Recombinant Lactobacillus casei Glucose-6-phosphate isomerase (pgi)

What is the role of phosphoglucose isomerase (Pgi) in L. casei metabolism?

Phosphoglucose isomerase (Pgi) catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate, representing a critical junction in carbohydrate metabolism. In L. casei, Pgi plays a fundamental role in glycolysis and functions at a key branching point between anabolic and catabolic pathways. The enzyme significantly influences carbon flux through the glycolytic pathway, particularly at the glucose-6P intermediate level, which affects both energy production and biosynthetic processes. Studies have demonstrated that the physiological amount of Pgi activity is limited for L. casei growth on certain carbon sources, particularly lactose and galactose, indicating its metabolic importance .

How can we identify and clone the pgi gene in L. casei?

The pgi gene encoding phosphoglucose isomerase activity in L. casei can be identified through genomic analysis and functional screening approaches. Researchers have successfully identified and cloned this gene from L. casei BL23. The methodology involves:

• Genomic DNA extraction from L. casei cultures

• PCR amplification using primers designed from conserved regions of known bacterial pgi genes

• Cloning the amplified gene into appropriate expression vectors

• Verification of functionality through complementation studies or enzyme activity assays

• Sequence confirmation through Sanger sequencing

The cloned pgi gene can then be used for homologous overexpression studies to evaluate its functional impact on metabolism .

What experimental controls are essential when studying recombinant L. casei pgi?

When investigating recombinant L. casei pgi, several controls are critical for robust experimental design:

• Vector-only control (L. casei transformed with empty vector) to account for effects of the transformation process and vector backbone

• Wild-type L. casei strain to establish baseline growth and metabolic parameters

• Growth on different carbon sources (glucose, lactose, galactose) to evaluate substrate-specific effects

• Time-course measurements to capture dynamic metabolic changes

• Multiple biological and technical replicates to ensure statistical validity

For transformation experiments specifically, controls should include competent cells without plasmid DNA and cells transformed with a known functional plasmid to validate transformation efficiency .

How does pgi overexpression affect growth characteristics of L. casei on different carbon sources?

Pgi overexpression in L. casei results in carbon source-dependent growth effects:

These findings demonstrate that the physiological amount of Pgi activity is a limiting factor for L. casei growth on lactose and galactose, and that limitation was overcome through pgi gene overexpression. The differential effects across carbon sources highlight the metabolic versatility and regulation of L. casei .

What changes in sugar-phosphate and nucleotide sugar levels result from pgi overexpression?

Pgi overexpression in L. casei induces significant changes in the intracellular concentrations of key metabolic intermediates:

• Glucose-6P levels: Reduced to 25% of control strain when cultured in glucose, and 59% when cultured in lactose

• Fructose-6P levels: Increased to 128% of control strain levels when cultured on glucose

• UDP-glucose levels: Reduced to 66% of control strain levels when cultured on galactose

• UDP-galactose levels: Reduced to 55% of control strain levels when cultured on galactose

These alterations in metabolite concentrations demonstrate the modulation capacity of carbon fluxes in L. casei at the level of the glycolytic intermediate glucose-6P, indicating that Pgi activity redirects carbon flow between anabolic and catabolic pathways .

How does pgi modification impact lactate production in L. casei?

Overexpression of pgi in L. casei results in increased lactate production, particularly when grown on galactose. Research has demonstrated that the lactate yield increased to 115% in the strain overproducing Pgi grown in galactose compared to control strains. This suggests that enhanced glucose-6-phosphate isomerase activity redirects carbon flux through glycolysis, ultimately increasing pyruvate availability for lactate production. The effect appears to be carbon source-dependent, highlighting the complex relationship between glucose-6-phosphate metabolism and fermentation end-products in L. casei .

What transformation methods are most effective for L. casei genetic modification?

