Recombinant Mannheimia succiniciproducens 3-phosphoshikimate 1-carboxyvinyltransferase (aroA)

What is Mannheimia succiniciproducens and why is it significant for research?

Mannheimia succiniciproducens is a capnophilic (CO₂-loving) rumen bacterium originally isolated from Korean cows. It has gained significant research attention due to its natural ability to produce succinic acid (SA) efficiently under anaerobic conditions in the presence of CO₂. This bacterium serves as an important industrial microorganism for bio-based production of succinic acid, which is a key building block for various value-added chemicals including 1,4-butanediol, γ-butyrolactone, tetrahydrofuran, and polymers such as polyesters and polyamides .

The complete genome sequence of M. succiniciproducens has been reported, enabling researchers to conduct genome-scale metabolic analyses and targeted genetic engineering to enhance its succinic acid production capabilities . The strain MBEL55E is one of the most well-studied variants and serves as the primary research model for metabolic engineering applications.

What is 3-phosphoshikimate 1-carboxyvinyltransferase (aroA) and what pathway does it function in?

3-phosphoshikimate 1-carboxyvinyltransferase, encoded by the aroA gene, is a critical enzyme in the shikimate pathway, which is responsible for the biosynthesis of aromatic amino acids (phenylalanine, tyrosine, and tryptophan) in bacteria, fungi, and plants. This enzyme catalyzes the sixth step in the shikimate pathway, specifically the transfer of the enolpyruvyl group from phosphoenolpyruvate (PEP) to 3-phosphoshikimate, forming 5-enolpyruvylshikimate-3-phosphate (EPSP) and inorganic phosphate .

The enzymatic reaction is as follows:

3-phosphoshikimate + phosphoenolpyruvate → 5-enolpyruvylshikimate-3-phosphate + phosphate

This reaction is particularly significant as it represents a critical link between primary and secondary metabolism in the organism. In M. succiniciproducens, this pathway is interconnected with the central carbon metabolism, potentially affecting the carbon flux distribution and, consequently, succinic acid production.

Why is recombinant aroA from M. succiniciproducens of interest to researchers?

Recombinant expression of M. succiniciproducens aroA is of interest to researchers for several reasons:

• Structural studies: Understanding the structure-function relationship of this enzyme provides insights into substrate specificity and catalytic mechanisms .

• Metabolic engineering applications: As M. succiniciproducens is an important industrial microorganism for succinic acid production, understanding and potentially modifying its aromatic amino acid biosynthesis pathway can help optimize carbon flux toward desired products .

• Comparative enzymology: Studying aroA from different organisms helps understand evolutionary relationships and species-specific adaptations in enzyme function.

• Drug target potential: In pathogenic relatives of Mannheimia, this enzyme could serve as a target for antimicrobial development, similar to how 3-phosphoshikimate 1-carboxyvinyltransferase from other organisms has been studied for drug design purposes .

What are the optimal conditions for cloning and expressing recombinant M. succiniciproducens aroA?

When cloning and expressing recombinant M. succiniciproducens aroA, researchers should consider the following protocol based on successful approaches with similar enzymes:

Cloning strategy:

• Amplify the aroA gene from M. succiniciproducens genomic DNA using PCR with high-fidelity polymerase and primers containing appropriate restriction sites.

• Digest both the PCR product and expression vector with selected restriction enzymes.

• Ligate the digested PCR product into the expression vector, preferably one with an N-terminal or C-terminal affinity tag (His-tag is commonly used).

• Transform the ligation mixture into a suitable E. coli cloning strain (e.g., DH5α).

• Confirm the correct insert by colony PCR and DNA sequencing.

Expression conditions:

• Transform the confirmed plasmid into an E. coli expression strain (BL21(DE3) or derivatives).

• Grow cultures in LB or TB medium at 37°C until OD₆₀₀ reaches 0.6-0.8.

• Induce protein expression with IPTG (typically 0.1-1.0 mM) at a reduced temperature (16-25°C) to enhance soluble protein production.

• Continue expression for 16-20 hours.

