Recombinant Lactobacillus plantarum Adenine deaminase (ade), partial

What is Lactobacillus plantarum and why is it significant for recombinant enzyme studies?

Lactobacillus plantarum is a versatile lactic acid bacterium widely recognized as both a potential starter culture and health-promoting probiotic. Its significance for recombinant enzyme studies stems from its GRAS (Generally Recognized As Safe) status and established genetic systems. L. plantarum WCFS1 is particularly valuable for genetic engineering due to its fully sequenced genome and the availability of various genetic tools. The bacterium's probiotic nature makes it an attractive candidate for industrial applications, especially when engineering it to produce bioactive compounds without introducing exogenous genes or plasmids .

How do adenine deaminases differ from adenosine deaminases?

Adenine deaminase (ADE) catalyzes the deamination of adenine to hypoxanthine, while adenosine deaminase (ADA) catalyzes the irreversible deamination of adenosine and 2'-deoxyadenosine to inosine and 2'-deoxyinosine, respectively. The key difference lies in their substrates - ADE acts on the nucleobase (adenine), while ADA acts on the nucleoside (adenosine). Both belong to the broader class of deaminases that catalyze the hydrolysis of C-NH₂ bonds in amino compounds, producing ammonia . ADA has been extensively studied in mammals with highest levels found in lymphoid tissues, while bacterial adenine deaminases play key roles in purine salvage pathways .

What genetic engineering approaches are most effective for L. plantarum recombinant enzyme expression?

For L. plantarum recombinant enzyme expression, CRISPR/Cas9-assisted genome editing combined with double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) recombineering has proven highly effective. This approach allows for seamless genome modifications including gene knockouts, insertions, and point mutations. The process typically involves:

• Introduction of recombinase genes (lp_0642, lp_0641, and lp_0640) from a prophage P1 locus

• Delivery of the CRISPR/Cas9 system for targeted genomic cleavage

• Provision of homologous repair templates (dsDNA or ssDNA)

• Selection of successful edits through CRISPR-based counterselection

This methodology eliminates the need for antibiotic markers or exogenous plasmids, making it ideal for producing recombinant enzymes in a food-grade manner .

How should researchers optimize CRISPR/Cas9-assisted recombineering for L. plantarum enzyme expression?

Optimizing CRISPR/Cas9-assisted recombineering for L. plantarum enzyme expression requires a systematic approach addressing multiple factors:

• sgRNA design: Select target sites with minimal off-target effects and optimal GC content for efficient Cas9 binding and cleavage.

• Homology arm design: For dsDNA recombineering, use homology arms of 500-1000 bp for optimal recombination efficiency. When direct substitution proves challenging, consider adopting a two-step recombineering method:

  • First insert the target DNA fragment with antibiotic resistance gene and loxP sites

  • Then excise the antibiotic marker using the Cre/loxP system

  • Finally, remove any remaining loxP sites using CRISPR/Cas9 targeting

• DNA protection: Apply phosphorothioate modifications to the 5' ends of dsDNA to protect against exonuclease degradation, significantly improving insertion efficiency.

• Methyltransferase overexpression: Overexpress the endogenous adenine-specific methyltransferase (Dam, lp_2243) to improve ssDNA recombination efficiency by temporarily disabling the mismatch repair (MMR) system. This approach has been shown to remarkably improve mutation efficiencies, especially with consecutive mismatches .

• Selection strategy: Design appropriate screening methods based on phenotype or PCR verification, with CRISPR counterselection eliminating non-edited cells.

What are the key considerations for characterizing recombinant adenine deaminase activity?

When characterizing recombinant adenine deaminase activity from L. plantarum, researchers should address:

• Enzyme purification: Develop a purification protocol that preserves enzyme structure and activity, typically involving affinity chromatography for histidine-tagged proteins.

• Activity assays: Employ spectrophotometric methods to monitor the conversion of adenine to hypoxanthine. Based on adenosine deaminase studies, measure activity by tracking changes in absorbance at 265 nm as adenine is converted to hypoxanthine.

