Recombinant Chromobacterium violaceum Acetyl-coenzyme A carboxylase carboxyl transferase subunit alpha (accA)

What is Chromobacterium violaceum and why is it significant for metabolic engineering studies?

Chromobacterium violaceum is a gram-negative bacterium known for its ability to accumulate polyhydroxyalkanoates (PHAs) such as poly(3-hydroxybutyrate) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) when grown on fatty acid carbon sources . It has attracted significant research interest due to its unique metabolic capabilities, including the production of violacein pigment and various antimicrobial compounds . The organism's metabolic versatility makes it an excellent candidate for studies focused on carbon metabolism, including pathways involving Acetyl-coenzyme A carboxylase (ACCase).

What is the function of Acetyl-coenzyme A carboxylase (ACCase) in bacterial metabolism?

Acetyl-coenzyme A carboxylase (ACCase) plays a crucial role in bacterial fatty acid biosynthesis by catalyzing the first committed step of this pathway - the carboxylation of acetyl-CoA to form malonyl-CoA . This reaction requires biotin as a cofactor and comprises two half-reactions: (1) the biotin carboxylase component transfers a carboxyl group to biotin, and (2) the carboxyltransferase component (which includes the alpha subunit, accA) transfers this carboxyl group from biotin to acetyl-CoA, forming malonyl-CoA. Malonyl-CoA serves as an essential building block for fatty acid synthesis and various secondary metabolites, making ACCase a central enzyme in bacterial metabolism and a potential target for antimicrobial drug development .

How is the ACCase enzyme structurally organized in Gram-negative bacteria like C. violaceum?

In Gram-negative bacteria, ACCase typically exists as a multi-subunit complex consisting of four distinct proteins: AccA, AccD (together forming the carboxyltransferase component), AccB (biotin carboxyl carrier protein), and AccC (biotin carboxylase) . The carboxyltransferase component (AccAD) catalyzes the transfer of the carboxyl group from biotin to acetyl-CoA, while AccB carries the biotin prosthetic group, and AccC catalyzes the ATP-dependent carboxylation of biotin . Unlike the single-polypeptide ACCase found in eukaryotes, this multi-subunit organization is characteristic of most bacteria, including C. violaceum. When expressing recombinant ACCase, all four subunits need to be correctly assembled to form a functional holoenzyme .

What are the recommended strategies for cloning the accA gene from C. violaceum?

For cloning the accA gene from C. violaceum, the following methodological approach is recommended:

• Genomic Library Construction: Create a genomic library of C. violaceum by digesting genomic DNA with appropriate restriction enzymes (such as BamHI) and ligating fragments into a suitable cloning vector such as pBBR1MCS-1 .

• Primer Design: Design specific primers based on conserved regions of the accA gene from related organisms or from available C. violaceum genome data. If the sequence is unknown, degenerate primers can be designed based on aligned sequences from related bacteria.

• PCR Screening: Screen the genomic library using PCR with the designed primers, or alternatively, use a labeled probe and colony hybridization technique. For hybridization screening, a digoxigenin (DIG)-labeled PCR product can be used as described for other C. violaceum genes .

• Sequence Verification: Once potential positive clones are identified, perform sequencing to confirm the identity of the accA gene. Compare with known accA sequences from other organisms using BLAST analysis to verify the gene's identity.

• Subcloning: For expression studies, subclone the verified accA gene into an appropriate expression vector with a suitable promoter for the host organism .

What expression systems are most effective for producing recombinant C. violaceum AccA protein?

The choice of expression system depends on research objectives, but several options have proven effective for similar bacterial proteins:

Expression Host Comparison:

When expressing the AccA subunit, consider these methodological recommendations:

• Co-expression Strategy: For functional studies, co-express accA with accD, accB, and accC to reconstitute the complete ACCase holoenzyme .

• Fusion Tags: Incorporate affinity tags (His6, GST, etc.) to facilitate purification and detection. Place these at either the N- or C-terminus based on structural considerations.

• Induction Conditions: Optimize expression by testing various induction conditions (temperature, inducer concentration, time) to maximize soluble protein yield.

