Recombinant Escherichia coli O17:K52:H18 Succinyl-CoA ligase [ADP-forming] subunit beta (sucC)

What is the role of the sucC gene in E. coli metabolism?

The sucC gene encodes the beta subunit of Succinyl-CoA ligase [ADP-forming] (EC 6.2.1.5), a critical enzyme in the tricarboxylic acid (TCA) cycle. This enzyme catalyzes the reversible conversion of succinyl-CoA to succinate coupled with the synthesis of ATP from ADP and inorganic phosphate . The reaction is represented as:
ATP + succinate + CoA ⟷ ADP + phosphate + succinyl-CoA

This enzyme participates in multiple metabolic pathways beyond the TCA cycle, including propanoate metabolism, C5-branched dibasic acid metabolism, and the reductive carboxylate cycle . The sucC gene product forms part of a heterodimeric complex with the alpha subunit (encoded by sucD), creating the functional enzyme that plays a central role in cellular energy production.

What are the advantages of using E. coli for expressing recombinant Succinyl-CoA ligase?

E. coli offers numerous advantages as an expression system for Succinyl-CoA ligase:

• Simpler and more cost-effective than alternative systems like mammalian cell culture

• Rapid growth rate and high cell density in simple growth media

• Well-established genetic manipulation techniques

• Straightforward fermentation processes

• Virus-free product production

• High product yields and cost-effective production

• Extensive toolkit of expression vectors with various promoters and fusion tags

• Availability of specialized host strains optimized for protein expression

These advantages make E. coli particularly suitable for recombinant protein production in both academic and industrial settings, despite some limitations that can often be addressed through optimization strategies .

What E. coli strains are recommended for optimal expression of recombinant sucC?

Several E. coli strains have been used successfully for recombinant protein expression, each with specific advantages:

• E. coli MG1655: A well-characterized K-12 strain used as a base for constructing succinate-producing strains

• E. coli DH5α: Commonly used for plasmid construction and maintenance

• BL21(DE3) and derivatives: Preferred for high-level expression due to T7 RNA polymerase system and reduced protease activity

• Rosetta or CodonPlus strains: Useful if the sucC sequence contains rare codons

• C41/C43 strains: Derived from BL21, better suited for expressing potentially toxic proteins

• Arctic Express: Engineered to co-express cold-adapted chaperones for improved protein folding at lower temperatures

For metabolic engineering applications focusing on succinate production, specialized strains have been developed through genetic modifications. For example, strain WCY-7 was created by deleting sdhAB (encoding succinate dehydrogenase), iclR (encoding the isocitrate lyase regulator), and maeB (encoding malic enzyme) in E. coli MG1655 .

What vectors are recommended for sucC gene expression in E. coli?

The choice of expression vector significantly impacts recombinant protein yield and quality. Recommended vectors include:

• pTrc99a: Used successfully for expressing TCA cycle enzymes including acetyl-CoA synthetase (acs)

• pCL1920: A low-copy number plasmid suitable for moderate expression levels

• pET series vectors: Offer strong T7 promoter-driven expression

• pBAD vectors: Provide arabinose-inducible expression with fine-tuned control

• Fusion tag vectors: Such as pGEX (GST tag) and pMAL (MBP tag) that can enhance solubility

When selecting a vector, consider the promoter strength, copy number, selection markers, and fusion tags based on your specific experimental requirements. For metabolic engineering applications like succinate production, combinations of genes (acs, gltA, acnB) have been successfully expressed from plasmids like pW-4, constructed from pTrc99a .

How can I verify successful expression of recombinant Succinyl-CoA ligase?

