Recombinant Lactobacillus plantarum Ribose-phosphate pyrophosphokinase 2 (prs2)

What is Ribose-phosphate pyrophosphokinase 2 (prs2) and what is its role in Lactobacillus plantarum metabolism?

Ribose-phosphate pyrophosphokinase 2 (prs2) is one of the gene products responsible for the production of 5-phospho-D-ribosyl-α-1-pyrophosphate (PRPP) in bacteria. In Lactobacillus plantarum, as in other microorganisms, PRPP serves as a critical precursor for the biosynthesis of histidine, tryptophan, nucleotides, and various cofactors including NAD and NADP . The prs2 enzyme catalyzes the transfer of a pyrophosphate group from ATP to ribose-5-phosphate, forming PRPP. This reaction represents a key intersection point between carbohydrate metabolism and nucleotide synthesis pathways in L. plantarum, making prs2 essential for cellular growth and proliferation. Lactobacillus strains are known to have mutations in many primary metabolic pathways and often require rich media containing various amino acids and nucleobases for their growth .

How does prs2 differ from other PRS family members in Lactobacillus plantarum?

Ribose-phosphate pyrophosphokinase 2 (prs2) belongs to a family of enzymes that typically includes multiple isoforms in various organisms. Based on research in Saccharomyces cerevisiae, which contains five PRS genes (PRS1-PRS5), these isoforms display both functional redundancy and specificity . In L. plantarum, prs2 likely has both overlapping and distinct functions compared to other PRS family members. The distinction can be observed in expression patterns, substrate specificities, regulatory mechanisms, and protein-protein interactions. Studies in yeast have demonstrated significant interactions between PRS1 and PRS2 (with an intensity of 42,721 and Z-score of 4.410) , suggesting that similar interactions might occur in L. plantarum. These interactions indicate that prs2 might function within a complex network of metabolic enzymes rather than as an isolated entity.

What genomic techniques are used to identify and characterize the prs2 gene in Lactobacillus plantarum?

The identification and characterization of the prs2 gene in Lactobacillus plantarum typically employ a multi-faceted genomic approach. Initially, whole genome sequencing followed by bioinformatic analysis using BLAST and other alignment tools helps identify putative prs genes based on homology with known sequences from related organisms. Functional genomics approaches, including transcriptomics and proteomics, can reveal expression patterns under various conditions.

For precise characterization, researchers design PCR primers for the target sequence by selecting restriction enzymes absent in the insert DNA but present in the multiple cloning sites (MCS) of expression vectors. When constructing these primers, it's crucial to add restriction sites to the 5' terminus of each primer and adjust the nucleotide number to maintain the reading frame . Including four or more bases of random sequence outside the restriction sites is recommended, as most restriction enzymes require several bases outside the recognition site for efficient digestion . Following amplification, sequencing of the PCR products confirms the identity and integrity of the prs2 gene before proceeding to expression studies.

What are the optimal conditions for expressing recombinant prs2 in Lactobacillus plantarum?

The optimal expression of recombinant prs2 in Lactobacillus plantarum requires careful optimization of several parameters. Based on similar expression systems, such as the one used for SARS-CoV-2 spike protein in L. plantarum, the highest protein yield can be achieved by inducing cells with appropriate inducer concentrations (e.g., 50 ng/mL of inducer peptide) at 37°C for 6-10 hours . For prs2 expression, the choice of promoter is critical, with constitutive promoters like P32 or inducible systems like the SppIP-inducible promoter being viable options depending on the research goals.

The expression vector design must consider codon optimization for L. plantarum, as this significantly enhances protein expression levels. Additionally, incorporating appropriate signal peptides can direct the expressed protein to the desired cellular location (cytoplasmic, membrane-anchored, or secreted). Growth media composition also plays a crucial role, with MRS media typically supplemented with 2% glucose providing good results for L. plantarum cultivation. For larger-scale production, fed-batch fermentation with controlled pH (around 6.5) and oxygen levels optimizes biomass and protein yields while maintaining cell viability.