• Strain Selection: Use specialized E. coli strains like BL21(DE3) for T7-based expression or Rosetta for rare codon supplementation if needed.

What are the recommended methods for purifying recombinant AccA protein?

Purification of recombinant AccA protein can be achieved through the following methodological approach:

• Cell Lysis: Disrupt cells using sonication, French press, or enzymatic methods in an appropriate buffer system (typically pH 7.5-8.0) containing protease inhibitors.

• Initial Clarification: Remove cell debris by centrifugation (15,000-20,000 × g, 30 minutes, 4°C).

• Affinity Chromatography: If the protein contains an affinity tag, use the corresponding affinity resin (Ni-NTA for His-tagged proteins, glutathione-agarose for GST-fusion proteins).

• Ion Exchange Chromatography: Further purify using anion or cation exchange chromatography based on the protein's theoretical isoelectric point.

• Size Exclusion Chromatography: As a final polishing step, apply size exclusion chromatography to separate the protein from remaining contaminants and assess its oligomeric state.

• Quality Assessment: Evaluate purity by SDS-PAGE and verify identity using Western blotting or mass spectrometry.

For ACCase holoenzyme reconstitution, purify all four subunits individually and then mix them in an appropriate buffer containing ATP, biotin, and magnesium to form the active complex .

How can I assess the enzymatic activity of recombinant C. violaceum ACCase?

ACCase enzymatic activity can be assessed using several complementary methods:

Direct Detection of Malonyl-CoA Formation:

The gold standard for ACCase activity measurement is direct detection of malonyl-CoA formation using liquid chromatography-tandem mass spectrometry (LC-MS/MS) . The reaction mixture typically contains:

• Purified ACCase components (AccA, AccD, AccB, AccC)

• Acetyl-CoA (substrate)

• ATP

• Biotin

• MgCl₂

• Appropriate buffer (e.g., HEPES pH 8.0)

After incubation at optimal temperature (typically 30-37°C), the reaction is quenched, and malonyl-CoA formation is quantified by LC-MS/MS.

ATP Depletion Assay:

A luminescence-based assay that monitors ATP depletion can be used as a higher-throughput alternative for inhibitor screening . This assay utilizes commercial kits that measure remaining ATP levels after the ACCase reaction.

Carboxyltransferase (AccAD) Specific Assay:

To assess specifically the carboxyltransferase activity of the AccAD subcomplex:

• Isolate the AccAD subcomplex (containing AccA)

• Set up a reaction with malonyl-CoA and an acyl-ACP acceptor

• Monitor the reverse reaction (decarboxylation of malonyl-CoA)

• Detect the formation of acetyl-CoA or free CoA using specific colorimetric reagents or HPLC analysis

What strategies can overcome expression challenges for C. violaceum AccA in heterologous hosts?

Recombinant expression of C. violaceum AccA may face several challenges in heterologous hosts. The following advanced strategies can help overcome these issues:

Codon Optimization:

C. violaceum has a different codon usage bias compared to common expression hosts like E. coli. Synthesize a codon-optimized accA gene based on the preferred codons of the expression host to improve translation efficiency and protein yield .

Solubility Enhancement:

• Fusion Partners: Use solubility-enhancing fusion partners such as MBP (maltose-binding protein), SUMO, or Thioredoxin.

• Chaperone Co-expression: Co-express molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE) to assist proper protein folding.

• Expression Conditions: Lower the expression temperature (15-25°C) and reduce inducer concentration to slow down protein synthesis and improve folding.

Alternative Host Systems:

If E. coli expression is problematic, consider alternative hosts that may provide a more suitable metabolic environment:

• K. aerogenes or R. eutropha: These organisms have been successfully used for expressing C. violaceum genes and may provide a more compatible cellular environment .

• Cell-Free Expression Systems: For proteins that are toxic to cells or prone to inclusion body formation, cell-free protein synthesis systems can be an effective alternative.

How can I investigate the interaction between AccA and other ACCase subunits in C. violaceum?

