Recombinant Lactobacillus plantarum Ribose-phosphate pyrophosphokinase 1 (prs1)

What is Ribose-phosphate pyrophosphokinase 1 (prs1) and what is its primary function in metabolic pathways?

Ribose-phosphate pyrophosphokinase 1 (PRPS1/prs1) is an essential enzyme that catalyzes the phosphoribosylation of ribose 5-phosphate to 5-phosphoribosyl-1-pyrophosphate (PRPP). This reaction is a critical step in the de novo pathway for purine, pyrimidine, histidine, and tryptophan biosynthesis . The enzyme functions at a key metabolic intersection, connecting the pentose phosphate pathway with nucleotide synthesis pathways. Research has demonstrated that PRPS1 is expressed across various developmental stages in organisms and plays fundamental roles in cellular metabolism by providing the PRPP substrate necessary for nucleic acid synthesis .

The catalytic activity of PRPS1 makes it particularly important in rapidly dividing cells where nucleotide demand is high. In eukaryotic systems, PRPS1 has been associated with various physiological processes beyond basic metabolism, including potential roles in resistance mechanisms to environmental stressors, as evidenced by studies in insect models .

Why is Lactobacillus plantarum considered a suitable expression system for recombinant proteins?

Lactobacillus plantarum has emerged as a valuable expression system for recombinant proteins due to several advantageous characteristics:

• Mucosal delivery capability: L. plantarum can survive passage through the gastrointestinal tract, making it an excellent vector for delivering proteins to mucosal surfaces where they can stimulate local immune responses .

• Safety profile: L. plantarum has Generally Recognized As Safe (GRAS) status, minimizing regulatory concerns for research applications.

• Immunomodulatory properties: Beyond serving as a protein delivery vehicle, L. plantarum itself possesses immunomodulatory properties that can enhance immune responses to recombinant antigens .

• Versatile expression systems: Multiple genetic tools and vector systems have been developed specifically for L. plantarum, facilitating the expression of heterologous proteins.

Studies have demonstrated successful application of recombinant L. plantarum for expressing various antigens, including viral components like influenza virus antigen HA1, making it particularly valuable for vaccine research and immunological studies .

What expression vectors are commonly used for developing recombinant L. plantarum strains?

Researchers working with L. plantarum typically utilize several established expression vector systems, each with distinct advantages depending on research objectives:

• pSIP vectors: These inducible expression vectors contain signal peptides for protein secretion and are regulated by a quorum-sensing mechanism, allowing controlled expression.

• pWCF vectors: As demonstrated in research with influenza virus antigens, pWCF vectors can be effectively used for surface display or secretion of heterologous proteins in L. plantarum .

• pIB/V5-His expression vectors: While primarily designed for insect cell expression, modified versions have been adapted for lactic acid bacteria including L. plantarum, providing options for His-tagged protein expression and simplified purification .

When selecting an appropriate vector, researchers should consider:

• Required expression levels (constitutive vs. inducible)

• Protein localization needs (intracellular, secreted, or surface-anchored)

• The presence of affinity tags for downstream purification

• Antibiotic resistance markers compatible with L. plantarum

The choice of vector significantly impacts expression efficiency, protein localization, and ultimately the success of the recombinant protein production strategy.

How can expression levels of prs1 be optimized in recombinant L. plantarum?

Optimizing prs1 expression in L. plantarum requires a multifaceted approach addressing several key factors:

Codon Optimization Strategies:
Adapting the prs1 sequence to L. plantarum codon usage bias significantly improves translation efficiency. Studies with other recombinant proteins in lactic acid bacteria have shown 2-10 fold expression increases following codon optimization. Key considerations include:

• Avoiding rare codons in L. plantarum

• Optimizing the 5' region of the coding sequence

• Eliminating internal Shine-Dalgarno-like sequences that may interfere with translation

Promoter Selection:
The choice of promoter dramatically affects expression levels. Options include:

• Constitutive promoters (P23, P59) - providing continuous expression

• Inducible systems (nisin-controlled, sakacin-regulated) - allowing controlled expression timing

• Stress-responsive promoters - enabling environment-dependent expression

Signal Peptide Optimization:
For secreted prs1 constructs, signal peptide selection affects secretion efficiency. Common signal peptides used with L. plantarum include:

• Usp45 (from L. lactis)

• SlpA (from L. brevis)

• Lp_0373 (native to L. plantarum)

Experimental data from expression studies with similar-sized proteins suggest that Usp45 typically yields 1.5-3 times higher secretion efficiency compared to other signal peptides in L. plantarum systems .

