Recombinant Pseudomonas putida Succinyl-CoA ligase [ADP-forming] subunit beta (sucC)

What is the biological function of Succinyl-CoA ligase in Pseudomonas putida metabolism?

Succinyl-CoA ligase (SCL) [ADP-forming] subunit beta (sucC) in P. putida serves as a critical enzyme in the tricarboxylic acid (TCA) cycle, catalyzing the reversible conversion of succinyl-CoA to succinate with the concurrent production of ATP (via ADP phosphorylation). This reaction represents a key step in energy metabolism where substrate-level phosphorylation occurs in the TCA cycle.

Unlike the well-characterized SCL from P. aeruginosa, the P. putida enzyme likely plays additional roles in cellular metabolism beyond the canonical TCA cycle function . The enzyme participates in:

• Energy conservation through ADP-dependent ATP generation

• Metabolic flux regulation between oxidative and reductive TCA cycle routes

• Anaplerotic pathway connections, particularly with methylmalonyl-CoA pathways

The sucC gene product forms a heterodimer with sucD (α-subunit), creating the functional enzyme complex that requires magnesium as a cofactor for catalytic activity .

How does the structure of P. putida sucC compare with homologous proteins from other Pseudomonas species?

Comparative analysis of P. putida sucC with its P. aeruginosa homolog reveals significant structural conservation with approximately 85-90% sequence identity in the core catalytic domains. The β-subunit (sucC) typically contains:

• An ATP-binding domain with conserved Walker A and B motifs

• CoA-binding pocket with preserved cysteine residues

• Dimerization interface for interaction with the α-subunit (sucD)

While the P. aeruginosa sucC protein sequence (388 amino acids) includes conserved regions like "MNLHEYQGKQLFAEYGLPVS" at the N-terminus , P. putida sucC shows species-specific variations in non-catalytic regions. These modifications may reflect adaptation to different metabolic requirements and environmental niches occupied by P. putida.

Key differences appear in the mitochondrial targeting sequences (when comparing to eukaryotic homologs) and in surface-exposed loops that likely mediate species-specific protein interactions. These structural variations may impact protein stability and activity under different environmental conditions typical of P. putida habitats .

What expression systems are most effective for producing recombinant P. putida sucC protein?

For optimal recombinant expression of P. putida sucC, several expression systems have demonstrated effectiveness, each with distinct advantages:

E. coli-based expression systems:

• BL21(DE3) strains with pET vectors show high yield but may require optimization of induction parameters

• Arctic Express or Rosetta strains improve folding of difficult domains

• Codon optimization for E. coli is recommended when expressing full-length protein

Baculovirus expression systems:

• Insect cell (Sf9 or Hi5) expression provides superior folding for complex proteins

• Post-translational modifications more closely resemble native conditions

• Higher yield of soluble protein compared to bacterial systems

Pseudomonas-based homologous expression:

• Using modified P. putida KT2440 as host provides native chaperones and cofactors

• I-SceI–mediated recombination systems allow precise genomic integration

• CRISPR-Cas9 techniques enable fine control of expression levels

The choice of expression tag significantly impacts purification success:

• N-terminal His6 tags generally maintain activity

• C-terminal tags may interfere with dimerization

• TEV protease cleavage sites allow tag removal while maintaining native structure

Optimal expression temperatures (16-25°C) and extended induction times (12-18 hours) typically maximize soluble protein yield regardless of the chosen system.

What are the recommended storage and handling protocols for recombinant P. putida sucC protein to maintain enzymatic activity?

Proper storage and handling of recombinant P. putida sucC protein is critical for preserving enzymatic activity and ensuring experimental reproducibility. Based on established protocols for similar Succinyl-CoA ligases, I recommend the following:

Short-term storage (1-2 weeks):

• Store at 4°C in stabilizing buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, and 1 mM DTT

• Avoid repeated freeze-thaw cycles by preparing working aliquots

• Include protease inhibitors (PMSF or commercial cocktail) for extended refrigeration

Long-term storage (months to years):

• Store at -20°C with 20-50% glycerol as cryoprotectant

• For extended archival storage, maintain at -80°C in small aliquots (50-100 μL)

• Flash freezing in liquid nitrogen before transfer to -80°C minimizes ice crystal formation

Reconstitution protocol:

• Centrifuge vial briefly before opening to collect contents

• Reconstitute lyophilized protein to 0.1-1.0 mg/mL using sterile deionized water

• Add glycerol to 5-50% final concentration for stability

• Allow complete dissolution by gentle rotation rather than vortexing

• Confirm protein concentration by Bradford or BCA assay before use

Activity preservation considerations:

• Maintain Mg²⁺ (1-5 mM) in all working buffers as essential cofactor

• pH stability is optimal between 7.0-8.0

• Avoid chelating agents (EDTA) that may sequester essential metal ions

• Shield from direct light during handling to prevent oxidative damage

Enzyme activity typically demonstrates a shelf life of approximately 6 months at -20°C for liquid preparations and 12 months for lyophilized forms .