Understanding the protein-protein interactions between AccA and other ACCase subunits is crucial for elucidating enzyme function and regulation. Advanced methodological approaches include:

Structural Biology Approaches:

• X-ray Crystallography: Co-crystallize AccA with AccD to determine the structure of the carboxyltransferase component.

• Cryo-electron Microscopy: Visualize the entire ACCase complex architecture, particularly useful for large multi-subunit complexes.

• NMR Spectroscopy: For studying dynamic interactions between smaller domains or subunits.

Protein-Protein Interaction Studies:

• Yeast Two-Hybrid Assays: Screen for interactions between AccA and other ACCase subunits.

• Pull-down Assays: Use tagged versions of AccA to pull down interacting partners from cell lysates.

• Surface Plasmon Resonance (SPR): Quantitatively measure binding kinetics between purified AccA and other ACCase subunits.

• Cross-linking Mass Spectrometry: Identify interaction interfaces by cross-linking followed by MS analysis.

Computational Approaches:

• Homology Modeling: Build structural models of C. violaceum AccA based on homologous proteins from other bacteria.

• Molecular Docking: Predict interaction interfaces between AccA and other ACCase subunits.

• Molecular Dynamics Simulations: Investigate the dynamic behavior of the AccA-AccD complex.

How can recombinant C. violaceum AccA be utilized in metabolic engineering applications?

Recombinant C. violaceum AccA, as part of the complete ACCase enzyme, can be leveraged in several metabolic engineering applications:

Production of Polyketides and Fatty Acid Derivatives:

ACCase catalyzes the formation of malonyl-CoA, a rate-limiting precursor for polyketide and fatty acid biosynthesis. Overexpression of optimized ACCase components (including AccA) can increase the malonyl-CoA pool, enhancing production of:

• 3-Hydroxypropionic Acid (3-HP): By combining ACCase with malonyl-CoA reductase (MCR), the malonyl-CoA pathway for 3-HP production can be established in host organisms .

• Fatty Acid-Derived Biofuels: Enhanced malonyl-CoA availability can improve production of fatty acid ethyl esters (FAEEs) and fatty alcohols.

• Polyketide Antibiotics: Increased malonyl-CoA can boost production of medicinally important polyketides.

Engineering Alternative Substrate Specificity:

Through protein engineering of the AccA-AccD carboxyltransferase component, it may be possible to alter substrate specificity to accept alternative acyl-CoA substrates, enabling the production of novel compounds.

What role does AccA play in antibiotic resistance, and how can this inform drug development?

ACCase is a promising target for antibiotic development due to its essential role in bacterial fatty acid biosynthesis . The AccA-AccD carboxyltransferase component offers several advantages as a drug target:

• Structural Uniqueness: The bacterial carboxyltransferase differs significantly from human ACCase, offering selectivity for antibacterial action.

• Essential Function: Inhibition of ACCase blocks fatty acid biosynthesis, which is lethal to most bacteria.

• Known Inhibitors: Several classes of compounds (e.g., certain herbicides, natural products) inhibit bacterial carboxyltransferase activity.

Methodological approaches for investigating AccA as an antibiotic target include:

High-Throughput Screening for Inhibitors:

• Enzyme-Based Assays: Use purified recombinant AccA-AccD to screen compound libraries for inhibitors using the ATP depletion luminescence assay .

• Fragment-Based Drug Discovery: Screen smaller chemical fragments that bind to AccA and can be developed into larger inhibitors.

Structure-Based Drug Design:

Using structural information (X-ray crystallography or homology models) of C. violaceum AccA to design specific inhibitors targeting this subunit.

Whole-Cell Assays:

Validate identified inhibitors in C. violaceum and pathogenic bacteria to assess antibacterial activity, membrane permeability, and potential resistance mechanisms.

How can the study of C. violaceum AccA contribute to understanding bacterial evolution and adaptation?

Studying C. violaceum AccA provides insights into bacterial evolution and adaptation through several research approaches:

Comparative Genomics and Phylogenetic Analysis:

• Compare AccA sequences across diverse bacterial species to understand evolutionary relationships and selective pressures.

• Analyze AccA sequence conservation in relation to habitat and metabolic adaptations of different bacteria.

• Investigate horizontal gene transfer events involving accA genes in bacterial communities.