• Harvest cells by centrifugation and proceed with protein purification.

The reduced temperature during induction is particularly important for obtaining soluble and active aroA, as higher temperatures often lead to inclusion body formation with this class of enzymes.

What purification methods are most effective for recombinant M. succiniciproducens aroA?

The most effective purification strategy for recombinant M. succiniciproducens aroA typically involves:

Affinity chromatography:

• Resuspend cell pellet in lysis buffer (typically 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT, and protease inhibitors).

• Lyse cells using sonication or high-pressure homogenization.

• Remove cell debris by centrifugation (20,000 × g, 30 min, 4°C).

• Load clarified lysate onto a Ni-NTA column pre-equilibrated with lysis buffer.

• Wash with buffer containing 20-30 mM imidazole.

• Elute protein with buffer containing 250-300 mM imidazole.

Secondary purification:

• Pool protein-containing fractions and dialyze against a lower-salt buffer (e.g., 20 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM DTT).

• Apply sample to an ion-exchange column (typically Q-Sepharose).

• Elute with a linear NaCl gradient (50-500 mM).

• Perform size-exclusion chromatography as a final polishing step.

The inclusion of Mg²⁺ ions (2-5 mM MgCl₂) in all buffers is recommended as they are often essential for maintaining enzyme stability and activity . Additionally, maintaining a reducing environment with DTT or β-mercaptoethanol helps prevent cysteine oxidation that might affect enzyme activity.

How can researchers assess the activity and kinetic parameters of purified recombinant aroA?

Researchers can assess the activity and kinetic parameters of purified recombinant M. succiniciproducens aroA using several complementary approaches:

Continuous spectrophotometric assay:

• Measure the release of inorganic phosphate during the enzymatic reaction using a coupled enzyme system with purine nucleoside phosphorylase and 2-amino-6-mercapto-7-methylpurine.

• Monitor absorbance change at 360 nm over time.

• Use various concentrations of substrates (3-phosphoshikimate and PEP) to determine Km and Vmax.

HPLC-based assay:

• Incubate enzyme with substrates for defined time periods.

• Stop reactions with acid or heat.

• Analyze the formation of EPSP by HPLC with UV detection.

• Quantify product using standard curves.

Typical reaction conditions:

• Buffer: 100 mM HEPES or Tris-HCl, pH 7.5-8.0

• MgCl₂: 5 mM (essential cofactor)

• KCl: 100 mM

• Temperature: 30-37°C

• Substrate ranges for kinetics: PEP (10-1000 μM), 3-phosphoshikimate (10-1000 μM)

Kinetic parameters calculation:

• Plot initial velocities versus substrate concentration

• Fit data to Michaelis-Menten equation using non-linear regression

• Determine Km, Vmax, kcat, and kcat/Km

For accurate kinetic measurements, it's crucial to ensure that:

• Initial rates are measured (typically <10% substrate conversion)

• Enzyme concentration is significantly lower than substrate concentration

• All cofactors are present at saturating concentrations

• pH and temperature are carefully controlled

How does the structure of M. succiniciproducens aroA compare to aroA enzymes from other organisms?

The structure of M. succiniciproducens aroA is expected to share the core structural features of other bacterial 3-phosphoshikimate 1-carboxyvinyltransferases while possessing unique species-specific adaptations. Based on structural data from related enzymes:

Core structural features:

• Two-domain structure with the active site located at the domain interface

• N-terminal domain consisting of a three-layer αβα sandwich

• C-terminal domain formed by a four-helix bundle

• Binding pocket for 3-phosphoshikimate involving residues from both domains

• Separate binding site for PEP adjacent to the 3-phosphoshikimate site

Comparative structural analysis with aroA enzymes from other organisms reveals both conserved features and potential differences:

While there is no directly reported crystal structure for M. succiniciproducens aroA in the provided search results, structural prediction based on sequence homology and comparing with the structure from C. burnetii (PDB: 3TR1) would provide insights into its unique features. Understanding these structural differences is crucial for designing specific inhibitors or engineering aroA for improved catalytic efficiency.

