Recombinant Gluconobacter oxydans Chorismate synthase (aroC)

What is the functional role of chorismate synthase (aroC) in G. oxydans?

Chorismate synthase (aroC) in Gluconobacter oxydans catalyzes the anti-1,4-elimination of the C-3 phosphate and the C-6 proR hydrogen from 5-enolpyruvylshikimate-3-phosphate (EPSP) to yield chorismate. This reaction introduces a second double bond into the aromatic ring system . The enzyme plays a critical role in the aromatic amino acid biosynthesis pathway as chorismate serves as the branch point compound that functions as the starting substrate for the three terminal pathways of aromatic amino acid biosynthesis . In G. oxydans, aroC is part of a metabolic network that connects to various other cellular processes, including the synthesis of important secondary metabolites.

How does the aromatic amino acid biosynthesis pathway in G. oxydans compare to other bacterial species?

The aromatic amino acid (AroAA) biosynthesis pathway in G. oxydans shares similarities with other bacterial species but has distinct characteristics:

Unlike some archaea like H. salinarum that may use non-canonical pathways for the first steps of aromatic amino acid biosynthesis, G. oxydans appears to use the canonical pathway where aroC (chorismate synthase) converts EPSP to chorismate . The pathway in G. oxydans is particularly relevant to its industrial applications, as it connects to various oxidative metabolic processes characteristic of acetic acid bacteria .

What expression systems are most effective for producing recombinant G. oxydans aroC protein?

Multiple expression systems have been employed for recombinant G. oxydans aroC production, each with distinct advantages:

What methodological approaches are most effective for characterizing the kinetic properties of recombinant G. oxydans aroC?

Characterizing the kinetic properties of recombinant G. oxydans aroC requires multiple complementary approaches:

• Spectrophotometric Assays: The enzyme activity can be monitored by following the conversion of EPSP to chorismate spectrophotometrically at 275 nm, corresponding to the formation of the second double bond in the aromatic ring.

• Coupled Enzyme Assays: Since chorismate synthase activity is often FMN-dependent and requires a reduced flavin cofactor, coupled enzyme systems using flavin reductase can be employed.

• HPLC-Based Quantification: For precise measurement of substrate consumption and product formation, HPLC methods can separate and quantify EPSP and chorismate.

A standardized protocol involves:

• Purifying the recombinant enzyme using affinity chromatography

• Determining optimal pH and temperature conditions (pH 7.0-7.5 is typical for G. oxydans enzymes)

• Measuring initial reaction rates at varying substrate concentrations

• Calculating kinetic parameters (Km, Vmax, kcat) using Michaelis-Menten or Lineweaver-Burk plots

• Evaluating the effects of potential inhibitors or activators

For G. oxydans aroC specifically, researchers should be aware that the enzyme may require specific ionic conditions - Mg²⁺ is often essential for activity , and the assay buffer should be optimized accordingly.

How can mutational studies be designed to investigate critical residues in G. oxydans aroC function?

Designing effective mutational studies for G. oxydans aroC involves several strategic considerations:

• Selection of Target Residues:

  • Identify conserved residues through multiple sequence alignment with chorismate synthases from other organisms

  • Focus on residues in the predicted active site based on homology modeling

  • Consider residues involved in FMN binding or substrate interaction

• Mutation Strategy:

  • Site-directed mutagenesis for targeted amino acid changes (alanine scanning is a common approach)

  • Creation of chimeric proteins by swapping domains with homologous enzymes

  • Random mutagenesis for unbiased functional screening

• Functional Assessment:

  • Compare kinetic parameters between wild-type and mutant enzymes

  • Assess substrate specificity changes

  • Measure thermal stability using differential scanning fluorimetry

• In vivo Validation:

  • Complement G. oxydans aroC knockout strains with mutated versions

  • Assess growth phenotypes under conditions requiring aroC function

When designing mutagenesis experiments, researchers should consider the observation from related studies that aroC functions within a metabolic context. For example, in H. salinarum, genes involved in aromatic amino acid biosynthesis showed coordinated expression patterns , suggesting that regulatory elements may also be important targets for mutagenesis studies.

What approaches can be used to develop G. oxydans aroC knockout strains for studying pathway dependencies?

Developing G. oxydans aroC knockout strains requires specialized approaches due to the characteristics of this bacterial species:

• Knockout Strategy Options:

  • Transposon mutagenesis: Random insertional mutagenesis followed by screening for aroC disruption

  • Homologous recombination: Targeted replacement of aroC with antibiotic resistance markers

  • CRISPR-Cas9 system: Precise genome editing with customized guide RNAs

• Selection System Design:

  • Growth media supplemented with aromatic amino acids to support auxotrophic mutants

  • Counter-selection markers for isolating double-crossover events

  • Fluorescent reporters for tracking successful recombination events

• Phenotypic Characterization:

  • Metabolite profiling using LC-MS to identify pathway intermediates

  • Growth analysis under varying nutritional conditions

  • Transcriptomic analysis to identify compensatory responses

A recent whole-genome knockout collection of single-gene transposon disruption mutants for G. oxydans B58 has been developed, which could serve as a resource for obtaining or designing aroC knockout strains . When working with G. oxydans knockout strains, researchers should be aware that the bacterium's obligate aerobic nature may influence cultivation conditions, and appropriate aeration must be maintained during all experiments.

How can researchers address discrepancies in the literature regarding aroC function across different bacterial species?

Addressing discrepancies in aroC function literature requires systematic comparative analyses:

• Standardized Experimental Protocols:

  • Develop and apply consistent enzyme assay conditions across species

  • Use identical substrate preparations and analytical methods

  • Control environmental variables (pH, temperature, ionic strength)

• Phylogenetic Context Analysis:

  • Construct phylogenetic trees of aroC sequences from diverse species

  • Correlate functional differences with evolutionary relationships

  • Identify species-specific adaptations in enzyme function

• Structural Biology Approaches:

  • Compare crystal structures or homology models across species

  • Identify structural determinants of functional differences

  • Perform molecular dynamics simulations to assess conformational behaviors

• Heterologous Complementation Studies:

  • Express aroC genes from different species in a common knockout host

  • Evaluate the degree of functional restoration

  • Identify species-specific requirements for activity

A significant discrepancy example comes from research on the homologous aromatic amino acid biosynthesis in Methanococcus maripaludis versus other organisms. Despite high sequence similarity (69% identity to M. jannaschii homolog), the MMP0006 gene was found not to be required for aromatic amino acid biosynthesis in M. maripaludis . This highlights the importance of experimental validation rather than relying solely on sequence homology when studying G. oxydans aroC.

What metabolic engineering strategies can be employed to enhance aromatic compound production through manipulation of G. oxydans aroC?

Metabolic engineering of G. oxydans aroC for enhanced aromatic compound production involves several sophisticated approaches:

• Flux Optimization Strategies:

  • Overexpression of aroC to remove pathway bottlenecks

  • Promoter engineering to modulate expression levels

  • RBS optimization for translation efficiency

  • Co-expression with other rate-limiting enzymes in the pathway

• Feedback Regulation Modification:

  • Engineer aroC variants resistant to end-product inhibition

  • Modify regulatory elements controlling aroC expression

  • Implement dynamic pathway regulation systems

• Cofactor Engineering:

  • Enhance availability of FMN/FMNH₂ for aroC activity

  • Optimize redox balance for sustained pathway function

  • Engineer protein to utilize alternative cofactors

• Integration with G. oxydans Metabolism:

  • Redirect carbon flux from central metabolism toward aromatic amino acid pathway

  • Couple aroC activity with the strong oxidative capacity of G. oxydans

  • Utilize the membrane-bound dehydrogenases characteristic of G. oxydans

What are the optimal conditions for enzymatic assays of recombinant G. oxydans aroC?

Optimal enzymatic assay conditions for recombinant G. oxydans aroC should be carefully determined:

A methodological approach should include:

• Buffer optimization: Test multiple buffer systems (phosphate, Tris, HEPES) to identify optimal conditions

• Temperature profiling: Measure activity at 5°C intervals from 20-40°C

• Metal ion screening: Test various divalent cations (Mg²⁺, Mn²⁺, Ca²⁺) at different concentrations

• Reducing agent evaluation: Compare effectiveness of DTT, β-mercaptoethanol, or sodium dithionite for maintaining FMN in reduced state

• Stability assessment: Monitor activity retention over time under storage conditions

Researchers should note that G. oxydans enzymes may have specific adaptations to the acidic environments this bacterium typically inhabits, which could influence optimal assay conditions .

How can researchers effectively perform heterologous expression and purification of G. oxydans aroC?

Effective heterologous expression and purification of G. oxydans aroC requires optimization at multiple stages:

Expression System Selection and Optimization:

• Vector Design:

  • Use pET system for E. coli expression with T7 promoter

  • Include purification tags (His₆-tag, preferably N-terminal)

  • Consider fusion partners (MBP, SUMO) to enhance solubility

• Host Strain Selection:

  • E. coli BL21(DE3) or derivatives for T7-based expression

  • Rosetta or CodonPlus strains if G. oxydans codon bias is an issue

  • SHuffle or Origami strains if disulfide bonds are present

• Culture Conditions:

  • Induction at lower temperatures (16-25°C) to enhance solubility

  • Extended expression time (overnight) at reduced inducer concentration

  • Supplementation with riboflavin to ensure FMN availability

Purification Protocol:

• Cell lysis in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM PMSF

• Ni-NTA affinity chromatography (imidazole gradient: 20-250 mM)

• Size exclusion chromatography to remove aggregates

• Optional tag removal using specific proteases if needed for activity

• Final polishing using ion exchange chromatography

Quality Control Checkpoints:

• SDS-PAGE to assess purity (>95% is ideal for enzymatic studies)

• Western blot to confirm identity

• Dynamic light scattering to evaluate monodispersity

• Circular dichroism to confirm proper folding

• Activity assays to verify functional integrity

Researchers working with G. oxydans aroC should be aware that the enzyme belongs to the chorismate synthase family , which may have specific stability requirements. Adding stabilizing agents like glycerol (10-20%) and reducing agents (1-5 mM DTT) to all buffers can help maintain enzyme activity throughout purification.

What approaches can resolve solubility and stability issues with recombinant G. oxydans aroC?

Addressing solubility and stability challenges with recombinant G. oxydans aroC requires systematic troubleshooting:

Solubility Enhancement Strategies:

• Expression Modifications:

  • Lower induction temperature (16°C)

  • Reduce inducer concentration (0.1 mM IPTG)

  • Use auto-induction media for gradual protein production

• Genetic Engineering:

  • Fusion with solubility tags (MBP, SUMO, GST, TrxA)

  • Codon optimization for expression host

  • Surface entropy reduction mutations

• Buffer Optimization:

  • Screen additives (glycerol, arginine, trehalose)

  • Test different salt concentrations (100-500 mM NaCl)

  • Include mild detergents (0.05% Tween-20, 0.1% Triton X-100)

Stability Enhancement Approaches:

• Storage Conditions:

  • Determine optimal buffer composition

  • Test cryoprotectants (glycerol, sucrose)

  • Evaluate flash-freezing vs. slow cooling

• Formulation Screening:

  • pH optimization (typically pH 7.0-8.0)

  • Addition of cofactors (FMN, Mg²⁺)

  • Inclusion of reducing agents (DTT, TCEP)

• Structural Stabilization:

  • Disulfide engineering if appropriate

  • Surface charge optimization

  • Ligand or substrate addition for conformational stabilization

Experimental Approach for Optimization:
Design a factorial experiment varying:

• Buffer type (HEPES, Tris, Phosphate)

• pH (6.5-8.5)

• NaCl concentration (100-500 mM)

• Additives (glycerol 5-20%, arginine 50-200 mM)

• Reducing agents (DTT 1-5 mM, β-mercaptoethanol 5-20 mM)

Monitor protein quality using analytical SEC, thermal shift assays, and activity measurements to identify optimal conditions.

How can researchers validate the in vivo function of recombinant G. oxydans aroC?

Validating the in vivo function of recombinant G. oxydans aroC requires multiple complementary approaches:

• Genetic Complementation Studies:

  • Generate aroC knockout in G. oxydans (transposon mutagenesis or homologous recombination)

  • Introduce recombinant aroC on expression plasmid

  • Assess restoration of growth without aromatic amino acid supplementation

  • Compare growth kinetics between wild-type, knockout, and complemented strains

• Metabolic Profiling:

  • Use LC-MS to quantify pathway intermediates

  • Monitor accumulation of shikimate pathway metabolites in knockout strain

  • Verify restoration of normal metabolite levels in complemented strain

  • Trace isotope-labeled precursors to confirm pathway flux

• Transcriptional Regulation Analysis:

  • Examine expression of aroC and related pathway genes using RT-qPCR

  • Investigate regulatory responses to aromatic amino acid limitation

  • Identify potential feedback mechanisms controlling aroC expression

  • Compare transcriptional profiles between wild-type and engineered strains

• Stress Response Characterization:

  • Test growth under various stress conditions (oxidative, pH, temperature)

  • Evaluate potential moonlighting functions beyond aromatic amino acid biosynthesis

  • Assess impact on membrane integrity and cellular physiology

When interpreting results, researchers should consider that aromatic amino acid biosynthesis genes in bacteria often show coordinated transcriptional regulation. In related systems, the transcription of genes involved in this pathway was compared between cells grown with and without aromatic amino acids, with significant regulation observed . Similar approaches would be valuable for characterizing G. oxydans aroC regulation.

What techniques can be used to investigate potential protein-protein interactions involving G. oxydans aroC?

Investigating protein-protein interactions involving G. oxydans aroC requires specialized techniques:

• In vitro Interaction Studies:

  • Pull-down Assays: Using tagged aroC as bait to identify interacting partners

  • Surface Plasmon Resonance (SPR): For quantitative binding kinetics

  • Isothermal Titration Calorimetry (ITC): For thermodynamic parameters

  • Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS): To determine oligomeric states

• In vivo Interaction Mapping:

  • Bacterial Two-Hybrid: Modified for use in G. oxydans

  • Protein-Fragment Complementation Assays: Split reporter proteins that restore activity when interaction occurs

  • Co-Immunoprecipitation: Using antibodies against aroC or interacting partners

  • Chemical Cross-linking coupled with Mass Spectrometry: To capture transient interactions

• Structural Approaches:

  • X-ray Crystallography: Of aroC in complex with partner proteins

  • Cryo-Electron Microscopy: For larger complexes

  • Small-Angle X-ray Scattering (SAXS): For solution structure of complexes

  • Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): To map interaction interfaces

• Computational Predictions:

  • Protein-Protein Docking: Using homology models if structures unavailable

  • Co-evolution Analysis: To identify residues that may be involved in interactions

  • Genomic Context Analysis: Examining gene neighborhood for functional associations