Recombinant Lactobacillus plantarum Xaa-Pro dipeptidyl-peptidase (pepX), partial

What is Xaa-Pro dipeptidyl-peptidase (pepX) and what is its role in Lactobacillus species?

Xaa-Pro dipeptidyl-peptidase (PepX) is a proline-specific peptidase that hydrolyzes dipeptides with proline in the second position. In Lactobacillus species, PepX plays a role in the proteolytic system, particularly in the breakdown of proline-rich peptides. The enzyme shows varying degrees of conservation across Lactobacillus species, with significant differences in sequence identity. For example, L. rhamnosus PepX shows only 40% and 39% identity with PepXs from L. helveticus and L. delbrueckii, respectively, which is only slightly higher than the 36% identity observed between L. rhamnosus and Lactococcus PepX proteins . This variation suggests species-specific adaptations of this enzyme across different bacterial hosts.

How has the pepX gene been characterized in different Lactobacillus species?

The pepX gene has been characterized through various molecular techniques including cloning, expression analysis, and gene inactivation studies. In L. rhamnosus, for instance, researchers have identified that pepX is part of a polycistronic transcript that includes glnRA, which is significant as it represents the first reported case of cotranscription of glnA with a downstream gene in gram-positive bacteria . Characterization typically involves:

• Genomic library construction in E. coli

• Screening for PepX activity using chromogenic substrates like Gly-Pro-pNA

• Sequence analysis to determine conservation levels across species

• Northern blot analysis to study transcriptional patterns

• Gene inactivation through deletion mutations to assess functional significance

In L. rhamnosus specifically, pepX inactivation resulted in complete loss of Gly-Pro-pNA-hydrolyzing activity, confirming its enzymatic role .

What expression systems are available for recombinant production of pepX in L. plantarum?

Several expression systems are available for recombinant protein production in L. plantarum, with the pSIP system being one of the most commonly used. This system has been successfully employed for expressing various proteins, including membrane proteins . For pepX expression specifically, potential systems include:

The choice of expression system depends on research goals - whether focused on protein characterization, enzymatic studies, or immunological applications. The pSIP system has demonstrated effectiveness in producing approximately 1 mg of pure protein per 3 g of wet-weight cell pellet for membrane proteins in L. plantarum, suggesting its potential utility for pepX expression .

How should one design constructs for optimal pepX expression in L. plantarum?

Designing optimal constructs for pepX expression in L. plantarum requires strategic consideration of several factors:

• Codon optimization: Adapt the pepX gene sequence to the codon usage bias of L. plantarum to enhance translation efficiency. This is particularly important when expressing genes from distantly related organisms.

• Promoter selection: Choose appropriate promoters based on expression goals:

  • For constitutive expression: Use strong constitutive promoters like P23

  • For inducible expression: The sakacin-based promoters in pSIP vectors allow controlled induction

• Signal sequence consideration: Include appropriate signal sequences if secretion or membrane anchoring is desired. The Usp45 signal sequence from Lactococcus lactis is commonly used.

• Fusion partners and tags: Consider adding:

  • Affinity tags (His6) for purification

  • Fluorescent protein fusions for localization studies

  • DC-targeting peptides for enhanced immunogenicity when developing vaccine vectors

• Terminator sequences: Include efficient transcriptional terminators to prevent read-through and ensure mRNA stability

For experimental validation, restriction enzyme analysis and PCR amplification using specific primers can confirm correct construct assembly, similar to the validation approaches used with other recombinant L. plantarum constructs .

What are the critical factors affecting the successful purification of recombinant pepX from L. plantarum?

Successful purification of recombinant pepX from L. plantarum depends on multiple critical factors:

• Cell disruption method: L. plantarum has a thick cell wall that requires efficient disruption techniques. Options include:

  • Mechanical disruption (French press, bead beating)

  • Enzymatic lysis with lysozyme combined with sonication

  • Multiple freeze-thaw cycles in appropriate buffer systems

• Buffer composition: Optimize buffer conditions to maintain protein stability:

  • pH maintenance (typically pH 7.0-8.0)

  • Inclusion of protease inhibitors to prevent degradation

  • Addition of stabilizing agents (glycerol 10-20%)

• Purification strategy: A multi-step approach is recommended:

  • Initial capture: Immobilized metal affinity chromatography (IMAC) for His-tagged proteins

  • Intermediate purification: Ion exchange chromatography based on pepX's predicted isoelectric point

  • Polishing: Size-exclusion chromatography to ensure homogeneity

This approach has proven effective for membrane proteins expressed in L. plantarum, yielding high purity compared to E. coli expression systems . For pepX specifically, activity assays using chromogenic substrates like Gly-Pro-pNA can be used to track purification efficiency .