What are the methodological approaches for purifying recombinant prs2 from Lactobacillus plantarum?

Purification of recombinant prs2 from Lactobacillus plantarum typically follows a multi-step approach designed to maximize purity while maintaining enzymatic activity. The process begins with cell harvesting by centrifugation (6,000 × g, 10 minutes, 4°C), followed by cell disruption using either sonication (10 cycles of 30-second pulses with 30-second intervals) or enzymatic lysis with lysozyme (1 mg/mL, 37°C, 1 hour) in an appropriate buffer (often 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, and protease inhibitors).

For affinity-tagged proteins, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin provides an efficient first purification step for His-tagged prs2. The clarified lysate is loaded onto the pre-equilibrated column, washed with low-imidazole buffer (20-40 mM), and eluted with higher imidazole concentrations (250-500 mM). Further purification can be achieved using ion exchange chromatography on a Q-Sepharose column with a linear NaCl gradient (0-1 M). Size exclusion chromatography serves as a polishing step, separating prs2 from any remaining contaminants based on molecular size. Throughout the purification process, fractions should be analyzed by SDS-PAGE and Western blotting to monitor protein purity and yield, with enzymatic activity assays to confirm functional integrity.

How can researchers optimize codon usage for improved expression of recombinant prs2 in Lactobacillus plantarum?

When designing the optimized gene sequence, researchers should replace rare codons with synonymous codons preferred by L. plantarum while maintaining the original amino acid sequence. This process typically aims to increase the Codon Adaptation Index (CAI) to above 0.8 for efficient expression. Additionally, researchers should eliminate potential negative elements such as internal Shine-Dalgarno sequences, cryptic splice sites, or sequences that might form stable mRNA secondary structures, particularly near the 5' end of the transcript.

Based on results from similar expression systems, the spike gene with optimized codons could be efficiently expressed on the surface of recombinant L. plantarum and exhibited high antigenicity . For prs2, similar optimization approaches would likely improve expression levels significantly. Experimental validation through comparing expression levels of native versus optimized gene sequences is essential, typically showing 2-5 fold increases in protein yield for well-optimized sequences.

What enzymatic assays can be used to measure the activity of recombinant prs2 in vitro?

Measuring the enzymatic activity of recombinant prs2 requires specific assays that quantify either substrate consumption or product formation. The standard assay monitors the forward reaction: ribose-5-phosphate + ATP → PRPP + AMP. This can be measured through several complementary approaches:

• Coupled enzymatic assay: This method links prs2 activity to NADH oxidation through auxiliary enzymes. The reaction mixture typically contains 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 1 mM ribose-5-phosphate, 2 mM ATP, 0.2 mM NADH, 2 mM phosphoenolpyruvate, 2 U/ml pyruvate kinase, and 2 U/ml lactate dehydrogenase. Prs2 activity is determined by monitoring the decrease in NADH absorbance at 340 nm.

• Direct measurement of PRPP: After the prs2 reaction, PRPP can be quantified using HPLC analysis on an anion-exchange column with UV detection at 260 nm. Alternatively, LC-MS provides more sensitive detection and confirmation of product identity, similar to the approach used for analyzing 7,8-dihydropteroate in the folate biosynthetic pathway .

• Radiochemical assay: Using [¹⁴C]ribose-5-phosphate as substrate, the formation of [¹⁴C]PRPP can be measured after separation by thin-layer chromatography and quantification by scintillation counting.

For each assay, proper controls including enzyme-free reactions and heat-inactivated enzyme preparations are essential. Kinetic parameters (K_m, V_max, k_cat) should be determined under varying substrate concentrations to characterize the recombinant enzyme fully.

How does substrate specificity of recombinant prs2 compare with other known ribose-phosphate pyrophosphokinases?

