Recombinant Lactobacillus reuteri Porphobilinogen deaminase (hemC)

What is Limosilactobacillus reuteri and why is it suitable for recombinant protein expression?

Limosilactobacillus reuteri (formerly Lactobacillus reuteri) is a gut symbiont species that has evolved to thrive in various vertebrate hosts, including humans. Its suitability for recombinant protein expression stems from several key characteristics:

• Demonstrated ability to survive gastrointestinal transit, making it an excellent candidate for delivery of therapeutics to the gut

• Availability of advanced genetic tools for manipulation, including recombineering techniques and CRISPR-Cas systems

• Multiple health-promoting characteristics that provide additional benefits beyond protein delivery

• Capacity to secrete therapeutic molecules efficiently, as demonstrated with anti-NetB nanobodies and murine IFN-β

• Genetic and phenotypic stability of recombinant strains over extended periods (>480 generations)

The species has been successfully exploited as a chassis to secrete various therapeutic molecules, establishing its credentials as a versatile delivery vehicle .

What is porphobilinogen deaminase (hemC) and why express it in L. reuteri?

Porphobilinogen deaminase (hemC) is an enzyme involved in tetrapyrrole biosynthesis. While traditionally expressed in systems like E. coli or yeast for high yields and rapid production cycles, expression in probiotic bacteria like L. reuteri offers unique advantages:

• In situ delivery capability via oral administration, avoiding the need for purification of the recombinant enzyme

• Protection of the enzyme during gastrointestinal transit through the bacterial cell envelope

• Potential for targeted delivery to specific gut regions through appropriate strain selection

• Possibility of combining hemC activity with intrinsic probiotic benefits of the L. reuteri carrier strain

Expression systems must be carefully selected based on requirements for post-translational modifications necessary for correct protein folding and enzymatic activity .

What genetic engineering tools are available for developing recombinant L. reuteri strains?

Several genetic tools have been developed specifically for L. reuteri engineering:

• Dual-recombineering schemes for efficient barcoding and gene modification

• Single-stranded DNA recombineering (SSDR) procedures, though efficacy varies between strains

• CRISPR-Cas systems, which are naturally abundant in lactobacilli (present in ~63% of sequenced genomes compared to 46% of total bacterial genomes)

• Combined CRISPR-SSDR approaches for simplified and accelerated development

The genetic toolbox for L. reuteri continues to expand, although researchers should note that transformation efficiencies can differ by as much as 100-fold between strains .

How should researchers select appropriate L. reuteri strains for hemC expression?

Strain selection is critical for successful hemC expression and depends on several factors:

Researchers should prioritize strains with established genetic tools, appropriate host adaptation for the target organism, and metabolic capabilities supportive of recombinant protein expression .

What expression systems optimize hemC production in L. reuteri?

While specific data on hemC expression in L. reuteri is limited, several strategies can be inferred from research on recombinant protein expression in lactic acid bacteria:

• Promoter selection is critical, with constitutive promoters (e.g., P11) useful for continuous expression and inducible systems for controlled production

• Signal peptide optimization can enhance secretion efficiency if extracellular delivery is desired

• Codon optimization should account for the specific codon usage bias of L. reuteri

• Integration site selection impacts expression levels and strain stability

For hemC specifically, researchers should consider whether the enzyme requires specific cofactors available in L. reuteri's intracellular environment to maintain functionality .

How can gene integration stability be assessed in recombinant L. reuteri?

Stability assessment is crucial for therapeutic applications and should include:

• Long-term culturing experiments: Recombinant L. reuteri strains have demonstrated genetic and phenotypic stability over 480 generations in previous studies

• Regular PCR verification of transgene presence during extended cultivation

• Enzyme activity assays to confirm continued functional expression

• Genome sequencing to identify any mutations or rearrangements affecting the expression cassette

• In vivo passage experiments to verify stability under physiological conditions

These assessments are especially important for strains intended for therapeutic applications to ensure consistent performance over time.

How can CRISPR-Cas systems enhance hemC expression in L. reuteri?

CRISPR-Cas systems offer several advantages for optimizing hemC expression in L. reuteri:

• Precise genomic integration of the hemC gene into optimal chromosomal locations

• Multiplexed modification of regulatory elements to enhance expression levels

• Knockout of competing metabolic pathways to redirect cellular resources

• Engineering of chaperone systems to improve protein folding efficiency

• Selection of low-efficiency recombinant genotypes using native Cas enzymes combined with user-defined CRISPR arrays

The natural abundance of CRISPR-Cas systems in Lactobacillus genomes (~63% compared to 46% in other bacteria) provides researchers with opportunities to utilize native systems rather than introducing heterologous components .

What methods can evaluate colonization potential of recombinant hemC-expressing L. reuteri?