How can researchers accurately measure the enzymatic activity of recombinant P. putida sucC protein?

Accurate measurement of P. putida sucC enzymatic activity requires carefully designed assays that account for both the forward and reverse reactions catalyzed by this enzyme. I recommend the following established methodologies:

Forward reaction (Succinyl-CoA → Succinate + ATP):

• Coupled spectrophotometric assay:

  • Couple ATP production to NADH oxidation via pyruvate kinase and lactate dehydrogenase

  • Monitor absorbance decrease at 340 nm (ε = 6,220 M⁻¹cm⁻¹)

  • Standard reaction mixture: 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 0.2 mM NADH, 1 mM PEP, 0.2 mM ADP, 0.5 mM succinyl-CoA, 2 U/mL pyruvate kinase, 2 U/mL lactate dehydrogenase

• Direct ATP quantification:

  • Measure ATP production using luciferase-based assays

  • Correlate luminescence signal to ATP concentration using standard curve

  • Terminate reaction at different timepoints to determine initial velocity

Reverse reaction (Succinate + ATP → Succinyl-CoA + ADP):

• DTNB-based assay:

  • Monitor CoA-SH incorporation using 5,5'-dithiobis-(2-nitrobenzoic acid)

  • Measure absorbance increase at 412 nm (ε = 14,150 M⁻¹cm⁻¹)

  • Reaction mixture: 50 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, 0.1 mM DTNB, 0.4 mM CoA, 1 mM ATP, 10 mM succinate

• HPLC analysis:

  • Quantify succinyl-CoA formation directly by reverse-phase HPLC

  • Use C18 column with gradient of acetonitrile in phosphate buffer

  • Monitor absorbance at 254 nm for CoA derivatives

Kinetic parameter determination:

• Vary substrate concentrations (0.1-10× Km) to generate Michaelis-Menten plots

• Calculate Km, Vmax, and kcat using non-linear regression analysis

• Determine substrate specificity by testing nucleotide analogs (GTP, ITP)

Controls and validation:

• Include enzyme-free reactions to account for non-enzymatic hydrolysis

• Use commercially available Succinyl-CoA ligase as positive control

• Run heat-inactivated enzyme as negative control

When reporting activity, express as μmol of product formed per minute per mg of enzyme (U/mg) under standard conditions (pH 7.5, 25°C).

What purification strategies yield the highest purity and specific activity for recombinant P. putida sucC?

Achieving high purity and specific activity of recombinant P. putida sucC requires a strategic multi-step purification approach. Based on successful protocols for homologous proteins, I recommend this optimized procedure:

Initial capture:

• Immobilized Metal Affinity Chromatography (IMAC)

  • Use Ni-NTA or TALON resin for His-tagged constructs

  • Equilibrate column with 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole

  • Apply stepped imidazole gradient (50, 100, 250 mM) to minimize contaminants

  • Expected purity after IMAC: 75-80%

Intermediate purification:
2. Ion Exchange Chromatography

• Apply IMAC-purified sample to Q-Sepharose (anion exchange) at pH 8.0

• Create linear gradient from 50-500 mM NaCl to separate charge variants

• Alternatively, use SP-Sepharose (cation exchange) at pH 6.0 if pI of construct permits

• Expected purity after ion exchange: 85-90%

• Hydrophobic Interaction Chromatography

  • Particularly effective for removing residual endotoxins

  • Equilibrate Phenyl Sepharose column with 50 mM phosphate buffer pH 7.0, 1.5 M ammonium sulfate

  • Create descending ammonium sulfate gradient (1.5-0 M)

  • Expected purity: >90%

Polishing:
4. Size Exclusion Chromatography

• Apply concentrated sample to Superdex 200 column

• Isocratic elution with 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol

• Collect fractions corresponding to expected molecular weight (~41 kDa for monomer)

• Expected final purity: >95% (SDS-PAGE)

Purification monitoring table:

Critical considerations:

• Maintain 5-10 mM MgCl₂ throughout purification to stabilize enzyme structure

• Add 5% glycerol to all buffers to prevent aggregation

• Include 1 mM DTT or 0.5 mM TCEP to protect thiol groups

• Keep temperature at 4°C during all steps

• Perform activity assays after each purification step to track specific activity

• Utilize a protease inhibitor cocktail in initial lysis buffer

Final specific activity should reach 15-20 μmol/min/mg with >95% homogeneity as assessed by SDS-PAGE and confirmed by mass spectrometry.

