Recombinant Lactobacillus johnsonii Serine--tRNA ligase (serS)

What is Lactobacillus johnsonii and why is it significant for recombinant protein studies?

Lactobacillus johnsonii is a Gram-positive, homofermentative, non-spore-forming rod-shaped host-adapted bacterium found naturally in the vaginal and gastrointestinal tracts of various vertebrates including humans, rodents, swine, and poultry. It has gained significant research interest due to its probiotic properties, including pathogen antagonism, immune response modulation, and enhancement of epithelial barrier function . L. johnsonii strains have been studied for their ability to partially survive gastric conditions, making them potential candidates for oral vaccine delivery systems and recombinant protein expression . The genome of L. johnsonii strains (such as ZLJ010) contains approximately 1,999,879 bp with a GC content of 34.91%, encoding 18 rRNA genes, 77 tRNA genes, and 1,959 protein coding sequences, providing a robust genomic foundation for recombinant protein expression studies .

What is Serine--tRNA ligase (serS) and what is its biological function in L. johnsonii?

Serine--tRNA ligase (serS) is an essential aminoacyl-tRNA synthetase responsible for catalyzing the attachment of serine to its cognate tRNA molecules during protein synthesis. This enzyme plays a crucial role in the translation process by ensuring accurate incorporation of serine amino acids into growing polypeptide chains. In L. johnsonii, serS is part of the core genome essential for protein synthesis and cellular function. The enzyme catalyzes a two-step reaction: first activating serine with ATP to form seryl-adenylate, then transferring the seryl group to the appropriate tRNA molecule. This process is fundamental to the organism's protein synthesis machinery and represents an essential housekeeping function in bacterial metabolism .

How does the genome organization of L. johnsonii influence recombinant serS expression?

The genome organization of L. johnsonii directly impacts recombinant protein expression strategies. L. johnsonii ZLJ010 contains a single circular chromosome of 1,999,879 bp with a relatively low GC content of 34.91% . This low GC content must be considered when designing expression constructs, as codon optimization may be necessary when expressing L. johnsonii serS in other host systems with different GC preferences. Additionally, L. johnsonii lacks complete biosynthetic pathways for certain amino acids but compensates with enhanced transport systems and unique amino acid permeases . These genomic characteristics necessitate careful consideration of growth media composition when expressing recombinant proteins. When designing vectors for serS expression, researchers should consider the native promoter elements and regulatory regions to ensure proper transcriptional control and expression levels in the chosen host system .

What expression systems are most suitable for recombinant L. johnsonii serS production?

For recombinant expression of L. johnsonii serS, several expression systems can be utilized depending on the research objectives:

• Homologous Expression: Using L. johnsonii itself as an expression host provides the advantage of native codon usage and post-translational modifications. This approach is particularly valuable when studying the protein in its natural cellular context. A vector system similar to that used for expressing PrtB fusion proteins in L. johnsonii can be adapted for serS expression .

• E. coli Expression Systems: For high-yield production, E. coli-based expression systems (pET, pBAD, or pGEX) can be employed with codon optimization to account for the low GC content (34.91%) of L. johnsonii . This approach typically provides higher protein yields but may require additional optimization for proper folding.

• Lactic Acid Bacteria (LAB) Expression Systems: Alternative LAB hosts like Lactococcus lactis may offer advantages for expression of L. johnsonii proteins due to similar cellular environments and secretion mechanisms .

The choice depends on research goals, required yield, and whether authentic post-translational modifications are essential. For structural studies requiring large protein quantities, E. coli systems typically offer the highest yields, while functional studies may benefit from expression in LAB hosts to maintain native protein characteristics.

What purification strategies yield the highest activity of recombinant L. johnsonii serS?

Optimal purification strategies for maintaining high activity of recombinant L. johnsonii serS include:

Multi-step Purification Protocol:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using a His-tag fusion is effective for initial capture, particularly with imidazole gradient elution (20-250 mM) to reduce non-specific binding.

• Intermediate Purification: Ion exchange chromatography (typically DEAE or Q-Sepharose) at pH 7.5-8.0 leverages the theoretical pI of serS.

• Polishing Step: Size exclusion chromatography using Superdex 200 to remove aggregates and achieve >95% purity.

Critical Buffer Considerations:

• Maintain 20-50 mM Tris-HCl or HEPES buffer (pH 7.5-8.0)

• Include 100-200 mM NaCl to maintain solubility

• Add 1-5 mM DTT or 2-mercaptoethanol to protect cysteine residues

• Include 5-10% glycerol as a stabilizing agent

• Add 0.1-0.5 mM EDTA to chelate metal ions that might promote oxidation

This strategy typically yields enzyme with specific activity >500 U/mg when measured by aminoacylation assays. Throughout purification, it's essential to monitor both protein concentration and enzymatic activity to calculate specific activity and recovery percentages at each step .

What analytical methods are most effective for characterizing the activity and specificity of recombinant L. johnsonii serS?

Several complementary analytical methods can effectively characterize recombinant L. johnsonii serS:

Enzymatic Activity Assays:

• ATP-PPi Exchange Assay: Measures the first step of aminoacylation (activation of serine with ATP). Typically yields Km values of 0.1-0.5 mM for serine and 0.2-1.0 mM for ATP.

• tRNA Charging Assay: Quantifies the complete aminoacylation reaction using either radioactive [³H]-serine or colorimetric detection methods. Expected kcat values range from 2-5 s⁻¹.

• Pyrophosphate Release Assay: A continuous assay coupling PPi release to enzymatic reactions with colorimetric/fluorometric detection.

Specificity Characterization:

• Substrate Specificity Panels: Test activity with serine analogs (e.g., threonine, cysteine) to assess amino acid discrimination.

• tRNA Isoacceptor Testing: Evaluate charging efficiency across different tRNASer isoacceptors to determine tRNA specificity patterns.

Structural Analysis:

• Circular Dichroism (CD): Analyze secondary structure elements (typically 30-40% α-helix, 20-30% β-sheet).

• Thermal Shift Assays: Determine protein stability and effects of buffer conditions on melting temperature (Tm typically 45-55°C).

• Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS): Determine oligomeric state and molecular weight in solution.

These methods provide comprehensive characterization of enzymatic parameters, substrate specificity, and structural properties essential for comparing wild-type and mutant serS variants or evaluating enzyme performance under different conditions .

How can recombinant L. johnsonii serS be utilized in mucosal vaccine delivery systems?

Recombinant L. johnsonii serS can be strategically utilized in mucosal vaccine delivery systems through several sophisticated approaches:

L. johnsonii has demonstrated partial survival under simulated gastric conditions, making it a promising vehicle for oral vaccination strategies . To leverage serS in this context, researchers can employ the following methodologies:

Surface Display Fusion Strategy:

• Create chimeric constructs linking immunogenic epitopes to serS using flexible linkers (GGGGS)n

• Alternatively, utilize the cell wall anchoring system demonstrated with proteinase PrtB to display serS-epitope fusions on the L. johnsonii surface

• Express these constructs using vector systems optimized for stable expression in L. johnsonii

Immunological Outcomes:
Research indicates that oral immunization with recombinant L. johnsonii expressing surface proteins can induce both systemic IgG responses and local mucosal IgA responses . By incorporating target epitopes into serS fusion constructs, researchers can potentially elicit:

• Systemic immunity (measured by serum IgG titers)

• Mucosal immunity (measured by secretory IgA in fecal samples)

• Targeted immune responses against the displayed epitopes

Optimization Parameters:
To maximize efficacy, researchers should consider:

• Epitope positioning within the serS structure to ensure proper folding and accessibility

• Expression levels to balance immunogenicity with L. johnsonii fitness

• Dosing regimens (typically 10⁸-10¹⁰ CFU/dose administered 3-5 times)

• Formulation enhancements to improve gastric transit (e.g., microencapsulation)

This approach offers advantages for targeting enteric pathogens or conditions requiring both mucosal and systemic immunity, with significantly less risk of side effects compared to attenuated pathogen vaccines .

What mechanisms explain the substrate specificity of L. johnsonii serS compared to serS from other bacterial species?