Electroporation has proven to be the most effective transformation method for L. casei genetic modification. Based on published protocols, the optimal procedure includes:

• Preparation of ice-cold competent L. casei cells (typically 1 × 10^8 CFU/mL)

• Mixing plasmid DNA with competent cells

• Transferring the mixture to a pre-cooled electroporation cuvette (0.2 cm interelectrode distance)

• Applying a single electrical pulse at specific parameters (1.5 V, 25 μF)

• Immediate recovery in specialized medium (e.g., MRS broth with 0.3 M sucrose)

• Incubation at 37°C for 3-3.5 hours to allow expression of antibiotic resistance genes

• Plating on selective media (e.g., MRS agar with appropriate antibiotic such as 10 μg/mL chloramphenicol)

Successful transformants can be verified through PCR, sequencing, and functional assays. This method has been effectively used for introducing various recombinant constructs into L. casei strains .

What vector systems are most suitable for pgi expression in L. casei?

For effective pgi expression in L. casei, several vector design considerations are critical:

• Origin of replication compatible with L. casei (typically derived from native L. casei plasmids)

• Appropriate selection markers (chloramphenicol resistance genes are commonly used)

• Promoter selection:

  • Constitutive promoters for continuous expression

  • Inducible promoters (such as the lactose operon) for controlled expression

• Signal sequences:

  • Surface-display vectors for anchoring proteins to the cell wall

  • Secretion vectors for protein release into the extracellular environment

Integration vectors that facilitate recombination with the L. casei chromosome are particularly valuable for stable expression without antibiotic selection. For example, integration into the chromosomal lactose operon has been successfully employed, allowing gene expression to follow the same regulation pattern as the lac genes (repressed by glucose and induced by lactose) .

How can gene integration be verified in recombinant L. casei strains?

Verification of successful gene integration in recombinant L. casei strains requires a multi-faceted approach:

• PCR verification:

  • Colony PCR using primers flanking the integration site

  • PCR amplification of the integrated gene with gene-specific primers

• Sequencing confirmation:

  • Sanger sequencing of PCR products to confirm correct sequence and integration site

  • Whole genome sequencing for comprehensive verification

• Functional validation:

  • Western blotting to confirm protein expression

  • Enzyme activity assays (e.g., Pgi activity measurements)

  • Metabolite analysis to detect changes in relevant compounds (glucose-6P, fructose-6P)

• Stability testing:

  • Serial passaging without selection to confirm stable integration

  • PCR verification after multiple generations

These approaches collectively ensure the genetic modification has been successfully implemented and is functionally expressed in the recombinant strain .

What techniques are optimal for measuring changes in sugar-phosphate levels in recombinant L. casei?

For precise quantification of sugar-phosphate metabolites in recombinant L. casei strains, several analytical techniques are recommended:

• Liquid Chromatography-Mass Spectrometry (LC-MS):

  • Provides high sensitivity for detection of sugar phosphates

  • Allows simultaneous measurement of multiple metabolites

  • Enables differentiation between isomeric compounds (e.g., glucose-6P vs. fructose-6P)

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • 13C-NMR analysis can track carbon flux through metabolic pathways

  • Provides structural information about metabolites

  • Can be used for real-time metabolic studies

• Enzymatic assays:

  • Coupled enzyme assays specifically measuring glucose-6P or fructose-6P

  • Provides targeted, sensitive, and specific quantification

• Sample preparation considerations:

  • Rapid quenching of metabolism (cold methanol or liquid nitrogen)

  • Efficient extraction protocols (perchloric acid or hot ethanol extraction)

  • Careful handling to prevent degradation of labile metabolites

These methods have successfully demonstrated that pgi overexpression in L. casei results in reduced glucose-6P levels (to 25-59% of control) and increased fructose-6P levels (to 128% on glucose) .

How can the impact of pgi modification on carbon flux be measured?

