Recombinant Lactobacillus johnsonii Thymidylate synthase (thyA)

What is the role of thymidylate synthase (thyA) in Lactobacillus metabolism?

Thymidylate synthase (thyA) catalyzes the conversion of dUMP to dTMP, which represents the key step in the de novo biosynthesis of thymidine tri-phosphate (dTTP). This methylation reaction requires methylene-tetrahydrofolate as a methyl donor. In lactic acid bacteria, the thyA gene encodes this essential enzyme required for DNA synthesis and cellular replication .

Methodological approach: When investigating thyA function in L. johnsonii, researchers should:

• Perform comparative genomic analysis with well-characterized thyA genes from related species like L. lactis

• Conduct growth experiments in minimal media with and without thymidine supplementation

• Measure enzyme activity using purified recombinant protein with spectrophotometric assays that monitor the conversion of dUMP to dTMP

The L. lactis thyA gene has been cloned and characterized, with the corresponding enzyme purified and studied in detail. This provides a valuable model for understanding the L. johnsonii enzyme .

How does thyA function as a biological containment system in recombinant lactic acid bacteria?

Thymidylate synthase can serve as an effective biological containment strategy for genetically modified organisms (GMOs). This approach was successfully demonstrated in L. lactis, where researchers exchanged the chromosomal thyA gene with a human IL-10 gene. This genetic modification created a strain that was critically dependent on external thymine or thymidine for survival .

Methodological approach for implementing thyA-based containment in L. johnsonii:

• Create a precise deletion of the chromosomal thyA gene using CRISPR-Cas9 or homologous recombination

• Replace the thyA gene with your gene of interest

• Verify thymine/thymidine dependency through growth assays in defined media

• Confirm environmental safety by testing survival in ecological conditions lacking thymine

This containment system allows the recombinant strain to survive inside the human body (where thymine/thymidine is available) but prevents environmental spread once excreted .

What expression systems provide optimal heterologous protein production in thyA-modified L. johnsonii?

Several expression systems have been optimized for Lactobacillus strains, with modifications that can be adapted for thyA-engineered L. johnsonii:

Methodological approach:

• Select an appropriate promoter system based on desired expression characteristics

• Engineer constructs with optimal codon usage for L. johnsonii

• Include appropriate secretion signals if extracellular protein production is desired

• Test multiple clones and expression conditions to identify optimal parameters

How can researchers troubleshoot growth limitations in thyA-deficient L. johnsonii strains?

ThyA-deficient strains often encounter growth challenges that require systematic troubleshooting:

Methodological approach:

• Media optimization: Supplement growth media with precise amounts of thymine or thymidine (typically 50-100 μg/mL)

• Growth conditions: Monitor growth curves at different temperatures (30-37°C) and pH values (5.5-6.5)

• Genetic stability: Regularly sequence the modified region to detect potential suppressor mutations

• Metabolic burden: Adjust expression levels of heterologous proteins if growth defects persist despite thymine supplementation

If growth remains suboptimal, investigate the potential interconnection between thyA deficiency and other metabolic pathways. Evidence indicates pyrimidine metabolism in lactic acid bacteria is regulated through an attenuator mechanism involving PyrR protein bound to UMP .

What analytical methods best quantify thyA expression and activity in recombinant strains?

For comprehensive characterization of thyA expression and activity:

• Transcriptional analysis:

  • RT-qPCR to measure thyA mRNA levels

  • RNA-seq for global transcriptional responses

• Protein quantification:

  • Western blotting with specific antibodies

  • Mass spectrometry for absolute quantification

• Enzymatic activity:

  • Spectrophotometric assays measuring the conversion of dUMP to dTMP

  • Tritium release assays using [5-3H]dUMP as substrate

• Growth phenotyping:

  • Thymine/thymidine dependency assays

  • Minimal inhibitory concentration determination for thymine analogs

How might thyA-modified L. johnsonii be developed as a therapeutic delivery system for Type 1 diabetes?

L. johnsonii N6.2 has demonstrated significant potential in Type 1 diabetes (T1D) applications. This strain mitigates T1D onset in Biobreeding Diabetes-Prone (BBDP) rats by improving epithelial barrier function, increasing tight junction protein expression, enhancing mucus production, and decreasing intestinal oxidative stress .

