Recombinant Lactobacillus plantarum Lysine--tRNA ligase (lysS)

What expression systems are most effective for recombinant lysS production in L. plantarum?

The pSIP expression system is widely recognized as one of the most efficient platforms for heterologous protein expression in L. plantarum. This system, particularly variants like pSIP401 and pSIP409, contains inducible promoters based on bacteriocin production regulatory elements, enabling controlled expression of target genes. For recombinant lysS expression, pSIP vectors offer several advantages including:

• Tight regulation through inducible promoters to control expression timing

• Compatibility with various signal peptides for either intracellular expression or secretion

• Established selection markers (erythromycin resistance) for stable plasmid maintenance

• Multiple cloning sites for flexible genetic engineering

L. plantarum WCFS1 has been extensively utilized as a host for intracellular expression, secretion, and cell-surface display of heterologous proteins using these pSIP expression systems . When expressing proteins like α-amylase, researchers have successfully employed the pSIP401/409 systems in combination with different signal peptides to achieve high-yield production .

For lysS expression specifically, these systems would provide a solid foundation, potentially requiring optimization of induction parameters and signal peptide selection based on the enzyme's characteristics.

What are the advantages of using L. plantarum as a host for recombinant protein expression compared to other systems?

L. plantarum offers several distinct advantages as an expression host for recombinant proteins, including lysS:

• Safety profile: L. plantarum possesses "Generally Recognized As Safe" (GRAS) status, making it suitable for various applications in food, pharmaceutical, and biotechnology sectors .

• Efficient secretion: Depending on the signal peptide utilized, L. plantarum can efficiently secrete proteins into the extracellular environment, facilitating downstream processing and purification .

• Growth characteristics: These bacteria grow robustly in simple media without complex nutritional requirements or aeration, typically reaching optical densities (OD600) around 8.0 after 18-24 hours of cultivation .

• Established genetic tools: Numerous genetic manipulation tools have been developed specifically for L. plantarum, including expression vectors, transformation protocols, and homologous recombination methods .

• Environmental adaptability: L. plantarum demonstrates remarkable tolerance to various environmental conditions, including the ability to "survive in gastrointestinal conditions," which can be advantageous for certain applications .

For lysS expression, these characteristics would potentially translate to cost-effective production, simplified purification (especially if secreted), and versatile application possibilities.

What growth conditions optimize recombinant protein expression in L. plantarum?

Optimal conditions for maximizing recombinant protein expression in L. plantarum typically include:

Research has shown that recombinant L. plantarum strains typically reach maximum culture density after 18-24 hours of cultivation, although strains containing certain signal peptide constructs (such as Lp_3050) may exhibit slower growth rates . Additionally, the timing of harvest can be critical, as some recombinant proteins show activity decline after specific timepoints - for example, AmyL activities dropped significantly after 12 hours of cultivation .

For recombinant lysS expression, these parameters would serve as a starting point, with potential refinement needed based on the specific characteristics of the enzyme and expression construct.

How do different signal peptides affect protein secretion efficiency in L. plantarum?

Signal peptide selection dramatically impacts both the total expression and secretion efficiency of recombinant proteins in L. plantarum. Comparative studies have evaluated several signal peptides:

• Lp_2145, Lp_3050, and Lp_0373 (derived from L. plantarum WCFS1)

• Native signal peptides from target proteins (e.g., SP_AmyL from L. plantarum S21)

• Signal peptides from related species (e.g., SP_AmyA from L. amylovorus)

Research findings reveal significant performance differences among these signal peptides:

These findings suggest that for recombinant lysS expression, testing multiple signal peptides would be advisable, with Lp_2145 likely optimizing total yield while Lp_0373 might offer better secretion efficiency if the native signal peptide is unavailable or underperforming.

What molecular mechanisms explain the correlation between signal peptide selection and mRNA levels in recombinant L. plantarum systems?

An intriguing and often overlooked aspect of heterologous protein expression in L. plantarum is the significant impact of signal peptide selection on transcription levels. Research utilizing real-time reverse-transcriptase quantitative PCR (RT-qPCR) has revealed that different signal peptides not only affect protein secretion but also influence mRNA abundance of the target gene .

