Customized Gene Expression Engineered Bacteria
Customized Gene Expression Engineered Bacteria
1. Research Background
In the synthetic biology and biotechnology industries, constructing efficient and stable "cell factories" is the core of realizing large-scale production of biomolecules. Whether for heterologous expression of recombinant proteins, reconstruction of metabolic pathways, or development of complex biosensors, precise customized genomic modification is crucial. Against this background, Sutong Biotech launches customized gene expression engineered bacteria construction services. Using CRISPR/Cas9 and multi-fragment recombination technology, we achieve precise integration of target fragments, precise regulation of expression intensity, and optimization of metabolic flux in a variety of chassis microorganisms (e.g., Escherichia coli, Bacillus subtilis, yeasts, etc.), delivering high-yield and stable customized mutant strains.
2. Host Characteristics and Chassis Biological Background
(1) Chassis Cells Selection: Provide diverse chassis options according to expression requirements.
(2) Escherichia coli (E. coli): It has the clearest genetic background and rapid growth, suitable for the expression of most primary metabolites and proteins.
(3) Bacillus subtilis (B. subtilis): A Gram-positive bacterium with strong protein secretion capacity (GRAS safety level).
(4) Metabolic Regulation Logic: Optimize the nutrient utilization and energy distribution of host bacteria for specific production goals.
(5) Environmental Adaptability: Enhance the stability of strains in industrial fermenter environments (high osmotic pressure, heat stress) through gene editing.
3. Reported Engineering Strategies in Literature
To address the challenges of low expression efficiency and instability of foreign genes in host cells, Sutong Biotech adopts the following strategies:
3.1 CRISPR/Cas9 Site-Directed Integration (Mainstream Strategy)
(1) Principle: Utilize Cas9-mediated double-strand breaks (DSB) to induce homologous recombination.
(2) Advantage: Achieve efficient integration of foreign fragments at chromosomal "safe harbor" loci, avoiding decreased expression caused by plasmid loss.
3.2 Promoter/RBS Engineering
By constructing promoter libraries and RBS sequences of different strengths, precise regulation of the transcription and translation levels of target genes is achieved.
3.3 Codon Optimization and Metabolic Flux Balancing
Perform whole-gene optimization according to host preferences to balance the supply of precursor substances and the synthesis rate of target products.
3.4 High Secretory Signal Peptide Screening
For secretory proteins, screen the optimal signal peptides to ensure efficient transmembrane transport of proteins.
4. Core Application Areas
(1) Recombinant Protein Production: Construct engineered strains for high-yield enzymes, antibody fragments, and cytokines.
(2) Metabolic Engineering Modification: Reconstruct metabolic pathways through multi-gene integration to produce high-value-added chemicals (e.g., amino acids, organic acids).
(3) Live Bacterial Drug Delivery: Insert therapeutic genes into probiotics to achieve in situ intestinal expression and drug delivery.
(4) Chassis Cell Streamlining: Remove redundant metabolic burden through large-fragment deletion to construct "minimalist" and efficient chassis.
5. Project Workflow & Validation
Sutong Biotech provides one-stop services from sequence design to engineered bacteria delivery to ensure expression performance:
(1) Sequence Analysis & Optimization: Conduct codon optimization and expression system design.
(2) Vector Construction & Transformation: Prepare high-efficiency electrocompetent or chemically competent cells.
(3) Genomic Site-Directed Integration: Use CRISPR technology for precise "positioning" insertion.
(4) Multilevel Validation & Evaluation: Genotypic validation includes PCR identification and Sanger sequencing. Phenotypic validation detects expression yield through Western Blot, ELISA, or HPLC.
6. Introduction to Gene Editing Projects
6.1 Core Services
(1) High-Yield Strain Construction/Optimization: Achieve overexpression through strong promoter replacement and increased gene copy number.
(2) Site-Directed Gene Integration: Realize scarless and stable integration of foreign DNA into the genome.
(3) Multigene Co-Expression: Enable simultaneous and efficient expression of multiple functional subunits in a single strain.
6.2 Technical Advantages
(1) High Stability: Chromosomally integrated strains still maintain extremely high genetic stability under antibiotic-free conditions.
(2) Customized Solutions: Design secretion, intracellular expression, or surface display strategies according to the structure of the target protein.
(3) Full Closed-Loop Delivery: Provide sequencing reports, protein quantification results, and fermentation performance evaluation.