Verification of recombinant Succinyl-CoA ligase expression can be accomplished through multiple complementary approaches:

• SDS-PAGE analysis to visualize protein bands at the expected molecular weight

• Western blotting using antibodies against Succinyl-CoA ligase or fusion tags

• Enzymatic activity assays measuring:

  • ADP formation using coupled enzyme systems

  • Consumption of substrates (succinate, ATP, CoA)

  • Production of products (succinyl-CoA, ADP, phosphate)

• Mass spectrometry to confirm protein identity and integrity

• N-terminal sequencing to verify correct processing

• FACS analysis if using GFP or other fluorescent fusion proteins

For quantitative assessment of expression levels, include appropriate controls and standards. Novel approaches like the FACS-based selection method described by researchers can help identify high-expressing constructs, where GFP is fused to the C-terminus of the protein of interest to enable fluorescence-based screening .

How can I optimize codon usage for maximum expression of sucC in E. coli O17:K52:H18?

Codon optimization is crucial for maximizing expression of recombinant proteins. For sucC in E. coli O17:K52:H18, consider the following strategies:

• Analyze the codon usage bias of the host strain and adapt the sucC sequence accordingly

• Pay special attention to the nucleotides immediately following the start codon, as they significantly impact translation efficiency

• Implement the directed evolution-based approach using FACS to screen N-terminal coding sequences

• Avoid rare codons (especially in clusters) that might cause ribosomal stalling

• Optimize the 5' mRNA region to minimize secondary structures that could impede translation initiation

• Consider both natural codons (CN) and optimized codons (CO) approaches

• Design libraries of N-terminal variants for screening if maximum expression is critical

Research has demonstrated that modifying the N-terminal sequences through directed evolution and FACS-based selection can increase protein production yields by over 30-fold . This approach is particularly valuable when rational design methods fail to provide satisfactory expression levels.

What metabolic engineering applications exist for modified sucC expression?

Modified expression of sucC offers several applications in metabolic engineering:

• Enhanced production of TCA cycle intermediates:

  • Succinate production from acetate (demonstrated with strain WCY-7, achieving 11.23 mM succinate from 50 mM sodium acetate)

  • Fumarate, malate, and other organic acids

• Design of synthetic metabolic pathways:

  • Integration with existing pathways for novel compound production

  • Creation of alternative carbon utilization routes

• Optimization of cellular energetics:

  • Improved ATP generation efficiency

  • Enhanced biomass production

• Development of strains for bioconversion:

  • Utilization of alternative carbon sources

  • Valorization of waste streams

The engineering approach often requires complementary modifications. For example, the succinate-producing strain WCY-7 combined deletion of sdhAB, iclR, and maeB with overexpression of acs, gltA, and acnB to redirect carbon flux toward succinate production .

How does deletion or modification of sucC affect central carbon metabolism in E. coli?

Deleting or modifying sucC creates significant metabolic effects throughout central carbon metabolism:

• Disruption of the TCA cycle at the succinyl-CoA to succinate conversion step

• Potential accumulation of succinyl-CoA and upstream metabolites

• Activation of alternative pathways to compensate for the metabolic block:

  • Increased flux through the glyoxylate bypass

  • Altered anaplerotic reactions

• Changes in redox balance (NADH/NAD+ ratio)

• Impacts on related pathways:

  • Amino acid biosynthesis (particularly those derived from TCA intermediates)

  • Porphyrin biosynthesis (uses succinyl-CoA)

  • Membrane lipid composition

These effects are similar to those seen with sdhAB deletion, which blocks the succinate to fumarate conversion step. In metabolic engineering applications, deletion of sdhAB in combination with other modifications has been used to accumulate succinate by preventing its further oxidation in the TCA cycle . Similar principles could apply to sucC modification, potentially creating novel metabolic states useful for biotechnological applications.

What approaches can be used to study protein-protein interactions involving Succinyl-CoA ligase?

