Recombinant Escherichia coli O8 Succinyl-CoA ligase [ADP-forming] subunit beta (sucC)

What is Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) and what is its role in E. coli metabolism?

Succinyl-CoA ligase [ADP-forming] subunit beta, encoded by the sucC gene, is a critical enzyme in the tricarboxylic acid (TCA) cycle of E. coli. This enzyme catalyzes the reversible conversion of succinyl-CoA to succinate, coupled with substrate-level phosphorylation that generates ATP. The reaction is particularly important as it represents one of the key energy-conserving steps in the TCA cycle.

In wild-type E. coli, sucC works in conjunction with the alpha subunit (encoded by sucD) to form the functional Succinyl-CoA synthetase complex. This enzyme serves as a critical junction in central metabolism, connecting the TCA cycle with various anabolic and catabolic pathways. When researchers target succinate production in E. coli, the sucC gene and its expression become particularly important focal points for metabolic engineering strategies .

How does sucC expression affect succinate production in recombinant E. coli strains?

As demonstrated in metabolic engineering studies, the manipulation of genes encoding TCA cycle enzymes, including those related to sucC function, can dramatically alter succinate production. For instance, when researchers deleted genes like sdhAB (encoding succinate dehydrogenase) and manipulated regulatory genes like iclR, succinate production increased from undetectable levels in wild-type strains to 4.62 mM in engineered strains . This represents a fundamental redirect of carbon flux toward succinate accumulation.

What genetic modifications complement sucC function to optimize succinate production?

Optimizing succinate production requires a systems biology approach that considers multiple pathway intersections. Research has shown that several complementary genetic modifications enhance sucC-related pathways:

• Deletion of sdhAB (encoding succinate dehydrogenase) prevents succinate conversion to fumarate, creating a metabolic block that allows succinate accumulation .

• Deletion of iclR (encoding the isocitrate lyase regulator) releases repression of the aceBAK operon, enhancing the glyoxylate shunt pathway .

• Deletion of maeB (encoding malic enzyme) prevents carbon flux diversion into gluconeogenesis .

• Overexpression of genes encoding upstream TCA cycle enzymes, such as citrate synthase (gltA) and aconitate hydratase (acnB), increases carbon flux toward succinate production .

These strategic modifications collectively redirect carbon metabolism to leverage sucC function for enhanced succinate production, as evidenced in the WCY-7 strain that accumulated 11.23 mM succinate from 50 mM sodium acetate .

What are the optimal conditions for expressing recombinant sucC in E. coli?

The optimal expression of recombinant sucC in E. coli requires careful consideration of several factors to ensure high yields of soluble, active protein:

Vector Selection: Vectors with moderate copy numbers often perform better than high-copy plasmids when expressing TCA cycle enzymes like sucC. This minimizes metabolic burden and potential toxicity. Research indicates that while high-copy plasmids like pTrc99a may enable faster initial growth, they can create substantial metabolic burden. Comparatively, moderate-copy vectors like pCL1920 may result in slower initial growth but better final cell density and protein yields .

Temperature Optimization: Lower induction temperatures (16-25°C) typically enhance the solubility of recombinant proteins in E. coli. This slows protein synthesis, allowing more time for proper folding and reducing inclusion body formation .

Induction Strategy: Gradual induction with lower IPTG concentrations (0.1-0.5 mM) often yields better results than strong induction, particularly for metabolic enzymes like sucC .

Host Strain Selection: BL21(DE3) derivatives often provide good expression platforms for sucC. The absence of certain proteases and the DE3 lysogen carrying the T7 RNA polymerase gene make these strains particularly suitable .

How can I improve the solubility of recombinant sucC when expression results in inclusion bodies?

Inclusion body formation is a common challenge when expressing recombinant proteins in E. coli. For sucC, several approaches have proven effective:

Co-expression with Chaperones: Chaperone proteins such as GroEL/GroES, DnaK/DnaJ/GrpE, or trigger factor can significantly enhance proper protein folding. These molecular chaperones assist in preventing protein aggregation during synthesis and folding .