7. Case Studies
Case: Biomarker-Responsive Engineered Probiotic for Diagnosing, Recording, and Ameliorating Inflammatory Bowel Disease (IBD) in Mice
Project Description: Developed an intelligent engineered probiotic i-ROBOT that can non-invasively monitor, record, and ameliorate inflammatory bowel disease (IBD) in mice.
(1) Research Background
Inflammatory Bowel Disease (IBD) is a chronic gastrointestinal inflammatory disease with a continuously rising global incidence. Traditional diagnostic methods (such as colonoscopy) are invasive and difficult to monitor disease dynamics in real time. Engineered microorganisms provide a new idea for IBD diagnosis and treatment through synthetic biology technology, but existing tools have the following limitations: insufficient detection sensitivity, difficulty in capturing low-concentration biomarkers (such as thiosulfate); lack of historical recording ability, as traditional sensor signals disappear with the disappearance of biomarkers, making it impossible to record disease history; lack of adaptive treatment, most drug delivery systems are constitutive or inducible, and cannot adjust drug release in real time according to disease status. In this study, Escherichia coli Nissle 1917 (EcN) was used as the chassis to develop the i-ROBOT system, which integrates sensing, recording, and therapeutic modules, aiming to achieve full-process management of IBD.
(2) Scheme Design
The i-ROBOT system is based on modular design, including three core components:
Sensing Module: A two-component system ThsS/R was used to respond to the inflammatory biomarker thiosulfate, controlling the inducible promoter PphsA to drive the expression of downstream genes. The promoter and ribosome binding site (RBS) were optimized to achieve a detection sensitivity of 0.016 mM and an induction ratio as high as 81-fold.
Recording Module: A CRISPR-Cas9 base editing system (BE2) was used to convert thiosulfate stimulation into permanent single-base mutations (ACG→ATG) in genomic DNA, activating the expression of the reporter gene (such as LacZ), and realizing genetic memory. The editing efficiency was verified by sequencing and color reaction (such as X-Gal plate turning blue), and the in vitro editing efficiency could reach 25.2%.
Therapeutic Module: The immunomodulatory protein AvCystatin (derived from parasites) was secreted, and the hemolysin (Hly) secretion system was used to achieve targeted release. The expression of the drug was regulated by the concentration of thiosulfate, avoiding side effects caused by constitutive expression. The overall design of the system allows the adaptation to different biomarkers by replacing the sensing element, forming a universal platform.
(3) Experimental Conclusions
In Vitro Validation: High sensitivity and specificity were demonstrated. The i-ROBOT showed a significantly better response to thiosulfate compared to other metabolites (e.g., sulfate, nitrite), with a minimum detection limit of 5 μM. Dual signal output was achieved, where the fluorescence intensity of sfGFP increased with the concentration of thiosulfate. The base editing efficiency was positively correlated with the stimulation concentration, and the signal could be inherited by progeny cells. Controllable drug release was realized, and the secretion of AvCystatin increased with the concentration of thiosulfate, reaching a maximum of 22 ng/mL.
In Vivo Validation in Mouse Models: In the DSS-induced colitis model, the i-ROBOT successfully detected the severity of inflammation through fecal fluorescent flow cytometry and genomic sequencing. The editing signal was still traceable in the late stage of the disease, realizing the recording of historical events. In the i-ROBOT treatment group, the mice showed significantly reduced weight loss, increased colon length, and decreased levels of inflammatory cytokines (e.g., TNF-α, IL-6). Histopathological examination indicated improved integrity of the colonic epithelium and reduced fibrosis. Drug release was matched to the degree of inflammation, avoiding over-treatment.
System Stability: Serial passage experiments showed that the signal output and drug secretion capacity of the i-ROBOT remained stable for more than 7 generations. In the complex intestinal environment, the engineered bacteria colonized for approximately 48 hours, requiring daily administration to maintain the effect.
(4) Application Significance
Clinical Value: Provide a non-invasive IBD diagnostic tool to replace traditional colonoscopy, suitable for long-term monitoring. The self-regulating drug release system improves the safety of treatment and reduces side effects.
Technology Promotion: The modular design allows adaptation to other disease biomarkers (e.g., eATP, metabolites), extending to fields such as metabolic diseases and cancer. Combined with the genetic recording function of base editing, it provides a new tool for the dynamic research of chronic diseases.
Biosafety: Probiotic EcN was used as the chassis, which has high clinical safety; in the future, plasmid systems can be replaced by genomic integration to reduce the risk of gene transfer.
Limitations and Prospects: The colonization ability is limited, and strategies for bacterial colonization in the colon (e.g., surface modification) need to be optimized. Further validation of the effectiveness and safety in large animal models is required to promote clinical translation.
