Recombinant Chromobacterium violaceum Phosphoenolpyruvate carboxylase (ppc), partial

What is the metabolic significance of PEPC in Chromobacterium violaceum?

Phosphoenolpyruvate carboxylase (PEPC) in C. violaceum plays a critical role in central carbon metabolism by catalyzing the carboxylation of phosphoenolpyruvate (PEP) to form oxaloacetate (OAA). This anaplerotic reaction replenishes TCA cycle intermediates and serves as a key control point in bacterial metabolism. Although C. violaceum-specific PEPC activity has not been extensively characterized compared to other bacteria, research in related organisms suggests PEPC contributes approximately 10% of the total oxaloacetate synthesis in glucose-growing cells . The enzyme forms part of the metabolic network connecting glycolysis, the TCA cycle, and amino acid biosynthesis pathways, including those leading to aromatic amino acids and violacein production.

To investigate PEPC's role in C. violaceum, researchers typically employ carbon flux analysis using 13C-labeled substrates, genetic knockout studies, and in vitro enzyme assays. Methodologically, creating PEPC-deficient mutants via targeted gene deletion or CRISPR-Cas9 editing, followed by metabolomic profiling, can help elucidate the specific metabolic consequences of PEPC activity in this organism.

How does PEPC regulation affect secondary metabolite production in C. violaceum?

PEPC activity indirectly influences the production of secondary metabolites, including the characteristic violacein pigment, by affecting the availability of precursors for aromatic amino acid biosynthesis. C. violaceum produces erythrose-4-phosphate (E4P), a precursor to aromatic amino acid biosynthesis, via the non-oxidative portion of the hexose monophosphate pathway since it lacks 6-phosphogluconate dehydrogenase . The genome contains all genes required for the pathway from E4P plus phosphoenolpyruvate to tryptophan, which is a precursor for violacein biosynthesis .

PEPC regulation may create metabolic branch points that direct carbon flux either toward central metabolism or secondary metabolite production. When studying these regulatory networks, researchers should implement metabolic flux analysis techniques combined with transcriptomics to understand how PEPC activity correlates with violacein production under various growth conditions. Key methodological approaches include measuring enzyme activities under different nutrient conditions and correlating PEPC expression levels with violacein production using quantitative RT-PCR and proteomics.

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

For recombinant expression of C. violaceum PEPC, E. coli-based expression systems typically provide the highest yield and simplest methodology. The pET expression system with T7 RNA polymerase control is particularly effective for controlled overexpression. When implementing this approach, researchers should optimize:

• Growth temperature: Lower temperatures (16-25°C) often improve proper folding of recombinant PEPC

• Induction conditions: IPTG concentration typically between 0.1-0.5 mM

• Host strain: BL21(DE3) or its derivatives like Rosetta for rare codon optimization

For purification, a two-step approach using nickel affinity chromatography followed by size exclusion chromatography typically yields highly pure enzyme preparations. When designing expression constructs, researchers should consider adding a cleavable His-tag to facilitate purification while allowing tag removal for structural or kinetic studies that require native protein conformations.

How can protein engineering overcome feedback inhibition of C. violaceum PEPC?

C. violaceum PEPC, like many bacterial PEPCs, is subject to allosteric regulation, particularly feedback inhibition by aspartate and malate . This regulatory mechanism can limit carbon flux through this pathway in metabolic engineering applications. To overcome these limitations, protein engineering approaches targeting specific regulatory sites can be implemented based on methodologies established for related organisms.

Based on studies in C. glutamicum, rational protein design focusing on the allosteric binding sites can generate PEPC variants with reduced sensitivity to feedback inhibitors. This approach has yielded mutants with significantly reduced sensitivity toward aspartate and malate in other bacterial species . For C. violaceum PEPC, researchers should:

A methodical approach would involve creating a library of PEPC variants with mutations in the allosteric domain, followed by high-throughput screening for variants that maintain catalytic activity in the presence of inhibitors. In vivo characterization of these variants would then determine their effect on central carbon metabolism and secondary metabolite production.

What is the relationship between PEPC activity, quorum sensing, and violacein production in C. violaceum?

The relationship between PEPC activity and violacein production intersects with quorum sensing (QS) regulation in C. violaceum. Violacein biosynthesis is controlled by a complex regulatory network that includes the CviR/CviI QS system and the VioS repressor protein . While PEPC affects the availability of metabolic precursors, QS controls the expression of the vioABCDE operon responsible for violacein synthesis .

