Customized Gene Expression Engineered Strains

Customized Gene Expression Engineered Strains

1. Research Background

In the synthetic biology and biotechnology industries, constructing efficient and stable "cell factories" is the cornerstone of large-scale biomolecule production. Whether for the heterologous expression of recombinant proteins, the reconstruction of metabolic pathways, or the development of complex biosensors, precise customized genomic modification is indispensable.

Against this backdrop, GeneRulor offers a comprehensive Customized Gene Expression Engineered Strain Construction Service. Leveraging CRISPR/Cas9 and multi-fragment recombination technologies, we achieve precise integration of target sequences, fine-tuned regulation of expression intensity, and metabolic flux optimization across various microbial chassis (e.g., E. coli, B. subtilis, and yeast). Our mission is to deliver high-yield, stable, and customized mutant strains tailored to your specific research and production needs.

2. Host Characteristics & Chassis BiologyChassis Cell Selection:

（1）Chassis Cell Selection： We provide a diverse range of chassis options tailored to specific expression requirements.

（2） Escherichia coli (E. coli): Features the most well-defined genetic background and rapid growth rates; ideal for the expression of most primary metabolites and proteins.

（3）Bacillus subtilis (B. subtilis): A Gram-positive bacterium with robust protein secretion capabilities; classified as GRAS (Generally Recognized as Safe).

（4）Metabolic Regulatory Logic: We optimize nutrient utilization and energy allocation of the host strain according to specific production targets.

（5）Environmental Adaptability: We utilize gene editing to enhance strain stability under industrial fermentation conditions, such as high osmotic pressure and thermal stress.

Figure 1 Schematic diagram of the custom expression engineered bacterium

3. Engineering Strategies Reported in Literature

To address common challenges such as low expression efficiency and genetic instability of exogenous genes in host strains, GeneRulor employs the following advanced strategies:

3.1 CRISPR/Cas9 Site-Specific Integration (Primary Protocol)

（1）Mechanism: Utilizes Cas9-mediated double-strand breaks (DSBs) to induce high-efficiency homologous recombination.

（2）Advantage: Enables the precise integration of exogenous fragments into chromosomal "safe harbor" sites, effectively eliminating expression decline caused by plasmid loss.

3.2 Promoter & RBS Engineering

By constructing libraries of promoters and RBS (Ribosome Binding Site) sequences with varying strengths, we achieve precise, tunable control over the target gene at both the transcriptional and translational levels.

3.3 Codon Optimization & Metabolic Flux Balancing

We perform full-gene optimization based on the specific codon usage bias of the host strain. This balances the supply of precursor metabolites with the synthesis rate of target products to maximize overall yield.

3.4 High-Efficiency Secretion Signal Peptide Screening

For secretory proteins, we screen and identify the optimal signal peptides to ensure efficient transmembrane transport and proper folding of the target protein.

4. Core Application Areas

（1）Recombinant Protein Production: Construction of high-yield engineered strains for enzymes, antibody fragments, and cytokines.

（2）Metabolic Engineering: Reconstruction of metabolic pathways through multi-gene integration to produce high-value chemicals, such as amino acids and organic acids.

（3）Live Biotherapeutic Delivery: Insertion of therapeutic genes into probiotics to achieve in situ expression and drug delivery within the gastrointestinal tract.

（4）Chassis Streamlining: Construction of "minimalist" high-efficiency chassis by removing redundant metabolic burdens through large-fragment deletions.

5. Project Workflow & Validation

GeneRulor provides a comprehensive one-stop service, from initial sequence design to the final delivery of the engineered strain, ensuring optimal expression performance:

（1）Sequence Analysis and Optimization: We perform professional codon optimization and strategic design of the expression system.

（2）Vector Construction and Transformation: Preparation of high-efficiency electrocompetent or chemically competent cells for seamless integration.

（3）Genomic Site-Specific Integration: Utilizing CRISPR technology for precise "locus-specific" insertion of target sequences.

（4）Multifaceted Verification and Evaluation:   Genotyping: Accurate verification via PCR identification and Sanger sequencing.

（5）Phenotypic Characterization: Quantitative assessment of expression yields and product quality through Western Blot, ELISA, or HPLC analysis.

Figure 2 Schematic diagram of the project process

6. Gene Editing Project Overview

6.1 Core Services

（1）High-Yield Strain Construction/Optimization: Achieving overexpression through strong promoter replacement and gene copy number amplification.

（2）Site-Specific Gene Integration: Enabling stable, marker-free (seamless) integration of exogenous DNA into the host genome.

（3）Multi-gene Co-expression: Realizing the simultaneous and high-efficiency expression of multiple functional subunits within a single microbial strain.

6.2 Technical Advantages

（1）High Stability: Chromosomal integration strains exhibit exceptional genetic stability, maintaining performance even under antibiotic-free conditions.

（2）Customized Strategies: Tailored design of secretory, intracellular, or surface-display strategies based on the specific structure of the target protein.

（3）Closed-Loop Delivery: We provide a comprehensive delivery package, including sequencing reports, protein quantification results, and fermentation performance assessments.