What mutations in aroA could potentially enhance carbon flux toward succinic acid production in M. succiniciproducens?

Strategic mutations in aroA could potentially enhance carbon flux toward succinic acid production in M. succiniciproducens by modulating the consumption of phosphoenolpyruvate (PEP), a critical metabolic intermediate at the intersection of glycolysis and the succinic acid production pathway. Based on the metabolic network of M. succiniciproducens , the following mutation strategies might be effective:

Mutation strategy 1: Reduced PEP consumption by aroA

• Introduce mutations in the PEP binding site to slightly reduce its affinity

• Target residues involved in PEP binding but not in the catalytic mechanism

• This would redirect more PEP toward the PEP carboxykinase (PckA) reaction, which is critical for succinic acid production

Mutation strategy 2: Feedback regulation modification

• Identify and modify allosteric sites that respond to aromatic amino acid levels

• Mutations that desensitize the enzyme to feedback inhibition while maintaining catalytic function

• This would allow better control of carbon flux distribution between aromatic amino acid synthesis and central metabolism

Mutation strategy 3: Activity modulation under production conditions

• Introduce mutations that optimize enzyme activity at the specific pH and Mg²⁺ concentrations used during succinic acid production

• Target surface residues affecting protein stability under fermentation conditions

• This approach would synchronize aroA activity with the production phase

A rational design approach would involve:

• Structural modeling of M. succiniciproducens aroA

• Computational docking of substrates and potential allosteric regulators

• Identification of key residues for mutagenesis

• In vitro assessment of mutant enzymes

• In vivo testing in M. succiniciproducens strains

These approaches must be carefully balanced to maintain sufficient aromatic amino acid biosynthesis for cell growth while optimizing carbon flux for succinic acid production.

How does metal ion dependency, particularly Mg²⁺, affect aroA activity and how can this be leveraged in metabolic engineering?

Mg²⁺ dependency of aroA:

• Mg²⁺ ions are essential for proper substrate binding, particularly PEP

• They stabilize the negative charges during the catalytic reaction

• They influence the enzyme's conformational dynamics during catalysis

• Proper Mg²⁺ concentration affects both activity and stability of the enzyme

The research results show that optimizing Mg²⁺ transport and availability significantly impacts succinic acid production in M. succiniciproducens . While this is not specifically linked to aroA in the provided data, the general principle can be applied to optimize aroA function within the metabolic network.

Leveraging Mg²⁺ dependency in metabolic engineering:

• Coordinated optimization of Mg²⁺ transport systems:

  • Overexpression of efficient Mg²⁺ transporters, such as the mgtB from Salmonella enterica, significantly enhanced succinic acid production (152.23 ± 0.99 g/L)

  • Similar approaches could be used to optimize aroA function in coordination with other Mg²⁺-dependent enzymes

• Medium and fermentation condition optimization:

  • Using a combination of NH₄OH and Mg(OH)₂ as neutralizing agents increased intracellular Mg²⁺ concentration from 3.46 ± 0.20 mM to 5.81 ± 0.36 mM

  • This approach could be fine-tuned to achieve optimal aroA activity while maintaining high succinic acid production

• Protein engineering of aroA:

  • Mutations could be introduced to optimize the Mg²⁺ binding site of aroA

  • Engineering the enzyme to function optimally at the Mg²⁺ concentrations that favor succinic acid production

• Integration with other metabolic engineering strategies:

  • Combining aroA optimization with enhancements in PEP carboxykinase (PckA), which showed increased activity (up to 7.11-fold) in the presence of Mg²⁺

  • This integrated approach would ensure coordinated enhancement of the entire metabolic pathway

By understanding and optimizing the Mg²⁺ dependency of aroA along with other key enzymes, researchers can develop more robust and efficient succinic acid-producing strains of M. succiniciproducens.

What are the challenges and solutions for expressing functional aroA in heterologous hosts for structural studies?