Research methodologies to investigate this relationship should include:

• Construction of dual reporter systems to simultaneously monitor PEPC activity and violacein operon expression

• Creation of strains with controlled expression of both PEPC and QS components

• Metabolic flux analysis under different quorum sensing states

In C. violaceum ATCC31532, the VioS protein negatively regulates violacein production by interfering with QS-mediated positive regulation of the vioA promoter . This creates a fine-tuning mechanism that modulates violacein production in response to population density. PEPC activity may influence this regulation by affecting the metabolic state of the cell, potentially altering the availability of resources for secondary metabolite production.

To experimentally address this question, researchers should implement transcriptomics and proteomics to identify regulatory connections between carbon metabolism and QS signaling. Cross-feeding experiments using co-cultures or conditioned media from strains with altered PEPC activity could reveal metabolic interactions that influence QS-dependent phenotypes.

How do environmental signals integrate with PEPC regulation to control metabolism in C. violaceum?

C. violaceum responds to various environmental signals that may affect PEPC regulation and subsequent metabolite production. For instance, sublethal concentrations of antibiotics that inhibit polypeptide elongation during translation (including blasticidin S, spectinomycin, hygromycin B, apramycin, tetracycline, erythromycin, and chloramphenicol) induce violacein production . This response represents a sophisticated integration of stress signals with metabolic regulation.

The methodological approach to study this integration should include:

• Transcriptomic analysis of C. violaceum under various environmental conditions to identify co-regulated genes

• ChIP-seq to identify transcription factors that bind to the PEPC promoter region

• Metabolomic profiling to identify shifts in central metabolism in response to environmental signals

What are the kinetic and structural differences between native and recombinant forms of C. violaceum PEPC?

Recombinant expression of C. violaceum PEPC may result in structural and kinetic differences compared to the native enzyme. These differences can arise from:

• Post-translational modifications present in the native but not recombinant form

• Structural alterations due to purification tags or expression conditions

• Differences in oligomeric state or protein-protein interactions

Methodologically, a comparative analysis requires:

• Purification of both native PEPC (from C. violaceum) and recombinant PEPC using identical final purification steps

• Detailed kinetic characterization including Km, Vmax, and response to allosteric effectors

• Structural analysis using circular dichroism, analytical ultracentrifugation, and potentially X-ray crystallography or cryo-EM

Data should be presented in tabular format comparing key parameters:

This comparative approach enables researchers to determine whether recombinant PEPC faithfully represents the native enzyme's properties and informs experimental design for subsequent metabolic engineering applications.

What are the optimal conditions for measuring C. violaceum PEPC activity in vitro?

Accurate measurement of C. violaceum PEPC activity requires careful optimization of assay conditions. The standard coupled enzyme assay utilizes malate dehydrogenase to monitor NADH oxidation spectrophotometrically at 340 nm. For optimal results:

• Buffer composition: 100 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 10 mM NaHCO3

• Substrate concentration: 2-5 mM PEP (determine optimal concentration empirically)

• Coupling system: 0.2 mM NADH and excess malate dehydrogenase (typically 5-10 U/ml)

• Temperature: 30°C (the optimal growth temperature for C. violaceum)

Common troubleshooting issues include:

• Loss of activity during purification: Add 10% glycerol and 1 mM DTT to all buffers

• Interfering background activity: Include appropriate control reactions without PEP

• Inconsistent results: Ensure CO2/HCO3- equilibrium is established before initiating the reaction

When designing experiments to characterize engineered variants, researchers should include wild-type PEPC as a control in each experimental batch to account for day-to-day variations in assay conditions.

How can metabolic flux through PEPC be accurately measured in vivo in C. violaceum?

Measuring in vivo PEPC flux in C. violaceum requires sophisticated metabolic flux analysis techniques. The most accurate approach utilizes 13C-labeled substrates (typically glucose) followed by mass spectrometry analysis of metabolic intermediates and amino acids derived from central metabolism.

Methodological steps include:

• Cultivation of C. violaceum in minimal media with 13C-labeled glucose as the sole carbon source

• Extraction of intracellular metabolites and derivatization for GC-MS or LC-MS/MS analysis

• Measurement of isotopic enrichment patterns in metabolites

• Computational flux modeling using software such as 13CFLUX2 or INCA

When implementing this approach, researchers should ensure steady-state metabolic conditions before sampling and include parallel experiments with different labeling patterns (e.g., [1-13C]glucose vs. [U-13C]glucose) to improve flux resolution. The resulting data can reveal how PEPC activity redistributes carbon flux between glycolysis, the TCA cycle, and biosynthetic pathways under various growth conditions.