7. Case Study

Case Title: Biomarker-Responsive Engineered Probiotics for the Diagnosis, Recording, and Improvement of Inflammatory Bowel Disease (IBD) in Mice

Project Overview: Development of i-ROBOT, a smart engineered probiotic designed to non-invasively monitor and record the onset and progression of Inflammatory Bowel Disease (IBD), while releasing therapeutic agents through a self-regulating mechanism.

(1) Research Background

Inflammatory Bowel Disease (IBD) is a chronic gastrointestinal inflammatory condition with a rising global incidence. Traditional diagnostic methods, such as colonoscopy, are invasive and struggle with real-time dynamic monitoring. While engineered microbes offer a synthetic biology-based solution, existing tools face limitations:

Insufficient Sensitivity: Difficulty in capturing low concentrations of early-stage biomarkers (e.g., thiosulfate).

Lack of Archival Capability: Signals from traditional sensors vanish once the biomarker disappears, failing to record disease history.

Lack of Adaptive Therapy: Most drug release systems are either constitutive or inducible, lacking real-time regulation based on disease severity.

This study utilizes Escherichia coli Nissle 1917 (EcN) as the chassis to develop the i-ROBOT system, integrating sensing, recording, and therapeutic modules for comprehensive IBD management.

(2) System Design

The i-ROBOT system is based on a modular design consisting of three core components:

Sensing Module: Utilizes the ThsS/R two-component system to respond to the inflammation marker thiosulfate, controlling the inducible promoter $P_{phsA}$ to drive downstream gene expression. Optimized promoters and Ribosome Binding Sites (RBS) achieved a sensitivity of 0.016 mM and an induction ratio of up to 81-fold.

Recording Module: Employs a CRISPR-Cas9 base editing system (BE2) to convert thiosulfate stimuli into permanent single-base mutations (ACG $\rightarrow$ ATG) in the genomic DNA. This activates reporter genes (e.g., LacZ) to create a genetic memory. An editing efficiency of 25.2% was verified in vitro through sequencing and colorimetric assays (e.g., X-Gal blue-spotting).

Therapeutic Module: Secretes the immunomodulatory protein AvCystatin (derived from parasites) using a hemolysin (Hly) secretion system for targeted release. Drug expression is regulated by thiosulfate concentration, avoiding the side effects of constitutive expression. The modular design allows for the replacement of sensing elements to adapt to different biomarkers.

(3) Experimental Conclusions

In Vitro Validation:

High Sensitivity & Specificity: i-ROBOT showed a significantly stronger response to thiosulfate than to other metabolites (sulfates, nitrites), with a detection limit of 5 $\mu$M.

Dual-Signal Output: sfGFP fluorescence intensity increased with thiosulfate concentration; base editing rates correlated positively with stimuli and were inheritable by daughter cells.

Controlled Drug Release: AvCystatin secretion scaled with thiosulfate levels, reaching up to 22 ng/mL.

In Vivo Mouse Model Validation:

In DSS-induced colitis models, i-ROBOT successfully detected inflammation severity via fecal flow cytometry and genomic sequencing. The editing signal remained traceable in the late stages of the disease, achieving historical event recording.

The i-ROBOT treatment group showed significantly reduced weight loss, increased colon length, and lower levels of inflammatory cytokines (e.g., TNFalpha, IL-6). Histopathology revealed improved epithelial integrity and reduced fibrosis.

System Stability: Continuous passage experiments confirmed that signal output and drug secretion remained stable for over 7 generations. In the complex gut environment, the engineered bacteria colonized for approximately 48 hours, requiring daily administration for sustained effect.

(4) Application Significance

Clinical Value: Provides a non-invasive diagnostic tool for IBD as an alternative to colonoscopy, suitable for long-term monitoring. The self-regulating drug release system enhances safety and minimizes side effects.

Technological Scalability: The modular design can be adapted to other disease markers (e.g., eATP, specific metabolites), extending its use to metabolic diseases and oncology.

Biosafety: The use of the probiotic EcN ensures high clinical safety. Future genomic integration can replace plasmid-based systems to further reduce the risk of horizontal gene transfer.

Limitations & Outlook: Current colonization is limited; optimization of "colonization strategies" (e.g., surface modification) is required. Further validation in large animal models is necessary to drive clinical translation.

8. References

[1] Tong, S. Y., et al. (2015). Staphylococcus aureus infections: Epidemiology, pathophysiology, clinical manifestations, and management. Clinical Microbiology Reviews.

[2] Bruckner, R. (1997). Gene replacement in Staphylococcus aureus via homologous recombination. Methods in Molecular Biology.

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

[4] Luo, M. L., et al. (2016). The CRISPR-Cas9 system as a tool for editing the genomes of Staphylococcus aureus. Applied and Environmental Microbiology.

[5] Zou ZP, et al. (2023). Biomarker-responsive engineered probiotic diagnoses, records, and ameliorates inflammatory bowel disease in mice. Cell Host Microbe.

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