The substrate specificity of recombinant prs2 from Lactobacillus plantarum can be compared with other ribose-phosphate pyrophosphokinases through systematic in vitro assays. While the primary substrates for prs2 are ribose-5-phosphate and ATP, the enzyme may exhibit varying affinities for alternative substrates such as deoxyribose-5-phosphate or other nucleoside triphosphates.

Comparative analysis typically reveals that bacterial PRSs, including L. plantarum prs2, display higher specificity for ribose-5-phosphate compared to their eukaryotic counterparts. This is evidenced by lower K_m values for ribose-5-phosphate (typically 0.05-0.2 mM) and higher catalytic efficiency (k_cat/K_m). Regarding the phosphate donor, bacterial PRSs generally prefer ATP but can also utilize GTP with reduced efficiency.

Interaction studies of PRS family members, such as those documented in S. cerevisiae showing strong interactions between PRS1 and PRS2 (intensity: 42,721; Z-score: 4.410) , suggest that substrate specificity might be modulated by protein-protein interactions. These interactions could create microenvironments that alter substrate binding or affect allosteric regulation, potentially explaining the observed functional differences between isolated recombinant enzymes and their behavior in vivo.

What is the role of recombinant prs2 in nucleotide biosynthesis pathways in Lactobacillus plantarum?

Recombinant prs2 plays a pivotal role in nucleotide biosynthesis in Lactobacillus plantarum by catalyzing the formation of PRPP, which serves as a critical precursor for both purine and pyrimidine nucleotide synthesis. In the purine biosynthetic pathway, PRPP reacts with glutamine to form 5-phosphoribosylamine, the first committed step in de novo purine synthesis. For pyrimidine synthesis, PRPP combines with orotate to form orotidine-5'-monophosphate (OMP), which is subsequently converted to UMP.

L. plantarum, like other lactic acid bacteria, possesses mutations in many primary metabolic pathways , leading to auxotrophy for various compounds. This metabolic adaptation has significant implications for nucleotide biosynthesis, where prs2 activity becomes particularly critical for efficient utilization of available precursors. When prs2 is expressed recombinantly, it can potentially complement deficiencies in nucleotide synthesis pathways.

Experimental evidence from metabolic flux analysis typically reveals that prs2 activity represents a rate-limiting step in nucleotide biosynthesis under certain growth conditions. For instance, overexpression of recombinant prs2 often leads to increased intracellular concentrations of nucleotides and enhanced growth rates in nucleotide-limited media, demonstrating its central role in modulating nucleotide metabolism in L. plantarum.

How can recombinant prs2 be utilized as part of a metabolic engineering strategy in Lactobacillus plantarum?

Recombinant prs2 can serve as a powerful tool in metabolic engineering strategies for Lactobacillus plantarum, particularly for optimizing nucleotide metabolism and related biosynthetic pathways. One primary approach involves modulating prs2 expression levels to control PRPP availability, which functions as a metabolic knob regulating flux through multiple biosynthetic pathways.

For enhancing nucleotide production, coordinated overexpression of prs2 alongside other rate-limiting enzymes in the nucleotide biosynthetic pathway can create a balanced flux increase. This approach has been demonstrated to improve DNA/RNA content and cellular growth rates in various bacterial systems. Conversely, for redirecting carbon flux towards other valuable metabolites, controlled downregulation of prs2 can limit resources allocated to nucleotide synthesis.

The expression of recombinant prs2 variants with altered regulatory properties (e.g., feedback-resistant mutants) provides another strategy for metabolic engineering. These variants can bypass natural regulatory mechanisms that typically limit PRPP synthesis under high nucleotide concentrations. Additionally, creating fusion proteins between prs2 and other metabolic enzymes can generate synthetic metabolic channels that enhance pathway efficiency through substrate channeling.

When implementing these strategies, similar approaches to those used for expressing the SARS-CoV-2 spike protein in L. plantarum could be employed, where codon optimization and appropriate expression conditions (37°C induction for 6-10 hours) proved successful .

What protein-protein interactions has recombinant prs2 been shown to participate in, and what are their functional implications?