How does PEPC function within the broader metabolic network of C. violaceum?

PEPC functions as a key node in the C. violaceum metabolic network, connecting glycolysis with the TCA cycle and amino acid biosynthesis. Understanding this integration requires a systems biology approach combining multiple omics technologies with computational modeling.

Genome-scale metabolic modeling (GSMM) provides a framework for predicting how changes in PEPC activity affect the entire metabolic network. To develop an accurate model:

• Integrate genomic, transcriptomic, and proteomic data to construct a comprehensive metabolic network

• Validate the model using 13C metabolic flux analysis data under various growth conditions

• Perform flux balance analysis (FBA) to predict optimal flux distributions

• Use the model to identify potential bottlenecks and targets for metabolic engineering

Experimental validation should include comparison of model predictions with experimental data from PEPC overexpression or knockout strains. This approach can reveal unexpected metabolic adaptations and regulatory mechanisms that compensate for altered PEPC activity.

What computational approaches can predict optimal PEPC mutations for enhanced activity or altered regulation?

Computational protein engineering offers powerful tools for designing PEPC variants with enhanced properties. These approaches leverage structural information, evolutionary analysis, and machine learning to predict beneficial mutations:

• Homology modeling and molecular dynamics simulations can identify flexible regions and allosteric sites that might be targeted for engineering

• Sequence alignment across diverse bacterial PEPCs can reveal conserved residues critical for function versus variable positions amenable to mutation

• Statistical coupling analysis (SCA) can identify co-evolving residue networks important for protein function

• Machine learning algorithms trained on existing mutational data can predict novel mutations likely to enhance desired properties

The methodological workflow includes:

• Building a reliable structural model of C. violaceum PEPC based on homologous structures

• Conducting in silico mutagenesis and energy calculations to predict stability changes

• Simulating enzyme-substrate interactions to identify residues affecting catalysis

• Predicting the effects of mutations on allosteric regulation

Top candidate mutations should be experimentally validated through site-directed mutagenesis followed by kinetic characterization. An iterative approach combining computational prediction with experimental validation provides the most efficient path to PEPC variants with desired properties for metabolic engineering applications.

How might PEPC engineering in C. violaceum enhance production of novel bioactive compounds?

Engineering PEPC activity in C. violaceum represents a promising approach for enhancing the production of bioactive compounds, including violacein and potentially novel secondary metabolites. By redirecting carbon flux through central metabolism, optimized PEPC variants could increase precursor availability for biosynthetic pathways.

Strategic approaches include:

• Developing feedback-resistant PEPC variants to maintain high anaplerotic flux regardless of cellular metabolic state

• Creating synthetic regulatory circuits that coordinate PEPC activity with the expression of biosynthetic gene clusters

• Engineering protein-protein interactions between PEPC and other metabolic enzymes to create substrate channeling effects

When implementing these strategies, researchers should monitor not only target compound production but also global metabolic changes using metabolomics and fluxomics. This comprehensive analysis can reveal unexpected consequences of PEPC engineering and identify additional targets for pathway optimization.

Potential applications extend beyond violacein to other tryptophan-derived compounds and polyketides that might benefit from increased availability of acetyl-CoA and malonyl-CoA precursors. The relationship between PEPC activity, quorum sensing, and secondary metabolism in C. violaceum suggests that engineering this enzyme could unlock novel bioactive compounds regulated by similar mechanisms .

What insights can comparative studies of PEPC from C. violaceum and other pigment-producing bacteria provide?

Comparative analysis of PEPC from C. violaceum and other pigment-producing bacteria can reveal evolutionary adaptations linking central metabolism with secondary metabolite production. Both C. violaceum and Pseudoalteromonas ferrooxidans produce violacein , making their PEPC enzymes particularly interesting for comparative studies.

Methodological approaches should include:

• Phylogenetic analysis of PEPC sequences across diverse pigment-producing bacteria

• Comparative biochemical characterization of purified PEPCs

• Heterologous expression studies swapping PEPC genes between species

• Analysis of transcriptional and metabolic responses to environmental conditions

This comparative approach can identify unique regulatory features or structural adaptations in C. violaceum PEPC that might be associated with its ecological niche or specific metabolic requirements for violacein production. For instance, the VioS repressor is found only in C. violaceum and P. ferrooxidans , suggesting these bacteria may share regulatory mechanisms linking central metabolism and secondary metabolite production.

The findings from such comparative studies would provide evolutionary context for understanding PEPC function in C. violaceum and potentially reveal novel regulatory mechanisms that could be exploited for metabolic engineering applications.