Recombinant Lactobacillus plantarum Xanthine phosphoribosyltransferase (xpt)

What is Xanthine phosphoribosyltransferase (xpt) and what is its function in Lactobacillus plantarum metabolism?

Xanthine phosphoribosyltransferase (xpt) is a key enzyme in the purine salvage pathway that catalyzes the conversion of xanthine and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to xanthine monophosphate (XMP) and pyrophosphate. In Lactobacillus plantarum, xpt plays a crucial role in purine nucleotide metabolism by allowing the organism to recycle xanthine derived from the degradation of nucleic acids.

The enzyme is part of a broader nucleotide salvage system that enables L. plantarum to efficiently utilize purines available in its environment, which is particularly important given that many lactic acid bacteria have limited de novo synthesis capabilities. In genetic studies of related organisms, xpt has been found to be co-regulated with purine permeases (like pbuX in Bacillus subtilis), which facilitate the uptake of xanthine from the extracellular environment .

How is recombinant L. plantarum expressing xpt typically constructed in laboratory settings?

Construction of recombinant L. plantarum expressing xpt typically involves the following methodology:

• Vector selection: Appropriate expression vectors for L. plantarum such as pSIP or pWCF series plasmids are selected based on desired expression characteristics .

• Gene optimization: The xpt gene coding sequence is optimized for L. plantarum codon usage to enhance expression efficiency. Similar to spike protein expression studies, codon optimization can significantly increase protein yields .

• Promoter selection: Inducible promoters (such as SppIP-responsive promoters) are often preferred to control expression timing and level .

• Fusion design: The xpt gene may be fused with appropriate signal peptides or anchoring domains if surface display is desired, similar to the method used for displaying SARS-CoV-2 spike protein or HA1 antigen on L. plantarum .

• Transformation: Competent L. plantarum cells are transformed using electroporation protocols optimized for this species.

• Selection and verification: Transformants are selected using appropriate antibiotics, and successful xpt expression is verified by Western blotting, flow cytometry, or enzymatic activity assays.

What expression systems are most effective for producing recombinant proteins in L. plantarum?

Several expression systems have proven effective for heterologous protein production in L. plantarum:

The most suitable system depends on specific research requirements:

• For xpt expression as a cytoplasmic enzyme, constitutive promoters may be sufficient

• For surface display applications, vectors containing anchoring motifs like pgsA' are preferable

• For secreted xpt, appropriate signal peptides must be incorporated

In studies with surface-displayed antigens, the pWCF system demonstrated excellent efficiency with 37.5% of cells showing positive expression compared to 2.5% in control strains .

What are the fundamental differences between expressing xpt in E. coli versus L. plantarum?

The expression of xpt in E. coli versus L. plantarum involves several key differences that researchers should consider:

Genetic context and regulation:

• E. coli: Typically employs T7 or lac-based promoter systems

• L. plantarum: Requires specific lactic acid bacteria-compatible promoters such as pSIP or SppIP-inducible systems

Codon usage optimization:

• E. coli: Standard laboratory strains have well-characterized codon preferences

• L. plantarum: Requires specific codon optimization; studies show this significantly impacts expression efficiency

Post-translational processing:

• E. coli: Limited capacity for certain modifications; may form inclusion bodies

• L. plantarum: Better suited for proteins requiring specific lactic acid bacteria-type modifications

Cultivation conditions:

• E. coli: Aerobic, relatively simple media requirements

• L. plantarum: Microaerophilic/anaerobic, specific media requirements (e.g., MRS medium)

Protein localization options:

• E. coli: Primarily cytoplasmic or periplasmic expression

• L. plantarum: Offers efficient surface display options through specialized anchoring domains

Application context:

• E. coli: Primarily for protein production and purification

• L. plantarum: Direct application as a food-grade or probiotic delivery system

What methodologies can be employed to optimize the enzymatic activity of recombinant xpt in L. plantarum?

Optimizing the enzymatic activity of recombinant xpt in L. plantarum requires a multifaceted approach:

Gene sequence optimization:

• Implement codon optimization specific to L. plantarum to enhance translation efficiency

• Consider incorporating rare codons strategically to modulate protein folding kinetics

Expression conditions optimization:

• Systematically test induction parameters (concentration of inducer, timing, temperature)

• For SppIP-based systems, optimal conditions have been established at 50 ng/mL SppIP at 37°C for 6-10 hours

Protein engineering strategies:

• Site-directed mutagenesis of active site residues based on structural models of xpt

• Creation of fusion proteins to enhance stability (consider that spike protein fusions maintain stability at 50°C, pH 1.5, and in high salt concentrations)

Metabolic engineering considerations:

• Co-expression of proteins involved in substrate provision (purine permeases)

• Deletion of competing pathways to channel metabolic flux through xpt

Activity measurement protocols:
A standardized assay for monitoring xpt activity includes:

• Cell lysis by sonication or enzymatic treatment

• Reaction mixture containing xanthine, PRPP, and appropriate buffers

• Detection of XMP formation by HPLC or coupled enzymatic assays

• Analysis of kinetic parameters (Km, Vmax) for wild-type versus optimized variants

Research has shown that proteins expressed in L. plantarum can maintain significant stability under challenging conditions, suggesting that proper optimization can yield highly active xpt enzyme .

How do the purine salvage pathways in L. plantarum interact with other metabolic networks, and what role does xpt play?

The purine salvage pathways in L. plantarum form a complex metabolic network with multiple interconnections:

Integration with nucleoside metabolism:
Xpt functions alongside other salvage enzymes such as nucleoside hydrolases (rihA, rihC) and nucleoside permeases (yxjA) identified in genome analyses . These enzymes collectively enable L. plantarum to utilize various purine sources from the environment.

Connection to de novo synthesis:
In L. plantarum, the purine salvage pathway interacts with remnants of the de novo pathway, though many lactic acid bacteria have incomplete de novo synthesis capabilities. PRPP synthetase (PRPS) provides a key connection point between these pathways .

Relationship with pyrimidine metabolism:
Parallel to xpt in purine salvage, the uracil phosphoribosyltransferase (UPRT) in the pyrimidine salvage pathway plays a similar role in recycling pyrimidine nucleobases. The upp-pyrP gene cluster encodes this functionality, with pyrP serving as a pyrimidine transporter .

Regulatory network interactions:
Expression of xpt in related organisms is regulated by purine availability, often through transcription attenuation mechanisms similar to those controlling the pyr operon. This suggests regulatory cross-talk between purine and pyrimidine pathways .

Metabolic impact of xpt activity:
Metabolomic studies of nucleoside metabolism in L. plantarum SQ001 demonstrated that purine nucleoside salvage significantly impacts intracellular metabolite compositions. When incubated with nucleoside solutions, L. plantarum showed decreased levels of inosine (p < 0.0001), guanosine (p < 0.0001), and adenosine (p < 0.0001), with corresponding increases in nucleobases like guanine (p < 0.0001) and xanthine (p < 0.0001) .

Impact on host metabolism:
L. plantarum with active purine salvage capabilities has been shown to affect host uric acid metabolism, suggesting potential applications in hyperuricemia treatment .

What immune responses are triggered by recombinant L. plantarum expressing xpt, and how can these be measured experimentally?

While specific immune responses to recombinant L. plantarum expressing xpt have not been directly characterized in the provided search results, we can extrapolate from studies of L. plantarum expressing other recombinant proteins:

Expected immune responses:

• Innate immune activation through pattern recognition receptors

• Potential humoral immune responses if xpt is surface-displayed

• Mucosal immune responses due to the probiotic nature of L. plantarum

Experimental measurement methodologies:

• Cellular immune response assessment:

  • Flow cytometry to quantify activation of dendritic cells in Peyer's patches

  • Measurement of CD4+IFN-γ+ and CD8+IFN-γ+ T cells in the spleen and mesenteric lymph nodes

  • T cell proliferation assays in response to xpt stimulation

• Humoral immune response evaluation:

  • ELISA to detect xpt-specific IgG, IgG1, and IgG2a antibodies in serum

  • ELISA for measurement of xpt-specific IgA antibodies in fecal samples

  • Western blotting to assess antibody specificity

• Mucosal immune response analysis:

  • Immunofluorescence staining to detect IgA in the lungs, duodenum, jejunum, and ileum

  • Flow cytometry to measure B220+IgA+ cells in Peyer's patches

  • Quantification of secretory IgA in intestinal washes

• Dendritic cell activation and maturation:

  • Assessment of co-stimulatory molecule expression (CD80, CD86, CCR7)

  • Measurement of cytokine production (IL-12, TNF-α, IL-10)

  • Evaluation of immunoregulatory factors (PD-L1, IDO)

Studies with recombinant L. plantarum expressing HA1 showed significant increases in CD4+IFN-γ+ cells in mesenteric lymph nodes (p < 0.001 compared to PBS controls) and specific IgG antibody levels at weeks 2 (p < 0.05), 4 (p < 0.01), and 10 (p < 0.0001) post-immunization .

What methodological challenges arise when purifying functional xpt from recombinant L. plantarum, and how can they be addressed?

Purification of functional xpt from recombinant L. plantarum presents several methodological challenges:

Cell lysis optimization:

• Challenge: L. plantarum has a thick peptidoglycan layer resistant to standard lysis methods

• Solution: Combined enzymatic (lysozyme, mutanolysin) and mechanical (sonication, bead-beating) approaches with optimized buffer compositions

Protein solubility issues:

• Challenge: Potential formation of inclusion bodies or membrane association

• Solution: Expression optimization through temperature modulation (6-10h induction at 37°C has shown good results for other proteins) and testing various solubilizing agents

Enzymatic activity preservation:

• Challenge: Maintaining structural integrity and catalytic activity during purification

• Solution: Inclusion of stabilizing agents (glycerol, specific ions) and minimizing exposure to extreme conditions

Purification strategy selection:
A comprehensive purification protocol might include:

Tag design and removal considerations:

• Challenge: Tags may interfere with enzymatic function or create artifacts

• Solution: Incorporate cleavable tags (TEV protease site) and compare activity before and after tag removal

Scale-up considerations:

• Challenge: Maintaining consistency when scaling up production

• Solution: Careful optimization of growth and induction parameters in bioreactors

Studies with other recombinant proteins from L. plantarum demonstrated that proteins can retain stability under various conditions (50°C, pH 1.5, high salt) , suggesting that with proper handling, functional xpt can be successfully purified.

How can genome editing techniques be applied to enhance xpt expression or activity in L. plantarum?

Advanced genome editing techniques offer promising approaches to enhance xpt expression or activity in L. plantarum:

CRISPR-Cas9 applications:

• Precise modification of native xpt promoter regions to increase expression

• Introduction of optimized xpt variants at specific genomic loci

• Deletion of competing metabolic pathways to channel metabolic flux through xpt

• Knockout of repressor genes that might limit xpt expression

Homologous recombination strategies:

• Integration of additional xpt copies into non-essential regions of the genome

• Replacement of native xpt with engineered variants with improved catalytic properties

• Introduction of constitutive promoters upstream of xpt to bypass natural regulation

Metabolic engineering approaches:

• Modification of PRPP synthetase to increase substrate availability for xpt

• Enhancement of purine permease systems for improved xanthine uptake

• Integration of heterologous enzymes to create novel metabolic routes supporting xpt function

Regulatory circuit engineering:

• Development of synthetic regulatory systems to fine-tune xpt expression

• Creation of feedback-responsive promoters tied to purine metabolite levels

• Implementation of orthogonal control systems for independent regulation of xpt

Experimental design for genome editing optimization:

• Construct multiple editing templates with varying modifications

• Transform L. plantarum using optimized protocols (electroporation is most common)

• Screen transformants using PCR, sequencing, and activity assays

• Characterize successful variants through growth studies and metabolic analysis

• Validate improvements through comparative enzymatic assays

When implementing these approaches, researchers should consider that genome modifications in L. plantarum typically show transformation efficiencies of 10^3-10^5 CFU/μg DNA, with homologous recombination frequencies varying significantly based on design parameters .

What are the current gaps in understanding xpt function in L. plantarum and how might these be addressed through future research?

Several significant knowledge gaps exist regarding xpt function in L. plantarum that warrant further investigation:

Structural and enzymatic characterization:
Current gap: Detailed structural information about L. plantarum xpt is lacking
Research direction: X-ray crystallography or cryo-EM studies of purified xpt to determine structure-function relationships and catalytic mechanisms

Regulatory networks:
Current gap: Precise understanding of how xpt expression is regulated in response to environmental conditions
Research direction: Transcriptomic and ChIP-seq analyses to identify transcription factors and regulatory elements controlling xpt expression

Metabolic integration:
Current gap: Comprehensive understanding of how xpt activity affects global metabolism
Research direction: Metabolomic studies comparing wild-type, xpt-deficient, and xpt-overexpressing strains under various growth conditions

Host-microbe interactions:
Current gap: Limited knowledge about how L. plantarum xpt activity influences host physiology
Research direction: In vivo studies examining the impact of L. plantarum with modified xpt expression on host purine metabolism and immune responses

Therapeutic potential:
Current gap: Unclear whether recombinant L. plantarum expressing modified xpt could have therapeutic applications
Research direction: Investigation of recombinant strains in models of hyperuricemia or other purine metabolism disorders

Experimental approaches to address these gaps:

• Comparative genomics and phylogenetic analysis:
  Compare xpt sequences and genetic contexts across Lactobacillus species to understand evolutionary conservation and specialization

• Systems biology framework:
  Integrate transcriptomic, proteomic, and metabolomic data to construct comprehensive models of purine metabolism in L. plantarum

• Advanced microscopy techniques:
  Employ fluorescently tagged xpt to track subcellular localization and potential protein-protein interactions

• Synthetic biology approaches:
  Design and test synthetic xpt variants with altered substrate specificity or catalytic efficiency

• Multi-omics analysis of host responses: Assess host transcriptomic, proteomic, and metabolomic changes in response to colonization with L. plantarum expressing various xpt variants