The substrate specificity of L. johnsonii serS involves sophisticated molecular mechanisms that distinguish it from serS enzymes in other bacterial species:

• The serine binding pocket, where L. johnsonii serS may have unique residues contributing to serine recognition and discrimination against similar amino acids like threonine

• The ATP binding region, which influences the kinetics of the first step in the aminoacylation reaction

• The tRNA acceptor stem interaction interface, determining tRNA specificity

Recognition Elements for tRNA Specificity:
L. johnsonii serS recognizes specific elements in tRNASer isoacceptors:

• The discriminator base (position 73, typically G73 in bacterial tRNASer)

• The variable arm (characteristic of tRNASer molecules)

• The acceptor stem base pairs

The molecular interactions at these recognition points may differ between L. johnsonii and other bacterial species due to co-evolution of the serS enzyme with the specific tRNA pool available in L. johnsonii .

Evolutionary Context:
Comparative genomic analysis between L. johnsonii and other species suggests evolutionary adaptations in serS that reflect:

• Codon usage patterns in the low GC content (34.91%) genome of L. johnsonii

• Adaptations to the nutritional environment of host-associated niches

• Possible horizontal gene transfer events that have shaped aminoacyl-tRNA synthetase evolution in Lactobacillus species

These molecular adaptations create a unique specificity profile that influences both amino acid and tRNA recognition, with potential implications for the fidelity of protein synthesis in L. johnsonii .

How can site-directed mutagenesis of L. johnsonii serS reveal insights about its catalytic mechanism?

Site-directed mutagenesis of L. johnsonii serS provides a powerful approach to dissect its catalytic mechanism at the molecular level:

Strategic Mutation Targets:

• ATP Binding Site Residues:

  • Conserved lysine residues in Motif 1 (typically K̲XXK) can be mutated to arginine or alanine

  • Expected outcome: Mutations typically reduce kcat by 100-1000 fold with minimal effect on Km for ATP

  • Methodology: Compare ATP-PPi exchange rates between wild-type and mutant enzymes

• Serine Recognition Pocket:

  • Key threonine/serine residues involved in hydrogen bonding with serine

  • Expected outcome: T→A or S→A mutations generally increase Km for serine by 10-50 fold

  • Methodology: Measure aminoacylation efficiency with varying serine concentrations (0.01-10 mM range)

• tRNA Recognition Interface:

  • Residues interacting with the variable arm or acceptor stem of tRNASer

  • Expected outcome: Altered charging efficiency for specific tRNASer isoacceptors

  • Methodology: In vitro transcription of tRNASer followed by charging assays

Experimental Design Table:

Interpretation Framework:
The results can be analyzed in the context of:

• Conservation patterns across bacterial serS enzymes

• Homology models based on known crystal structures of bacterial serS

• Molecular dynamics simulations to visualize the effects of mutations on substrate binding

This systematic mutagenesis approach provides mechanistic insights that can inform both fundamental understanding of aminoacylation mechanisms and potential applications in protein engineering or antibiotic development targeting aminoacyl-tRNA synthetases .

How can recombinant L. johnsonii serS be employed in synthetic biology applications?

Recombinant L. johnsonii serS offers several innovative applications in synthetic biology:

Orthogonal Translation Systems:
Engineered serS variants with altered specificity can enable incorporation of non-canonical amino acids into proteins via:

• Engineering the amino acid binding pocket to accept serine analogs

• Co-evolving the serS with modified tRNASer to create orthogonal pairs

• Implementing in systems requiring selective labeling of proteins with serine analogs

Biosensor Development:
serS-based biosensors can be developed by:

• Coupling serS activity to reporter systems (fluorescent or colorimetric)

• Creating fusion constructs with split reporter proteins that assemble upon serS-tRNA interaction

• Enabling detection of serine levels in complex biological samples with sensitivity in the micromolar range

Cell-Free Protein Synthesis Enhancement:
L. johnsonii serS can improve cell-free protein synthesis systems by:

• Supplementing reaction mixtures with purified serS to prevent serine charging from becoming rate-limiting

• Optimizing the aminoacylation efficiency for tRNASer isoacceptors matching the codon usage of target proteins

• Enabling more efficient synthesis of serine-rich proteins

Biotechnological Parameters for serS Applications:

These applications leverage the unique properties of L. johnsonii serS, including its potential stability characteristics and specificity profile, to create novel biotechnological tools .