Analyzing carbon flux in pgi-modified L. casei requires sophisticated metabolic flux analysis techniques:

• 13C-metabolic flux analysis:

  • Growth on 13C-labeled substrates (e.g., [1-13C]glucose or [U-13C]glucose)

  • Analysis of isotope distribution in metabolic intermediates and end products

  • Mathematical modeling to determine flux distributions

• Metabolomics approaches:

  • Comprehensive profiling of intracellular metabolites

  • Time-course measurements to capture dynamic changes

  • Correlation analysis between metabolite levels

• Transcriptomic and proteomic analysis:

  • RNA-seq to measure gene expression changes

  • Proteomics to quantify enzyme abundance

  • Integration of multi-omics data for comprehensive pathway analysis

• Enzyme activity measurements:

  • In vitro assays of key enzymes in related pathways

  • Determination of kinetic parameters (Km, Vmax)

  • Correlation between enzyme activities and metabolic flux

These approaches collectively provide a comprehensive understanding of how pgi modification alters carbon flow through central metabolic pathways in L. casei .

What are the recommended methods for measuring Pgi enzyme activity in recombinant L. casei?

For accurate assessment of Pgi enzyme activity in recombinant L. casei strains, the following methodological considerations are important:

• Cell preparation:

  • Harvesting cells in exponential growth phase

  • Careful cell lysis methods (sonication, mechanical disruption, or enzymatic lysis with lysozyme)

  • Preparation of cell-free extracts with appropriate buffer systems

• Enzyme assay formats:

  • Spectrophotometric assays measuring NADPH production in a coupled system

  • Forward reaction: Glucose-6P → Fructose-6P (coupled with phosphofructokinase and aldolase)

  • Reverse reaction: Fructose-6P → Glucose-6P (coupled with glucose-6P dehydrogenase)

• Assay optimization:

  • Determination of optimal pH, temperature, and buffer conditions

  • Linearity verification with respect to time and enzyme concentration

  • Inclusion of appropriate controls (heat-inactivated extracts, known standards)

• Data analysis:

  • Calculation of specific activity (μmol/min/mg protein)

  • Determination of kinetic parameters (Km, Vmax)

  • Statistical analysis comparing different strains or growth conditions

These methods enable quantitative comparison of Pgi activity levels between wild-type and recombinant strains, confirming successful overexpression and correlating enzyme activity with observed metabolic changes .

How can pgi-modified L. casei be utilized for metabolic engineering applications?

Pgi-modified L. casei strains offer several promising avenues for metabolic engineering:

• Enhanced production of valuable metabolites:

  • Redirecting carbon flux toward target compounds

  • Combining pgi modification with other genetic changes for synergistic effects

  • Fine-tuning glycolytic flux for optimal productivity

• Strain optimization for industrial fermentations:

  • Improving growth rates on specific carbon sources

  • Enhancing substrate utilization efficiency

  • Developing strains with customized metabolic profiles

• Probiotic applications:

  • Engineering strains with enhanced survival in the gastrointestinal tract

  • Developing strains that produce beneficial compounds in situ

  • Creating recombinant probiotics with specific health-promoting functions

• Vaccine delivery vehicles:

  • Using L. casei as a vector for antigen delivery

  • Exploiting metabolic engineering to enhance immunogenicity

  • Developing oral vaccine candidates with improved efficacy

Knowledge of the role of key enzymes like Pgi in metabolic fluxes at branching points between anabolic and catabolic pathways allows for rational design of engineering strategies in L. casei for these various applications .

What strategies can combine pgi modification with other genetic changes for enhanced functionality?

Integrating pgi modification with other genetic changes can create L. casei strains with enhanced functionality through several strategies:

• Multi-enzyme pathway engineering:

  • Coordinated modification of multiple glycolytic enzymes

  • Combined overexpression of pgi with α-phosphoglucomutase (α-Pgm)

  • Engineering of branching pathways to direct flux toward desired products

• Redox balance optimization:

  • Inactivation of L-lactate dehydrogenase gene combined with pgi overexpression

  • Engineering alternative NAD+ regeneration pathways

  • Creation of strains with modified NADH/NAD+ ratios for specific applications

• Regulatory network modifications:

  • Engineering carbon catabolite repression systems

  • Modifying transcriptional regulators controlling pgi expression

  • Implementing synthetic regulatory circuits for dynamic control

• Adaptive laboratory evolution:

  • Directed evolution of pgi-modified strains under selective pressure

  • Selection for improved growth or product formation

  • Identification of compensatory mutations that enhance phenotype

One particularly successful example combines pgi overexpression with L-lactate dehydrogenase inactivation, which leads to increased production of alternative products as the engineered route provides an alternative pathway for NAD+ regeneration .

