Recombinant Lactobacillus plantarum Phosphoserine aminotransferase (serC)

What is Lactiplantibacillus plantarum and why is it significant as a recombinant expression system?

Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) is a lactic acid bacterium widely used in recombinant protein expression due to its "Generally Regarded as Safe" (GRAS) status, potential adjuvant properties, and tolerogenicity to the host. It has emerged as a promising alternative to other expression systems, particularly for mucosal delivery of antigens and therapeutic proteins . The bacterium offers several advantages including its ability to survive in gastrointestinal conditions and improve both local and distal immune responses in vivo. Studies have shown that L. plantarum-based vaccines demonstrate higher immunogenicity compared to Lactococcus lactis when orally administered to mouse models . This makes it particularly valuable for developing novel biotherapeutics and vaccine candidates.

What is phosphoserine aminotransferase (serC) and what is its biochemical function?

Phosphoserine aminotransferase (PSAT or serC) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the conversion of 3-phosphohydroxypyruvate (3-PHP) to 3-phosphoserine (PSer) through an L-glutamate-linked reversible transamination reaction . This reaction proceeds through a bimolecular ping-pong mechanism. The enzyme is part of the phosphorylated pathway of serine biosynthesis, one of three recognized routes in plant organisms that yield serine. In this pathway, 3-phosphoglycerate (3-PGA) is first oxidized by 3-PGA dehydrogenase to form 3-PHP, which is then subjected to transamination with glutamate by PSAT to yield PSer and α-ketoglutarate. In the final step, serine is produced by phosphoserine phosphatase . The serC gene encodes this essential enzyme involved in amino acid metabolism.

Why would researchers express serC in recombinant L. plantarum?

Researchers express serC in recombinant L. plantarum for several strategic reasons. First, this approach enables fundamental studies of phosphoserine metabolism in a well-characterized probiotic organism. Second, it provides a potential platform for enhancing amino acid production, particularly serine and its derivatives, which have applications in metabolic engineering. Third, the recombinant system can be used to investigate the role of serC in bacterial adaptation and stress responses. Expressing serC in L. plantarum leverages the bacterium's GRAS status while creating a research tool to study this metabolic pathway in a controlled manner . Additionally, the recombinant system could potentially be developed for therapeutic applications where targeted delivery of the enzyme or its products to mucosal surfaces is desired.

What are the most effective vector systems for expressing serC in L. plantarum?

The most effective vector systems for expressing serC in L. plantarum include shuttle vectors that can replicate in both E. coli and L. plantarum. Based on recent research, the pLP-S vector system has shown particular promise for heterologous protein expression in L. plantarum . For optimal expression, vectors should contain:

• A strong constitutive promoter (such as P23 from Lactococcus lactis) or an inducible promoter system (such as the nisin-inducible system)

• A Gram-positive origin of replication compatible with L. plantarum

• Appropriate selection markers (erythromycin or chloramphenicol resistance genes work well in L. plantarum)

• A multiple cloning site with diverse restriction enzyme options

• Signal peptides for targeting (if secretion or surface display is desired)

For surface display of serC, fusion with the pgsA anchor motif has proven effective, as demonstrated in similar recombinant L. plantarum constructs . The pMG36e backbone also provides a reliable expression system with strong constitutive promoters suitable for heterologous protein expression in L. plantarum .

What transformation protocols yield the highest efficiency when introducing recombinant serC constructs into L. plantarum?