How does sucC function differ between Pseudomonas putida and clinically relevant Pseudomonas aeruginosa strains?

The functional distinctions between P. putida and P. aeruginosa sucC reflect evolutionary adaptations to their respective ecological niches and metabolic requirements:

Catalytic efficiency differences:
P. putida sucC typically exhibits higher catalytic efficiency (kcat/Km) under aerobic conditions compared to P. aeruginosa homologs, consistent with P. putida's strictly aerobic metabolism. In contrast, P. aeruginosa sucC demonstrates greater versatility across oxygen-limited environments, reflecting its ability to use nitrate as an alternative electron acceptor during anaerobic respiration .

Regulatory mechanisms:
P. aeruginosa sucC expression is significantly upregulated during infection and biofilm formation, whereas P. putida sucC regulation appears more responsive to carbon source availability and environmental stress conditions . This difference aligns with P. aeruginosa's pathogenic lifestyle versus P. putida's environmental adaptability.

Structural adaptations:
Comparative sequence analysis reveals conserved catalytic domains but divergent surface-exposed regions between species:

Biofilm-specific functions:
P. aeruginosa sucC shows upregulation during biofilm formation and contributes to the characteristic metabolic rewiring observed in chronic infections . While P. putida forms biofilms in environmental settings, its sucC expression pattern during biofilm formation suggests different metabolic priorities focused on nutrient scavenging rather than virulence.

Antibiotic resistance connections:
Recent research has identified unexpected links between TCA cycle enzymes and antibiotic resistance mechanisms. P. aeruginosa sucC mutations can affect susceptibility to aminoglycosides through altered membrane potential , whereas P. putida sucC perturbations more significantly impact efflux pump activity and intrinsic resistance to aromatic antimicrobials.

These functional differences make P. putida sucC particularly valuable for biotechnological applications, while P. aeruginosa sucC remains important for understanding metabolic adaptations during infection processes.

What genetic engineering approaches can be used to modify sucC expression in Pseudomonas putida for metabolic engineering applications?

Genetic manipulation of sucC expression in P. putida offers powerful opportunities for metabolic engineering. Advanced techniques provide precise control over this key metabolic node:

Chromosomal modification approaches:

• I-SceI-mediated homologous recombination

  • Enables markerless, precise genomic modifications

  • Requires construction of suicide vectors carrying homology regions flanking sucC

  • Two-step process with counterselection using I-SceI double-strand breaks

  • Efficiency: ~60-80% success rate for sucC modifications

• CRISPR-Cas9 genome editing

  • Allows direct, scarless modification of the sucC locus

  • Design sgRNAs targeting sucC with appropriate PAM sites

  • Co-deliver repair template containing desired modifications

  • Enables multiplexed editing when modifying sucC alongside other TCA cycle genes

  • Efficiency: ~70-90% editing efficiency with optimized protocols

Expression tuning methodologies:

• Promoter engineering

  • Replace native sucC promoter with synthetic, tunable promoters

  • Options include:

    • Constitutive promoters of varying strengths (e.g., Ptac, PEM7)

    • Inducible systems (XylS/Pm, lacIq/Ptrc)

    • Environment-responsive promoters (stress-activated, oxygen-sensitive)

• Ribosome binding site (RBS) modification

  • Predictive algorithms (e.g., RBS Calculator) enable precise translation efficiency

  • Library-based approaches can generate expression range spanning 2-3 orders of magnitude

  • Combinatorial RBS-promoter libraries allow fine-tuned expression optimization

Advanced control systems:

• CRISPRi transcriptional repression

  • dCas9-based repression enables dynamic, reversible control of sucC expression

  • Titratable repression through inducible dCas9 expression

  • Multiple sgRNAs can provide graded control levels

  • Particularly useful for studying essential gene functions

• RNA-based expression control

  • Riboswitches responsive to metabolic intermediates

  • Small RNA regulators for post-transcriptional regulation

  • Theophylline-responsive riboswitches show excellent performance in P. putida

Implementation efficiency comparison:

When engineering sucC, researchers should consider potential metabolic bottlenecks created by altering TCA cycle flux. Complementary modifications to related pathways or substrate availability may be necessary to achieve desired phenotypes .