How does one optimize culture conditions for maximal pepX expression in recombinant L. plantarum?

Optimizing culture conditions for maximal pepX expression requires systematic evaluation of multiple parameters:

For inducible systems like pSIP, the timing of induction is critical - typically, induction during mid-logarithmic phase (OD600 of 0.3-0.6) yields optimal results. When using constitutive promoters, harvest time optimization becomes even more important.

Additionally, anaerobic or microaerophilic growth conditions may improve expression levels in L. plantarum compared to aerobic conditions. Supplementation with specific amino acids, particularly proline, may also enhance pepX expression due to its substrate specificity .

What enzymatic assays are most suitable for characterizing recombinant pepX activity?

Several enzymatic assays can effectively characterize recombinant pepX activity, with selection depending on specific research questions:

• Chromogenic substrate assays:

  • Gly-Pro-pNA hydrolysis is the gold standard for PepX activity detection

  • Measurement at 410 nm to detect released p-nitroaniline

  • Allows for kinetic studies (Km, Vmax determination)

• Fluorogenic peptide substrates:

  • Substrates like Gly-Pro-AMC offer higher sensitivity

  • Useful for detecting low enzyme concentrations

  • Requires fluorescence detection capabilities

• HPLC-based assays:

  • Analysis of dipeptide hydrolysis from longer peptides

  • Provides insight into substrate preference beyond model substrates

  • Allows physiologically relevant substrate testing

• Coupled enzyme assays:

  • Linking PepX activity to secondary reactions with spectrophotometric readouts

  • Useful for continuous monitoring in complex mixtures

The absence of Gly-Pro-pNA-hydrolyzing activity in PepX deficiency mutants confirms this assay's specificity for PepX function, making it particularly reliable for recombinant enzyme characterization .

How can one assess the impact of pepX expression on L. plantarum physiology and metabolism?

Assessing the impact of pepX expression on L. plantarum physiology and metabolism requires multi-faceted approaches:

• Growth kinetics analysis:

  • Compare growth rates of wild-type, pepX-deficient, and recombinant pepX-overexpressing strains

  • Measure in different media conditions (complex vs. defined media)

  • Assess acid production rates through pH monitoring

• Metabolomic profiling:

  • NMR or LC-MS/MS analysis of cellular metabolites

  • Focus on amino acid and peptide profiles

  • Comparative analysis between wild-type and modified strains

• Transcriptomic analysis:

  • RNA-Seq to identify differentially expressed genes

  • RT-qPCR validation of key pathway components

  • Special attention to nitrogen metabolism and proteolytic system genes

• Proteolytic capability assessment:

  • Growth on media with different protein/peptide sources

  • Analysis of peptide utilization profiles

  • Milk coagulation tests if relevant to application

It's noteworthy that in L. rhamnosus, pepX deficiency did not affect growth rate or acid production in MRS or milk, suggesting possible functional redundancy in the proteolytic system in some Lactobacillus species . Similar studies in L. plantarum would be valuable for understanding pepX's specific role in this organism.

What bioinformatic approaches help identify potential physiological substrates for pepX in L. plantarum?

Several bioinformatic approaches can help identify potential physiological substrates for pepX in L. plantarum:

• Sequence-based substrate prediction:

  • Analysis of known protein sequences in L. plantarum for Xaa-Pro motifs

  • Ranking of potential substrates based on accessibility of target bonds

  • Integration of structural information where available

• Comparative genomic analysis:

  • Identification of conserved peptides/proteins across Lactobacillus species

  • Correlation with pepX conservation patterns

  • Evolutionary analysis of substrate-enzyme co-evolution

• Protein-protein interaction network analysis:

  • Prediction of functional associations between pepX and other proteins

  • Integration of experimental interactome data where available

  • Pathway enrichment analysis for potential substrates

• Machine learning approaches:

  • Training models on known substrates from related enzymes

  • Feature extraction from peptide sequences

  • Validation through targeted enzymatic assays

• Structural modeling and docking:

  • Homology modeling of pepX based on crystallized homologs

  • Virtual screening of peptide libraries

  • Molecular dynamics simulations to assess binding energetics

These approaches generate testable hypotheses about physiological substrates that can then be validated experimentally through targeted enzymatic assays or peptidomic analyses comparing wild-type and pepX-deficient strains.

