Recombinant Lactobacillus plantarum 6,7-dimethyl-8-ribityllumazine synthase (ribH)

What expression systems are available for recombinant ribH production in L. plantarum?

Several expression systems have been developed for recombinant protein production in L. plantarum. The most effective systems utilize constitutive promoters of varying strengths coupled with appropriate plasmid backbones. Based on comparative studies, the P11 promoter demonstrates strong constitutive expression capabilities, particularly when combined with high-copy-number plasmids . Additional promoter options include Ptuf33 and Ptuf34, which offer alternative expression levels for researchers seeking fine-tuned protein production .

For optimal expression, researchers should consider the following methodological approach:

• Select an appropriate promoter (P11 for high expression, Ptuf33/Ptuf34 for moderate expression)

• Choose between high-copy (pCDLbu-1ΔEc-based) or low-copy (p256-based) plasmid backbones

• Optimize the ribosomal binding site (RBS) design and spacing

• Consider the impact of codon usage on translation efficiency

The choice between these systems should be dictated by the experimental requirements, as higher expression levels may lead to increased protein yield but potentially compromise bacterial growth rates or proper protein folding .

What are the optimal storage conditions for maintaining recombinant ribH stability?

Recombinant L. plantarum 6,7-dimethyl-8-ribityllumazine synthase exhibits optimal stability when stored at -20°C to -80°C in its liquid form, with an expected shelf life of approximately 6 months under these conditions . For long-term storage, researchers should consider the following recommendations:

• Store purified protein in a buffer containing stabilizing agents (e.g., glycerol at 10-20%)

• Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Include reducing agents if the protein contains critical cysteine residues

• Consider lyophilization for extended storage periods

• Monitor activity periodically using standardized enzyme assays

When working with the enzyme, gradual temperature transitions and maintenance of optimal pH conditions (typically pH 7.0-7.5) will help preserve catalytic activity during experimental procedures.

How can the ribosomal binding site be optimized to enhance ribH expression in L. plantarum?

Optimizing the ribosomal binding site (RBS) represents a sophisticated approach to fine-tuning recombinant protein expression in L. plantarum. Research demonstrates that both the sequence complementarity to the Shine-Dalgarno sequence (SDS) and the spacing between the SDS and start codon significantly impact translation efficiency .

For optimal translation of ribH in L. plantarum, consider the following evidence-based approach:

• Utilize an RBS that closely matches the consensus sequence for L. plantarum (derived from highly expressed genes like slpB)

• Experiment with SDS-start codon spacing between 5-12 nucleotides, with optimal spacing typically falling between 7-9 nucleotides

• Consider the sequence context surrounding the RBS, as secondary structures can impede ribosome binding

Data from experimental studies suggests that optimal SDS design can yield up to 2-fold increases in protein expression compared to suboptimal sequences, even when using identical promoters and vector backbones . When designing constructs, researchers should prioritize RBS optimization alongside promoter selection to achieve desired expression levels.

What structural and functional characteristics distinguish L. plantarum ribH from homologs in other species?

While specific structural data for L. plantarum ribH is limited in the provided search results, comparative analysis with homologous enzymes provides valuable insights. The Schizosaccharomyces pombe 6,7-dimethyl-8-ribityllumazine synthase functions as a homopentamer with 17-kDa subunits forming a complex with an apparent molecular mass of 87 kDa . This quaternary structure is likely conserved in the L. plantarum enzyme.

Key structural-functional relationships observed in homologous enzymes include:

• Critical tryptophan residues (like Trp27 in S. pombe) that influence substrate binding and catalytic efficiency

• Distinct binding sites for substrate and product molecules

• Conformational changes upon ligand binding that affect catalytic activity

Experimental approaches to investigate these characteristics in L. plantarum ribH would include:

• X-ray crystallography to determine three-dimensional structure (similar to the S. pombe homolog crystals that diffract to 2.4Å resolution)

• Site-directed mutagenesis studies targeting conserved aromatic residues

• Spectroscopic analyses to examine substrate and product binding (noting that riboflavin binding to the S. pombe enzyme shows altered fluorescence properties and distinctive absorption characteristics)

What methodological approaches are most effective for purifying active recombinant ribH from L. plantarum?

