Recombinant Lactobacillus plantarum 1- (5-phosphoribosyl)-5-[ (5-phosphoribosylamino)methylideneamino] imidazole-4-carboxamide isomerase (hisA)

What expression systems are most effective for recombinant HisA production in Lactobacillus plantarum?

The pSIP expression system has emerged as one of the most effective platforms for recombinant protein expression in L. plantarum. This system utilizes the regulatory elements from quorum sensing-based bacteriocin regulation operons in L. sakei and is induced by autoinducer peptides (AIP) . For HisA expression specifically, the pSIP411 vector has demonstrated high efficiency, capable of producing significant yields of other recombinant proteins (up to 1800 Miller Unit equivalents of β-glucuronidase) . The system enables dose-dependent expression control, which is particularly valuable for enzymes like HisA where expression level optimization may be necessary to prevent metabolic burden.

For optimal results, consider:

• Using the pSIP401/411 expression vectors with erythromycin resistance markers

• Incorporating appropriate signal peptides for desired localization (intracellular vs. secreted)

• Optimizing inducer concentration (typically 50 ng/mL SppIP for maximum yield)

• Induction timing (typically at early to mid-logarithmic phase)

How does codon optimization affect HisA expression in L. plantarum?

Codon optimization is critical for efficient HisA expression in L. plantarum due to its specific codon usage bias. Studies with other recombinant proteins have demonstrated that codon optimization can significantly enhance expression efficiency . For example, in a study with recombinant spike protein expression, researchers found that codon optimization according to L. plantarum's preferences resulted in 2-3 fold higher expression levels .

For HisA optimization:

• Analyze the codon usage bias of L. plantarum strain being used (typically WCFS1)

• Replace rare codons in the hisA gene with preferred synonymous codons

• Avoid introducing unwanted secondary structures in the mRNA

• Optimize the 5' region of the gene, which has a particularly strong impact on translation efficiency

• Consider GC content adjustments to match host preferences

What signal peptides are most efficient for HisA secretion or localization in L. plantarum?

Signal peptide selection significantly impacts recombinant protein yield and localization. For HisA expression in L. plantarum, specific signal peptides have shown superior performance:

What growth and induction conditions optimize recombinant HisA expression in L. plantarum?

Optimizing growth and induction conditions is essential for maximizing functional HisA production:

Growth Medium and Conditions:

• MRS broth for initial cultivation at 37°C without agitation

• For defined studies, chemically defined medium (CDM) can eliminate interference from complex media components

• Anaerobic conditions typically yield higher biomass for L. plantarum

Induction Parameters:

• Induce at early-mid logarithmic phase (OD600 ≈ 0.3-0.5)

• Optimal inducer concentration: 50 ng/mL SppIP for pSIP-based systems

• Induction temperature: 37°C shows highest protein yield

• Harvest timing: 6-10 hours post-induction typically yields maximum protein

Research has shown that mRNA levels of recombinant genes in L. plantarum peak at approximately 3 hours post-induction, while protein accumulation continues until 6-10 hours . Extended cultivation may lead to proteolytic degradation of the target protein.

What methods are most reliable for measuring HisA enzymatic activity in L. plantarum?

For accurate assessment of HisA activity in recombinant L. plantarum:

Direct Enzymatic Assay:

• Measure conversion of ProFAR to PRFAR spectrophotometrically at 300 nm

• Alternatively, couple the reaction with downstream histidine pathway enzymes and measure product formation

• Use cell-free extracts for intracellular enzyme or culture supernatants for secreted variants

Complementation Assay:

• Transform HisA-deficient bacterial strains with the recombinant L. plantarum hisA

• Assess growth restoration on histidine-free medium

• Compare growth rates as semi-quantitative measure of HisA activity

Protein Quantification:

• Western blot analysis using anti-His tag antibodies for His-tagged HisA

• SDS-PAGE with Coomassie staining for visual comparison

• Advanced MS-based absolute quantification methods for precise measurements

Ensure consistent biomass normalization (e.g., by OD600) when comparing different strains or conditions to obtain reliable specific activity values.