How does recombinant L. plantarum expressing pepX influence gut microbiota composition?

Recombinant L. plantarum strains can significantly influence gut microbiota composition, though specific effects of pepX expression remain to be fully characterized. Based on studies with other recombinant L. plantarum strains, several patterns may be anticipated:

• Diversity impacts:

  • Recombinant L. plantarum strains have been shown to increase species diversity in gut bacteria based on Shannon-Wiener index measurements

  • Beta diversity analysis demonstrates that microbial community structure can be altered following administration

• Functional shifts:

  • Enhanced metabolism-related functions in gut microbiota

  • Improved immune regulation capabilities

• Species-specific effects:

  • Differential impacts on various bacterial phyla

  • Potential increases in beneficial microbes

  • Possible competitive inhibition of pathogenic species

• Persistence factors:

  • Duration of recombinant strain colonization impacts long-term microbiota changes

  • Expression of specific proteins like pepX may contribute to adaptation and persistence in the gut environment

What immune responses are triggered by recombinant L. plantarum expressing pepX?

While specific immune responses to pepX-expressing L. plantarum require direct experimental investigation, insights can be drawn from studies of other recombinant L. plantarum strains:

• Humoral immunity:

  • Increased levels of serum IgG and IgG1 antibodies

  • Enhanced mucosal immunity with elevated sIgA in intestinal secretions

  • Potential for pepX-specific antibody development

• Cellular immunity:

  • Activation of dendritic cells in Peyer's patches

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

  • Enhanced proliferation capability of CD4+ and CD8+ cells

• Mucosal immune system effects:

  • Increased B220+IgA+ cells in Peyer's patches

  • Elevated IgA levels in lungs and different intestinal segments

  • Local immune modulation at mucosal surfaces

• Antigen-presenting cell activation:

  • Upregulation of co-stimulatory molecules on DCs

  • Enhanced cytokine production

  • Improved antigen processing and presentation

The immunomodulatory effects of recombinant L. plantarum appear to involve both innate and adaptive immune responses, with particular enhancement of mucosal immunity. This makes these strains particularly valuable for vaccine delivery applications .

How can recombinant L. plantarum pepX be utilized in vaccine development?

Recombinant L. plantarum expressing pepX could be utilized in vaccine development through several strategic approaches:

• Adjuvant properties:

  • pepX could serve as an immunomodulatory adjuvant when co-expressed with vaccine antigens

  • Its proteolytic activity might enhance antigen processing

  • Potential for synergistic effects with other adjuvants

• Antigen delivery platform design:

  • Construction of fusion proteins combining pepX with target antigens

  • pepX could potentially be fused with dendritic cell-targeting peptides (DCpep) to enhance immunogenicity

  • Expression as surface-anchored or secreted protein depending on desired immune response

• Vaccination protocols:

  • Oral administration schedules typically involve multiple doses (e.g., primary immunization on days 1-3, followed by boosters on days 10-12 and 21-23)

  • Dosage optimization (typical effective doses around 1 × 10^9 CFU)

  • Route of administration considerations (oral vs. intranasal)

• Immune response assessment:

  • Measurement of serum antibodies (IgG, IgG1, IgG2a)

  • Detection of mucosal antibodies (sIgA) in feces and at mucosal surfaces

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

  • Analysis of T cell responses through flow cytometry

This approach has proven successful with other recombinant L. plantarum strains expressing viral antigens like influenza HA1, where significant increases in specific antibodies and protective immunity were observed following oral administration .

What are the common challenges in expressing functional pepX in L. plantarum and how can they be overcome?

Several challenges may arise when expressing functional pepX in L. plantarum, each requiring specific troubleshooting approaches:

• Low expression levels:

  • Challenge: Insufficient protein production for detection or purification

  • Solutions:

    • Optimize codon usage for L. plantarum

    • Test different promoter systems (constitutive vs. inducible)

    • Adjust induction parameters (timing, concentration)

    • Incorporate signal peptides optimized for L. plantarum

• Protein misfolding:

  • Challenge: Expression of non-functional protein due to improper folding

  • Solutions:

    • Reduce expression rate to allow proper folding

    • Include molecular chaperones as co-expression partners

    • Optimize growth temperature (often lower temperatures improve folding)

    • Add stabilizing agents to growth media

• Proteolytic degradation:

  • Challenge: Rapid degradation of recombinant pepX

  • Solutions:

    • Use protease-deficient L. plantarum strains

    • Incorporate protease inhibitors during extraction

    • Express as fusion with stabilizing protein partners

    • Optimize harvest timing to capture maximum yield

• Loss of plasmid stability:

  • Challenge: Plasmid loss during prolonged cultivation

  • Solutions:

    • Maintain selective pressure throughout culture

    • Use food-grade selection systems for long-term applications

    • Consider chromosomal integration for stable expression

    • Monitor plasmid retention through regular plating checks

Comparative studies have shown that L. plantarum can sometimes provide advantages over E. coli for certain proteins, including better folding and higher purity of the final product .

How can researchers validate the structural integrity of purified recombinant pepX?

Validating the structural integrity of purified recombinant pepX involves multiple complementary techniques:

• Spectroscopic methods:

  • Circular dichroism (CD) spectroscopy to assess secondary structure content

  • Fluorescence spectroscopy to evaluate tertiary structure integrity

  • FTIR for additional structural information

  CD spectroscopy has proven effective for confirming structural integrity of proteins expressed in L. plantarum .

• Activity-based validation:

  • Enzymatic assays using synthetic substrates (Gly-Pro-pNA)

  • Kinetic parameter determination (Km, Vmax)

  • Comparison with native enzyme parameters where available

  • Temperature and pH stability profiles

• Biophysical characterization:

  • Size-exclusion chromatography to assess oligomeric state

  • Dynamic light scattering for homogeneity assessment

  • Thermal shift assays to determine stability

  • Limited proteolysis to probe domain structure

• Structural biology approaches:

  • X-ray crystallography for definitive structural validation

  • Cryo-EM for larger assemblies

  • NMR for dynamic information

• Mass spectrometry:

  • Intact mass analysis to confirm correct translation

  • Peptide mapping to verify sequence coverage

  • HDX-MS to probe solution dynamics and solvent accessibility

A combination of these approaches provides comprehensive validation of structural integrity, with functional enzymatic assays being particularly important for confirming that the recombinant pepX retains its native activity.

What strategies can improve pepX stability and activity in different experimental conditions?

Improving pepX stability and activity across various experimental conditions requires tailored stabilization strategies:

• Buffer optimization:

  • Systematic screening of buffer components:

    • pH range (typically 6.0-8.0)

    • Salt concentration (50-300 mM)

    • Addition of divalent cations if required for activity

  • Inclusion of stabilizing agents:

    • Glycerol (10-20%)

    • Reducing agents (DTT, β-mercaptoethanol) if disulfide bonds are present

    • Specific substrate analogs as stabilizers

• Protein engineering approaches:

  • Targeted mutagenesis based on sequence alignments with thermostable homologs

  • Disulfide bond introduction to enhance conformational stability

  • Surface charge optimization to improve solubility

  • Domain fusion with stability-enhancing protein partners

• Formulation strategies:

  • Lyophilization with appropriate cryoprotectants

  • Immobilization on solid supports for enhanced stability

  • Encapsulation in nanoparticles or liposomes

  • Addition of non-ionic detergents for membrane-associated forms

• Storage conditions:

  • Optimal temperature determination (-80°C, -20°C, 4°C)

  • Effect of freeze-thaw cycles on activity

  • Addition of protease inhibitors for long-term storage

  • Aliquoting to minimize repeated freeze-thaw cycles

Stability assessments should include both activity measurements and structural characterization techniques over time under various storage and experimental conditions. This approach has been successful with other proteins expressed in L. plantarum systems, resulting in functional proteins with maintained structural integrity .