Several complementary approaches can elucidate protein-protein interactions involving Succinyl-CoA ligase:

• Co-immunoprecipitation (Co-IP):

  • Use antibodies against sucC or fusion tags to pull down protein complexes

  • Identify interacting partners via mass spectrometry

• Affinity purification coupled with mass spectrometry:

  • Express tagged sucC in E. coli

  • Purify under native conditions to maintain interactions

  • Identify co-purifying proteins

• Bacterial two-hybrid systems:

  • Specifically designed for bacterial proteins

  • Can detect direct binary interactions

• Crosslinking studies:

  • Chemical or photo-crosslinking to capture transient interactions

  • MS/MS analysis to identify crosslinked peptides

• Surface plasmon resonance (SPR):

  • Quantitative measurement of binding kinetics

  • Determination of affinity constants

• Structural studies:

  • X-ray crystallography of protein complexes

  • Cryo-EM for larger assemblies

These methods can reveal interactions with other TCA cycle enzymes, regulatory proteins, or previously unidentified protein partners, providing insights into the functional organization of metabolic pathways in E. coli.

How does the E. coli O17:K52:H18 strain differ from other E. coli strains for recombinant protein expression?

The E. coli O17:K52:H18 strain belongs to clonal group A, which has been identified in various extraintestinal infections . Some key differences relevant to recombinant protein expression include:

• Genomic profile:

  • Contains specific virulence genes characteristic of extraintestinal pathogenic E. coli (ExPEC)

  • May harbor plasmids that could affect expression system compatibility

• Metabolic characteristics:

  • Potentially different central carbon metabolism regulation

  • May exhibit altered growth characteristics under laboratory conditions

• Expression considerations:

  • May require different antibiotic selection markers due to intrinsic resistance profiles

  • Could display different stress responses affecting recombinant protein yield

  • May have unique codon usage preferences

• Safety considerations:

  • Biosafety level requirements may differ from standard laboratory strains

  • Additional containment measures might be necessary

• Recombinant protein production:

  • May offer advantages for specific proteins due to unique cellular environment

  • Could provide different post-translational modifications

When working with this strain, researchers should consider its pathogenic potential and implement appropriate safety measures while adapting standard protocols to accommodate its unique characteristics.

What purification protocol yields the highest activity for recombinant Succinyl-CoA ligase?

An optimized purification protocol for maintaining high enzymatic activity includes:

• Cell lysis:

  • Buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM DTT, protease inhibitors

  • Gentle lysis methods (sonication with cooling or French press) to preserve activity

  • Addition of low concentrations of substrate components (0.1 mM ATP, 0.5 mM succinate) to stabilize the enzyme

• Initial purification:

  • Affinity chromatography (if tagged) using optimized binding and elution conditions

  • For His-tagged constructs: Ni-NTA with imidazole gradient elution (20-250 mM)

  • For GST-tagged constructs: Glutathione-Sepharose with reduced glutathione elution

• Intermediate steps:

  • Ion exchange chromatography (Q-Sepharose) with salt gradient elution

  • Optional tag removal using specific proteases (if tag affects activity)

  • Buffer exchange to remove components that might interfere with activity

• Final purification:

  • Size exclusion chromatography to separate oligomeric states and remove aggregates

  • Activity testing of different fractions to identify the most active form

• Storage:

  • Addition of glycerol (20-25%) to prevent freezing damage

  • Flash freezing in small aliquots to avoid freeze-thaw cycles

  • Storage at -80°C for long-term preservation of activity

Throughout the purification process, maintain enzyme stability by including reducing agents, glycerol, and potentially key metal ions (Mg²⁺) in all buffers. Purify both alpha and beta subunits together or reconstitute the complex after purification to ensure maximal activity.

How can I measure the enzymatic activity of Succinyl-CoA ligase in vitro?