How can HisA purification from L. plantarum be optimized?

Efficient purification of recombinant HisA from L. plantarum requires a strategic approach:

For His-tagged HisA:

• Cell disruption: Use French press or sonication with appropriate buffer (typically phosphate buffer pH 7.4 with 1 mM PMSF)

• Clarification: Centrifuge at 9,300 × g, 10 min, at 4°C to remove cell debris

• Immobilized metal affinity chromatography (IMAC): Use Ni-NTA resin with imidazole gradient elution

• Polishing step: Size exclusion chromatography to remove aggregates and achieve >95% purity

For non-tagged HisA:

• Ammonium sulfate precipitation (typically 40-60% saturation)

• Ion exchange chromatography (HisA pI-dependent)

• Hydrophobic interaction chromatography

• Gel filtration as a final polishing step

Protein stability during purification can be enhanced by including 10% glycerol and 5 mM β-mercaptoethanol in all buffers. For His-tagged constructs, the C-terminal His6-tag has been shown to be more effective than N-terminal tagging in L. plantarum expression systems .

How can transcriptional analysis of the hisA gene be performed in recombinant L. plantarum?

Accurate transcriptional analysis of hisA expression requires careful methodology:

Real-time RT-qPCR approach:

• Total RNA extraction: Use RNeasy Mini Kit (Qiagen) or similar, with DNase treatment

• cDNA synthesis: Employ reverse transcriptase with random hexamers or specific primers

• Reference gene selection: Use validated genes in L. plantarum like 16S rRNA, recA, ldhD

• Primer design: Target hisA-specific regions, avoiding vector sequences

• Data analysis: Apply relative quantification using the 2^-ΔΔCt method with REST2009 software

Critical considerations:

• Harvest cells at multiple time points post-induction (0h, 1h, 3h, 6h, 9h) to capture expression dynamics

• Include non-induced controls to determine baseline expression

• Verify primer specificity through melting curve analysis and amplicon sequencing

• Normalize to multiple reference genes for robust quantification

Studies with other recombinant proteins in L. plantarum have shown that transcript levels typically peak at approximately 3 hours post-induction, reaching up to 46-58 fold upregulation compared to non-induced controls .

What strategies address protein misfolding and insolubility issues with recombinant HisA in L. plantarum?

When confronting HisA misfolding or insolubility in L. plantarum:

Experimental approaches:

• Lower induction temperature (30°C instead of 37°C) to slow folding rate

• Reduce inducer concentration (25 ng/mL instead of 50 ng/mL) to prevent overwhelming folding machinery

• Co-express chaperones (GroEL/GroES) to assist proper folding

• Introduce fusion partners (thioredoxin, MBP) to enhance solubility

• Test multiple signal peptides for secreted variants - Lp_0373 may provide better folding despite lower yield

Analysis methods:

• Compare soluble vs. insoluble fractions via SDS-PAGE and Western blot

• Employ differential scanning calorimetry to assess protein stability

• Use circular dichroism to evaluate secondary structure integrity

• Apply size exclusion chromatography to detect aggregation states

Notably, environmental conditions of the L. plantarum cytoplasm (pH, redox potential) significantly impact recombinant protein folding. L. plantarum has adapted multiple mechanisms to maintain pH homeostasis, including upregulation of phosphofructokinase (pfk) and pyruvate-kinase (pyk) genes, which can be leveraged to optimize HisA folding conditions .

How can genetic stability of recombinant L. plantarum expressing HisA be monitored and maintained?

Maintaining genetic stability is critical for consistent HisA production:

Monitoring approaches:

• Regular PCR verification of plasmid presence using vector-specific primers

• Restriction enzyme analysis of recovered plasmids to detect rearrangements

• Sequencing of the expression cassette to identify mutations

• Routine activity assays to detect functional changes in the expressed enzyme

• Flow cytometry to quantify the percentage of L. plantarum cells retaining expression capacity

Stability enhancement strategies:

• Maintain selective pressure (antibiotic) during all cultivation steps

• Avoid extended cultivation (>24h) which selects for non-producing mutants

• Optimize codon usage to reduce metabolic burden

• Consider genomic integration for ultra-stable expression

• Use food-grade selection systems instead of antibiotic markers for long-term applications

Research with L. plantarum has shown that recombinant strains can maintain stable protein expression for at least 3 months with appropriate selection pressure . Host-derived adaptations during this period may actually improve expression efficiency through mutations that enhance carbohydrate utilization and acid tolerance .

How does HisA overexpression impact the metabolome of L. plantarum?

HisA overexpression creates significant metabolic perturbations that can be analyzed through comprehensive metabolomic approaches:

Expected metabolic changes:

• Altered histidine pathway metabolite concentrations (increased PRFAR, potential decrease in downstream metabolites)

• Changes in amino acid pools, particularly in related pathways (aromatic amino acids, purine metabolism)

• Potential impact on stress response metabolites (altered levels of amino acids like alanine and arginine that play role in acid stress response)

• Shifts in central carbon metabolism to accommodate increased protein production demand

Analytical techniques:

• Targeted LC-MS/MS for histidine pathway metabolites

• Untargeted metabolomics for global metabolic impact assessment

• 13C metabolic flux analysis to determine redistribution of carbon flows

• Transcriptomics to identify metabolic adjustments in response to HisA overexpression

Similar metabolomic studies with L. plantarum strains have revealed that recombinant protein expression often alters nucleoside metabolism and can increase production of beneficial compounds like linolenic acid and proline . These changes may impact both the industrial utility and potential probiotic applications of the strain.

What genomic integration approaches can be used for stable HisA expression in L. plantarum?

For applications requiring maximum stability, genomic integration provides advantages over plasmid-based expression:

Integration methodologies:

• Homologous recombination targeting non-essential genes

  • Design constructs with 1-2 kb homology arms flanking the hisA expression cassette

  • Use temperature-sensitive plasmids for delivery and selection of integrants

  • Screen for double-crossover events using negative selection markers

• CRISPR-Cas9 mediated integration

  • Design sgRNA targeting safe harbor sites in L. plantarum genome

  • Include homology-directed repair template containing the hisA expression cassette

  • Employ counterselection to eliminate CRISPR plasmid after integration

• Site-specific recombination systems

  • Engineer attB sites into L. plantarum genome

  • Deliver hisA cassette on plasmid containing corresponding attP sites

  • Express phage integrase to catalyze site-specific integration

Integration site selection criteria:

• Target intergenic regions between convergent genes

• Avoid disrupting operons or regulatory regions

• Consider transcriptionally active regions for higher expression

• Assess impact on fitness through growth rate comparison

While genomic integration typically results in lower expression levels than high-copy plasmids, the increased stability makes it preferable for long-term applications or when antibiotic selection is not possible.

How can CRISPR-Cas9 technology optimize HisA production in L. plantarum?

CRISPR-Cas9 technology offers revolutionary approaches to enhance HisA production:

Genetic optimization strategies:

• Knockout competing metabolic pathways

  • Target genes from branched amino acid pathways that compete for metabolic precursors

  • Delete proteases (Clp, DnaK) that may degrade recombinant HisA

  • Inactivate carbon sinks that divert resources from protein production

• Promoter engineering

  • Replace native promoters of folding assistants (chaperones) with stronger versions

  • Modify the pSIP expression system for enhanced transcription

  • Engineer constitutive promoters with varying strengths for optimal expression

• Multiplex genome editing

  • Simultaneously modify multiple targets affecting HisA production

  • Create libraries of variant strains for high-throughput screening

  • Integrate production enhancement modifications with biosafety features

• Base editing applications

  • Fine-tune ribosome binding sites for optimal translation initiation

  • Modify codons at critical positions without introducing double-strand breaks

  • Alter regulatory elements controlling stress response to enhance tolerance to HisA overproduction

The luxS gene represents a promising target for CRISPR modification, as its deletion has been shown to alter the expression of proteins involved in carbohydrate metabolism, amino acid metabolism, and two-component regulatory systems in L. plantarum , potentially enhancing recombinant protein production.