What are the current challenges in studying recombinant L. casei pgi and how might they be addressed?

Research on recombinant L. casei pgi faces several challenges that require innovative solutions:

• Genetic stability issues:

  • Challenge: Loss of plasmid-based expression systems without selection pressure

  • Solution: Chromosomal integration strategies, such as integration into the lactose operon

  • Implementation: Using homologous recombination or CRISPR-Cas9 techniques for precise genomic integration

• Expression level control:

  • Challenge: Achieving optimal expression levels for metabolic balance

  • Solution: Development of tunable promoter systems specific for L. casei

  • Implementation: Testing promoter libraries with varying strengths or inducible systems

• Strain-specific variability:

  • Challenge: Different L. casei strains may respond differently to pgi modification

  • Solution: Comparative studies across multiple strains (e.g., BL23, KACC92338)

  • Implementation: Standardized protocols for cross-strain comparison

• Metabolic burden of recombinant protein production:

  • Challenge: Overexpression may cause growth defects or metabolic imbalances

  • Solution: Fine-tuning expression levels and growth conditions

  • Implementation: Systematic optimization of culture conditions for each recombinant strain

• Translation to in vivo applications:

  • Challenge: Laboratory performance may not predict functionality in actual applications

  • Solution: Development of relevant model systems for testing

  • Implementation: Testing recombinant strains in simulated gastrointestinal conditions or animal models

Addressing these challenges will require interdisciplinary approaches combining molecular biology, systems biology, and metabolic engineering techniques .

What growth conditions should be tested when evaluating recombinant L. casei pgi strains?

When evaluating recombinant L. casei pgi strains, comprehensive testing across multiple growth conditions is essential:

Each condition should be tested with appropriate controls, including wild-type and empty vector strains. Time-course sampling is recommended to capture dynamic metabolic changes throughout growth. This comprehensive approach has revealed that pgi overexpression in L. casei results in different growth rates and metabolite profiles depending on the carbon source used .

How should experiments be designed to assess the survivability of recombinant L. casei?

Designing experiments to assess the survivability of recombinant L. casei requires consideration of both in vitro and in vivo conditions:

• In vitro survival assessment:

  • Acid tolerance tests (pH 2.0-4.0 for varying time periods)

  • Bile salt resistance (0.1-0.5% bile salts)

  • Temperature stress (4°C storage, 50-55°C heat shock)

  • Simulated gastrointestinal transit (sequential exposure to artificial gastric juice and intestinal fluid)

  • Long-term storage stability at different temperatures

• In vivo survival assessment:

  • Animal model studies with controlled feeding of recombinant strains

  • Time-course sampling from different intestinal segments

  • Quantification methods:

    • Selective plating with antibiotic markers

    • Strain-specific PCR for identification

    • Colony counts from intestinal samples

• Data analysis and reporting:

  • Recovery rates expressed as CFU/ml or CFU/g of intestinal content

  • Statistical comparison between different intestinal segments

  • Persistence monitoring over multiple days

Published studies have successfully used these approaches to demonstrate that recombinant L. casei can effectively colonize intestinal environments, with quantifiable presence in the fore-, mid-, and hind-gut regions, showing particularly high numbers in the hind-gut (4.3 × 10^5 CFU/ml for surface-displayed constructs) .

What controls and replicates are necessary for rigorous pgi expression studies?

For rigorous scientific investigation of pgi expression in L. casei, a comprehensive set of controls and replicate designs is essential:

• Strain controls:

  • Wild-type L. casei (unmodified parent strain)

  • Empty vector control (transformed with vector backbone only)

  • Positive control (strain with known phenotype)

  • Multiple independently derived transformants of the same construct

• Experimental controls:

  • No-template controls for PCR verification

  • Enzyme activity blanks and standards

  • Growth medium blanks

  • Heat-inactivated samples for enzyme assays

• Replication strategy:

  • Minimum of three biological replicates (independent cultures)

  • Three technical replicates per biological replicate

  • Independent repetition of key experiments on different days

• Validation approaches:

  • Multiple measurement techniques for critical parameters

  • Independent verification of key findings using alternative methods

  • Time-course measurements to ensure reproducibility across growth phases

• Statistical analysis:

  • Appropriate statistical tests (t-tests, ANOVA, etc.)