Rigorous evaluation of colonization potential is essential, particularly for strains with reduced colonization desired for safety reasons:

• Competitive colonization experiments using strain mixtures with subsequent molecular typing (e.g., leuS gene sequencing) to determine which strains become enriched

• Quantitative culture on selective media from fecal samples following standardized dose administration

• Adhesion assays using human cell lines (HT-29) and enteroid models to assess attachment capabilities

• Systematic testing of adhesin gene knockouts to identify key proteins involved in colonization

• Animal studies comparing wild-type and engineered strains for persistence in different gastrointestinal regions

Research has identified CmbA as a key protein in L. reuteri adhesion to intestinal cells, with nonuple mutants (strains with nine adhesin genes inactivated) showing significantly reduced adhesion to enteroid monolayers .

How does the metabolic versatility of L. reuteri impact hemC expression?

Understanding metabolic capabilities is crucial for optimizing expression conditions:

• Genome-scale metabolic models (GEMs) have been developed for multiple L. reuteri strains, revealing strain-specific metabolic capabilities

• The GEM of L. reuteri ATCC PTA 6475 (iHL622) contains 894 reactions and 726 metabolites linked to 622 metabolic genes

• Core and pan models of L. reuteri strains show metabolic capacity differences both between and within host groups

• These models can help predict optimal carbon sources and amino acid supplementation for maximizing recombinant protein production

• Metabolic engineering can redirect carbon flux toward protein synthesis pathways

Researchers should leverage these metabolic insights to design optimal growth media and conditions for hemC expression .

What delivery strategies maximize efficacy of recombinant L. reuteri expressing hemC?

Effective delivery strategies should consider:

• Administration route: Options include oral, intranasal, in ovo (for poultry applications), or via drinking water

• Dosage and timing: Standardized doses with appropriate timing can significantly impact colonization success

• Formulation: Protection from stomach acid may be necessary depending on the strain's acid resistance

• Host microbiome status: Administration to germfree or antibiotic-treated hosts reduces colonization resistance

• Host specificity: Strains perform significantly better in hosts from which they were originally isolated

Research has demonstrated that a two-dose regimen (in ovo followed by drinking water administration) of recombinant L. reuteri was effective in poultry applications , suggesting similar approaches could be effective for hemC delivery.

How can host adaptation influence the efficacy of L. reuteri as a hemC delivery vehicle?

Host adaptation significantly impacts delivery efficacy:

• L. reuteri has evolved distinct genetic lineages corresponding to different host origins (rodents, humans, pigs, poultry)

• Experimental evidence confirms strains perform better in their original host species

• In competitive colonization experiments, rodent strains constituted 66-76% of recovered colonies from mice by day 5, significantly outcompeting strains from other lineages

• The host-specific adaptation involves differences in adhesion capabilities and metabolic functions

These findings indicate that researchers should select strains from the appropriate host-adapted lineage for the target organism to maximize efficacy of hemC delivery .

What safety considerations apply to recombinant L. reuteri strains engineered to express hemC?

Several safety considerations must be addressed:

• Colonization potential: Engineering strains with reduced colonization capabilities can mitigate risks associated with persistent genetically modified organisms

• Genetic containment: Systems preventing horizontal gene transfer should be incorporated

• Antibiotic resistance: Selection markers should be removed or replaced with food-grade alternatives

• Immunogenicity: The potential for immune responses to the recombinant protein must be assessed

• Unintended effects: Comprehensive characterization of metabolic changes resulting from genetic modification is essential

Recent advances have focused on creating engineered L. reuteri with reduced colonization potential while maintaining therapeutic efficacy, as demonstrated by a nonuple mutant strain (with nine adhesin genes inactivated) that maintained efficacy in mitigating radiation toxicity in mice despite reduced colonization capacity .

What approaches can address poor expression levels of hemC in L. reuteri?

When facing challenges with expression levels:

• Perform codon optimization specific to the L. reuteri strain being used

• Test multiple promoters with different strengths and induction characteristics

• Optimize the ribosome binding site sequence and spacing

• Consider fusion partners that may enhance stability or solubility

• Investigate metabolic bottlenecks using genome-scale metabolic models

• Experiment with different culture conditions (temperature, pH, carbon source)

Researchers should systematically evaluate these parameters, ideally using a design of experiments (DOE) approach to identify optimal combinations.

How can enzyme activity of L. reuteri-expressed hemC be maintained and assessed?

Maintaining and assessing enzyme activity requires:

• Validation that the expression system provides any necessary cofactors for hemC activity

• Optimization of culture conditions to favor proper protein folding

• Development of appropriate activity assays adapted for use with bacterial cultures

• Consideration of fusion tags that can be removed post-expression if they interfere with activity

• Comparison of activity in different cellular compartments (cytoplasmic, cell-wall associated, or secreted)

While specific data on hemC expression in L. reuteri is limited, general principles from recombinant protein expression suggest these approaches would be valuable for optimizing active enzyme production.