Purification of recombinant ribH from L. plantarum requires a strategic approach that preserves enzymatic activity while achieving high purity. Based on properties of similar recombinant proteins, the following methodological workflow is recommended:

• Cell disruption optimization: Gentle lysis methods (e.g., lysozyme treatment combined with osmotic shock) may be preferable to mechanical disruption to maintain protein structure

• Initial capture: Affinity chromatography using incorporated tags (His-tag or Strep-tag) as demonstrated in related recombinant protein systems

• Intermediate purification: Ion exchange chromatography to separate based on charge properties

• Polishing step: Size exclusion chromatography to isolate the pentameric form from monomers or aggregates

• Activity preservation: Incorporation of stabilizing agents (e.g., glycerol, reducing agents) throughout the purification process

When designing expression constructs, researchers should consider incorporating tags like the 6×His-tag or Strep-tag demonstrated to be effective in similar recombinant L. plantarum systems . These tags facilitate detection and purification while typically maintaining native protein structure and function.

What kinetic parameters should be measured to characterize recombinant L. plantarum ribH?

Comprehensive kinetic characterization of recombinant L. plantarum ribH should include determination of the following parameters:

• Michaelis-Menten constants (Km) for both substrates:

  • 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione

  • 3,4-dihydroxy-2-butanone 4-phosphate

• Maximum reaction velocity (Vmax) and catalytic efficiency (kcat/Km)

• Product inhibition constants for 6,7-dimethyl-8-ribityllumazine

• Binding affinity (Kd) for riboflavin and other potential ligands

Homologous enzymes (e.g., from S. pombe) exhibit Km values of approximately 5 μM and 67 μM for 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate, respectively, with a Vmax of 13,000 nmol·mg-1·h-1 . These values provide reference points for comparative analysis of the L. plantarum enzyme.

Methodology for determining these parameters should include:

• Steady-state kinetic analysis using spectrophotometric assays

• Isothermal titration calorimetry for binding studies

• Surface plasmon resonance for interaction dynamics

How can different promoter systems be evaluated for ribH expression in L. plantarum?

Systematic evaluation of promoter performance for ribH expression requires a methodical comparative approach. The following experimental design is recommended based on successful strategies in L. plantarum expression systems:

• Construct preparation:

  • Generate expression vectors with ribH under control of various promoters (P11, Ptuf33, Ptuf34)

  • Maintain identical RBS, plasmid backbone, and terminator sequences to isolate promoter effects

  • Include reporter constructs (e.g., mCherry) for rapid visual assessment

• Expression analysis methods:

  • Quantitative PCR for transcript-level comparison

  • Western blotting for protein-level quantification

  • Enzymatic activity assays for functional protein assessment

  • Flow cytometry for single-cell expression analysis when using fluorescent reporters

• Data collection and analysis:

  • Monitor growth curves to assess metabolic burden

  • Determine specific productivity (protein per cell)

  • Calculate relative promoter strengths

Experimental data from L. plantarum expression systems indicates that P11 typically outperforms other constitutive promoters, especially when combined with high-copy-number plasmids, yielding approximately two-fold higher product levels compared to low-copy-number systems .

What strategies can address protein folding challenges for recombinant ribH?

Recombinant ribH production in L. plantarum may encounter folding challenges, particularly at high expression levels. The following evidence-based strategies can help overcome these limitations:

• Optimization of growth conditions:

  • Reduced growth temperature (20-25°C) to slow translation and facilitate folding

  • Controlled induction rates if using inducible systems

  • Media supplementation with cofactors or substrates that may stabilize the native conformation

• Co-expression approaches:

  • Introduction of chaperone proteins to assist folding

  • Expression of protein disulfide isomerases if disulfide bonds are present

• Protein engineering solutions:

  • Fusion to solubility-enhancing tags (e.g., thioredoxin, MBP)

  • Codon optimization to match L. plantarum preferences

  • Removal of rare codons that may cause translational pausing

• Process development considerations:

  • Batch vs. fed-batch cultivation strategies

  • Harvest timing optimization to maximize soluble protein yield

These strategies should be implemented systematically, evaluating protein solubility, activity, and yield at each stage to identify the most effective combination for ribH production in L. plantarum.