Expressing functional M. succiniciproducens aroA in heterologous hosts for structural studies presents several challenges, but also has established solutions based on experience with similar enzymes:

Challenges:

• Protein solubility issues:

  • aroA often forms inclusion bodies when overexpressed

  • Native folding may require specific chaperones not present in the host

• Cofactor requirements:

  • Proper folding and activity depend on Mg²⁺ and potentially other cofactors

  • Heterologous hosts may have different intracellular ion concentrations

• Post-translational modifications:

  • Any native modifications in M. succiniciproducens may be absent in the heterologous host

  • This could affect protein stability and crystallization properties

• Protein stability for crystallization:

  • Obtaining diffraction-quality crystals requires highly stable and homogeneous protein

  • aroA may have flexible regions that impede crystallization

Solutions and strategies:

• Expression system optimization:

  Expression Host	Advantages	Disadvantages	Recommended Tags
  E. coli BL21(DE3)	High yield, simple	May form inclusion bodies	N-terminal His₆ with TEV site
  E. coli Arctic Express	Better folding at low temperature	Slower growth, lower yield	His₆-SUMO fusion
  E. coli Rosetta	Supplies rare codons	Similar issues as standard BL21	C-terminal His₈
  Insect cells	Superior folding, PTMs	Complex, expensive	Dual Strep-tag II

• Solubility enhancement:

  • Fusion partners: MBP, SUMO, or Trx tags can dramatically improve solubility

  • Co-expression with chaperones (GroEL/ES, DnaK/J) from M. succiniciproducens

  • Addition of osmolytes (glycerol, arginine) to lysis and purification buffers

• Crystallization strategies:

  • Surface entropy reduction: mutate surface clusters of high-entropy residues

  • Limited proteolysis to remove flexible regions

  • Co-crystallization with substrates, products, or inhibitors to stabilize conformation

  • Screening at various Mg²⁺ concentrations (1-20 mM) to identify optimal conditions

• Alternative structural approaches:

  • Cryo-electron microscopy for challenging targets

  • Nuclear magnetic resonance for dynamics studies

  • Small-angle X-ray scattering for solution structure

Based on the success with similar enzymes, such as the C. burnetii enzymes studied for structural genomics , a combination of these approaches would likely yield functional protein suitable for structural studies.

How do environmental factors affect aroA expression and activity in the context of industrial succinic acid production?

pH effects:

The pH of the fermentation medium affects aroA in multiple ways:

• Direct impact on enzyme stability and catalytic activity

• Influence on the ionization state of active site residues

• Effect on substrate availability and binding

Oxygen availability:

M. succiniciproducens is a facultative anaerobe, and oxygen availability affects its metabolism:

• Under anaerobic conditions, carbon flux through PEP is primarily directed toward succinic acid production via PEP carboxykinase (PckA)

• Oxygen levels may influence the expression and activity of aroA, potentially affecting the balance between aromatic amino acid biosynthesis and central carbon metabolism

• Strictly controlled anaerobic conditions are essential for optimal succinic acid production

Carbon source effects:

• Glucose is the primary carbon source, but using dual carbon sources (glucose and glycerol) has shown enhanced succinic acid production

• Carbon source affects the intracellular PEP pool, which is a substrate for both aroA and PckA

• Optimizing carbon source feeding strategies can help balance aroA activity with succinic acid production

Metal ion availability:

As previously discussed, Mg²⁺ ions play a crucial role:

Practical strategies for industrial optimization:

• Implement precise pH control systems using Mg(OH)₂-containing neutralizers

• Maintain strict anaerobic conditions with controlled CO₂ supply

• Develop fed-batch strategies with optimal carbon source mixtures

• Engineer strains with enhanced Mg²⁺ uptake capabilities

• Monitor and adjust environmental parameters in real-time based on metabolic indicators

By carefully controlling these environmental factors, researchers can optimize aroA function within the broader metabolic context, leading to improved succinic acid production in industrial settings .

What are the emerging techniques for real-time monitoring of aroA activity in living M. succiniciproducens cells?

Emerging techniques for real-time monitoring of aroA activity in living M. succiniciproducens cells represent a frontier in metabolic engineering research. These approaches provide dynamic insights into enzyme function during fermentation processes, enabling more precise control and optimization strategies.