Fusion Tag Strategies: N-terminal fusion partners like MBP (maltose-binding protein), GST (glutathione S-transferase), or SUMO (small ubiquitin-like modifier) can dramatically increase sucC solubility. These fusion partners often act as solubility enhancers and can be removed post-purification with specific proteases .

Design Considerations: Remove or modify problematic regions in the protein sequence. For sucC, careful analysis of low complexity regions (LCRs) and hydrophobic patches, particularly at the N- and C-termini, can guide construct design to enhance solubility .

Chemical Additives: Including compounds like sorbitol, glycerol (5-10%), or mild detergents in the growth medium can help stabilize folding intermediates and improve solubility.

What purification approach yields the highest activity for recombinant sucC?

A multi-step purification strategy typically yields the highest activity for recombinant sucC:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using a His-tag is often the first choice, providing good initial purification while maintaining enzyme activity.

• Intermediate Purification: Ion exchange chromatography (IEX) separates sucC from similarly sized contaminants based on charge differences.

• Polishing Step: Size exclusion chromatography (SEC) ensures isolation of properly assembled sucC complexes (particularly important since functional sucC requires both alpha and beta subunits).

• Buffer Optimization: The final enzyme preparation should be stored in buffers containing 20-50 mM phosphate or Tris (pH 7.5-8.0), 100-150 mM NaCl, 1-5 mM MgCl₂ (essential for activity), and 1-5 mM DTT to maintain reduced cysteines.

Throughout purification, it's critical to monitor enzymatic activity, as some high-yield methods may compromise function. Researchers should perform activity assays after each purification step to ensure the final preparation retains catalytic efficiency.

What are the established methods for measuring Succinyl-CoA ligase enzymatic activity?

Several reliable methodologies exist for measuring Succinyl-CoA ligase activity:

Spectrophotometric Coupled Assay: This approach monitors ADP formation by coupling the reaction to pyruvate kinase and lactate dehydrogenase, measuring NADH oxidation at 340 nm. The reaction mixture typically contains:

• 50 mM HEPES buffer (pH 7.5)

• 10 mM MgCl₂

• 0.1 mM CoA

• 0.2 mM ATP

• 10 mM succinate

• 0.2 mM phosphoenolpyruvate

• 0.2 mM NADH

• 2 units pyruvate kinase

• 2 units lactate dehydrogenase

Radioactive Assay: Using ¹⁴C-labeled succinate or ATP provides highly sensitive measurement of activity, particularly useful for kinetic studies.

Direct Product Quantification: HPLC or LC-MS based quantification of succinate formation offers a direct measurement without coupling enzymes, eliminating potential interference.

When interpreting activity data, researchers should consider that substrate concentrations significantly impact measured activities. The enzyme shows typical Michaelis-Menten kinetics with respect to succinate, CoA, and ATP, with Km values in the submillimolar range.

How does sucC interact with other components of the TCA cycle in E. coli?

The interaction of sucC with other TCA cycle components involves both enzymatic connections and regulatory relationships:

Enzymatic Pathway Integration: sucC functions directly downstream of α-ketoglutarate dehydrogenase (converting α-ketoglutarate to succinyl-CoA) and upstream of succinate dehydrogenase (converting succinate to fumarate). The reaction catalyzed by sucC is reversible, allowing flux in both directions depending on metabolic conditions.

Protein-Protein Interactions: Beyond its catalytic role, sucC has been shown to participate in metabolic enzyme complexes. These physical associations create microenvironments that enhance substrate channeling and reaction efficiency within the TCA cycle.

In metabolic engineering applications targeting succinate production, the disruption of succinate dehydrogenase (deleting sdhAB) creates a metabolic block that allows succinate to accumulate rather than continuing through the TCA cycle. This strategic interruption, combined with enhancements to upstream flux through overexpression of citrate synthase (gltA) and aconitate hydratase (acnB), has been shown to significantly increase succinate production .

What factors affect sucC function in experimental settings?

