Recombinant Lactobacillus plantarum Serine hydroxymethyltransferase (glyA)

What is the glyA gene and what is its significance in recombinant systems?

The glyA gene encodes serine hydroxymethyl transferase (SHMT), a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the reversible conversion of serine to glycine with the concurrent conversion of tetrahydrofolate to 5,10-methylenetetrahydrofolate. In bacterial systems like Streptococcus thermophilus, SHMT also exhibits threonine aldolase activity, stereospecifically catalyzing the interconversion of L-threonine to glycine and acetaldehyde . The enzyme's catalytic versatility makes it valuable for recombinant systems, particularly for biocatalytic applications.

When expressed in recombinant systems, purified glyA-encoded enzymes show remarkable stability. For instance, lyophilized and precipitated enzymes from recombinant expression systems have demonstrated stability for at least 10 weeks when stored at -20°C and 4°C . This stability is a critical factor for biotechnological applications.

How does L. plantarum function as a recombinant expression host?

Lactobacillus plantarum serves as an excellent expression host for several reasons:

• It is recognized as a food-grade microorganism with GRAS (Generally Recognized As Safe) status

• It can effectively survive passage through the gastrointestinal tract

• It possesses probiotic properties that can enhance immune responses

• It has robust capabilities for heterologous protein expression

The L. plantarum genome contains multiple genes that contribute to its suitability as an expression host, including genes for acid tolerance (dltA&D, gadB), bile tolerance (bsh), and adhesion to intestinal epithelial layers (Mucin22, fbp) . These genes help recombinant L. plantarum survive both in vitro environmental stresses and in vivo conditions within the human gastrointestinal tract .

When used for recombinant protein expression, L. plantarum can display foreign proteins on its surface using anchor motifs such as polyglutamate synthase A (pgsA), enabling effective presentation of antigens or enzymes .

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

Several expression vectors have been developed specifically for L. plantarum. Common examples include:

• pMG36e-pgsA: Used for surface display of proteins by fusion with the pgsA anchor protein

• pWCF: An E. coli-Lactobacillus shuttle expression vector utilizing antibiotic-free screening markers like the aspartic acid-β-semialdehyde dehydrogenase (asd) gene and the alanine racemase (alr) gene

• pLuc2: Successfully used for transformation of L. plantarum ULAG11 strain

The choice of vector depends on specific research goals. For example, the pWCF vector system has been used with pgsA as an attachment matrix for displaying proteins on the L. plantarum surface, as demonstrated in studies expressing influenza virus antigen HA1 .

What are the optimal protocols for recombinant protein expression in L. plantarum?

Optimal protocols for recombinant protein expression in L. plantarum involve several key steps:

Genome Engineering:

• Construction of a suitable expression vector with appropriate promoters and signal peptides

• Selection of antibiotic-free screening markers (e.g., asd gene and alr gene) for environmental safety

• Use of an appropriate intermediate host (e.g., asd gene-deficient E. coli χ6212)

• Selection of appropriate host strain (e.g., alr gene deletion L. plantarum strain NC8Δ)

Expression Optimization:

• Use of surface display elements like pgsA for effective presentation of recombinant proteins

• Optimization of culture conditions, including medium composition, temperature, and induction parameters

• Verification of expression through techniques such as:

  • Immunoblotting with specific antibodies

  • Flow cytometry for surface-displayed proteins

  • Indirect immunofluorescence analysis

Example Protocol for Verification:
As demonstrated in recombinant L. plantarum expressing influenza virus antigen HA1, expression can be verified through:

• Sonication of recombinant bacteria followed by immunoblotting

• Subjecting recombinant L. plantarum to repeated freeze-thaw cycles followed by immunoblotting

• Flow cytometry with specific antibodies and fluorescent-conjugated secondary antibodies

How can enzyme activity and stability be maximized in recombinant systems?

Maximizing enzyme activity and stability in recombinant L. plantarum systems requires careful consideration of multiple factors:

Purification Strategy:
The choice of purification method significantly impacts enzyme recovery and activity. For instance, SHMT with threonine aldolase activity has been successfully purified to homogeneity using a single chromatographic step with Ni-nitrilotriacetic acid affinity, achieving a high activity-recovery yield of 83% .

Stability Enhancement:

• Lyophilization has proven effective for long-term storage

• Storage at appropriate temperatures (either -20°C or 4°C) can maintain stability for extended periods

• Optimization of buffer compositions and additives can enhance stability

Activity Optimization:
Understanding the optimal conditions for enzyme function is crucial. For instance, the threonine aldolase activity of recombinant SHMT demonstrates optimal activity in the pH range of 6-7 . Knowledge of substrate specificity is also important—the Km for L-allo-threonine can be significantly higher (38-fold) than that for L-threonine .