For achieving high transformation efficiency when introducing recombinant serC constructs into L. plantarum, the following electroporation protocol has demonstrated superior results:

• Grow L. plantarum culture in MRS medium supplemented with 1% glycine at 30°C until OD600 reaches 0.6-0.8

• Harvest cells by centrifugation at 4,000 × g for 10 minutes at 4°C

• Wash cells twice with ice-cold electroporation buffer (0.5 M sucrose, 7 mM potassium phosphate, pH 7.4)

• Resuspend cells in 1/100 volume of the same buffer

• Mix 50 μL of cell suspension with 1-5 μg of plasmid DNA

• Transfer to pre-chilled 0.2 cm electroporation cuvette and apply pulse (2.5 kV, 200 Ω, 25 μF)

• Immediately add 950 μL of recovery medium (MRS with 0.5 M sucrose and 20 mM MgCl2)

• Incubate at 30°C for 3 hours

• Plate on selective media containing appropriate antibiotics

This protocol typically yields transformation efficiencies of 10^5-10^6 transformants per μg of DNA when using the pLP-S vector system. For recalcitrant strains, increasing the glycine concentration to 2% and adding threonine (40 mM) to the growth medium can further improve competence .

How can researchers verify successful expression and functionality of serC in recombinant L. plantarum?

Verification of successful expression and functionality of serC in recombinant L. plantarum requires a multi-faceted approach:

• Protein expression verification:

  • Western blotting using anti-serC or anti-tag antibodies (if a fusion tag is incorporated)

  • Flow cytometry for surface-displayed serC (as demonstrated with other recombinant proteins in L. plantarum)

  • Mass spectrometry for protein identification and quantification

• Enzymatic activity assay:

  • Measure the conversion of 3-phosphohydroxypyruvate to 3-phosphoserine

  • Spectrophotometric assay monitoring the decrease in NADH (coupled reaction with lactate dehydrogenase)

  • HPLC analysis of reaction products

• Functional complementation:

  • Growth assays in serine-deficient media comparing wild-type, serC-knockout, and recombinant strains

  • Complementation of E. coli serC auxotrophs with the recombinant L. plantarum serC

• Structural analysis:

  • Circular dichroism to confirm proper protein folding

  • Thermal shift assays to assess protein stability

  • Crystal structure analysis of the purified enzyme (if pursuing in-depth structural studies)

The combination of these methods ensures comprehensive verification of both expression and functionality of the recombinant serC enzyme in L. plantarum.

How can recombinant L. plantarum expressing serC be applied in metabolic engineering research?

Recombinant L. plantarum expressing serC offers several valuable applications in metabolic engineering research:

• Enhanced serine biosynthesis pathway:

  • Overexpression of serC can increase flux through the phosphorylated serine biosynthesis pathway

  • This could lead to improved production of serine and its derivatives (glycine, cysteine)

  • Potential for developing strains with enhanced capabilities for synthesizing serine-rich proteins

• Metabolic flux analysis:

  • Recombinant strains can be used to study the regulation of carbon flux between glycolysis and amino acid biosynthesis

  • Modulation of serC expression levels allows investigation of rate-limiting steps in serine biosynthesis

  • Isotope labeling experiments with recombinant strains can reveal flux distribution patterns

• Biocatalyst development:

  • L. plantarum expressing serC can serve as a whole-cell biocatalyst for transamination reactions

  • Potential applications in the production of non-natural amino acids and pharmaceutical intermediates

  • The GRAS status of L. plantarum makes it advantageous for food-grade biotransformations

• Stress response engineering:

  • Amino acid metabolism is linked to bacterial stress responses

  • Modulating serC expression can be used to engineer strains with enhanced resistance to environmental stresses

  • This has applications in improving L. plantarum performance in various biotechnological processes

These applications demonstrate how recombinant L. plantarum expressing serC can advance both fundamental understanding of metabolism and applied biotechnological processes.

What are the key challenges in optimizing serC expression levels in L. plantarum, and how can they be addressed?