Several critical factors influence sucC function in experimental contexts:

Divalent Cation Requirements: Mg²⁺ is essential for sucC activity, with optimal concentration typically between 5-10 mM. Mn²⁺ can substitute but usually results in lower activity.

pH Sensitivity: The enzyme shows a bell-shaped pH-activity profile with optimum activity between pH 7.2-7.8. Activity decreases significantly below pH 6.5 or above pH 8.5.

Substrate Inhibition: High concentrations of succinyl-CoA (>0.5 mM) can inhibit enzyme activity through negative feedback mechanisms.

Oxidation Sensitivity: The enzyme contains cysteine residues susceptible to oxidation, which can significantly impair activity. Including reducing agents like DTT or β-mercaptoethanol in reaction buffers is recommended.

Temperature Effects: While the temperature optimum for activity is around 37°C, stability studies show that prolonged exposure to temperatures above 30°C can lead to activity loss if stabilizing agents are not present.

In recombinant expression systems, the ratio of alpha (sucD) to beta (sucC) subunits is critical for proper complex formation and maximal activity. Imbalanced expression can result in partial complexes with reduced activity.

How can sucC be engineered for improved succinate production?

Engineering sucC for enhanced succinate production involves several sophisticated approaches:

Protein Engineering Strategies:

• Site-directed mutagenesis to enhance catalytic efficiency by modifying key residues in the active site

• Directed evolution approaches to select for variants with improved kinetic properties

• Fusion of protein domains that enhance stability or substrate channeling

Metabolic Context Optimization:
When engineering sucC, researchers must consider the broader metabolic context. Research has demonstrated that a systems approach yields the best results. For example, the WCY-7 strain developed by researchers integrated several genetic modifications:

This integrated approach resulted in 11.23 mM succinate production from 50 mM sodium acetate after 48 hours of batch fermentation , representing a significant advancement in metabolic engineering for succinate production.

What are the current metabolic engineering strategies involving sucC in E. coli?

Current metabolic engineering strategies involving sucC focus on systems-level approaches for maximizing succinate production:

Pathway Balancing: Modern approaches optimize the expression levels of multiple enzymes simultaneously to balance carbon flux. This prevents bottlenecks and metabolite accumulation that could inhibit growth or production.

Regulatory Network Manipulation: Beyond direct pathway enzymes, manipulation of global regulators affects multiple pathways simultaneously. For instance, deletion of the isocitrate lyase regulator (iclR) enhances the glyoxylate shunt pathway, providing an alternative route for succinate formation .

Alternative Carbon Source Utilization: Recent research demonstrates that engineered E. coli strains can produce succinate from non-traditional carbon sources like acetate. The WCY-7 strain achieved 11.23 mM succinate production from acetate through strategic pathway optimization . This represents an important advancement in utilizing low-cost or waste carbon sources.

Growth-Production Decoupling: Advanced strategies include separating the growth phase from the production phase, often through inducible promoters or by engineering strains that naturally shift metabolism between these phases.

How do mutations in sucC affect E. coli metabolism and what can they teach us about TCA cycle regulation?

Mutations in sucC provide valuable insights into TCA cycle regulation and central metabolism:

Flux Distribution Effects: Mutations that alter sucC kinetic properties cause ripple effects throughout central metabolism. Studies of such mutations reveal how cells redistribute carbon flux in response to TCA cycle perturbations.

Regulatory Network Responses: When sucC function is compromised, cells activate compensatory regulatory mechanisms. Analyzing these responses has identified previously unknown regulatory connections between the TCA cycle and other metabolic pathways.

Metabolic Resilience Mechanisms: Some sucC mutations are lethal while others are tolerated through alternative metabolic routes. The pattern of viability helps map the essential versus dispensable aspects of TCA cycle function.

Evolutionary Conservation: Comparative studies of sucC variants across bacterial species reveal highly conserved regions critical for function versus regions that tolerate substitutions. This evolutionary perspective helps predict which modifications might be successfully engineered.

Energy Homeostasis Insights: Since sucC catalyzes a substrate-level phosphorylation step generating ATP, mutations affecting this function provide insights into how cells maintain energy homeostasis when key ATP-generating reactions are compromised.