What analytical methods are most effective for characterizing recombinant enzymes from L. plantarum?

Effective characterization of recombinant enzymes from L. plantarum requires a comprehensive analytical approach:

Genomic Analysis:

• Whole genome sequencing using platforms such as Illumina MiSeq

• Sequence similarity searches using BLASTP for identification of genes of interest

• Specialized databases (e.g., BAGEL4) for analysis of gene organization

Protein Characterization:

• SDS-PAGE for molecular weight determination and purity assessment

• Western blotting for identity confirmation

• Mass spectrometry for precise molecular characterization

• N-terminal sequencing for confirmation of protein processing

Functional Analysis:

• Enzyme kinetics studies to determine Km, Vmax, and catalytic efficiency

• Substrate specificity assays

• pH and temperature optima determination

• Stability studies under various conditions

Example Characterization Data for Recombinant SHMT:

How does recombinant L. plantarum affect immune responses in animal models?

Recombinant L. plantarum has demonstrated significant immunomodulatory effects in animal models:

Cellular Immune Response Activation:
Recombinant L. plantarum can activate dendritic cells in Peyer's patches (PPs) and increase the numbers of CD4+IFN-γ+ and CD8+IFN-γ+ cells in the spleen and mesenteric lymph nodes (MLNs) . This activation of T cells is crucial for mounting effective cellular immune responses against pathogens.

Flow cytometry analysis has shown that recombinant L. plantarum expressing antigens such as influenza virus HA1 significantly increases the numbers of CD4+IFN-γ+ and CD8+IFN-γ+ T cells compared to control groups . For example, when mice were immunized with recombinant L. plantarum expressing HA1 with dendritic cell-targeting peptide (DCpep), the following results were observed:

Humoral Immune Response Induction:
Recombinant L. plantarum can elicit strong antibody responses. Studies have shown that oral administration of recombinant L. plantarum can induce:

• Increased serum levels of specific IgG, IgG1, and IgG2a antibodies

• Elevated levels of secretory IgA (sIgA) in feces, bile, and duodenal lavages

• Enhanced hemagglutination inhibition (HI) potency

For instance, mice immunized with recombinant L. plantarum expressing HA1-DCpep showed significantly higher levels of specific IgG antibodies in serum compared to control groups at 2, 4, and 10 weeks post-immunization (P<0.0001) .

What factors influence the efficacy of recombinant L. plantarum as an expression system?

Several critical factors determine the effectiveness of L. plantarum as a recombinant expression system:

Genetic Factors:

• Choice of promoter and signal sequences

• Codon optimization for efficient translation

• Vector stability and copy number

• Presence of genetic elements that enhance protein folding and secretion

Physiological Factors:

• Growth conditions including media composition, pH, and temperature

• Metabolic state of the cells

• Cell wall and membrane characteristics that affect protein translocation

Strain-Specific Factors:
Different L. plantarum strains possess varying capabilities for recombinant protein expression. For example, genomic analysis has revealed strain-specific differences in key metabolic pathways. Some strains possess genes like dltA&D and gadB (responsible for acid tolerance), bsh (for bile tolerance), and clpL (for acid and bile tolerance), which can significantly impact the strain's viability and functionality as an expression host .

Example of Strain-Specific Genomic Differences:
Comparative genomic analysis of L. plantarum strains (e.g., DHCU70 and DKP1) has identified various probiotic genes that contribute to their adaptation to environmental stresses and survival in the gastrointestinal tract. These include genes related to:

• Stress response

• Adherence ability

• Microbe-microbe interaction

• Epithelial barrier protection

• Immune modulatory effects

How can recombinant L. plantarum expressing glyA be utilized in biocatalytic applications?

Recombinant L. plantarum expressing glyA offers several promising biocatalytic applications:

Stereoselective Synthesis:
The SHMT enzyme encoded by glyA demonstrates potential as a biocatalyst for the stereoselective synthesis of β-hydroxy-α-amino acids . When tested for aldol addition reactions with non-natural aldehydes (e.g., benzyloxyacetaldehyde and (R)-N-Cbz-alaninal), the enzyme can produce two possible β-hydroxy-α-amino acid diastereoisomers, albeit with moderate stereospecificity .

Enzymatic Conversion:
The ability of SHMT to catalyze the interconversion of L-threonine to glycine and acetaldehyde can be harnessed for various biotransformation processes . This capability is valuable for the production of chiral building blocks for pharmaceutical compounds.

Whole-Cell Biocatalysis:
Using recombinant L. plantarum as a whole-cell biocatalyst offers advantages over purified enzymes:

• Enhanced stability

• Simplified reaction processes (no need for enzyme purification)

• Potential for continuous processes

• Protection of the enzyme in the cellular environment

What are the emerging research areas for recombinant L. plantarum in vaccine development?