What are the challenges and solutions for scaling up production of recombinant L. johnsonii serS?

Key Challenges and Strategic Solutions for serS Scale-Up:

Expression Host Limitations:

• Challenge: L. johnsonii has restricted biosynthetic capabilities, lacking complete pathways for amino acid synthesis

• Solution: Implement fed-batch fermentation with controlled addition of amino acids and peptides; alternatively, transition to more robust expression hosts like L. lactis or engineered E. coli strains with compatible codon usage

Protein Solubility and Folding:

• Challenge: Aminoacyl-tRNA synthetases often show reduced solubility when overexpressed

• Solution: Employ fusion tags (MBP, SUMO) with demonstrated solubility enhancement; implement chaperone co-expression strategies; optimize induction conditions (typically 18-25°C, 0.1-0.5 mM inducer)

Purification Bottlenecks:

• Challenge: Scale-up of affinity chromatography faces limitations in binding capacity and flow rates

• Solution: Develop capture step alternatives such as expanded bed adsorption or precipitation methods followed by ion exchange chromatography; implement continuous chromatography methods for higher throughput

Scale-Up Performance Indicators:

Stability Enhancement Strategies:
To maintain enzyme activity throughout the production process:

• Add stabilizing excipients (10% glycerol, 100-200 mM NaCl, 1-5 mM DTT)

• Implement controlled proteolysis prevention (add protease inhibitors or use protease-deficient strains)

• Develop lyophilization protocols with appropriate cryoprotectants for long-term storage

These integrated strategies address the multi-factorial challenges of scaling up recombinant serS production while maintaining enzyme quality and activity .

What potential exists for using L. johnsonii serS in antimicrobial drug discovery?

Aminoacyl-tRNA synthetases, including serS, represent promising targets for antimicrobial development due to their essential role in protein synthesis. L. johnsonii serS offers unique opportunities in this domain:

Drug Target Potential:
L. johnsonii serS can serve as a model for studying serS inhibition in related pathogenic bacteria. The structural and functional similarities between synthetases across bacterial species, combined with differences from human counterparts, create a window for selective inhibition . By elucidating the specific binding pockets and catalytic mechanisms of L. johnsonii serS, researchers can design inhibitors that may be effective against related pathogens while maintaining selectivity over human serS.

Screening Platform Development:
Recombinant L. johnsonii serS can be employed in high-throughput screening platforms to identify potential inhibitors:

• Biochemical Assays:

  • ATP-PPi exchange assays adapted to 384-well format

  • Fluorescence-based tRNA charging assays using labeled tRNASer

  • Thermal shift assays to detect compound binding

• Structure-Based Approaches:

  • Homology modeling of L. johnsonii serS based on existing bacterial serS structures

  • Virtual screening against identified binding pockets

  • Fragment-based drug design targeting the ATP binding site or serine pocket

Comparative Inhibition Analysis:
By studying inhibition profiles of L. johnsonii serS alongside serS from pathogenic bacteria, researchers can identify:

Translational Potential:
The insights gained from L. johnsonii serS studies could lead to:

• Novel broad-spectrum antibiotics targeting serS in multiple bacterial species

• Narrow-spectrum agents selective for specific bacterial groups

• Combination therapies targeting multiple aminoacyl-tRNA synthetases to reduce resistance development

This approach leverages L. johnsonii as a non-pathogenic model system while contributing to the critical need for new antimicrobial discovery pathways .

How might genome engineering techniques improve recombinant serS expression in L. johnsonii?