What methodologies are most effective for evaluating immune responses to recombinant L. plantarum expressing heterologous proteins?

Assessment of immune responses to recombinant L. plantarum requires comprehensive evaluation across multiple immune parameters:

Humoral Immune Response Assessment:

• Quantification of serum antibodies (IgG, IgG1, IgG2a) through ELISA

• Functional antibody assays such as hemagglutination inhibition (HI) for influenza antigens

• B-cell population analysis in lymphoid tissues via flow cytometry (B220+IgA+ cells)

Cellular Immune Response Evaluation:

• Flow cytometric analysis of T-cell subsets (CD4+IFN-γ+ and CD8+IFN-γ+ cells)

• Lymphocyte proliferation assays upon antigen stimulation

• Cytokine profiling using ELISA or intracellular cytokine staining

Mucosal Immunity Assessment:

• Secretory IgA measurement in mucosal secretions (intestinal, respiratory)

• Analysis of immune cell populations in mucosal-associated lymphoid tissues

• Activation status of dendritic cells in Peyer's patches

Research with recombinant L. plantarum expressing influenza antigens demonstrated significant increases in:

• CD4+IFN-γ+ and CD8+IFN-γ+ cells in spleen and mesenteric lymph nodes

• B220+IgA+ cells in Peyer's patches

• IgA levels in lung and intestinal tissues

• Serum levels of specific IgG, IgG1, and IgG2a antibodies

These comprehensive assessment approaches provide insight into the multifaceted immune responses generated by recombinant L. plantarum constructs.

How does prs1 function influence nucleotide metabolism in bacterial expression systems?

PRPS1/prs1 functions at a critical metabolic junction, and its expression level can significantly impact nucleotide metabolism in bacterial systems:

Metabolic Impact of prs1 Expression:
PRPS1 catalyzes the synthesis of phosphoribosylpyrophosphate (PRPP), which serves as an essential precursor for:

• De novo purine synthesis

• De novo pyrimidine synthesis

• Histidine biosynthesis

• Tryptophan biosynthesis

When prs1 is overexpressed in bacterial systems, several effects may occur:

The ramifications of altered PRPP synthesis extend beyond simple nucleotide availability, potentially affecting:

• Cell wall synthesis through pentose phosphate pathway intermediates

• Energy metabolism through ATP consumption in PRPP synthesis

• Amino acid metabolism through histidine and tryptophan biosynthesis

Research in insect models has suggested that increased PRPS1 expression can support enhanced transcriptional responses, which may similarly apply to bacterial systems with elevated prs1 expression .

What are the optimal PCR and cloning strategies for isolating and inserting the prs1 gene into L. plantarum expression vectors?

Successful cloning of prs1 into L. plantarum expression vectors requires careful attention to several technical aspects:

PCR Amplification Strategy:
For efficient amplification of prs1, consider the following approach:

• Design primers with appropriate restriction sites compatible with the target vector

• Include a Kozak-like sequence (GAGATGG) before the start codon to enhance translation efficiency

• Consider adding two additional nucleotides after the Kozak sequence to maintain the reading frame

• Remove the stop codon if C-terminal fusion is planned

A successful primer design strategy based on similar gene cloning would include:

• Forward primer: 5′-GGACTAGTGAGATGGAAATG[prs1-specific sequence]-3′ (with SpeI site)

• Reverse primer: 5′-CCCTCGAG[prs1-specific sequence without stop codon]-3′ (with XhoI site)