Several methods can accurately measure Succinyl-CoA ligase activity:

• Forward reaction (succinyl-CoA → succinate):

  • Coupled enzyme assay linking ADP formation to NADH oxidation

  • Components: succinyl-CoA, ADP, Pi, MgCl₂, pyruvate kinase, lactate dehydrogenase, PEP, NADH

  • Monitor absorbance decrease at 340 nm as NADH is oxidized

  • Calculate activity using NADH extinction coefficient (6,220 M⁻¹cm⁻¹)

• Reverse reaction (succinate → succinyl-CoA):

  • Direct measurement of CoA consumption using DTNB (Ellman's reagent)

  • Components: succinate, ATP, CoA, MgCl₂, DTNB

  • Monitor absorbance increase at 412 nm as thiols react with DTNB

  • Calculate activity using TNB extinction coefficient (14,150 M⁻¹cm⁻¹)

• Direct product analysis:

  • HPLC separation and quantification of reaction components

  • LC-MS for more sensitive detection of intermediates and products

  • Allows observation of side reactions and partial activities

When performing kinetic measurements, establish linear reaction rates with respect to time and enzyme concentration, and conduct controls to account for background activity and spontaneous hydrolysis.

What are the key considerations when designing site-directed mutagenesis experiments for sucC?

Effective site-directed mutagenesis of sucC requires careful planning:

• Target selection rationale:

  • Catalytic residues identified from structural studies or sequence alignments

  • Substrate binding sites that determine specificity

  • Interface residues involved in alpha/beta subunit interactions

  • Regulatory sites that affect enzyme activity or allosteric regulation

• Mutation design strategy:

  • Conservative substitutions to probe specific chemical properties

  • Alanine scanning to identify essential residues

  • Introduction of non-natural amino acids for specialized functions

  • Homology-guided mutations based on related enzymes

• Technical considerations:

  • Primer design with appropriate melting temperatures (58-62°C)

  • Verification methods including sequencing the entire gene

  • Controls to distinguish mutation effects from expression artifacts

  • Expression testing to ensure the mutant protein is produced

• Functional analysis plan:

  • Enzymatic activity measurements for each mutant

  • Thermal stability assessment

  • Structural analysis when possible

  • In vivo complementation tests in sucC-deficient strains

Computational modeling approaches, similar to those used for succinate dehydrogenase , can help predict the effects of mutations and guide experimental design. This is particularly valuable for understanding how mutations might affect redox centers and electron transfer pathways in metabolic enzymes.

How can I troubleshoot low expression levels of recombinant sucC?

When facing low expression levels of recombinant sucC, consider this systematic troubleshooting approach:

• Vector and sequence analysis:

  • Verify the complete sequence for errors or mutations

  • Check the promoter region, ribosome binding site, and start codon

  • Examine the N-terminal sequence, which significantly impacts expression

  • Consider optimizing the sequence using FACS-based selection of N-terminal libraries

• Expression conditions optimization:

  • Test multiple temperatures (37°C, 30°C, 25°C, 18°C)

  • Vary inducer concentration and induction timing

  • Try different media formulations (LB, TB, minimal media with supplements)

  • Adjust aeration and growth phase at induction

• Strain selection:

  • Test multiple E. coli strains (BL21, Rosetta, C41/C43)

  • Consider strains with additional tRNAs for rare codons

  • Use strains with reduced protease activity

• Protein solubility enhancement:

  • Co-express with molecular chaperones

  • Add fusion tags known to enhance solubility (MBP, SUMO, GST)

  • Express with the partner subunit (sucD) to promote proper folding

  • Include appropriate cofactors or stabilizing agents in the growth medium

• Expression construct redesign:

  • Create a new construct with optimized N-terminal sequence

  • Consider expressing the complete sucCD operon to maintain natural stoichiometry

  • Try dual expression systems for both subunits

Research has shown that modifying the N-terminal sequence alone can increase protein yield by over 30-fold . This directed evolution approach, using GFP as a reporter and FACS for screening, provides a powerful method to overcome expression limitations when rational approaches fail.

What advanced research methods can be applied to study the role of sucC in E. coli metabolic networks?

Several advanced research methods can provide deeper insights into sucC function:

These advanced methods can be applied similarly to approaches used in studying succinate dehydrogenase , where computational modeling helped elucidate the mechanism of superoxide and hydrogen peroxide production, providing insight into the relationship between metabolic enzymes and cellular redox state.