Adaptive Laboratory Evolution Studies:

• Subject C. violaceum to various selective pressures (carbon sources, ACCase inhibitors) and monitor genetic changes in accA.

• Analyze how accA mutations affect fitness under different environmental conditions.

Structure-Function Relationships:

• Identify conserved domains and variable regions in AccA across bacterial species.

• Correlate structural features with functional adaptations in different bacterial lineages.

What are the most sensitive methods for detecting and quantifying AccA expression levels?

Several complementary methodological approaches can be used to detect and quantify AccA expression with high sensitivity:

Protein-Level Detection:

• Western Blotting: Using specific antibodies against C. violaceum AccA or against epitope tags if recombinant protein is tagged.

• Mass Spectrometry:

  • Selected Reaction Monitoring (SRM): Targeted MS approach for quantifying specific AccA peptides.

  • SWATH-MS: For label-free quantification of AccA in complex protein mixtures.

• ELISA: Development of sandwich ELISA using anti-AccA antibodies for quantitative detection in complex samples.

Transcript-Level Detection:

• RT-qPCR: Quantify accA mRNA levels using specific primers and appropriate reference genes.

• RNA-Seq: For global transcriptomic analysis that includes accA expression in different conditions.

Activity-Based Detection:

Measure the carboxyltransferase activity of AccA-AccD as a proxy for expression levels using the enzymatic assays described earlier .

What bioinformatic tools are most effective for analyzing and modeling C. violaceum AccA?

Several specialized bioinformatic tools can assist in the analysis and modeling of C. violaceum AccA:

Sequence Analysis Tools:

• Multiple Sequence Alignment: MUSCLE, Clustal Omega, or MAFFT for aligning AccA sequences from different organisms.

• Phylogenetic Analysis: MEGA, RAxML, or MrBayes for constructing phylogenetic trees.

• Domain Prediction: InterProScan or SMART for identifying functional domains within AccA.

• Secondary Structure Prediction: PSIPRED or JPred for predicting α-helices and β-sheets.

Structural Modeling:

• Homology Modeling: SWISS-MODEL, I-TASSER, or AlphaFold for generating 3D models based on homologous proteins.

• Model Validation: PROCHECK, VERIFY3D, or MolProbity for assessing model quality.

• Molecular Dynamics: GROMACS or AMBER for simulating protein dynamics in solution.

Protein-Protein Interaction:

• Molecular Docking: HADDOCK, ClusPro, or ZDOCK for modeling interactions between AccA and other ACCase subunits.

• Hot Spot Prediction: KFC2, HotPoint, or PredHS for identifying critical residues at protein interfaces.

How can I develop a high-throughput screening system for C. violaceum AccA modulators?

Development of a high-throughput screening (HTS) system for AccA modulators requires a multi-faceted methodological approach:

In vitro Enzyme-Based Assays:

• ATP Depletion Luminescence Assay: This assay monitors the ATP consumption by ACCase and can be adapted to 384-well plate format for HTS .

• Malonyl-CoA Production Assay: Coupling malonyl-CoA formation to a fluorescent or colorimetric readout using auxiliary enzymes.

• Thermal Shift Assay: Screen for compounds that bind to AccA by monitoring changes in protein thermal stability.

Cell-Based Assays:

• Growth Inhibition: Screen for compounds that inhibit growth of bacteria dependent on C. violaceum AccA.

• Reporter Systems: Develop genetic constructs that link AccA activity to expression of fluorescent or luminescent reporters.

Virtual Screening:

• Structure-Based Virtual Screening: Use the AccA 3D structure (experimental or modeled) to screen virtual compound libraries.

• Pharmacophore-Based Screening: Develop a pharmacophore model based on known AccA modulators for virtual screening.

Assay Optimization Considerations:

• Miniaturization: Optimize reaction volumes for 384 or 1536-well plates.

• Signal Stability: Ensure signal stability over the screening period.

• DMSO Tolerance: Determine maximum tolerable DMSO concentration as compounds are typically dissolved in DMSO.

• Statistical Validation: Calculate Z' factor to ensure assay robustness.