Cloning Procedure:

• Digest both PCR product and vector with appropriate restriction enzymes (e.g., SpeI and XhoI)

• Purify digested products to remove enzymes and buffer components

• Ligate using T4 DNA ligase with an optimal insert:vector ratio (typically 3:1 to 5:1)

• Transform into an appropriate E. coli strain (e.g., TOP10) for plasmid propagation

• Screen transformants by colony PCR using vector-specific and gene-specific primers

• Sequence verify positive clones before transformation into L. plantarum

For vector selection, consider plasmids with strong constitutive promoters for metabolic enzymes like prs1, unless regulated expression is specifically required.

What methods are most effective for quantifying prs1 expression levels in recombinant L. plantarum?

Accurate quantification of prs1 expression levels is crucial for experimental consistency and data interpretation:

Transcript Level Quantification:
Real-time quantitative PCR (qPCR) provides precise measurement of prs1 mRNA levels:

• Extract total RNA using specialized kits for Gram-positive bacteria

• Synthesize cDNA using reverse transcriptase

• Perform qPCR with prs1-specific primers (product size ~150-200 bp is optimal)

• Normalize against a reference gene (β-actin or 16S rRNA) using the 2^(-ΔΔCT) method

Example primer design for prs1 qPCR:

• Forward: 5′-CAGCCTCGGCAAACATCAT-3′

• Reverse: 5′-ATTCTGACCCACGGCATCTT-3′
  (Product size: ~150-160 bp)

Protein Level Quantification:
Western blotting provides direct measurement of PRPS1 protein:

• Extract total protein from L. plantarum cultures

• Perform SDS-PAGE separation

• Transfer to membrane and probe with:

  • Anti-PRPS1 antibodies (if available)

  • Anti-tag antibodies (if fusion protein)

• Quantify band intensity relative to a loading control

Enzyme Activity Assay:
Functional quantification through PRPS1 enzyme activity:

• Prepare cell-free extracts from recombinant L. plantarum

• Measure PRPP formation through coupled enzymatic assays

• Calculate specific activity (μmol PRPP formed/min/mg protein)

This multi-level quantification approach (transcript, protein, activity) provides comprehensive assessment of prs1 expression and functionality in the recombinant system.

What strategies can minimize metabolic burden when expressing prs1 in L. plantarum?

Expressing metabolic enzymes like prs1 in bacterial systems can create significant metabolic burdens that must be carefully managed:

Expression System Optimization:

• Inducible promoters: Utilize inducible systems (nisin, sakacin) rather than constitutive promoters to control expression timing and level

• Balanced plasmid copy number: Choose medium to low copy number vectors to prevent excessive gene dosage

• Codon harmonization: Adjust codon usage to match L. plantarum's natural translation rhythm rather than simply using the most frequent codons

Culture Condition Strategies:

• Two-phase cultivation:

  • Initial growth phase: Focus on biomass accumulation (no induction)

  • Production phase: Induce expression under optimized conditions

• Nutrient supplementation: Provide additional nucleotide precursors to support increased PRPP demand:

  • Ribose or ribose-5-phosphate

  • Adenine and guanine bases

  • Amino acid supplements (particularly histidine and tryptophan)

• Environmental parameters:

  • Temperature reduction during expression phase (25-30°C)

  • pH control within optimal range for L. plantarum (5.5-6.5)

  • Controlled oxygen levels based on expression requirements

Genetic Background Considerations:
Consider using L. plantarum strains with enhanced metabolic capacity or deleted competing pathways that might divert metabolic flux away from desired processes.

These strategies can be implemented individually or in combination to minimize metabolic burden while maintaining adequate prs1 expression levels.

How can recombinant L. plantarum expressing prs1 be applied in metabolic engineering studies?