How can recombinant L. plantarum expressing ribH be utilized in metabolic engineering?

L. plantarum strains engineered to express recombinant ribH at elevated levels present opportunities for metabolic engineering applications, particularly in riboflavin biosynthesis enhancement. Potential research directions include:

• Riboflavin overproduction:

  • Overexpression of ribH alongside other riboflavin biosynthetic enzymes

  • Relief of rate-limiting steps in the pathway

  • Creation of riboflavin-enriched probiotic strains

• Pathway optimization:

  • Fine-tuning expression levels using the characterized promoter systems (P11, Ptuf33, Ptuf34)

  • Balancing metabolic flux through intermediate accumulation analysis

  • Cofactor regeneration enhancement

• Synthetic biology applications:

  • Development of riboflavin-responsive biosensors

  • Creation of genetic circuits utilizing riboflavin-dependent components

  • Engineering of novel riboflavin derivatives with altered properties

• Whole-cell biocatalysis:

  • Utilizing recombinant L. plantarum as a cell factory for producing riboflavin or derivatives

  • Development of immobilized cell systems for continuous production

The successful expression of other recombinant proteins in L. plantarum, such as viral antigens , suggests that similar methodologies could be applied to ribH expression for metabolic engineering purposes.

What comparative analyses should be performed between ribH from L. plantarum and other bacterial sources?

Comprehensive comparative analysis between L. plantarum ribH and homologs from other bacterial sources should include:

• Sequence-structure-function relationships:

  • Multiple sequence alignment to identify conserved and variable regions

  • Homology modeling based on crystallized homologs (e.g., S. pombe enzyme)

  • Identification of species-specific structural features

• Biochemical property comparison:

  • Kinetic parameter analysis (Km, kcat, substrate specificity)

  • Stability under various pH and temperature conditions

  • Cofactor requirements and binding affinities

  • Oligomeric state confirmation (expected homopentamer like S. pombe enzyme)

• Biotechnological potential assessment:

  • Expression efficiency in heterologous hosts

  • Activity in non-native cellular environments

  • Stability during purification and storage

• Evolutionary analysis:

  • Phylogenetic relationships among ribH proteins

  • Selective pressures on conserved catalytic residues

  • Horizontal gene transfer events

Data from the S. pombe enzyme, which shows a Kd of 1.2 μM for riboflavin binding and displays distinctive spectroscopic properties when complexed with riboflavin , provides valuable comparative reference points for characterizing the L. plantarum enzyme.

What are common challenges in expressing functional ribH in L. plantarum and how can they be addressed?

Researchers frequently encounter several challenges when expressing recombinant ribH in L. plantarum. The following table outlines these issues and evidence-based solutions:

When troubleshooting expression issues, a systematic approach addressing each potential cause is recommended. Studies on L. plantarum expression systems indicate that fine-tuning the space between the Shine-Dalgarno sequence and the start codon (optimally 7-9 nucleotides) can significantly impact expression levels .

How can enzyme activity assays for ribH be optimized for high-throughput screening?

Development of optimized high-throughput screening assays for ribH activity requires careful consideration of reaction conditions and detection methods. The following methodological approach is recommended:

• Assay principle selection:

  • Direct measurement of 6,7-dimethyl-8-ribityllumazine formation via fluorescence

  • Coupled enzyme assays linking product formation to detectable signal

  • Binding assays using fluorescently-labeled substrates or products

• Miniaturization strategies:

  • Adaptation to 96/384-well plate format

  • Reduction of reaction volumes (50-100 μL)

  • Automated liquid handling implementation

• Detection optimization:

  • Fluorescence-based detection (λex = 408 nm, λem = 490 nm for 6,7-dimethyl-8-ribityllumazine)

  • Coupling to NAD(P)H-dependent reactions for spectrophotometric detection

  • Development of riboflavin-based FRET systems

• Validation parameters:

  • Signal-to-noise ratio optimization

  • Z'-factor determination (aim for >0.7)

  • Coefficient of variation assessment (<10%)

• Controls and standards:

  • Purified enzyme standards for calibration

  • Substrate/product standards for quantification

  • Positive and negative controls for each plate

These optimized assays can be employed for mutant screening, inhibitor discovery, or comparative analysis of ribH variants from different bacterial sources.