By systematically studying sucC mutations and their effects, researchers gain deeper understanding of metabolic network properties that inform rational strain design for biotechnological applications.

What experimental controls are essential when studying recombinant sucC function?

Rigorous experimental controls are critical for meaningful research on recombinant sucC:

Enzyme Activity Controls:

• Positive Control: Commercial succinyl-CoA ligase or well-characterized laboratory preparation

• Negative Control: Heat-inactivated enzyme preparation

• Background Control: Reaction mixture lacking one essential substrate

• Host Background Control: Extract from E. coli containing empty vector to account for endogenous activity

Expression System Controls:

• Expression Vector Control: Cells transformed with the same vector lacking the sucC insert

• Induction Control: Uninduced cultures containing the sucC expression construct

• Toxicity Control: Growth curve comparison between sucC-expressing and control strains

Purification Controls:

• Column Matrix Control: Passing extract from non-expressing cells through the purification protocol

• Tag-Only Control: Expression and purification of the tag portion alone (without sucC)

When manipulating sucC in the context of metabolic engineering, additional controls should include the parent strain without any modifications and strains with individual genetic modifications to isolate the effect of each change. This systematic approach enables researchers to attribute phenotypic changes specifically to sucC-related modifications .

What are common challenges in sucC research and how can they be addressed?

Researchers working with sucC frequently encounter several challenges:

Expression Problems:

• Challenge: Low expression levels or inclusion body formation

• Solution: Optimize codon usage for E. coli, adjust induction conditions (lower temperature, reduced inducer concentration), co-express with chaperones, or use solubility-enhancing fusion tags

Activity Loss During Purification:

• Challenge: Significant reduction in specific activity after purification steps

• Solution: Include stabilizing agents (glycerol, reducing agents), minimize purification steps, and maintain appropriate divalent cation concentrations throughout purification

Subunit Association Issues:

• Challenge: Imbalanced expression of alpha and beta subunits affecting complex formation

• Solution: Use bicistronic expression constructs to ensure proper stoichiometry, or co-purify using tags on different subunits

Metabolic Burden in Engineered Strains:

• Challenge: Growth defects in strains engineered for increased succinate production

• Solution: Use moderate-copy plasmids instead of high-copy vectors to reduce metabolic burden, as demonstrated in comparative studies of the WCY-6 and WCY-7 strains

Competing Metabolic Pathways:

• Challenge: Carbon flux diverted away from succinate production

• Solution: Implement multiple genetic modifications to block competing pathways, as demonstrated in the systematic development of succinate-producing strains

How should experiments be designed to study sucC function in vivo?

Effective in vivo studies of sucC function require careful experimental design:

Genetic Approach:

• Create clean deletion mutants using scarless methods to avoid polar effects on adjacent genes

• Complement with controlled expression constructs to verify phenotypes are specifically due to sucC

• Use chromosomal integration for stable expression rather than plasmid-based systems when possible

Physiological Characterization:

• Monitor growth parameters under various carbon sources (glucose, acetate, succinate)

• Measure intracellular metabolite concentrations using metabolomics approaches

• Track flux distributions using 13C-labeled substrates and metabolic flux analysis

Regulatory Studies:

• Use reporter fusions (lacZ, gfp) to monitor sucC promoter activity under various conditions

• Employ ChIP-seq to identify transcription factors binding to the sucC promoter region

• Integrate transcriptomics and proteomics to understand system-wide effects of sucC manipulation

Stress Response Analysis:

• Examine sucC function under various stress conditions (oxidative stress, acid stress)

• Compare wild-type and mutant strains for stress tolerance phenotypes

• Research suggests connections between metabolic enzymes and stress responses, as seen with ligase B in E. coli, which appears to mitigate damage from oxidative stress

When studying sucC in the context of succinate production, implementing a systematic approach that progressively builds on successful modifications has proven effective. For example, researchers developed the WCY-7 strain through sequential genetic modifications, each evaluated for its contribution to succinate production .

What emerging technologies might advance sucC research and applications?