Recombinant L. plantarum expressing prs1 presents several valuable applications in metabolic engineering:

Nucleotide Production Enhancement:
By overexpressing prs1, researchers can create strains with enhanced nucleotide biosynthetic capacity, useful for:

• Improved DNA/RNA yield in molecular biology applications

• Enhanced nucleotide-derived metabolite production

• Increased resistance to nucleotide synthesis inhibitors

Metabolic Flux Redirection:
Modulating PRPP availability through prs1 expression can redirect carbon flux through different metabolic pathways:

• Increased flux toward nucleotide biosynthesis

• Enhanced production of histidine and tryptophan

• Altered pentose phosphate pathway utilization

Growth Optimization in Nutrient-Limited Conditions:
Enhanced prs1 expression can potentially improve growth under:

• Purine or pyrimidine limitation

• Amino acid-restricted conditions

• Environments requiring rapid adaptation to metabolic stress

Potential Applications in Resistance Studies:
Based on findings in insect models, where increased PRPS1 expression was associated with deltamethrin resistance, engineered L. plantarum could serve as a model system for studying metabolic aspects of resistance mechanisms . The enhanced nucleotide synthesis capacity may support upregulation of detoxification enzymes and stress response proteins.

What challenges exist in maintaining stable prs1 expression in L. plantarum over multiple generations?

Maintaining stable expression of heterologous genes like prs1 in L. plantarum presents several challenges that researchers must address:

Genetic Stability Challenges:

• Plasmid Loss Without Selection Pressure:

  • Plasmids carrying prs1 may be lost over multiple generations without continuous selective pressure

  • Strategies to address: Implement post-segregational killing systems or balanced-lethal systems for plasmid maintenance without antibiotics

• Recombination and Genetic Rearrangements:

  • Homologous regions may lead to recombination events

  • Direct repeats in expression cassettes can result in deletion of the inserted gene

  • Mitigation: Minimize sequence repeats and monitor genetic stability through regular sequencing

• Metabolic Burden Leading to Suppressor Mutations:

  • High prs1 expression may select for mutations reducing expression

  • Consequences: Gradual decline in expression levels over generations

  • Solution: Moderate expression levels and regular assessment of expression stability

Monitoring Strategies:

Stabilization Approaches:

• Chromosomal Integration:
  Consider integrating prs1 into the L. plantarum chromosome for enhanced stability, though typically at lower expression levels than plasmid-based systems

• Selective Medium Development:
  Design cultivation media that provide selective advantage to prs1-expressing cells, potentially leveraging the metabolic effects of enhanced PRPP production

• Strain Engineering:
  Develop host strains with reduced homologous recombination capacity or engineered dependency on the prs1 expression system

These approaches can be combined to develop stable expression systems suitable for long-term studies or applications of recombinant L. plantarum expressing prs1.

How should researchers address inconsistent or contradictory results when studying prs1 expression effects?

When encountering inconsistent results in prs1 expression studies, a systematic troubleshooting approach is essential:

Experimental Variability Assessment:

• Expression Level Verification:

  • Quantify prs1 expression across experimental replicates using qPCR and Western blotting

  • Determine whether inconsistencies correlate with expression level variations

  • Standardize induction parameters and harvest timing to minimize variability

• Growth Condition Documentation:

  • Maintain detailed records of media composition, including lot numbers

  • Monitor and record growth parameters (OD, pH, temperature) throughout experiments

  • Standardize inoculum preparation and growth phase at harvest

• Strain Stability Verification:

  • Sequence verify plasmids from inconsistent experimental samples

  • Screen for spontaneous mutations affecting expression or function

  • Assess plasmid copy number variation across replicates

Data Analysis Approaches for Conflicting Results:

• Multivariate Analysis:
  Apply principal component analysis (PCA) or partial least squares discriminant analysis (PLS-DA) to identify variables contributing to experimental inconsistency

• Meta-Analysis Techniques:

  • Forest plots to visualize effect sizes across experiments

  • Random-effects models to account for between-study heterogeneity

  • Subgroup analysis to identify conditions under which effects are consistent

• Bayesian Analysis Framework:
  Implement Bayesian approaches to incorporate prior knowledge and uncertainty when interpreting contradictory results

Resolution Strategies:

• Independent Validation:

  • Develop alternative assays measuring the same parameters

  • Use complementary techniques to verify observations

  • Consider collaborations for external validation

• Hypothesis Refinement:

  • Revisit initial assumptions about prs1 function

  • Consider context-dependent effects based on metabolic state

  • Develop testable models explaining apparently contradictory results

This structured approach enables researchers to distinguish between technical variability and genuine biological phenomena when studying complex metabolic effects of prs1 expression.