How can researchers investigate the role of P. putida sucC in metabolic flux redirection for biotechnology applications?

Investigating P. putida sucC's role in metabolic flux redirection requires sophisticated approaches that combine molecular manipulation with advanced analytical methods:

Experimental design framework:

• Targeted genetic modifications

  • Create precise sucC variants with altered kinetic properties

  • Generate controlled expression systems (tunable promoters, inducible systems)

  • Design sucC protein engineering for altered substrate specificity

  • Construct reporter fusions to monitor sucC expression dynamics

• Metabolic flux analysis methodologies

  • ¹³C-Metabolic Flux Analysis (¹³C-MFA)

    • Feed cultures with isotopically labeled substrates (e.g., [1-¹³C]glucose)

    • Measure isotopomer distributions in downstream metabolites

    • Apply computational models to quantify flux distributions

    • Compare wild-type vs. sucC-modified strains

  • Flux Balance Analysis (FBA)

    • Develop genome-scale metabolic models incorporating sucC parameters

    • Constrain models with experimental data (uptake/secretion rates)

    • Perform in silico predictions of flux redistributions

    • Validate predictions with experimental measurements

• Real-time metabolite monitoring

  • Implement biosensor systems for key metabolites

  • Apply metabolomics approaches (LC-MS/MS, GC-MS)

  • Monitor cofactor ratios (ATP/ADP, NADH/NAD⁺)

  • Track secreted metabolites as indicators of flux changes

Experimental protocol for comprehensive sucC flux analysis:

• Generate defined sucC variants with expression levels spanning 10-200% of wild-type

• Cultivate strains in minimal media with defined carbon sources

• Introduce isotopically labeled substrates at steady state

• Collect samples at defined time points for:

  • Intracellular metabolite analysis

  • Protein expression quantification

  • Transcript abundance measurement

• Analyze data through integrated computational modeling

Key metrics to evaluate sucC impact on flux distribution:

Advanced approaches for specific applications:

• For bioremediation applications:

  • Monitor aromatic compound degradation pathways

  • Measure flux through β-ketoadipate pathway

  • Analyze expression coordination between sucC and peripheral degradation pathways

• For bioproduction platforms:

  • Determine optimal sucC expression for precursor availability

  • Analyze competing pathway fluxes

  • Identify metabolic bottlenecks created by sucC modification

• For understanding stress responses:

  • Track flux redistributions under environmental stressors

  • Measure energetic efficiency during adaptation

  • Correlate sucC activity with survival under challenging conditions

This systematic approach enables researchers to precisely characterize how sucC modulation affects metabolic flux distribution, providing essential insights for rational strain engineering and optimization .

What are common experimental challenges when working with recombinant P. putida sucC and how can they be addressed?

Researchers frequently encounter several technical challenges when working with recombinant P. putida sucC. Here are the most common issues and evidence-based solutions:

Low soluble protein expression

Poor enzymatic activity

Technical challenges in activity assays

Protein stability issues

Practical implementation note: When encountering multiple issues simultaneously, adopt a systematic troubleshooting approach by addressing protein expression challenges first, followed by purification optimization, and finally activity assay refinement. Document conditions carefully to establish reproducible protocols for your specific construct .

How can researchers differentiate between native and recombinant P. putida sucC activity in experimental systems?

Distinguishing between native and recombinant P. putida sucC activity requires carefully designed experimental approaches that exploit subtle differences between these enzyme forms. The following methodologies provide robust differentiation strategies:

Molecular tagging approaches:

• Epitope tag-based discrimination

  • Engineer recombinant sucC with epitope tags (His, FLAG, Strep)

  • Implement tag-specific antibody detection in Western blots

  • Perform immunoprecipitation to selectively isolate recombinant enzyme

  • Quantify contribution using tag-specific ELISA formats

  • Sensitivity: Can detect recombinant protein at 0.01-0.1% of total protein

• Activity-based protein profiling

  • Design activity-based probes targeting active site residues

  • Incorporate click chemistry handles for selective labeling

  • Visualize via fluorescence or perform enrichment via affinity capture

  • Specificity: >95% selective labeling of active enzyme forms

Genetic differentiation methods:

• Codon-optimized variant expression

  • Express recombinant sucC with altered codon usage but identical amino acid sequence

  • Design primers to distinguish native vs. recombinant transcripts

  • Quantify relative expression using RT-qPCR

  • Resolution: Can detect 2-fold differences in expression levels

• Silent mutation markers

  • Introduce restriction sites via silent mutations in recombinant gene

  • Perform restriction fragment length polymorphism analysis

  • Design allele-specific PCR primers spanning mutation sites

  • Detection limit: 1-5% recombinant DNA in mixed populations

Biochemical discrimination strategies:

• Kinetic parameter differentiation

  • Engineer recombinant sucC with altered kinetic parameters

  • Compare Km and Vmax values under standardized conditions

  • Utilize substrate analogs with differential affinity

  • Discrimination power: Can distinguish enzymes with >20% difference in kinetic parameters

• Thermal stability profiling

  • Introduce stability-enhancing mutations in recombinant sucC

  • Perform thermal shift assays (Thermofluor)

  • Measure activity retention after controlled heat treatment

  • Resolution: Can separate variants with ΔTm of 2-3°C

Experimental protocol for comprehensive differentiation:

Data interpretation framework:

• Establish baseline native sucC activity in wild-type P. putida

• Characterize recombinant sucC activity in heterologous expression system

• In mixed systems, apply at least two orthogonal discrimination methods

• Quantify relative contributions using standard curves

• Validate with genetic knockout controls when possible

This systematic approach enables researchers to precisely attribute observed enzymatic activities to either native or recombinant sucC forms, essential for accurate interpretation of metabolic engineering experiments and functional studies .

What are the primary limitations of using sucC as a metabolic engineering target in Pseudomonas putida?

While sucC represents an attractive target for metabolic engineering in P. putida, researchers must recognize several inherent limitations that can impact experimental success and strain performance:

Metabolic network constraints:

• TCA cycle essentiality

  • SucC functions at a critical junction in central metabolism

  • Complete inactivation is typically lethal under aerobic conditions

  • Severe downregulation leads to growth impairment (>70% reduction causes >50% growth rate decrease)

  • Necessity for complex complementation strategies when targeting the native gene

• Metabolic rigidity

  • Strong homeostatic regulation buffers against expression changes

  • Compensatory mechanisms activate when flux is perturbed

  • Requires multi-target approaches for effective flux redirection

  • Limited success with single-gene modifications in isolation

Technical engineering challenges:

• Protein complex formation requirements

  • SucC requires association with SucD (α-subunit) for activity

  • Stoichiometric expression necessary for optimal function

  • Overexpression of single subunit can create imbalance effects

  • Simultaneous engineering of both subunits increases complexity

• Regulatory network interference

  • Modification affects interconnected signaling pathways

  • Unexpected phenotypes due to regulatory cross-talk

  • Stress response activation with non-physiological expression levels

  • Adaptation mechanisms eventually counteract engineered changes

Performance limitations in biotechnology applications:

Strain stability issues:

• Evolutionary instability

  • Strong selection pressure against non-optimal sucC expression

  • Genetic drift in continuous cultivation (mutation accumulation after ~50 generations)

  • Insertion sequence mobilization under stress conditions

  • Requirement for selection pressure maintenance

• Heterologous protein expression challenges

  • Codon usage bias affecting translation efficiency

  • Protein misfolding with high expression levels

  • Inclusion body formation at >5× native expression levels

  • Limited capacity of cellular quality control systems

Practical research recommendations:

• Implement inducible rather than constitutive expression systems

• Consider dynamic regulatory circuits responsive to metabolic state

• Engineer entire sucCD operon rather than sucC alone

• Validate strain stability through extended cultivation studies

• Apply adaptive laboratory evolution to improve strain fitness

• Develop synthetic consortia approaches to distribute metabolic burden

Researchers can maximize success by acknowledging these limitations early in experimental design, implementing appropriate controls, and developing strategies that work with rather than against the intrinsic metabolic architecture of P. putida .

How might CRISPR-Cas9 techniques be optimized for studying sucC function in Pseudomonas putida strains?