Methodological approach for developing thyA-modified L. johnsonii as a T1D therapeutic:

• Strain selection: Use the well-studied L. johnsonii N6.2 strain as the chassis

• Genetic modification:

  • Replace thyA with therapeutic genes that complement its natural immunomodulatory properties

  • Engineer constructs to express proteins that modulate the Th17/Treg balance

  • Consider IL-10 expression (as demonstrated with L. lactis)

• Preclinical testing:

  • Evaluate in BBDP rat models

  • Monitor tryptophan:kynurenine ratios as a biomarker

  • Assess dendritic cell phenotypes and T cell polarization

• Clinical translation:

  • A Phase 2 clinical trial (NCT03961347) is currently evaluating L. johnsonii N6.2 supplementation in adults with T1D

  • Study endpoints include safety, tolerability, and immunological responses

Recent findings show that L. johnsonii N6.2 administration increases tryptophan levels, which correlates positively with Lactobacillus counts. Clinical data demonstrates increased monocytes, NK cells, CD4+ T cells, and serum IgA levels following L. johnsonii N6.2 supplementation .

What are the current research findings on recombinant Lactobacillus strains for immunomodulation?

Recombinant Lactobacillus strains have demonstrated significant potential for immunomodulatory applications:

• L. lactis expressing anti-inflammatory proteins:

  • Superoxide dismutase (SOD) from B. subtilis reduced inflammation in TNBS models

  • 15-lipoxygenase-1 (15-LOX-1) was effective in treating DSS-induced colitis

  • Anti-TNFα antibodies reduced inflammation in mouse models

  • TGF-β expression ameliorated clinical symptoms like weight loss and diarrhea

• IL-10 expressing strains:

  • L. lactis producing IL-10 showed promising results in preclinical models

  • A clinical trial with thyA-replaced L. lactis expressing human IL-10 was conducted for Crohn's disease

  • The biological containment strategy using thyA replacement allowed for regulatory approval

Methodological approach for immunomodulation studies:

• Select target cytokines or immunomodulatory molecules based on disease mechanism

• Design precise genetic modifications using thyA as a containment system

• Validate expression and bioactivity in vitro

• Test efficacy in appropriate animal models before clinical translation

What advances in thyA-based containment systems could improve recombinant L. johnsonii safety?

Future enhancements to thyA-based containment systems should focus on:

• Redundant containment mechanisms:

  • Combine thyA deletion with additional auxotrophies

  • Implement toxin-antitoxin systems as secondary containment

• Inducible thyA expression:

  • Develop systems where thyA is under strict environmental control

  • Create conditional expression systems that respond to gut-specific signals

• Enhanced detection methods:

  • Develop rapid assays to monitor containment efficacy

  • Implement real-time detection of potential containment failures

• Regulatory considerations:

  • Generate standardized safety assessment protocols

  • Establish clear guidelines for clinical application of thyA-modified strains

These advances would build upon the successful thyA-based containment strategy demonstrated in L. lactis, where the gene encoding thymidylate synthase was exchanged for the human IL-10 gene, making the strain dependent on thymine or thymidine that was artificially provided in culture medium .

How can researchers effectively study the systemic distribution of molecules produced by thyA-modified L. johnsonii?

To track the systemic distribution of molecules produced by recombinant L. johnsonii:

Methodological approach:

• Biomarker identification:

  • Characterize nanovesicles (NV) derived from L. johnsonii as potential biomarkers

  • Identify specific components that may serve as tracers in host-microbe interactions

• Advanced imaging techniques:

  • Use fluorescently labeled molecules or bacteria

  • Implement intravital microscopy for real-time tracking

• Molecular tracing:

  • Develop isotope labeling strategies for metabolites

  • Employ next-generation sequencing to trace bacterial DNA/RNA

• Omics approaches:

  • Utilize metabolomics to track bacterial-derived molecules in host circulation

  • Apply proteomics to identify bacterial proteins in host tissues

Research has shown that L. johnsonii N6.2 produces bioactive components that stimulate the innate immune response, including activation of TLR7 and TLR9. These molecules and their distribution can serve as indicators of bacterial activity in the host .