Recombinant Lactobacillus plantarum Rhamnulokinase (rhaB)

What is the functional role of Rhamnulokinase (rhaB) in L. plantarum metabolism?

Rhamnulokinase (rhaB) is a key enzyme in the rhamnose fermentation pathway of Lactobacillus plantarum. It functions primarily as a kinase that phosphorylates L-rhamnulose to L-rhamnulose-1-phosphate, which is an essential step in rhamnose catabolism. In L. plantarum, the rhamnose fermentation pathway involves several genes, with rhaB (LSL_1752) being identified as encoding rhamnulokinase . This enzyme is part of the metabolic flexibility that allows L. plantarum to utilize different carbon sources, including plant-derived rhamnose-containing compounds, which is particularly important for its ecological versatility in plant material and fermented vegetable substrates .

The ability to metabolize rhamnose provides L. plantarum with a competitive advantage in environments where this sugar is available. Experimental confirmation has shown that strains possessing functional rhamnose utilization genes can effectively assimilate and ferment rhamnose . This capability appears to be encoded on megaplasmids in some L. plantarum strains, suggesting that these extra-chromosomal elements contribute significantly to the metabolic repertoire of these bacteria.

What expression systems have been successfully used for recombinant production of L. plantarum Rhamnulokinase?

Several expression systems have been developed for the recombinant production of proteins in L. plantarum, which can be applied to Rhamnulokinase production. The pSIP expression system has emerged as particularly effective for heterologous protein expression in L. plantarum. This system allows for controlled gene expression and has been successfully used for various enzymes .

For recombinant protein expression in L. plantarum, researchers have employed several strategies:

• Native Signal Peptides: Using the natural signal peptides from the target protein or from other well-expressed proteins in L. plantarum.

• Non-Cognate Signal Peptides: Employing signal peptides from other proteins, such as Lp_2145, Lp_3050, and Lp_0373, which are Sec-type signal peptides derived from L. plantarum WCFS1 .

• Inducible Promoters: Utilizing inducible systems like the SppIP-inducible promoter, which allows for controlled expression at specific time points .

A comparative study of signal peptides for recombinant protein expression in L. plantarum WCFS1 demonstrated that:

The Lp_2145 signal peptide produced the highest total and extracellular enzymatic activities, representing a 6.2-fold and 5.4-fold increase respectively compared to using the native signal peptide .

How can researchers optimize the production of recombinant Rhamnulokinase in L. plantarum?

Optimizing recombinant Rhamnulokinase production in L. plantarum requires careful consideration of several factors:

• Selection of Signal Peptide: The choice of signal peptide significantly impacts expression levels and secretion efficiency. For example, the Lp_2145 signal peptide has shown superior performance for recombinant protein expression in L. plantarum WCFS1 .

• Induction Conditions: For SppIP-inducible systems, optimal conditions include:

  • Inducer concentration: 50 ng/mL SppIP

  • Temperature: 37°C

  • Induction time: 6-10 hours

• Codon Optimization: Adapting the codons of the target gene to match the codon usage bias of L. plantarum can significantly enhance expression efficiency .

• Growth Media and Conditions: L. plantarum strains are typically grown anaerobically in MRS broth at 37°C without agitation, with cultures reaching OD600 of around 8.0 after 18-24 hours .

• Transcript Levels: RT-qPCR analysis has shown that the choice of signal peptide can affect mRNA levels of the target gene. Constructs with the Lp_2145 signal peptide exhibited the highest mRNA levels, reaching peak expression at 3 hours post-induction .

• Stability Enhancement: The stability of recombinant proteins in L. plantarum can be maintained under various conditions:

  • Temperature stability: up to 50°C for 20 minutes

  • pH stability: as low as pH 1.5 for 30 minutes

  • Bile salt tolerance: up to 0.2% bile salt concentration

What are the recommended methods for assaying Rhamnulokinase activity in recombinant systems?

Assaying Rhamnulokinase activity requires specific methodologies to accurately measure its kinase function:

• Spectrophotometric Coupled Assays: Rhamnulokinase activity can be measured by coupling the production of ADP to NADH oxidation through pyruvate kinase and lactate dehydrogenase, monitoring the decrease in absorbance at 340 nm.

• Verification Techniques:

  • Western Blotting: Using specific antibodies to confirm expression, as demonstrated in studies with other recombinant proteins in L. plantarum .

  • SDS-PAGE Analysis: To visualize the recombinant protein in cell lysates and supernatants .

  • Flow Cytometry: To assess surface display efficiency when the protein is expressed on the bacterial surface .

• Stability Testing: Evaluating enzyme stability under different conditions:

  • Temperature variations (37°C or 50°C)

  • pH variations (pH 1.5 or 7)

  • Bile salt concentrations (0-0.5%)

• Real-time RT-qPCR: To estimate mRNA levels of the target gene transcript and correlate with protein expression levels .

What are the potential biotechnological applications of recombinant L. plantarum Rhamnulokinase?

Recombinant L. plantarum Rhamnulokinase has several potential applications in biotechnology:

• Biocatalysis: Rhamnulokinase can be used in biocatalytic processes for the production of rare sugars and sugar derivatives, particularly L-rhamnulose-1-phosphate, which is a valuable precursor for various biochemical syntheses.

• Metabolic Engineering: The enzyme can be employed in metabolic engineering strategies to enhance rhamnose utilization in industrial microorganisms, potentially improving the production of valuable metabolites.