Advanced genome engineering techniques offer promising avenues to enhance recombinant serS expression in L. johnsonii:

CRISPR-Cas9 Genome Editing Applications:

• Promoter Engineering: Replace native promoters with stronger constitutive or inducible promoters precisely calibrated for serS expression

• Ribosome Binding Site (RBS) Optimization: Engineer optimal RBS sequences to increase translation efficiency by 5-10 fold

• Genome Streamlining: Remove non-essential gene clusters (e.g., prophage elements identified in L. johnsonii ZLJ010 ) to redirect cellular resources toward serS production

Metabolic Engineering Strategies:

• Amino Acid Metabolism Enhancement: Address the limited amino acid biosynthetic pathways in L. johnsonii by introducing missing enzymes from related species

• Energy Metabolism Optimization: Enhance ATP production pathways to support the energy-intensive aminoacylation reaction

• Stress Response Modulation: Upregulate chaperones and attenuate stress responses to accommodate high-level recombinant protein expression

Systematic Genomic Integration Approach:
Rather than relying solely on plasmid-based expression (which can be unstable), develop genomic integration systems that allow:

• Single-copy integration at defined neutral sites

• Multi-copy integration at dispersed genomic locations

• Integration with inducible control elements for regulated expression

Expected Performance Improvements:

These approaches, particularly when combined in rational designs based on systems biology models of L. johnsonii metabolism, have the potential to significantly improve recombinant serS production while maintaining protein quality and function .

What insights could structural biology provide about L. johnsonii serS function and evolution?

Structural biology approaches would yield profound insights into L. johnsonii serS function and evolution:

Structural Determination Priorities:

• Full-Length serS Crystal Structure: Determine the atomic-level structure at <2.0 Å resolution to reveal:

  • Precise active site geometry and substrate binding determinants

  • Conformational changes during catalysis

  • Domain organization and interface regions

• Complex Structures with Substrates and Products:

  • serS:ATP complex to elucidate binding mode and catalytic residues

  • serS:Ser-AMP complex to capture the reaction intermediate

  • serS:tRNASer complex to identify recognition elements

Evolutionary Insights from Structural Analysis:
Comparing L. johnsonii serS structure with those from other species would reveal:

• Conserved catalytic cores representing ancestral aminoacyl-tRNA synthetase features

• Variable regions reflecting adaptation to different cellular environments

• Lineage-specific structural features correlating with the phylogenomic analysis of L. johnsonii strains

Structure-Function Relationships:
Structural data would enable mapping of functional properties to specific structural elements:

Integration with Biophysical Techniques:
Complementing structural studies with:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map protein dynamics

• Nuclear magnetic resonance (NMR) to analyze solution-state behavior

• Single-molecule FRET to capture conformational changes during catalysis

This multi-faceted structural biology approach would reveal how L. johnsonii serS has evolved within the constraints of maintaining essential aminoacylation function while adapting to the specific cellular environment of L. johnsonii, including its low GC content genome (34.91%) and host-adapted lifestyle .

How could systems biology approaches enhance our understanding of serS function within the L. johnsonii proteome network?

Systems biology approaches provide a comprehensive framework for understanding serS function within the broader L. johnsonii cellular context:

Multi-omics Integration Strategy:

• Transcriptomics Analysis:

  • RNA-seq under various growth conditions to identify co-expressed gene clusters with serS

  • Determine transcriptional regulation patterns in response to amino acid availability

  • Map operon structures and potential regulatory elements controlling serS expression

• Proteomics Networks:

  • Quantitative proteomics to determine serS abundance relative to other translation machinery components

  • Protein-protein interaction mapping using proximity labeling techniques (BioID, APEX)

  • Post-translational modification analysis to identify regulatory PTMs on serS

• Metabolomics Connections:

  • Track serine and related amino acid pools under different growth conditions

  • Measure tRNA charging levels across the aminoacyl-tRNA synthetase family

  • Identify metabolic bottlenecks affecting serS function

Genome-Scale Metabolic Modeling:
Incorporating serS function into genome-scale metabolic models of L. johnsonii to:

Network Analysis Frameworks:

Integration with Comparative Genomics:
Placing serS in the context of the pan-genome and core-genome analysis of L. johnsonii strains (1,324 core-genome orthologous gene clusters identified across strains) to understand:

• Conservation of serS network connections across strains

• Strain-specific adaptations in serS regulation

• Co-evolution patterns with interacting partners

This systems-level understanding would provide holistic insights into how serS functions within the complex cellular network of L. johnsonii, revealing both direct functional interactions and emergent properties that cannot be discerned through reductionist approaches alone .