Optimizing serC expression levels in L. plantarum presents several sophisticated challenges that researchers must address:

• Promoter strength and regulation:

  • Constitutive promoters may lead to metabolic burden if serC is overexpressed

  • Solution: Develop and characterize a library of promoters with varying strengths

  • Implement inducible systems with fine-tunable expression, such as nisin-controlled or xylose-dependent promoters

  • Consider growth phase-dependent promoters to synchronize expression with metabolic needs

• Codon optimization challenges:

  • L. plantarum has distinct codon usage preferences compared to other organisms

  • Solution: Perform species-specific codon optimization of the serC gene

  • Analyze GC content and rare codons in the target gene

  • Consider the impact of mRNA secondary structure on translation efficiency

  • Test multiple codon-optimized variants experimentally to identify optimal sequence

• Protein folding and solubility:

  • serC is a PLP-dependent enzyme requiring proper folding for activity

  • Solution: Co-express molecular chaperones like GroEL/GroES if misfolding occurs

  • Consider fusion partners that enhance solubility (e.g., thioredoxin, SUMO)

  • Optimize growth temperature (lower temperatures often improve folding)

  • Supplement media with pyridoxal 5'-phosphate to ensure cofactor availability

• Metabolic burden and toxicity:

  • Overexpression may deplete cellular resources or disrupt metabolic balance

  • Solution: Implement feedback-regulated expression systems

  • Develop metabolic models to predict optimal expression levels

  • Use ribosome binding sites of varying strengths to fine-tune translation rates

  • Consider chromosomal integration for stable, moderate expression levels

• Enzyme assay limitations:

  • Traditional enzyme assays may have low sensitivity or high background

  • Solution: Develop selective high-throughput screening methods

  • Consider synthetic biology approaches with serC activity linked to reporter systems

  • Implement metabolomic analysis to detect pathway intermediates and products

Addressing these challenges requires an integrated approach combining molecular biology, protein engineering, and systems biology tools to achieve optimal serC expression and activity in L. plantarum.

How does the structural biology of serC inform expression strategies in L. plantarum?

The structural biology of phosphoserine aminotransferase (serC) provides critical insights that should inform expression strategies in L. plantarum:

• Cofactor requirements and stability:

  • SerC is a pyridoxal 5'-phosphate (PLP)-dependent enzyme with the cofactor covalently bound to a conserved lysine residue (equivalent to K265 in AtPSAT1)

  • Expression strategy: Supplement growth media with pyridoxal 5'-phosphate (40-100 μM) to ensure proper cofactor incorporation during protein folding

  • Consider co-expression of enzymes involved in PLP biosynthesis to enhance cofactor availability

• Quaternary structure considerations:

  • SerC functions as a dimer with an S-shaped structure, as observed in the AtPSAT1 crystal structure

  • Expression strategy: Avoid fusion tags at interfaces that might disrupt dimerization

  • If surface display is desired, the anchor fusion should be positioned to preserve the dimeric interface

  • C-terminal fusions are generally preferable as the N-terminal region is often involved in dimerization

• Catalytic mechanism implications:

  • SerC undergoes conformational changes during catalysis, transitioning through multiple intermediate states:

    • Internal aldimine state (PLP covalently bound to catalytic lysine)

    • PMP complex after amino group transfer

    • Geminal diamine intermediate (cofactor bound to both lysine and substrate)

  • Expression strategy: Design constructs with sufficient flexibility to accommodate these conformational changes

  • Avoid rigid linkers that might constrain the necessary structural dynamics

• Domain architecture awareness:

  • SerC contains distinct domains that must maintain proper spatial relationships

  • Expression strategy: If domain truncations are considered, use structural information to preserve intact domains

  • Design fusion constructs based on natural domain boundaries

  • Consider the use of flexible glycine-serine linkers between functional domains

• Active site accessibility:

  • The active site of SerC must remain accessible for substrate binding

  • Expression strategy: For surface display, orient the protein so the active site faces away from the cell wall

  • Use structural modeling to predict the optimal configuration for maintaining enzymatic activity in recombinant constructs

By integrating these structural insights into expression strategies, researchers can enhance the likelihood of obtaining properly folded, active serC enzyme in recombinant L. plantarum systems.

What strategies can resolve common issues in the purification of recombinant serC from L. plantarum lysates?