CRISPR-Cas9 technologies offer unprecedented opportunities for precise genetic manipulation of sucC in P. putida. Optimization strategies should address species-specific challenges while leveraging cutting-edge advances in genome editing:

CRISPR-Cas9 delivery optimization:

• Improved vector systems

  • Develop specialized vectors with P. putida-optimized promoters for Cas9 expression

  • Implement temperature-sensitive replicons for transient Cas9 expression

  • Design broad-host-range vectors compatible with diverse P. putida strains

  • Incorporate toxin-antitoxin systems for stable maintenance without selection

• Delivery mechanisms

  • Electroporation protocols optimized for P. putida competence

  • Conjugation-based approaches using specialized donor strains

  • Transient expression systems to minimize Cas9 toxicity

  • Phage-based delivery for difficult-to-transform strains

sgRNA design and implementation:

• P. putida-specific sgRNA optimization

  • Analyze PAM site distribution in sucC locus across P. putida strains

  • Develop machine learning algorithms trained on P. putida editing outcomes

  • Optimize guide length (20-24 nucleotides) for P. putida specificity

  • Incorporate chemical modifications to enhance stability

• Multi-guide approaches

  • Simultaneous targeting of both sucC and sucD for operon engineering

  • Multiplexed editing of sucC with related TCA cycle genes

  • Combinatorial guide libraries for functional domain mapping

  • Sequential editing approaches for complex modifications

Advanced editing strategies:

• Base editing without double-strand breaks

  • Implement cytidine and adenine base editors for point mutations

  • Create sucC variants with altered catalytic properties

  • Engineer codon changes without template DNA requirements

  • Reduce off-target effects associated with Cas9 cleavage

• Prime editing adaptation

  • Develop pegRNA designs optimized for P. putida

  • Enable precise insertions, deletions, and substitutions

  • Create seamless modifications without selection markers

  • Improve editing efficiency through Pseudomonas-specific optimizations

Performance comparison of CRISPR-Cas9 variants in P. putida:

Implementation protocols for key applications:

• For sucC knockout studies:

  • Design sgRNAs targeting early coding sequence

  • Include homology arms (500-1000 bp) for marker insertion

  • Screen using phenotypic and PCR verification

  • Expected efficiency: 40-60% with proper optimization

• For precise mutations:

  • Implement cytosine base editor for catalytic site modifications

  • Target conserved residues identified in homology models

  • Screen using activity-based assays

  • Expected efficiency: 20-35% with optimized protocols

• For expression modulation:

  • Deploy CRISPRi with sgRNAs targeting promoter region

  • Implement inducible dCas9 expression

  • Monitor using RT-qPCR and activity assays

  • Expected repression: 70-95% depending on guide design

• For tagging applications:

  • Design repair templates with fluorescent or affinity tags

  • Include silent mutations to prevent re-cutting

  • Screen using fluorescence or Western blotting

  • Expected efficiency: 20-40% with selection

These optimized CRISPR-Cas9 approaches enable unprecedented precision in studying sucC function, creating opportunities to explore subtle aspects of enzyme function and metabolic integration with minimal disruption to cellular physiology .

What are the emerging applications of P. putida sucC in synthetic biology and metabolic engineering?

Pseudomonas putida sucC is emerging as a versatile target in synthetic biology and metabolic engineering, with applications extending beyond traditional metabolic role modifications. Current research reveals several innovative directions:

Biosensor development and metabolic monitoring:

• Succinyl-CoA sensing systems

  • Engineer sucC-based protein switches responsive to succinyl-CoA levels

  • Develop fluorescent reporters coupled to sucC promoter activity

  • Create split-protein complementation assays using sucC domains

  • Applications in high-throughput screening for metabolic engineering

• TCA cycle flux indicators

  • Utilize sucC as component of metabolic state biosensors

  • Implement real-time monitoring of central carbon metabolism

  • Develop feedback-controlled expression systems tied to TCA cycle activity

  • Enable dynamic regulation of engineered pathways

Novel bioproduction platforms:

• Precursor overproduction systems

  • Engineering sucC for enhanced dicarboxylic acid production

  • Modulating ATP/ADP ratios through controlled sucC expression

  • Creating push-pull strategies with sucC as central control point

  • Targeted production of succinyl-CoA-derived compounds

• Biopolymer synthesis applications

  • Utilizing sucC in polyhydroxyalkanoate (PHA) production pathways

  • Engineering novel polymers with succinyl-CoA-derived monomers

  • Developing dynamic monomer supply control through sucC regulation

  • Creating biodegradable plastics with tunable properties

Emerging synthetic biology frameworks:

• Orthogonal metabolism construction

  • Implementing non-native sucC variants in synthetic pathways

  • Creating metabolic isolation through compartmentalization

  • Developing specialized sucC variants with altered cofactor specificity

  • Establishing metabolic circuits with minimal cross-talk

• Metabolic toggle switches

  • Designing bistable genetic circuits controlling sucC expression

  • Creating switchable metabolic states for fermentation processes

  • Implementing digital-like metabolic responses to environmental conditions

  • Enabling programmed transitions between growth and production phases

Technological application potential:

Future integration possibilities:

• Multi-enzyme scaffolding systems

  • Co-localization of sucC with pathway partners on synthetic scaffolds

  • Creation of metabolic microcompartments for enhanced pathway efficiency

  • Synthetic enzyme assemblies mimicking natural metabolons

  • Expected efficiency improvement: 2-5 fold increased pathway flux

• Genome-minimized cell factories

  • Integration of optimized sucC variants into reduced-genome P. putida

  • Creation of specialized production strains with streamlined metabolism

  • Elimination of competing pathways to maximize precursor availability

  • Potential for 30-50% improvement in product yields

• Computer-designed sucC variants

  • Implementation of non-natural enzyme properties through computational design

  • Altered substrate specificity for novel product formation

  • Optimized catalytic parameters for specific applications

  • Integration with machine learning for iterative improvement

These emerging applications demonstrate how sucC is evolving from a simple metabolic enzyme target to a sophisticated component in designer biological systems with diverse technological applications .

What are the most significant recent advances in understanding P. putida sucC function and how might they impact future research?

The study of P. putida sucC has advanced significantly in recent years, revealing nuanced aspects of its function beyond the canonical role in the TCA cycle. These insights are reshaping our understanding of bacterial metabolism and opening new research avenues:

Key scientific breakthroughs:

• Metabolic integration complexity

  • Recognition of sucC as a metabolic hub connecting carbon, nitrogen, and sulfur metabolism

  • Identification of non-canonical substrates and reactions catalyzed under specific conditions

  • Discovery of unexpected protein-protein interactions influencing sucC regulation

  • Demonstration of sucC's role in metabolic adaptation to environmental stressors

• Regulatory network elucidation

  • Characterization of transcriptional and post-translational control mechanisms

  • Identification of small RNA regulators modulating sucC expression

  • Discovery of allosteric regulation by unexpected metabolites

  • Understanding of sucC's role in global metabolic rewiring during adaptation

• Structural and functional insights

  • High-resolution structures revealing species-specific catalytic mechanisms

  • Identification of critical residues governing substrate specificity

  • Elucidation of protein dynamics during catalytic cycle

  • Understanding subunit interaction interface with sucD and other proteins

Technological and methodological advances:

• Engineering toolkit expansion

  • Development of CRISPR-Cas9 systems optimized for P. putida

  • Creation of specific genetic tools for precise sucC modification

  • Standardization of expression systems for controlled sucC studies

  • Implementation of high-throughput phenotyping methods for sucC variants

• Analytical capabilities

  • Advanced metabolomics approaches for tracking sucC-dependent metabolite changes

  • Improved protein interaction mapping techniques

  • Development of activity-based probes for in vivo enzyme monitoring

  • Implementation of biophysical methods for studying conformational dynamics

Research trajectory and future impacts:

Implications for future research paradigms:

• Shift toward multi-omics approaches

  • Integration of transcriptomics, proteomics, and metabolomics for comprehensive sucC function analysis

  • Temporal studies capturing dynamic regulation during environmental transitions

  • Single-cell approaches revealing population heterogeneity in sucC expression

  • Expected outcome: Holistic understanding of sucC's role in cellular physiology

• Expanded engineering horizons

  • Creation of synthetic metabolic modules centered on redesigned sucC

  • Development of novel bioproduction pathways leveraging sucC-dependent reactions

  • Implementation of dynamic feedback control systems for metabolic optimization

  • Expected outcome: Precisely tunable metabolism for biotechnological applications

• Interdisciplinary collaboration acceleration

  • Increased integration of computational biology with experimental approaches

  • Collaboration between biochemists, synthetic biologists, and bioprocess engineers

  • Application of machine learning for predicting optimal sucC variants

  • Expected outcome: Accelerated translation of fundamental knowledge to applications