  • Multiple testing correction where applicable

  • Effect size reporting in addition to p-values

This rigorous experimental design ensures that observed differences in growth rates, metabolite levels, or enzyme activities can be confidently attributed to pgi modification rather than experimental variability or artifacts .

How does research on recombinant L. casei pgi relate to broader microbiome studies?

Research on recombinant L. casei pgi has significant implications for microbiome studies through several interconnected dimensions:

• Metabolic interactions:

  • Altered carbon metabolism in engineered L. casei may affect interactions with other microbiome members

  • Modified sugar utilization can influence competitive dynamics in mixed communities

  • Changes in metabolic end-products may serve as substrates for other microorganisms

• Ecological considerations:

  • Engineered strains with enhanced growth capabilities may have altered colonization potential

  • Modification of key metabolic enzymes may affect niche adaptation

  • Persistence studies in complex environments help predict behavior in natural microbiomes

• Functional capabilities:

  • Understanding metabolic engineering in L. casei provides insights into manipulating other microbiome members

  • Recombinant probiotics represent a potential approach for microbiome modulation

  • Metabolic studies in L. casei can serve as models for other lactic acid bacteria in microbiomes

• Translational applications:

  • Engineered L. casei strains may serve as targeted probiotics for microbiome modulation

  • Knowledge of metabolic pathways enables rational design of synbiotic approaches

  • Understanding colonization dynamics informs strategies for successful microbiome interventions

These connections highlight how fundamental research on L. casei metabolism contributes to our broader understanding of microbiome function and manipulation strategies .

What implications does pgi research have for developing L. casei as a probiotic platform?

Research on pgi in L. casei offers several important insights for developing enhanced probiotic platforms:

• Metabolic optimization:

  • Tailoring carbon metabolism for specific niches in the gastrointestinal tract

  • Engineering strains with enhanced survival through metabolic adaptations

  • Optimizing growth on available substrates in the intestinal environment

• Functional enhancements:

  • Using pgi modification as part of larger metabolic engineering strategies

  • Combining metabolic optimization with delivery of bioactive compounds

  • Developing strains with enhanced competitive advantage against pathogens

• Delivery system development:

  • Using metabolically engineered L. casei as delivery vehicles for antigens or therapeutic proteins

  • Optimizing expression systems for in situ production of beneficial compounds

  • Developing stable recombinant strains that maintain function without selection pressure

• Safety considerations:

  • Understanding metabolic consequences of genetic modifications

  • Evaluating stability and containment of recombinant constructs

  • Assessing potential for horizontal gene transfer in complex environments

The L. casei KACC92338 strain has been identified as a potential probiotic candidate for producing functional fermented foods, health care products, and skin care products, highlighting the translational potential of this research .

How might findings from L. casei pgi studies translate to other lactic acid bacteria?

Findings from L. casei pgi studies have broad implications for research on other lactic acid bacteria (LAB) through several mechanisms:

• Methodological transfers:

  • Genetic engineering techniques optimized for L. casei can be adapted for other LAB

  • Analytical methods for metabolite quantification can be applied across species

  • Experimental designs for studying carbon metabolism are broadly applicable

• Metabolic principles:

  • Understanding of glycolytic regulation through pgi may apply to related pathways in other LAB

  • Insights into carbon flux control points can inform metabolic engineering in diverse species

  • Strategies for redirecting metabolism toward valuable products may be transferable

• Comparative genomics approaches:

  • Identification of conserved and divergent aspects of pgi function across LAB

  • Leveraging genomic information to predict metabolic capabilities in less-studied species

  • Understanding species-specific adaptations in central carbon metabolism

• Biotechnological applications:

  • Development of expression systems that function across multiple LAB species

  • Creation of metabolic engineering toolkits with broad host range

  • Establishment of design principles for manipulating central carbon metabolism