Several cutting-edge technologies show promise for advancing sucC research:

CRISPR-Cas9 Applications: Precise genome editing enables multi-locus modifications without marker genes, facilitating complex metabolic engineering strategies involving sucC and related pathways.

Cell-Free Systems: Studying sucC function in reconstituted cell-free systems allows precise control over reaction conditions and eliminates cellular complexity that can confound results.

Synthetic Biology Approaches: Modular design principles and standardized parts for controlling sucC expression offer new possibilities for rational engineering of metabolism.

Systems Biology Integration: Multi-omics data integration (combining transcriptomics, proteomics, and metabolomics) provides comprehensive understanding of how sucC modifications affect the entire metabolic network.

Computational Modeling: Genome-scale metabolic models with enhanced kinetic parameters enable more accurate prediction of how sucC modifications will affect metabolism, guiding more efficient experimental design.

Protein Structure Prediction: Advanced tools like AlphaFold provide detailed structural predictions that can guide rational engineering of sucC for enhanced catalytic properties or stability.

How might sucC research contribute to understanding fundamental aspects of bacterial metabolism?

Research on sucC offers insights into several fundamental aspects of bacterial metabolism:

Metabolic Flexibility: Studies of sucC regulation help explain how bacteria rapidly adapt their central metabolism to changing environmental conditions.

Energy Conservation Strategies: As a key enzyme in substrate-level phosphorylation, sucC research illuminates how bacteria balance their energy budget under different growth conditions.

Evolutionary Conservation: Comparative studies of sucC across species reveal evolutionary constraints on central metabolism and identify core metabolic functions versus specialized adaptations.

Metabolic Network Robustness: Analysis of how cells compensate for altered sucC function reveals redundancy and regulatory mechanisms that ensure metabolic homeostasis.

Growth-Metabolism Relationships: The connection between sucC function and growth rate helps elucidate how bacteria coordinate metabolism with cell division and biomass production.

This research not only advances our fundamental understanding but also provides the knowledge foundation for applications in metabolic engineering, synthetic biology, and biotechnology.

How should researchers address data inconsistencies in sucC enzymatic assays?

When faced with data inconsistencies in sucC enzymatic assays, researchers should implement the following methodological approaches:

Systematic Troubleshooting Protocol:

• Verify enzyme stability by testing freshly prepared samples versus stored preparations

• Check for interfering compounds in the reaction mixture through control experiments

• Determine sensitivity to reaction conditions by systematically varying pH, temperature, and ionic strength

• Evaluate potential substrate competition or inhibition effects by varying substrate concentrations

Statistical Analysis Recommendations:

• Apply appropriate statistical tests (ANOVA, t-tests) to determine if differences are statistically significant

• Use multiple technical and biological replicates (minimum n=3 for each)

• Implement outlier analysis but apply conservative criteria for data exclusion

Reproducibility Enhancement:

• Standardize enzyme preparation methods across experiments

• Document detailed protocols including specific lot numbers of reagents

• Implement internal standards when possible

• Cross-validate results using complementary assay methods

When interpreting enzymatic data, researchers should consider that natural variations in enzyme activity can occur due to subtle differences in protein folding, post-translational modifications, or experimental conditions. Rigorous controls and standardization are essential for meaningful comparisons across experiments.

What approaches help differentiate between direct effects of sucC modifications and indirect metabolic consequences?

Distinguishing direct from indirect effects requires sophisticated experimental approaches:

Temporal Analysis: Monitor metabolic changes immediately following induced expression or inhibition of sucC to separate primary from secondary effects.

Isolated System Studies: Reconstitute sucC function in defined in vitro systems to observe direct effects without cellular complexity.

Metabolic Flux Analysis: Use 13C-labeled substrates to track carbon flow through pathways, identifying where flux patterns change following sucC modification.

Complementary Enzyme Manipulations: Compare metabolic effects of sucC modifications with manipulations of enzymes catalyzing adjacent reactions to identify pathway-specific versus enzyme-specific effects.

Targeted Metabolomics: Focus on immediate substrate/product pools (succinyl-CoA, succinate, CoA, ATP/ADP) to identify primary metabolic responses before compensatory mechanisms activate.