Biosensor-based approaches:

• Transcriptional biosensors:

  • Engineer transcription factors that respond to aroA substrates or products

  • Couple these with fluorescent reporter genes

  • Monitor fluorescence changes as indicators of metabolic flux through aroA

• FRET-based protein sensors:

  • Develop Förster resonance energy transfer (FRET) sensors that change conformation upon binding aroA substrates/products

  • Insert these sensors into cells via expression vectors

  • Monitor real-time changes in FRET efficiency during fermentation

Advanced analytical techniques:

• In vivo NMR spectroscopy:

  • Use ¹³C-labeled substrates to track carbon flux through the aroA reaction

  • Implement flow NMR systems for non-invasive, continuous monitoring

  • Correlate spectral changes with alterations in fermentation conditions

• Mass spectrometry with rapid sampling:

  • Develop microfluidic sampling systems coupled to MS detection

  • Implement stable isotope labeling to distinguish between metabolic pools

  • Create computational models to infer aroA activity from metabolite profiles

Integrated monitoring systems:

These emerging techniques would enable researchers to understand how aroA activity fluctuates throughout the fermentation process and how it interacts with other key enzymes such as PEP carboxykinase (PckA). This dynamic understanding is crucial for developing advanced control strategies that maintain optimal balance between aromatic amino acid biosynthesis and succinic acid production.

How might synthetic biology approaches be used to create novel aroA variants for enhanced metabolic engineering applications?

Synthetic biology approaches offer unprecedented opportunities to create novel aroA variants with enhanced properties for metabolic engineering applications in M. succiniciproducens. These approaches go beyond traditional protein engineering to reimagine aroA functionality within the broader metabolic context.

Design strategies for novel aroA variants:

• Directed evolution with high-throughput screening:

  • Create large libraries of aroA variants using error-prone PCR or DNA shuffling

  • Develop selection strategies that link aroA performance to cell growth or reporter output

  • Iterate selection under conditions mimicking industrial fermentation

  • Identify variants with optimal properties for succinic acid production

• Computational design approaches:

  • Use machine learning algorithms trained on aroA sequence-function relationships

  • Apply molecular dynamics simulations to predict effects of mutations

  • Design variants with altered substrate specificity, cofactor requirements, or regulation

  • Validate computational predictions experimentally

• Domain swapping and chimeric enzymes:

  • Create hybrid enzymes combining domains from aroA of different organisms

  • Design chimeras with enhanced stability under industrial fermentation conditions

  • Develop variants with reduced feedback inhibition by aromatic amino acids

  • Engineer altered PEP binding properties to optimize carbon flux distribution

Advanced synthetic biology applications:

• Metabolic toggle switches:

  • Design aroA variants with altered regulation that can be externally controlled

  • Create genetic circuits that modulate aroA activity in response to fermentation conditions

  • Develop systems that balance aroA activity with PckA activity for optimal carbon flux

• Enzyme co-localization strategies:

  • Create scaffold-bound aroA that co-localizes with other enzymes in the shikimate pathway

  • Design aroA variants that can be selectively isolated from PEP carboxylation pathways

  • Develop compartmentalized systems that segregate competing metabolic processes

• Orthogonal aroA systems:

  • Introduce completely redesigned aroA enzymes that use alternative substrates

  • Create parallel pathways for aromatic amino acid biosynthesis that minimize competition with succinic acid production

  • Develop aroA variants that function optimally under specific phases of fermentation

Integration with whole-cell optimization:

The development of novel aroA variants should be integrated with broader strain engineering efforts:

• Combine aroA modifications with optimized Mg²⁺ transport systems

• Coordinate with engineering of PEP carboxykinase and related enzymes

• Implement dynamic regulation systems that adjust aroA activity based on cell density and fermentation phase

• Develop feedback loops between aroA activity and carbon source utilization

By applying these synthetic biology approaches, researchers can create M. succiniciproducens strains with precisely tailored aroA variants that contribute to enhanced succinic acid production while maintaining optimal cellular function.

