.plantarum.

(1) ResearchBackground

Lactobacillusplantarum is a lactic acidbacterium naturally found in the human body.

Due to its safety,probiotic properties, and protein expression capacity, it is regarded as apromising vaccine delivery vehicle.

However, thedevelopment of bacterial delivery systems faces major limitations:

Traditionalengineered strains often rely on antibiotic resistance markers, which areundesirable in clinical applications.

Foreignplasmid-based expression systems carry excess exogenous DNA and may compromisestrain stability.

While chromosomalinsertion enables permanent gene expression, editing efficiency is low.

Existing tools suchas the loxP/Cre system suffer from complex operation, low efficiency, and scarsequence retention.

Although CRISPR/Cas9is well-established in other microbes, its application in L. plantarumis hindered by Cas9 toxicity and low homologous recombination efficiency.

This study aimed todevelop an optimized CRISPR/Cas9 system for efficient gene knock-in in L.plantarum, providing a tool for vaccine development and metabolicengineering.

(2) ExperimentalDesign

Two-PlasmidCRISPR/Cas9 System Construction

• Plasmid 1 (pSIPSh71_LpRec): Carries an inducible recombinase     operon (lp0640-42) under the P_sppA promoter to enhance     homology-directed repair (HDR) efficiency.

• Plasmid 2 (pCRISPR series): Contains the Cas9 gene,     targeting sgRNA, and HDR template, with a modular design for convenient     gene replacement.

Target LocusSelection:

Genes were insertedinto a non-coding region (between lp_2071 and lp_2074) to avoiddisrupting native gene function.

Inserted Genes:

• mCherry (fluorescent protein) for expression     efficiency evaluation.

• SARS-CoV-2 spike protein     receptor-binding domain (RBD), expressed either cytoplasmically or as a     lipoprotein-anchored surface display.

Optimized Workflow:

The recombinaseplasmid was first introduced into L. plantarum. After recombinaseinduction, the CRISPR plasmid was transformed.

The P_sppApromoter regulated Cas9 and recombinase expression to reduce toxicity fromconstitutive expression.

1 kb homology armswere used to balance editing efficiency and fragment length compatibility.

(3) ExperimentalConclusions

Knock-in Efficiency:

The systemsuccessfully inserted four expression cassettes (800–1,300 bp) withefficiencies ranging from 40% to 100%.

Inducible cassettes(e.g., P_sppA-mCherry) achieved 42%–50% efficiency, whileconstitutive cassettes (P_slpA-mCherry) yielded only 3%.

Recombinase Effect:

Almost no knock-inoccurred without recombinase induction, confirming its essential role inpromoting HDR.

Protein ExpressionValidation:

All knock-in strainsexpressed target proteins, though at lower levels than plasmid-based systems.

Inducible knock-instrains showed twofold higher signals than constitutive strains (p < 0.01).

RBD was anchored onthe cell surface via lipoprotein fusion, and surface antigen was detected byflow cytometry — representing the first CRISPR/Cas9-mediated surface display ina Gram-positive bacterium.

System Stability:

Inserted genes werestably maintained after multiple passages as verified by PCR.

Helper plasmids weresuccessfully cured under antibiotic-free conditions, yielding engineeredstrains free of exogenous vector DNA.

(4) ApplicationSignificance

Technical Innovation:

This work presentsthe first efficient, modular CRISPR/Cas9 knock-in system for L. plantarum,with an editing workflow shortened to 4–5 days.

The modular designallows rapid replacement of target genes and homology arms, facilitatingpersonalized strain development.

Vaccine DevelopmentPotential:

Successfulexpression and surface display of SARS-CoV-2 RBD pave the way for oral vaccinevector design.

Marker-freeengineered strains better meet clinical safety requirements.

Industrial andResearch Utility:

The system can beextended to other lactic acid bacteria for metabolic engineering (e.g., enzymeproduction) or probiotic enhancement.

It provides a modelfor studying position effects on chromosomal gene expression (e.g., insertionsite optimization).

Limitations andFuture Perspectives:

Protein expressionlevels in knock-in strains are lower than in plasmid systems, requiring furtherpromoter or integration site optimization.

Future integrationwith base editing or multiplex editing will expand application scope.

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.