• Kinetic parameters: Determine key parameters including:

  • K₍m₎ (substrate affinity)

  • V₍max₎ (maximum reaction velocity)

  • k₍cat₎ (turnover number)

  • pH and temperature optima

• Inhibition studies: As with other deaminases, characterize competitive inhibitors and their inhibition constants (K₍I₎) using Lineweaver-Burk or similar plots.

Table based on data from adenosine deaminase inhibition studies

• Metal ion dependency: Evaluate the dependence on Zn²⁺ or other metal cofactors, as deaminases typically require metal ions for catalysis .

How can adenine-specific methyltransferase improve recombination efficiency in L. plantarum?

Adenine-specific methyltransferase (Dam) significantly enhances recombination efficiency in L. plantarum through a precise molecular mechanism. When overexpressed, Dam methylates adenine residues in DNA, which temporarily disables the mismatch repair (MMR) system during genetic engineering. The process works as follows:

• Dam binds to mismatched bases or adjacent sequences faster than the MMR machinery can detect them

• Methylation of adenine residues in GATC sequences creates hemimethylated DNA

• The MMR system cannot efficiently repair mismatches in methylated DNA chains

• This window of opportunity allows introduced mutations to be established before repair occurs

Experimental data shows that Dam overexpression remarkably improves ssDNA recombineering efficiency, particularly with sequences containing three consecutive mismatches. When implemented via the plasmid p411-RecT-Dam in L. plantarum WCFS1, this approach can increase editing efficiency by several fold compared to standard methods .

What strategies can overcome the challenges of seamless genome editing in L. plantarum?

Seamless genome editing in L. plantarum faces several challenges, including limited antibiotic selection options and low recombination frequencies. Advanced strategies to overcome these limitations include:

• CRISPR/Cas9 counterselection: Instead of relying solely on antibiotic selection, use CRISPR/Cas9 to eliminate non-edited cells by targeting the unmodified sequence. This powerful negative selection allows recovery of rare recombination events.

• Two-step recombineering for complex edits: For insertions or large modifications:

  • First perform antibiotic-dependent dsDNA recombineering to insert the target fragment with selectable markers and loxP sites

  • After selection, excise the antibiotic resistance gene using the Cre/loxP system

  • Finally, remove any remaining loxP sites using CRISPR/Cas9-assisted editing

• ssDNA recombineering for small modifications: For point mutations or small insertions/deletions, use single-stranded DNA oligonucleotides (60-90 nt) with the mutation centered. This approach is highly efficient for targeted changes.

• DNA protection strategies: Incorporate phosphorothioate modifications at the 5' ends of dsDNA fragments to protect against exonuclease degradation, significantly improving insertion efficiency.

• MMR evasion techniques: Besides Dam overexpression, designing recombineering oligonucleotides to avoid creating mismatches recognized by the MMR system or introducing multiple consecutive mismatches can increase efficiency .

How should researchers analyze the structural and functional differences between native and recombinant partial adenine deaminase?

When comparing native and recombinant partial adenine deaminase from L. plantarum, researchers should implement a comprehensive analytical approach:

• Sequence analysis:

  • Confirm the sequence of the partial recombinant protein

  • Identify truncations or modifications relative to the native enzyme

  • Use bioinformatics to predict structural impacts of partial sequences

• Structural characterization:

  • Employ circular dichroism (CD) spectroscopy to assess secondary structure elements

  • Use X-ray crystallography or cryo-EM for high-resolution structural determination

  • Apply molecular dynamics simulations to predict conformational flexibility

• Functional comparison:

  • Compare kinetic parameters (K₍m₎, V₍max₎, k₍cat₎) between native and recombinant forms

  • Evaluate substrate specificity profiles across various adenine derivatives

  • Assess pH and temperature stability profiles

• Metal binding analysis:

  • Determine metal ion content using atomic absorption spectroscopy

  • Measure enzyme activity after metal chelation and reconstitution

  • Compare metal binding affinity between native and recombinant forms

• In silico analysis:

  • Use homology modeling based on known deaminase structures

  • Predict active site architecture and substrate binding residues

  • Identify potential differences in catalytic mechanisms

Native deaminases typically have rigid requirements for substrate binding, with variations in structure resulting in significant loss of important interactions . For recombinant partial enzymes, this sensitivity may be even more pronounced, requiring careful characterization of functional parameters.