Purifying recombinant serC from L. plantarum lysates presents several challenges that require specialized approaches:

• Cell wall disruption optimization:

  • L. plantarum has a robust cell wall that can be difficult to lyse

  • Solution: Combine enzymatic treatment (lysozyme, mutanolysin) with mechanical disruption

  • Optimized protocol: Treat cells with lysozyme (10 mg/mL) in a hypertonic buffer (50 mM Tris-HCl pH 8.0, 25% sucrose, 1 mM EDTA) for 30 minutes at 37°C, followed by sonication (10 cycles of 30s on/30s off at 40% amplitude)

  • For larger scale, use homogenization with zirconia beads in a BeadBeater device

• Protein stability during purification:

  • serC requires PLP cofactor for stability and can be sensitive to oxidation

  • Solution: Include the following in all purification buffers:

    • 50 μM PLP to maintain cofactor saturation

    • 1-5 mM reducing agent (TCEP preferred over DTT or β-mercaptoethanol due to stability)

    • 10% glycerol to enhance protein stability

    • Protease inhibitor cocktail specific for Gram-positive bacteria

• Affinity tag selection and positioning:

  • Tag position can affect folding, activity, and purification efficiency

  • Solution: Test both N- and C-terminal tags, with the following considerations:

    • His6-tag works well with IMAC purification but may bind native metal-binding proteins

    • Strep-tag II offers higher specificity but at higher cost

    • FLAG-tag provides high specificity for immunoaffinity purification

  • Include TEV protease cleavage sites to remove tags after purification

• Contaminating host proteins:

  • L. plantarum lysates contain numerous proteins that may co-purify with serC

  • Solution: Implement a multi-step purification strategy:

    1. Initial capture step (affinity chromatography)

    2. Intermediate purification (ion exchange chromatography)

    3. Polishing step (size exclusion chromatography)

  • Consider heat treatment (55°C for 10 minutes) if recombinant serC demonstrates higher thermostability than host proteins

• Activity preservation:

  • Enzymatic activity can diminish during purification

  • Solution: Monitor activity throughout purification process

  • Optimize buffer conditions using a thermal shift assay to identify stabilizing additives

  • Consider purifying as a fusion with a solubility-enhancing partner (MBP, SUMO) if activity loss is significant

These strategies provide a comprehensive approach to overcome common challenges in purifying active recombinant serC from L. plantarum lysates, ensuring both high yield and preserved enzymatic activity.

What innovative approaches could enhance serC functionality in L. plantarum for metabolic engineering applications?

Several innovative approaches hold promise for enhancing serC functionality in L. plantarum for advanced metabolic engineering applications:

• Protein engineering for improved catalytic properties:

  • Directed evolution of serC to enhance catalytic efficiency or alter substrate specificity

  • Structure-guided rational design based on crystal structures of homologous enzymes

  • Creation of chimeric enzymes by domain swapping with serC homologs from thermophilic organisms to improve stability

  • Computational design of active site residues to modify the kinetic parameters for specific metabolic objectives

• Synthetic biology approaches:

  • Implementation of dynamic sensor-regulator systems to control serC expression in response to metabolic needs

  • Design of synthetic metabolic valves to redirect carbon flux through the serine biosynthesis pathway

  • Integration of serC into synthetic operons that coordinate expression with other enzymes in serine-dependent pathways

  • Development of orthogonal translation systems for serC incorporating non-canonical amino acids with enhanced catalytic properties

• Advanced genomic integration strategies:

  • CRISPR-Cas9 mediated precise genome editing for optimal chromosomal integration of serC

  • Creation of synthetic genomic islands containing serC and associated metabolic genes

  • Implementation of recombineering approaches for markerless, scarless integration

  • Development of tunable integration systems with varying copy numbers to optimize expression levels

• Systems biology-guided optimization:

  • Genome-scale metabolic modeling to predict optimal serC expression levels and identify potential bottlenecks

  • Multi-omics analysis (transcriptomics, proteomics, metabolomics) to understand system-wide effects of serC modulation

  • Flux balance analysis to optimize media composition and culture conditions for enhanced pathway performance

  • Machine learning approaches to predict optimal genetic and environmental parameters for serC functionality

These innovative approaches represent the cutting edge of metabolic engineering and could significantly advance the application of recombinant L. plantarum expressing serC in various biotechnological contexts.

What are the potential implications of serC expression in L. plantarum for microbiome modulation and therapeutic applications?

The expression of serC in recombinant L. plantarum opens intriguing possibilities for microbiome modulation and therapeutic applications:

• Microbiome structure and function modulation:

  • serC-enhanced L. plantarum could influence gut microbial composition through altered metabolite production

  • Recent research shows recombinant L. plantarum can significantly boost gut bacterial diversity based on Shannon-Wiener indices

  • Modified serine metabolism may create unique ecological niches, favoring beneficial commensal bacteria

  • Potential to develop "precision probiotics" with metabolic capabilities designed to correct specific microbiome imbalances

• Immune system modulation potential:

  • L. plantarum strains have demonstrated ability to increase serum IgG and IgG1 levels and stimulate CD4+ T cells

  • serC-dependent metabolites could enhance these immunomodulatory properties

  • Metabolic interactions between recombinant L. plantarum and immune cells may be fine-tuned through serC expression

  • Potential applications in immunotherapy as an adjuvant to enhance vaccine responses or modulate autoimmune conditions

• Metabolite-based therapeutic approaches:

  • Enhanced serine production could yield increased levels of beneficial metabolites

  • Potential for treating disorders of serine metabolism or amino acid deficiencies

  • L. plantarum's GRAS status facilitates translation to clinical applications

  • Recombinant strains could be developed as living biotherapeutics for targeted amino acid delivery

• Dual-function therapeutic systems:

  • Integration of serC expression with surface display of therapeutic proteins

  • Combined metabolic and immunological intervention capabilities

  • Development of programmable probiotics responding to specific gut environmental cues

  • Potential for personalized microbiome modulation based on individual metabolic profiles

The convergence of metabolic engineering and microbiome science offers promising avenues for developing next-generation therapeutic approaches using serC-expressing L. plantarum. These approaches could potentially address conditions ranging from metabolic disorders to immune dysregulation through targeted microbiome modulation.

What are the most promising research trajectories for recombinant L. plantarum expressing serC in the next five years?

The most promising research trajectories for recombinant L. plantarum expressing serC over the next five years encompass several interconnected areas:

• Integration with synthetic biology platforms:

  • Development of genetically encoded biosensors for in situ monitoring of serC activity and metabolite production

  • Design of genetic circuits that enable conditional expression of serC in response to specific gut environmental signals

  • Creation of minimal synthetic gene clusters for optimized serine metabolism

  • These approaches will likely lead to programmable probiotic systems with enhanced metabolic capabilities

• Advanced applications in gut-brain axis research:

  • Investigation of serine metabolism's impact on neurotransmitter precursor availability

  • Exploration of serC-expressing L. plantarum for modulating tryptophan metabolism and serotonin production

  • Potential applications in neurological and psychiatric disorders with metabolic components

  • This trajectory connects microbial metabolism with neurological function in novel therapeutic paradigms

• Expansion to metabolic disease interventions:

  • Development of serC-enhanced L. plantarum as an intervention for inborn errors of serine metabolism

  • Exploration of applications in diabetes management through amino acid metabolism modulation

  • Investigation of serC's role in oxidative stress management in metabolic syndrome

  • These applications leverage the metabolic capabilities of engineered probiotics for precision medicine approaches

• Translation to clinical applications:

  • Optimization of formulations for enhanced survival and colonization in the human gut

  • Development of regulatory frameworks for recombinant probiotics as living biotherapeutics

  • Initiation of early-phase clinical trials for well-defined indications

  • This trajectory will address the critical gaps between laboratory research and clinical implementation