What are the emerging applications of recombinant L. plantarum expressing pepX in gut health research?

Several promising research directions are emerging for recombinant L. plantarum expressing pepX in gut health:

• Microbiome modulation:

  • Investigation of pepX's role in processing bioactive peptides in the gut

  • Effects on microbiome composition and metabolic functions

  • Potential prebiotic-like activities through specific peptide generation

  • Cross-feeding interactions with beneficial gut bacteria

• Intestinal barrier function:

  • Impact on tight junction protein expression and integrity

  • Role in processing peptides that affect epithelial cell function

  • Potential therapeutic applications in conditions with compromised barrier function

  • Interactions with mucus layer and associated proteins

• Immunomodulatory applications:

  • Development of pepX-expressing strains as adjuvants for oral vaccines

  • Investigation of tolerogenic vs. immunogenic effects based on expression levels

  • Potential applications in autoimmune and inflammatory conditions

  • Interaction with gut-associated lymphoid tissue (GALT)

• Bioactive peptide generation:

  • Targeted release of bioactive peptides from dietary proteins

  • Engineering pepX variants with modified substrate specificity

  • In situ generation of health-promoting peptides

  • Synergistic effects with other peptidases

These applications build upon observed effects of other recombinant L. plantarum strains on gut microbiota and immune function , with pepX offering unique capabilities through its specific proteolytic activity.

How might genetic engineering of pepX improve its applications in biotechnology?

Genetic engineering of pepX offers several avenues for enhancing its biotechnological applications:

• Substrate specificity modification:

  • Site-directed mutagenesis of active site residues

  • Creation of libraries with altered specificity profiles

  • Directed evolution for novel substrate recognition

  • Computational design of active site variants

• Stability engineering:

  • Thermostabilization through consensus design approaches

  • pH stability enhancement for gastric survival

  • Protease resistance improvement for in vivo applications

  • Engineering for cosolvent compatibility

• Fusion protein strategies:

  • Creation of bifunctional enzymes combining pepX with complementary activities

  • Design of cell surface display systems for immobilized applications

  • Development of stimulus-responsive fusion proteins

  • Integration with binding domains for targeted delivery

• Regulatory element optimization:

  • Inducible expression systems responsive to gut environmental cues

  • Stress-responsive promoters for condition-specific expression

  • Fine-tuning expression levels through synthetic biology approaches

  • Tissue-specific expression strategies

These engineering approaches can be implemented using CRISPR-Cas9 systems adapted for Lactobacillus species, site-directed mutagenesis, and synthetic biology platforms. The construction methodologies would be similar to those used for other recombinant proteins in L. plantarum, involving PCR-based cloning, restriction enzyme digestion, and electrotransformation .

What interdisciplinary research opportunities exist for recombinant L. plantarum pepX systems?

Recombinant L. plantarum pepX systems offer numerous interdisciplinary research opportunities:

• Synthetic biology and microbiome engineering:

  • Development of pepX-based biosensors for gut environment monitoring

  • Creation of synthetic microbial consortia with defined peptide processing capabilities

  • Engineering communication systems between recombinant L. plantarum and other gut microbes

  • Design of microbiome-modulating probiotics with targeted activities

• Biopharmaceutical applications:

  • Development of oral peptide delivery systems using pepX processing

  • Engineering of immunomodulatory live biotherapeutics

  • Design of combined prebiotic-probiotic systems (synbiotics)

  • Creation of recombinant vaccines targeting multiple pathogens

• Nutritional science interfaces:

  • Investigation of pepX's role in protein digestibility enhancement

  • Development of functional foods with targeted peptide release

  • Studies on allergenicity reduction through specific peptide processing

  • Bioavailability enhancement of dietary bioactive peptides

• Computational biology integration:

  • Systems biology modeling of pepX's impact on host-microbe interactions

  • Machine learning approaches to predict pepX substrate preferences

  • Pathway engineering for optimized peptide processing

  • Multi-scale modeling of pepX activity in the gut environment

These interdisciplinary approaches build upon established methodologies for recombinant L. plantarum studies, including animal models for immunological assessment , microbiome analysis techniques , and protein engineering platforms , while expanding into new areas of application through innovative combinations of technologies.