What are the implications of HisA expression on the probiotic properties of L. plantarum?

Understanding how HisA expression affects probiotic functionality is crucial for applications where both enzyme production and probiotic benefits are desired:

Potential impacts on probiotic properties:

• Stress tolerance alterations

  • HisA overexpression may affect acid and bile tolerance through metabolic burden

  • Changes in membrane composition may occur as adaptation to protein production

  • Expression system (particularly signal peptides) may impact cell wall integrity

• Immunomodulatory effects

  • L. plantarum naturally engages TLR2/TLR6 heterodimers to promote regulatory T cell responses

  • Recombinant protein expression may alter surface properties affecting this interaction

  • HisA itself may have immunogenic properties when exposed on bacterial surface

• Adhesion capabilities

  • Surface protein composition changes may alter binding to intestinal mucosa

  • Metabolic alterations could affect production of factors promoting adhesion

  • Physiological stress from recombinant expression may downregulate native adhesins

• Metabolic output modifications

  • Redirected metabolism may alter production of beneficial compounds like GABA

  • HisA expression could impact production of antimicrobial compounds

  • Changes in amino acid metabolism may affect health-promoting metabolites

Analysis methodologies should include comparative genomics, transcriptomics, and in vitro models assessing adhesion, immunomodulation, and stress tolerance. Animal models would ultimately be required to confirm retention of probiotic benefits alongside successful HisA expression.

What synthetic biology approaches could revolutionize HisA production in L. plantarum?

Emerging synthetic biology tools offer exciting possibilities for next-generation HisA production systems:

Advanced expression control systems:

• Riboswitches responsive to metabolic signals for auto-regulated expression

• Orthogonal RNA polymerases and promoters for expression independent of host machinery

• Genetic circuits enabling dynamic regulation based on cellular state

• Light-controlled promoters for non-invasive induction without chemical additives

Genome minimization projects:

• Creation of chassis strains with reduced genomes optimized for recombinant protein production

• Elimination of competing pathways and mobile genetic elements

• Engineering of synthetic metabolic modules specifically supporting HisA production

• Development of "plug-and-play" expression platforms with standardized parts

New delivery technologies:

• Engineering of outer membrane vesicles to carry HisA for targeted delivery

• Development of controlled lysis systems for programmable enzyme release

• Cell surface display approaches for immobilized enzyme applications

• Self-cleaving signal peptides for enhanced secretion efficiency

These approaches represent the frontier of L. plantarum engineering, moving beyond individual gene modifications to holistic redesign of cellular functions for optimal protein production.

How might directed evolution approaches improve HisA functionality when expressed in L. plantarum?

Directed evolution offers powerful methods to enhance HisA properties:

Directed evolution methodologies:

• Error-prone PCR libraries of hisA gene with varying mutation rates

• DNA shuffling with homologous hisA genes from related organisms

• Semi-rational design focusing on active site residues and substrate binding regions

• Continuous evolution systems coupling HisA activity to bacterial survival

Screening strategies:

• High-throughput colorimetric assays for HisA activity

• Growth-based selection in histidine auxotrophs

• FACS-based screening using fluorescent reporters linked to activity

• Microfluidic droplet sorting for ultra-high throughput evaluation

Target improvements:

• Enhanced catalytic efficiency (increased kcat/Km)

• Improved thermostability for industrial applications

• Extended pH tolerance matching L. plantarum growing conditions

• Reduced product inhibition for higher conversion rates

• Altered substrate specificity for novel biotechnological applications

Success with similar approaches has been demonstrated with other enzymes in L. plantarum, including the engineering of α-amylase variants with enhanced thermostability and specific activity .