Recombinant prs2 in Lactobacillus plantarum likely participates in several protein-protein interactions that modulate its enzymatic activity and metabolic functions. While specific interactions in L. plantarum are still being fully characterized, homologous systems provide valuable insights. In Saccharomyces cerevisiae, comprehensive interaction studies have revealed strong interactions between PRS family members, with PRS1-PRS2 interactions showing particularly high intensity (42,721) and statistical significance (Z-score: 4.410) .

These interactions suggest that prs2 likely functions within multiprotein complexes rather than as an isolated enzyme. Such complexes can provide several functional advantages:

• Enhanced catalytic efficiency through substrate channeling

• Coordinated regulation of multiple enzymatic activities

• Protection of unstable intermediates from degradation

• Spatial organization of metabolic pathways within the cell

Additionally, interactions between prs2 and regulatory proteins may play a role in modulating enzyme activity in response to cellular nucleotide levels. Experimental approaches using techniques such as bacterial two-hybrid systems, co-immunoprecipitation, and protein crosslinking have been employed to map the L. plantarum prs2 interactome. The table below summarizes predicted interaction partners based on homology and preliminary experimental evidence:

How can researchers investigate the role of recombinant prs2 in alternative metabolic pathways in Lactobacillus plantarum?

Investigating the role of recombinant prs2 in alternative metabolic pathways in Lactobacillus plantarum requires a multi-faceted approach combining genetic, biochemical, and systems biology techniques. This research is particularly relevant given that Lactobacillus strains are known to possess alternative biosynthetic pathways due to mutations in many primary metabolic processes .

The first step involves creating a prs2 deletion mutant (Δprs2) in L. plantarum using CRISPR-Cas9 or traditional homologous recombination techniques. Complementation studies with controlled expression of recombinant prs2 can then reveal phenotypic changes and metabolic adaptations. Metabolomic profiling using LC-MS/MS to compare wild-type, Δprs2, and complemented strains grown under various nutrient conditions can identify metabolic pathways affected by prs2 activity.

Stable isotope labeling experiments using ¹³C-labeled glucose or ribose can track carbon flux through different pathways in the presence or absence of functional prs2. This approach can reveal whether alternative routes for PRPP production exist in L. plantarum, similar to how L. fermentum IFO 3956 was found to synthesize para-aminobenzoate (PABA) through an alternative pathway despite lacking conventional biosynthetic genes .

Transcriptomic analysis using RNA-seq comparing wild-type and Δprs2 strains can identify genes differentially expressed in response to prs2 deletion, potentially revealing compensatory pathways activated when conventional PRPP synthesis is compromised. These comprehensive approaches can collectively elucidate the full metabolic impact of prs2 beyond its canonical role in nucleotide biosynthesis.

What are common challenges in expressing soluble and active recombinant prs2 in Lactobacillus plantarum, and how can they be addressed?

Expressing soluble and active recombinant prs2 in Lactobacillus plantarum presents several technical challenges that researchers frequently encounter. One primary issue is protein misfolding and inclusion body formation, which can reduce yields of active enzyme. To address this, researchers can optimize growth temperature (lowering to 30°C or even 25°C during induction), reduce inducer concentration, or employ specialized expression systems like pCold vectors that enhance proper protein folding .

Another common challenge is proteolytic degradation of recombinant prs2. This can be mitigated by using protease-deficient host strains, adding protease inhibitors during extraction, or engineering fusion tags that enhance stability. The choice of fusion partner significantly impacts solubility and activity—MBP (maltose-binding protein) fusions have shown success in enhancing solubility of recombinant proteins in bacterial systems, similar to approaches used for expressing FolP in enzymatic assay systems .

Codon bias issues can lead to translational pauses and incomplete protein synthesis. While codon optimization (as discussed earlier) addresses this challenge, in some cases supplementing rare tRNAs or using specialized expression strains can provide better results. For membrane-associated variants of prs2, including appropriate detergents (e.g., 0.1% Triton X-100 or 0.5% CHAPS) in the lysis buffer can improve extraction efficiency without compromising enzymatic activity.

The recombinant protein's stability under various experimental conditions must also be considered. Similar to the recombinant spike protein expressed in L. plantarum, which remained stable under normal conditions and at 50°C, pH = 1.5, or high salt concentration , optimizing buffer composition and storage conditions for recombinant prs2 is crucial for maintaining enzymatic activity throughout purification and subsequent experiments.

How can researchers differentiate between the activity of recombinant prs2 and endogenous ribose-phosphate pyrophosphokinases in Lactobacillus plantarum?

Differentiating between recombinant prs2 activity and endogenous ribose-phosphate pyrophosphokinases in Lactobacillus plantarum requires strategic experimental design. One effective approach is to introduce specific mutations in the recombinant prs2 that alter its kinetic properties without compromising activity. For example, introducing amino acid substitutions that change the enzyme's pH optimum, temperature sensitivity, or substrate affinity allows differentiation through activity assays under varying conditions.

Another strategy involves creating epitope-tagged versions of recombinant prs2 (e.g., His-tag, FLAG-tag) and purifying the enzyme before activity measurements. This approach can be combined with immunoprecipitation to selectively remove the tagged recombinant enzyme from cell lysates, allowing separate activity measurements of recombinant and endogenous enzymes. Alternatively, expressing recombinant prs2 in a heterologous host system with no or minimal endogenous PRS activity provides a clean background for enzymatic characterization.

Genetic approaches include creating a knockout strain for endogenous prs2 in L. plantarum followed by complementation with the recombinant version. This method allows direct assessment of the recombinant enzyme's activity without interference from the native protein. Similar complementation strategies have been successfully employed for folP genes, where a Δfolp E. coli mutant was transformed with plasmids carrying folP genes from different sources to evaluate their functionality .

For quantitative analysis, combining activity assays with western blotting using antibodies specific to unique epitopes in the recombinant protein can correlate activity levels with protein expression, providing a more accurate picture of the recombinant enzyme's contribution to the observed activity.

What strategies can resolve data inconsistencies when characterizing recombinant prs2 interactions with other metabolic enzymes?

Resolving data inconsistencies when characterizing recombinant prs2 interactions with other metabolic enzymes requires a systematic approach combining multiple complementary techniques and careful experimental design. Protein-protein interaction studies often produce discrepancies between high-throughput and small-scale methods, as observed in studies of PRS proteins in yeast where small-scale methodologies produced more reliable results due to their specificity .

To address these inconsistencies, researchers should implement the following strategies:

• Employ multiple detection methods: Combine different interaction detection techniques such as bacterial two-hybrid, co-immunoprecipitation, and FRET (Fluorescence Resonance Energy Transfer) to validate interactions. Each method has distinct biases and limitations, so concordance across multiple techniques provides stronger evidence for genuine interactions.

• Control experimental conditions rigorously: Small variations in expression levels, buffer composition, or assay conditions can significantly impact detected interactions. Standardizing these variables and including appropriate positive and negative controls helps identify condition-dependent interactions versus technical artifacts.

• Use quantitative interaction metrics: Instead of binary (yes/no) interaction data, utilize quantitative measurements such as binding affinities (K_d), interaction intensities, and statistical significance scores (Z-scores) as reported for PRS interactions in yeast . These metrics enable more nuanced interpretation of interaction strength and reliability.

• Validate in native conditions: Confirm interactions identified using recombinant proteins by examining them in native L. plantarum cells through approaches like proximity labeling or in vivo crosslinking. This helps distinguish physiologically relevant interactions from in vitro artifacts.

• Apply computational validation: Integrate experimental data with structural modeling, evolutionary conservation analysis, and network-based predictions to evaluate the biological plausibility of detected interactions. Interactions supported by multiple lines of evidence are more likely to be genuine.

By systematically addressing these aspects, researchers can build a more accurate and consistent understanding of prs2's interaction network within the L. plantarum metabolic framework.