• Food-Grade Enzyme Production: As L. plantarum is a food-grade organism, recombinant enzymes produced in this host can be used in food applications without extensive purification .

• Vaccine Development: The L. plantarum expression system used for Rhamnulokinase can be adapted for developing oral vaccines, as demonstrated with other recombinant proteins such as the SARS-CoV-2 spike protein and influenza virus antigens .

How does Rhamnulokinase function compare to other enzymes in the rhamnose metabolism pathway?

Rhamnose metabolism involves several enzymes working in concert:

• Rhamnulokinase (rhaB): Phosphorylates L-rhamnulose to L-rhamnulose-1-phosphate using ATP.

• Rhamnosidases: These enzymes hydrolyze rhamnose from various substrates like rutinosides and neohesperidosides. While distinct from Rhamnulokinase, they function upstream in rhamnose metabolism .

Comparative analysis of rhamnose-metabolizing enzymes in different Lactobacillus species:

The rhamnose metabolism pathway in Lactobacillus is of particular interest because it contributes to the bacteria's ability to utilize plant-derived compounds, potentially enhancing their ecological fitness in plant environments and fermented food products .

What methodologies are available for structural and functional analysis of recombinant Rhamnulokinase?

Several approaches can be employed for the structural and functional characterization of recombinant Rhamnulokinase:

• Protein Purification Techniques:

  • Affinity chromatography using tagged recombinant proteins

  • Ion exchange chromatography

  • Size exclusion chromatography

• Structural Analysis:

  • X-ray crystallography to determine three-dimensional structure

  • Circular dichroism (CD) spectroscopy to analyze secondary structure

  • Mass spectrometry for protein identification and modification analysis

• Functional Characterization:

  • Enzyme kinetics studies to determine Km, Vmax, and other kinetic parameters

  • Substrate specificity analysis using various rhamnose derivatives

  • Inhibition studies to identify potential regulators of enzyme activity

• Immunological Methods:

  • Development of specific antibodies for detection and localization

  • Immunofluorescence assays to visualize protein localization in bacterial cells

  • ELISA for quantitative analysis of protein expression

• Molecular Visualization:

  • Transmission electron microscopy (TEM) to examine protein structure and bacterial morphology

  • Computational modeling to predict protein structure and function

What are the common challenges in producing and studying recombinant L. plantarum Rhamnulokinase?

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

• Expression Efficiency:

  • Codon usage bias affecting translation efficiency

  • Variation in protein yield based on signal peptide selection

  • Decline in protein activity after certain culture periods (e.g., AmyL activities dropping after 12 hours)

• Secretion Barriers:

  • Limited secretion capacity of L. plantarum for heterologous proteins

  • Potential protein misfolding during secretion

  • Proteolytic degradation of secreted proteins

• Functional Validation:

  • Ensuring proper protein folding and activity

  • Developing sensitive and specific assays for Rhamnulokinase activity

  • Distinguishing recombinant enzyme activity from native host enzymes

• Stability Issues:

  • Maintaining enzyme stability during expression and purification

  • Optimizing conditions to prevent denaturation

  • Addressing potential inhibition by fermentation byproducts

• Scale-Up Considerations:

  • Translating laboratory-scale production to larger volumes

  • Maintaining consistency in protein quality and activity

  • Developing cost-effective purification strategies

Research has shown that optimal expression conditions and careful selection of signal peptides can significantly improve recombinant protein production in L. plantarum. For instance, the Lp_2145 signal peptide has demonstrated superior performance in expressing recombinant proteins, with mRNA levels approximately 3-fold higher than with native signal peptides .

How does L. plantarum compare to other expression systems for Rhamnulokinase production?

L. plantarum offers several advantages and disadvantages compared to other expression systems:

L. plantarum has been successfully used to express various recombinant proteins, including the SARS-CoV-2 spike protein , influenza virus antigens , and various enzymes like α-amylase . The safety profile and ability to survive gastrointestinal conditions make L. plantarum particularly attractive for applications requiring oral delivery or food-grade status.

What are the promising future research areas for recombinant L. plantarum Rhamnulokinase?

Several promising research directions could advance our understanding and application of recombinant L. plantarum Rhamnulokinase:

• Enzyme Engineering:

  • Protein engineering to enhance catalytic efficiency and stability

  • Development of fusion proteins for improved expression or novel functions

  • Structure-guided mutagenesis to alter substrate specificity

• Pathway Integration:

  • Engineering complete rhamnose utilization pathways in industrial strains

  • Coupling Rhamnulokinase activity with production of valuable metabolites

  • Creating synthetic pathways for novel rhamnose-derived compounds

• Expression System Optimization:

  • Development of novel, more efficient signal peptides

  • Creation of food-grade selection systems not requiring antibiotics

  • Engineering L. plantarum strains with enhanced secretion capacity

• Applications Development:

  • Enzymatic modification of plant flavonoids to improve bioavailability

  • Development of enzymatic processes for rare sugar production

  • Creation of enzymatic biosensors for rhamnose detection

• Comparative Genomics and Evolution:

  • Analysis of rhamnose metabolism across different Lactobacillus species

  • Understanding the evolutionary acquisition of rhamnose utilization genes

  • Investigation of the role of megaplasmids in rhamnose metabolism

Recent advances in genome editing tools like CRISPR-Cas9 offer exciting opportunities for precise genetic manipulation of L. plantarum, potentially allowing for more efficient production and engineering of recombinant Rhamnulokinase and related enzymes.