What statistical approaches are recommended for analyzing variable prs1 expression data from L. plantarum cultures?

Statistical analysis of prs1 expression data requires consideration of the biological variability inherent in bacterial cultures:

Recommended Statistical Frameworks:

• Data Normalization Approaches:

  • Quantile normalization for high-throughput expression data

  • Log transformation to address skewed distributions

  • Normalization to multiple reference genes for qPCR data using geometric averaging

• Statistical Tests for Comparative Analysis:

  • Student's t-test for simple two-group comparisons with normally distributed data

  • ANOVA with appropriate post-hoc tests for multi-group comparisons

  • Non-parametric alternatives (Mann-Whitney, Kruskal-Wallis) for non-normal distributions

• Advanced Modeling Approaches:

  • Mixed-effects models to account for batch variation and repeated measures

  • Regression analysis to identify factors influencing expression levels

  • Time-series analysis for expression dynamics over growth periods

Sample Size and Power Considerations:

Practical Implementation:

• Software Recommendations:

  • R with specialized packages (limma, DESeq2, edgeR) for expression data

  • GraphPad Prism for accessible statistical analysis and visualization

  • SPSS or SAS for complex experimental designs

• Reporting Standards:

  • Always include measures of central tendency AND dispersion (mean ± SD or median with IQR)

  • Report exact p-values rather than significance thresholds

  • Provide transparency about outlier handling and data exclusions

• Visualization Approaches:

  • Box plots showing distribution characteristics

  • Scatter plots with individual data points for small sample sizes

  • Heat maps for visualizing patterns across multiple experimental conditions

What are the future research directions for recombinant L. plantarum expressing prs1?

The investigation of recombinant L. plantarum expressing prs1 opens several promising research avenues:

Metabolic Engineering Applications:

• Development of L. plantarum strains with enhanced nucleotide production capacity for biotechnological applications

• Creation of probiotic strains with improved stress resistance through modulated nucleotide metabolism

• Engineering strains with enhanced capacity for synthesis of secondary metabolites dependent on PRPP availability

Immunological Research:

• Investigation of potential adjuvant effects associated with altered bacterial metabolism

• Studies on how metabolically engineered L. plantarum might influence host-microbe interactions

• Development of novel mucosal vaccine delivery systems leveraging metabolically optimized L. plantarum

Fundamental Research:

• Elucidation of the complete metabolic impact of prs1 overexpression using systems biology approaches

• Comparative studies across different Lactobacillus species to identify species-specific effects

• Investigation of potential resistance mechanisms associated with enhanced nucleotide synthesis capacity

Future studies would benefit from integrating multiple omics approaches (transcriptomics, proteomics, metabolomics) to fully characterize the systemic effects of prs1 expression in L. plantarum and its potential applications in biotechnology and health research.

How does current research on recombinant L. plantarum compare with other bacterial expression systems for heterologous protein production?

L. plantarum offers distinct advantages and limitations compared to other bacterial expression systems:

Comparative Analysis of Expression Systems:

Unique Advantages of L. plantarum:

• Survival through GI tract enabling mucosal delivery applications

• Natural immunomodulatory properties that can enhance responses to heterologous antigens

• Ability to deliver proteins to mucosal surfaces while minimizing systemic exposure

• Potential for use as a live vector in food and feed applications due to GRAS status

Current Limitations:

• Lower protein yields compared to industrial E. coli systems

• More limited genetic toolkit compared to model organisms

• Variable expression levels requiring optimization for each protein

• Challenges in large-scale cultivation compared to industrial production strains