Several molecular mechanisms potentially explain this phenomenon:

For lysS expression optimization, comparative transcriptional analysis of various signal peptide constructs would be essential to identify the optimal configuration for maximizing both transcription and secretion.

How can real-time PCR methods be optimized for monitoring lysS expression in recombinant L. plantarum?

Optimizing real-time PCR methods for accurate quantification of lysS expression requires careful consideration of several methodological aspects:

• RNA extraction optimization: L. plantarum, being a Gram-positive bacterium, requires specialized extraction protocols due to its thick peptidoglycan layer. Methods employing enzymatic cell wall degradation (lysozyme treatment) followed by phenol-chloroform extraction or commercial kits designed for Gram-positive bacteria yield superior results.

• Reference gene selection: Proper normalization requires stable reference genes. For L. plantarum, several housekeeping genes have proven effective:

• Primer design considerations: Primers for lysS should:

  • Target unique regions to avoid amplification of native aminoacyl-tRNA synthetases

  • Span exon-exon junctions if possible to prevent genomic DNA amplification

  • Maintain optimal GC content (40-60%) for efficient amplification

  • Generate amplicons of 80-150 bp for optimal qPCR efficiency

• Validation approaches: Employ standard curves with known quantities of template to determine amplification efficiency, and use melt curve analysis to confirm amplicon specificity.

Previous research has demonstrated that RT-qPCR can effectively correlate mRNA levels with protein production in L. plantarum recombinant systems, with highest mRNA levels typically predicting highest protein yields . This approach enables rapid screening of different expression constructs and optimization of induction parameters.

What strategies can address the challenge of proteolytic degradation during recombinant lysS production?

Proteolytic degradation presents a significant challenge in recombinant protein production in L. plantarum. For complex enzymes like lysS, maintaining structural integrity throughout expression and purification requires multifaceted approaches:

• Host strain engineering options:

  • Deletion or disruption of major extracellular protease genes

  • Overexpression of protease inhibitors during recombinant protein production

  • Selection of naturally low-protease variants through screening

• Expression vector design considerations:

  • Fusion with stabilizing partners (e.g., thioredoxin, SUMO) that increase proteolytic resistance

  • Incorporation of protease-resistant linker sequences between domains

  • Strategic elimination of recognized protease cleavage sites through silent mutations

• Cultivation parameter optimization:

  • Temperature reduction during expression phase (30-32°C) to slow proteolytic activity

  • pH adjustment to suboptimal conditions for L. plantarum proteases (typically pH 6.5-7.0)

  • Supplementation with amino acids to reduce nutrient-limitation stress responses

• Harvest and purification considerations:

  • Addition of protease inhibitor cocktails immediately upon cell disruption

  • Rapid processing at reduced temperatures (4°C) to minimize degradation

  • Implementation of affinity purification strategies to quickly isolate target protein

Research has demonstrated that protein stability can vary significantly between different recombinant proteins in L. plantarum, with some proteins maintaining stability over extended periods while others show rapid activity decline . For lysS, which has complex structural requirements for enzymatic activity, optimization of these parameters would be essential to maintain functional integrity.

How does codon optimization influence recombinant lysS expression in L. plantarum systems?

Codon optimization represents a powerful strategy for enhancing recombinant lysS expression in L. plantarum by addressing several key factors that affect translation efficiency:

• Codon usage adaptation:
  L. plantarum exhibits distinct codon preferences that differ from other organisms. Analyzing the codon adaptation index (CAI) of native lysS versus L. plantarum-optimized sequences reveals:

• mRNA secondary structure modification:

  • Optimization can eliminate strong secondary structures, particularly near the start codon

  • Reduction of ΔG values in the first 40-50 nucleotides can significantly improve translation initiation

  • Strategic introduction of silent mutations can disrupt hairpin formations that impede ribosome progression

• GC content adjustment:

  • L. plantarum WCFS1 has a genomic GC content of approximately 44.5%

  • Adapting the lysS gene to similar GC content improves mRNA stability and processing

  • Extreme GC content in localized regions can be eliminated through synonymous substitutions

• Elimination of problematic sequence elements:

  • Removal of internal Shine-Dalgarno-like sequences that cause translational pausing

  • Elimination of internal transcription termination signals

  • Avoidance of repeated sequence elements that might promote recombination

Transcriptional analysis has demonstrated that codon-related factors can significantly influence mRNA levels and stability . For lysS expression, codon optimization should be considered especially if the native gene originates from an organism with substantially different codon usage patterns than L. plantarum.