Recombinant L. plantarum has shown promising results as a vaccine delivery system in several emerging research areas:

Mucosal Vaccine Development:
Recombinant L. plantarum can effectively induce mucosal immune responses, making it an excellent candidate for vaccine development against pathogens that infect through mucosal surfaces. For example, oral immunization with recombinant L. plantarum expressing influenza virus antigens resulted in significantly elevated IgA levels in the lungs, duodenum, jejunum, and ileum of mice .

Adjuvant Properties:
L. plantarum can function as both an adjuvant and a vector for recombinant vaccines, enhancing the immunogenicity of the expressed antigens . Studies have shown that recombinant L. plantarum can significantly trigger specific IgG and IgA antibodies against avian leukosis virus subgroup J (ALV-J) and enhance the levels of IgG and sIgA compared to control groups .

Surface Display Technology:
Using surface display elements like pgsA to present antigens on the surface of L. plantarum has proven effective for enhancing immune responses. For instance, recombinant L. plantarum expressing the influenza virus antigen HA1 with dendritic cell-targeting peptide (DCpep) demonstrated enhanced immune responses compared to strains without DCpep .

Protection Against Viral Challenges:
Studies have shown that immunization with recombinant L. plantarum can provide protection against viral challenges. For example, chickens immunized with recombinant L. plantarum expressing viral antigens showed significantly lower positive viremia ratios following viral challenge compared to control groups :

What are common challenges in expressing and purifying recombinant proteins from L. plantarum?

Researchers face several challenges when working with recombinant L. plantarum:

Expression Challenges:

• Low yield of recombinant proteins

• Formation of inclusion bodies

• Improper folding of complex proteins

• Proteolytic degradation

• Toxicity of the expressed protein to the host

Purification Challenges:

• Cell lysis difficulties due to the robust cell wall of L. plantarum

• Co-purification of native L. plantarum proteins

• Loss of activity during purification steps

• Scale-up issues for larger preparations

Solutions and Optimization Strategies:

• Vector optimization: Selection of appropriate promoters, signal sequences, and fusion tags

• Host strain selection: Different L. plantarum strains may offer varying expression capabilities based on their genomic features

• Culture condition optimization: Adjusting media composition, temperature, pH, and induction parameters

• Purification strategy refinement: For example, using affinity chromatography with Ni-nitrilotriacetic acid can achieve high activity-recovery yields (83% as demonstrated with SHMT)

• Stability enhancement: Proper storage conditions (e.g., lyophilization and storage at -20°C or 4°C) can maintain enzyme stability for extended periods

How can genetic stability be maintained in recombinant L. plantarum strains?

Maintaining genetic stability is crucial for consistent expression of recombinant proteins in L. plantarum:

Key Challenges to Genetic Stability:

• Plasmid loss during continuous cultivation

• Genetic rearrangements

• Insertion sequence element activation

• Selection pressure in the absence of antibiotics

Strategies for Enhancing Genetic Stability:

• Antibiotic-free selection systems: Using auxotrophic markers like the asd gene and alr gene instead of antibiotic resistance genes

• Chromosomal integration: Integrating the gene of interest into the chromosome rather than using plasmid-based expression

• Balanced expression systems: Avoiding metabolic burden through controlled expression levels

• Reduced cultivation time: Minimizing the number of generations to reduce the chance of genetic drift

• Media optimization: Providing essential nutrients to reduce selective pressure for genetic changes

Example of Antibiotic-Free Selection System:
Using an E. coli-Lactobacillus shuttle expression vector with the aspartic acid-β-semialdehyde dehydrogenase (asd) gene and the alanine racemase (alr) gene as antibiotic-free screening markers has proven effective. This approach uses asd gene-deficient E. coli (E. coli χ6212) as the plasmid donor and alr gene deletion L. plantarum strain NC8Δ as the host strain .

What are promising areas for future research in recombinant L. plantarum expressing glyA?

Several promising research directions could advance the field:

Enzyme Engineering:

• Structure-guided mutagenesis to enhance the catalytic efficiency of SHMT

• Protein evolution approaches to expand substrate specificity

• Fusion protein designs to combine multiple enzymatic activities

Metabolic Engineering:

• Integration of glyA into synthetic pathways for production of valuable compounds

• Optimization of metabolic flux to enhance precursor availability

• Investigation of the relationship between glutamine metabolism and enzyme production capacity

Advanced Applications:

• Development of recombinant L. plantarum as a living therapeutic

• Creation of multi-functional recombinant strains expressing both glyA and other therapeutic proteins

• Exploration of synergistic effects between glyA expression and other probiotic features of L. plantarum

Regulatory and Delivery Research:

• Development of inducible expression systems for controlled production

• Investigation of targeted delivery mechanisms to specific tissues or organs

• Exploration of novel formulations to enhance stability and efficacy