What are the most significant recent advances in understanding and engineering M. succiniciproducens aroA?

Recent research has significantly advanced our understanding of M. succiniciproducens metabolism, particularly in relation to succinic acid production. While the specific advances in aroA research are not directly covered in the provided search results, the broader context of metabolic engineering in this organism has seen remarkable progress.

The most significant recent advances include:

• Complete genome sequencing and metabolic modeling of M. succiniciproducens, which has enabled comprehensive understanding of metabolic pathways including those involving aroA .

• Identification of key CO₂-fixing reactions including phosphoenolpyruvate (PEP) carboxykinase, PEP carboxylase, and malic enzyme, which interact with the aroA-utilizing pathways in the competition for PEP .

• Development of highly engineered strains through systematic gene knockouts, particularly the PALK strain (ΔldhA and Δpta-ackA), which achieved remarkable succinic acid production (66.14 g/L) .

• Discovery of the critical role of Mg²⁺ ions in enhancing enzyme activities, including those in central carbon metabolism, which likely affects aroA function indirectly .

• Engineering of Mg²⁺ transport systems by introducing efficient transporters from other organisms (e.g., mgtB from Salmonella enterica), resulting in unprecedented levels of succinic acid production (152.23 g/L) .

These advances create a strong foundation for future specific research on aroA in M. succiniciproducens, particularly in understanding how this enzyme can be optimized within the context of an engineered metabolic network focused on succinic acid production.

How does current knowledge of M. succiniciproducens aroA contribute to broader applications in industrial biotechnology?

Current knowledge of M. succiniciproducens metabolism and the potential role of aroA within its metabolic network contributes significantly to broader applications in industrial biotechnology:

• Bio-based production of succinic acid as a platform chemical:
  The understanding of M. succiniciproducens metabolism has enabled the development of strains capable of producing succinic acid at industrial levels (152.23 g/L with productivity of 11.71 g/L/h) . This bio-based production offers a sustainable alternative to petroleum-based processes for producing this versatile chemical building block.

• Template for engineering other industrial microorganisms:
  The strategies developed for M. succiniciproducens, including balancing competing metabolic pathways (which would include aroA-dependent pathways), provide valuable blueprints for engineering other industrial microorganisms.

• Development of biorefinery concepts:
  Knowledge of aromatic amino acid biosynthesis pathways involving aroA contributes to the development of integrated biorefinery concepts where multiple valuable products can be produced from renewable feedstocks.

• Advanced fermentation strategies:
  The optimization of fermentation conditions, including the use of Mg²⁺-enriched neutralizers , provides insights applicable to other industrial fermentation processes where enzyme function needs to be optimized.

• Sustainable production of valuable chemicals:
  The high yields of succinic acid achieved with engineered M. succiniciproducens strains (yield of 1.30 mol/mol glucose) demonstrate the potential for sustainable production of industrially important chemicals from renewable resources.

By continuing to advance our understanding of specific enzymes like aroA within the context of the broader metabolic network, researchers can further refine and improve these industrial biotechnology applications, making them more economically competitive with traditional chemical processes while reducing environmental impact.

What are the critical research gaps that need to be addressed to fully understand and optimize M. succiniciproducens aroA for biotechnological applications?

Despite significant progress in metabolic engineering of M. succiniciproducens for succinic acid production, several critical research gaps remain in understanding and optimizing aroA for biotechnological applications:

Research priorities to address these gaps:

• Determine the crystal structure of M. succiniciproducens aroA under various ligand-bound states

• Develop biosensors to monitor aroA activity and aromatic amino acid concentrations in vivo

• Create comprehensive datasets of aroA kinetic parameters under various fermentation conditions

• Integrate aroA-specific knowledge into genome-scale metabolic models with enzyme constraints

• Design and test aroA variants with properties optimized for succinic acid production

• Develop dynamic regulatory systems to modulate aroA activity throughout the fermentation process

• Investigate the interplay between aroA and the Mg²⁺-dependent enzymes identified as crucial for succinic acid production