What purification strategies yield highest recovery of active recombinant lysS from L. plantarum cultures?

Purification of recombinant lysS from L. plantarum requires a strategic approach to maintain enzymatic activity while achieving high purity. Based on the biochemical properties of aminoacyl-tRNA synthetases and L. plantarum expression characteristics, the following methodological workflow is recommended:

• Initial processing strategies:

  For intracellular expression:

  • Cell harvesting at optimal time point (typically early stationary phase)

  • Resuspension in buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 5 mM DTT

  • Cell disruption via sonication or high-pressure homogenization

  • Clarification by centrifugation (15,000×g, 30 min, 4°C)

  For secreted expression:

  • Culture supernatant collection after centrifugation

  • Concentration via tangential flow filtration or ammonium sulfate precipitation

  • Buffer exchange into purification-compatible conditions

• Chromatographic purification sequence:

• Activity preservation considerations:

  • Inclusion of essential cofactors (Mg²⁺, ATP) in purification buffers

  • Addition of stabilizing agents (10% glycerol, reducing agents)

  • Maintenance of optimal pH range (typically 7.0-7.5 for lysS)

  • Temperature control (4°C throughout purification)

• Quality assessment methods:

  • SDS-PAGE analysis for purity evaluation

  • Aminoacylation assay measuring formation of Lys-tRNA^Lys

  • ATP-PPi exchange assay for catalytic activity

  • Thermal shift assay to assess protein stability

Studies with other recombinant proteins in L. plantarum have demonstrated that proper purification strategy selection significantly impacts retention of enzymatic activity . For lysS, which requires proper folding and cofactor binding for activity, preservation of these structural features throughout purification is essential.

What protein engineering approaches can enhance the stability and catalytic efficiency of recombinant L. plantarum lysS?

Protein engineering offers powerful approaches to enhance recombinant lysS stability and catalytic properties. Based on established methodologies and the structural-functional relationship of aminoacyl-tRNA synthetases, the following strategies can be implemented:

• Rational design approaches based on structural analysis:

  • Introduction of surface-exposed charged residues to enhance solubility

  • Engineering disulfide bridges at strategic positions to stabilize tertiary structure

  • Modification of flexible loops to reduce proteolytic susceptibility

  • Optimization of active site residues to improve substrate binding or catalysis

• Directed evolution methodologies:

  • Error-prone PCR to generate lysS variant libraries

  • DNA shuffling between lysS genes from thermophilic and mesophilic organisms

  • Screening protocols using high-throughput aminoacylation assays

  • Selection systems based on complementation of lysS-deficient strains

• Computational design strategies:

  • Molecular dynamics simulations to identify destabilizing regions

  • In silico prediction of stabilizing mutations using algorithms like CUPSAT or FoldX

  • Automated design of consensus sequences from multiple lysS homologs

  • Energy minimization calculations to optimize protein core packing

• Domain engineering approaches:

  • Creation of chimeric lysS by combining domains from different species

  • Truncation analysis to identify minimal functional units

  • Addition of stabilizing domains from thermophilic organisms

  • Integration of protein-protein interaction domains for immobilization

These engineering approaches would require careful structural characterization of the modified proteins. Successful protein engineering has been demonstrated with other enzymes expressed in L. plantarum, such as α-amylase, which has been extensively characterized regarding its enzymatic properties and stability under various conditions .

For lysS specifically, engineering goals might include enhancing thermostability for longer shelf-life, improving catalytic efficiency under suboptimal conditions, and increasing tolerance to oxidative conditions frequently encountered during processing and storage.