What are common challenges in expression and purification of recombinant L. plantarum adenine deaminase?

Researchers frequently encounter several challenges when expressing and purifying recombinant L. plantarum adenine deaminase:

• Expression levels: Low protein yield is common due to codon usage differences or protein toxicity. This can be addressed by:

  • Optimizing codon usage for L. plantarum

  • Using inducible expression systems like the sakacin P-inducible system

  • Carefully controlling induction conditions to balance protein production and cell health

• Protein solubility: Recombinant deaminases often form inclusion bodies. Strategies to improve solubility include:

  • Lowering expression temperature (20-25°C)

  • Adding solubility tags (MBP, SUMO, etc.)

  • Co-expressing molecular chaperones

  • Including zinc or other metal ions in growth media to facilitate proper folding

• Activity retention: Ensuring the recombinant enzyme maintains catalytic function requires:

  • Careful selection of purification buffers to maintain metal cofactors

  • Avoiding chelating agents like EDTA

  • Including reducing agents to maintain cysteine residues in reduced state

  • Testing activity at each purification step to track activity loss

• Partial protein expression: When expressing a partial adenine deaminase, additional challenges include:

  • Determining the correct boundaries to maintain fold integrity

  • Potential exposure of hydrophobic regions normally buried in the full-length protein

  • Loss of stabilizing interactions from missing domains

How can researchers interpret and resolve contradictory data when characterizing recombinant adenine deaminase?

When facing contradictory data in recombinant adenine deaminase characterization, researchers should implement a systematic troubleshooting approach:

• Validate protein identity and integrity:

  • Confirm protein sequence by mass spectrometry

  • Check for post-translational modifications or truncations

  • Verify metal content, as incorrect metal incorporation can dramatically alter activity

• Standardize assay conditions:

  • Ensure consistent buffer composition, pH, and temperature

  • Validate substrate quality and purity

  • Use appropriate controls, including commercially available deaminases

• Cross-validate with multiple methods:

  • Compare activity measurements using different detection techniques

  • Correlate spectrophotometric results with HPLC-based product detection

  • Verify kinetic parameters using different mathematical models

• Address experimental artifacts:

  • Test for interfering compounds in enzyme preparations

  • Evaluate potential product inhibition

  • Check for enzyme aggregation or precipitation during assays

• Systematic data analysis:

  • Perform statistical analysis to determine significance of differences

  • Use global fitting of data rather than transformed plots when possible

  • Consider alternative kinetic models beyond Michaelis-Menten if data doesn't fit

An example of systematic troubleshooting can be seen in adenosine deaminase studies, where varying inhibition constants across different studies were reconciled by standardizing experimental conditions and carefully controlling enzyme source and purity .

What novel applications could emerge from engineered L. plantarum expressing modified adenine deaminase?

Engineered L. plantarum expressing modified adenine deaminase opens several promising research directions:

• Nucleoside analog prodrug activation: Modified adenine deaminases could activate nucleoside prodrugs in targeted tissue environments, offering site-specific drug delivery for cancer or antiviral therapies.

• Purine metabolism modulation: Engineered probiotics expressing modified deaminases could help regulate adenine/adenosine levels in the gut, potentially benefiting conditions like inflammatory bowel disease or colorectal cancer.

• RNA editing applications: Adenine deaminases can be engineered to target specific RNA sequences, creating programmable RNA editing tools that could correct genetic mutations at the RNA level.

• Biosensor development: Modified deaminases with altered substrate specificity could serve as biosensors for detecting specific nucleobase analogs or environmental toxins.

• Synthetic biology circuits: Deaminases could function as molecular switches in synthetic gene circuits, responding to specific nucleobase inputs and producing defined outputs.

The seamless genome editing techniques developed for L. plantarum make these applications particularly attractive, as they allow precise modifications without introducing exogenous genes or plasmids, maintaining the probiotic's GRAS status .

How might combining adenine deaminase engineering with other metabolic modifications enhance L. plantarum's capabilities?

Integrating adenine deaminase engineering with broader metabolic modifications in L. plantarum could create multifunctional probiotic strains with enhanced capabilities: