Recombinant Actinobacillus succinogenes ATP synthase subunit beta (atpD)

What is the role of ATP synthase subunit beta (atpD) in Actinobacillus succinogenes metabolism?

The ATP synthase subunit beta (atpD) in A. succinogenes plays a crucial role in energy conservation during cellular metabolism. As a catalytic component of the F1F0-ATP synthase complex, atpD is directly involved in ATP synthesis through oxidative phosphorylation. In A. succinogenes, which is a facultative anaerobe, this protein contributes significantly to energy balance during both aerobic respiration and anaerobic fermentation pathways . The atpD subunit contains nucleotide binding sites and catalyzes the reversible reaction of ADP + Pi ⟷ ATP, which is essential for maintaining energy homeostasis during the mixed-acid fermentation that produces succinate as a major end product.

How does atpD expression correlate with succinic acid production in A. succinogenes?

The expression of atpD in A. succinogenes demonstrates a complex relationship with succinic acid production. Studies indicate that under anaerobic conditions where succinate production is highest, the expression pattern of atpD adjusts to accommodate the energetic demands of the cell . During mixed-acid fermentation, A. succinogenes directs carbon flux through the reductive branch of the TCA cycle, producing succinate while regenerating NAD+ from NADH. This process affects the proton motive force that drives ATP synthase, thereby establishing an indirect but significant correlation between atpD activity and succinate production capabilities .

What are the standard methods for isolating and purifying recombinant A. succinogenes atpD?

Recombinant A. succinogenes atpD is typically isolated and purified through a multi-step protocol:

• Gene Cloning and Expression:

  • PCR amplification of the atpD gene from A. succinogenes genomic DNA

  • Ligation into an expression vector (commonly pET series vectors)

  • Transformation into an E. coli expression host (BL21(DE3) or similar strains)

  • Induction of protein expression using IPTG (typically 0.5-1.0 mM)

• Cell Lysis and Initial Purification:

  • Harvesting of cells by centrifugation (6,000 × g, 15 min, 4°C)

  • Resuspension in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole)

  • Cell disruption via sonication or high-pressure homogenization

  • Removal of cell debris by centrifugation (15,000 × g, 30 min, 4°C)

• Affinity Chromatography:

  • For His-tagged constructs: Nickel affinity chromatography

  • Washing with increasing imidazole concentrations

  • Elution with high imidazole buffer (300-500 mM)

• Additional Purification Steps:

  • Size exclusion chromatography for higher purity

  • Ion exchange chromatography for removal of contaminants

• Quality Assessment:

  • SDS-PAGE to verify purity

  • Western blotting for identity confirmation

  • Activity assays to confirm functionality

This methodology provides high yields of functional protein suitable for structural and functional studies .

How does the sequence and structure of A. succinogenes atpD compare to other bacterial species, and what implications does this have for its function in succinate production?

Comparative analysis of A. succinogenes atpD with homologs from other bacteria reveals significant insights into its specialized function:

The high conservation of catalytic domains suggests a preserved fundamental function, but the specific differences in regulatory regions may contribute to the unique metabolic behavior of A. succinogenes during succinate production . The particular arrangement of charged residues at the catalytic interface appears optimized to function efficiently under the capnophilic conditions that favor succinate production, allowing ATP synthesis to proceed even as the cell diverts significant carbon flux toward succinate rather than typical energy-yielding pathways . These structural adaptations likely represent evolutionary specialization for A. succinogenes' ecological niche.

What are the most effective strategies for overexpressing functional atpD in recombinant systems without disrupting cellular energy balance?

Overexpression of functional atpD requires careful consideration of cellular energy homeostasis. The most effective strategies include:

• Inducible Expression Systems with Fine Control:

  • Use of tightly regulated promoters (like tetO or araBAD) rather than strong constitutive promoters

  • Titration of inducer concentrations to prevent excessive expression

  • Temperature-sensitive expression systems for gradual induction

• Co-expression with Partner Subunits:

  • Simultaneous expression of other F1 subunits (particularly alpha) to ensure proper complex formation

  • Use of polycistronic constructs that maintain natural stoichiometric ratios

  • Expression of chaperones (GroEL/GroES) to assist proper folding

• Physiological Adaptations During Expression:

  • Cultivation at lower temperatures (16-25°C) during induction phase

  • Supplementation with additional carbon sources to maintain energy balance

  • Aeration control to accommodate shifts in respiratory chain function

• Strain Engineering Approaches:

  • Use of host strains with attenuated native ATP synthase expression

  • Engineering of strains with enhanced capacity to handle protein overexpression

  • Integration of compensatory pathways to maintain redox balance

These approaches have demonstrated success rates of 60-85% depending on the specific experimental context, with the combined strategy of lower temperature induction and chaperone co-expression showing particularly robust results .

What interplay exists between atpD expression and the phosphoenolpyruvate (PEP) carboxylation pathway in carbon dioxide fixation during succinate production?

The interplay between atpD expression and the PEP carboxylation pathway represents a critical nexus in A. succinogenes metabolism:

ATP synthase activity, regulated by atpD expression, affects the cellular energy state, which in turn modulates the activity of key enzymes in the PEP carboxylation pathway. Research indicates a sophisticated regulatory relationship whereby:

• Energetic Coupling:

  • ATP synthase activity influences the intracellular ATP/ADP ratio, which acts as an allosteric regulator of phosphoenolpyruvate carboxykinase (PEPCK)

  • Under conditions of high ATP demand, decreased atpD activity can redirect phosphoenolpyruvate toward carboxylation rather than ATP-generating pathways

• Proton Motive Force (PMF) Effects:

  • ATP synthase consumes PMF during ATP synthesis

  • The same PMF drives certain transmembrane CO2/bicarbonate transporters

  • Modulation of atpD expression affects CO2 availability for PEP carboxylation

• Redox Balance Coordination:

  • ATP synthase activity influences NADH oxidation via respiratory chain

  • The reductive branch of TCA cycle (essential for succinate production) requires NADH

  • atpD expression levels help maintain optimal NADH/NAD+ ratios for maximum carbon flux to succinate

• Carbon Flux Distribution:

  • In engineered strains with altered atpD expression, carbon flux through PEPCK increases by 15-30%

  • CO2 fixation rates correlate with changes in ATP synthase activity

  • Studies show a direct correlation between atpD expression levels and CO2 consumption rates

This sophisticated metabolic interplay explains why attempts to engineer A. succinogenes for improved succinate production must consider the broader implications of modifying energy metabolism components like atpD .

What are the recommended protocols for site-directed mutagenesis of A. succinogenes atpD to investigate structure-function relationships?

The following protocol outlines a comprehensive approach for site-directed mutagenesis of A. succinogenes atpD:

• Target Selection and Primer Design:

  • Identify conserved residues through multiple sequence alignment with homologous proteins

  • Design mutagenic primers (25-35 bp) with the desired mutation centered and 10-15 bp of correct sequence on each side

  • Ensure primers have a GC content of 40-60% and Tm ≥78°C

  • Incorporate silent restriction sites for screening when possible

• PCR-Based Mutagenesis:

  • Use a high-fidelity polymerase with proofreading capability (Q5, Pfu Ultra, or KAPA HiFi)

  • Perform PCR with the following parameters:

    • Initial denaturation: 98°C for 30 seconds

    • 18 cycles of: 98°C for 10 seconds, 55-65°C for 30 seconds, 72°C for 30 seconds/kb

    • Final extension: 72°C for 10 minutes

  • Treat PCR product with DpnI (10U) at 37°C for 1 hour to digest methylated template DNA

• Transformation and Screening:

  • Transform 5 μl of DpnI-treated product into competent E. coli cells

  • Plate on selective media and incubate at 37°C overnight

  • Screen colonies by restriction digestion or colony PCR

  • Verify mutations by DNA sequencing

• Functional Validation:

  • Express wild-type and mutant proteins under identical conditions

  • Purify proteins using the same protocol to minimize variability

  • Conduct comparative activity assays:

    • ATP synthesis rate measurement

    • ATP hydrolysis assays with colorimetric phosphate detection

    • Binding affinity studies for substrates and inhibitors

• Structural Analysis:

  • Perform circular dichroism to verify similar secondary structure

  • If possible, determine crystal structures of mutant proteins

  • Use molecular dynamics simulations to analyze subtle conformational changes

This methodology has been successfully employed to identify critical residues in the catalytic site and regulatory domains of ATP synthase beta subunits from various organisms, with application potential for A. succinogenes atpD .

How can researchers effectively incorporate recombinant atpD into liposomes for in vitro bioenergetic studies?

Incorporating recombinant atpD into liposomes for bioenergetic studies requires a methodical approach:

• Preparation of Proteoliposomes:

  • Lipid Selection and Preparation:

    • Use a mixture of phosphatidylcholine (70%), phosphatidylethanolamine (20%), and cardiolipin (10%)

    • Dissolve lipids in chloroform, evaporate under nitrogen gas, and rehydrate in buffer

    • Sonicate or extrude through polycarbonate membranes (100 nm) to form unilamellar vesicles

  • Protein Reconstitution:

    • Solubilize purified atpD (and other ATP synthase subunits if studying the complex) in a mild detergent (0.5% n-dodecyl-β-D-maltoside)

    • Mix with preformed liposomes at a lipid-to-protein ratio of 20:1 to 50:1

    • Remove detergent by dialysis or adsorption to Bio-Beads

    • Concentrate proteoliposomes by ultracentrifugation (100,000 × g, 1 hour)

• Validation of Reconstitution:

  • Microscopy Verification:

    • Negative staining electron microscopy to confirm protein incorporation

    • Freeze-fracture electron microscopy to assess protein distribution

  • Functional Assessment:

    • Measure proton pumping using pH-sensitive fluorescent dyes (ACMA or pyranine)

    • Assess membrane integrity with carboxyfluorescein leakage assays

• Bioenergetic Measurements:

  • ATP Synthesis Assays:

    • Establish proton gradient by acid-base transition or using bacteriorhodopsin

    • Initiate reaction with ADP and Pi

    • Monitor ATP formation with luciferase-based luminescence assays

  • ATP Hydrolysis Measurements:

    • Couple ATP hydrolysis to NADH oxidation through enzymatic reactions

    • Monitor absorbance changes at 340 nm in real-time

    • Calculate activity rates under varying conditions

• Environmental Parameter Testing:

  • Study effects of pH, temperature, and ionic strength

  • Evaluate inhibitor sensitivity compared to native enzyme

  • Measure activity under varying Δψ and ΔpH gradients

This methodology provides a controlled system for studying the bioenergetic properties of recombinant atpD alone or within the ATP synthase complex, enabling detailed analysis of its contribution to A. succinogenes energy metabolism .

What are the most reliable methods for measuring ATP synthase activity in whole-cell A. succinogenes during succinate fermentation?

Measuring ATP synthase activity in whole-cell A. succinogenes during succinate fermentation presents unique challenges due to the complex metabolic background. The following methods have proven most reliable:

These methods have demonstrated reproducible results with standard deviations typically <15% across biological replicates, with the combined approach of membrane potential measurements and inhibitor studies offering the most comprehensive assessment of ATP synthase activity in fermenting cells .

What are common pitfalls in recombinant A. succinogenes atpD expression systems and how can they be addressed?

Researchers frequently encounter several challenges when working with recombinant A. succinogenes atpD. Here are the most common issues and their solutions:

• Protein Misfolding and Inclusion Body Formation:

  • Issue: atpD often forms inclusion bodies when overexpressed, especially at higher temperatures.

  • Solutions:

    • Reduce expression temperature to 16-20°C

    • Use specialized strains like Rosetta-gami or Arctic Express

    • Co-express molecular chaperones (GroEL/GroES, trigger factor)

    • Add 5-10% glycerol or 0.1-0.5 M sorbitol to growth media

    • Try fusion tags that enhance solubility (SUMO, MBP, or TrxA)

• Low Expression Levels:

  • Issue: Poor expression yields despite optimized conditions.

  • Solutions:

    • Codon optimization for expression host

    • Use strong inducible promoters with fine-tuned inducer concentrations

    • Screen multiple expression vectors and signal sequences

    • Optimize media composition (consider using terrific broth or auto-induction media)

    • Extend expression time at lower temperatures (24-48 hours at 18°C)

• Loss of Catalytic Activity:

  • Issue: Purified protein shows low or no enzymatic activity.

  • Solutions:

    • Ensure buffers contain stabilizing agents (glycerol, ATP, or Mg²⁺)

    • Avoid freeze-thaw cycles by storing in single-use aliquots

    • Maintain reducing environment with 1-5 mM DTT or β-mercaptoethanol

    • Use mild detergents (0.05% DDM) to mimic membrane environment

    • Co-purify with alpha subunit to maintain proper conformation

• Protein Degradation:

  • Issue: Significant proteolysis during expression or purification.

  • Solutions:

    • Use protease-deficient expression strains

    • Include multiple protease inhibitors in all buffers

    • Minimize handling time during purification

    • Optimize purification strategy for speed rather than yield

    • Consider on-column refolding protocols if working with inclusion bodies

• Non-specific Binding During Purification:

  • Issue: Contaminants co-purify with the target protein.

  • Solutions:

    • Increase imidazole in washing buffers (30-50 mM) for His-tagged constructs

    • Add low concentrations of non-ionic detergents to reduce hydrophobic interactions

    • Implement multi-step purification strategy (affinity followed by ion exchange or SEC)

    • Consider alternative tag systems (Strep-tag II or FLAG tag)

    • Pre-clear lysates with appropriate resins before affinity chromatography

By addressing these common pitfalls, researchers can significantly improve the yield and quality of recombinant A. succinogenes atpD, with success rates improving from typical initial yields of 0.5-1 mg/L to optimized yields of 5-15 mg/L of culture .

How can researchers differentiate between effects caused by atpD mutations versus other metabolic shifts in A. succinogenes?

Differentiating between direct effects of atpD mutations and secondary metabolic adaptations requires a systematic experimental design:

• Complementation Analysis:

  • Methodology:

    • Create a clean deletion of native atpD

    • Complement with wild-type or mutant atpD variants on plasmids

    • Use inducible promoters to achieve controlled expression levels

    • Compare phenotypes at equivalent protein expression levels

  • Controls:

    • Express catalytically inactive atpD (e.g., mutation in Walker A/B motifs)

    • Monitor growth and fermentation profiles across different expression levels

    • Measure the copy number of complementing plasmids to ensure comparable dosage

• Targeted Metabolomics:

  • Direct vs. Indirect Markers:

    • Identify metabolites directly linked to ATP synthase function (ATP/ADP ratio, Pi levels)

    • Monitor secondary metabolites that respond to energetic shifts

    • Track time-course metabolite changes after conditional expression of atpD variants

  • Statistical Analysis:

    • Apply principal component analysis to metabolite profiles

    • Use partial least squares discriminant analysis to identify key differentiating metabolites

    • Calculate correlation coefficients between atpD activity levels and metabolite concentrations

• Multi-omics Integration:

  • Workflow:

    • Combine transcriptomics, proteomics, and metabolomics data

    • Identify regulatory networks responding to atpD mutations

    • Apply network analysis to distinguish primary effects from compensatory responses

  • Time-resolved Analysis:

    • Sample at multiple time points after induction of atpD variants

    • Identify immediate vs. delayed responses in gene expression and metabolism

    • Map temporal changes to known regulatory networks

• Kinetic Modeling:

  • Model Construction:

    • Develop kinetic models incorporating experimentally determined parameters

    • Simulate the effects of atpD mutations on metabolic fluxes

    • Compare model predictions with experimental observations

  • Sensitivity Analysis:

    • Identify pathways most sensitive to changes in ATP synthase activity

    • Quantify the threshold of atpD activity change needed to trigger metabolic shifts

    • Predict compensatory mechanisms and verify experimentally

• Chemical Genetic Approach:

  • Methodology:

    • Use specific inhibitors of ATP synthase (oligomycin, DCCD) at sub-lethal doses

    • Compare metabolic profiles between inhibitor treatment and atpD mutations

    • Identify shared vs. unique responses to distinguish direct effects

This integrated approach allows researchers to create a causality map that differentiates primary effects of atpD mutations from secondary metabolic adaptations with approximately 85-90% confidence, based on statistical validation across multiple experimental conditions .

What experimental designs best elucidate the relationship between ATP homeostasis and redox balance during succinate production?

The relationship between ATP homeostasis and redox balance in A. succinogenes requires sophisticated experimental designs:

• Continuous Culture Systems with Controlled Parameters:

  • Chemostat Setup:

    • Maintain constant growth rate at different dilution rates (0.05-0.3 h⁻¹)

    • Vary one parameter (e.g., CO₂ availability) while monitoring ATP and redox indicators

    • Implement pH-stat to maintain constant pH during acid production

    • Collect samples at steady state for comprehensive analysis

  • Data Collection:

    • Measure intracellular ATP/ADP and NADH/NAD⁺ ratios

    • Monitor succinate and other organic acid concentrations

    • Quantify biomass yield coefficient (Yx/s)

    • Calculate maintenance energy requirements at different production rates

• Perturbation Studies with Multi-parameter Monitoring:

  • Experimental Design:

    • Establish steady-state cultures with stable succinate production

    • Introduce specific perturbations:

      • Transient shift in CO₂ availability

      • Addition of uncouplers at sub-inhibitory concentrations

      • Switch in carbon source (glucose to xylose or mixed sugars)

    • Monitor immediate responses and adaptation trajectory

  • Real-time Analysis:

    • Track cytoplasmic pH using ratiometric fluorescent proteins

    • Monitor membrane potential with potential-sensitive dyes

    • Measure ATP synthesis rates using luciferase reporters

    • Quantify NAD(P)H levels via autofluorescence

• Genetic Toggle Systems for ATP Synthase Modulation:

  • Construct Design:

    • Engineer strains with inducible atpD expression systems

    • Create conditional knockdowns using CRISPR interference

    • Design sensor-actuator systems that respond to ATP or NADH levels

    • Generate reporter strains with fluorescent indicators for ATP and redox state

  • Experimental Protocol:

    • Induce different expression levels of atpD

    • Monitor metabolic shifts during transition to new steady states

    • Calculate flux control coefficients for key pathways

    • Correlate atpD expression with redox cofactor balances

• 13C Metabolic Flux Analysis with ATP Balancing:

  • Labeling Strategy:

    • Use position-specific 13C-labeled substrates

    • Apply parallel labeling experiments (1-13C and U-13C glucose)

    • Collect samples at multiple time points during steady-state production

    • Analyze isotopomer distributions in key intermediates

  • Computational Analysis:

    • Construct metabolic models with ATP and redox constraints

    • Calculate flux distributions accounting for all energy-generating and consuming reactions

    • Determine the relative contributions of substrate-level phosphorylation vs. ATP synthase

    • Quantify the energetic cost of maintaining redox balance during succinate production

These experimental designs collectively provide a systems-level understanding of how ATP homeostasis interacts with redox balance to influence succinate production pathways in A. succinogenes, revealing that a 15-20% shift in ATP availability can redirect up to 30-40% of carbon flux between competing fermentation pathways .

What promising approaches exist for engineering atpD to enhance A. succinogenes ATP efficiency during anaerobic fermentation?

Several promising approaches for engineering atpD to enhance ATP efficiency during anaerobic fermentation include:

• Rational Protein Engineering Based on Structural Insights:

  • Targeted Approaches:

    • Modify catalytic residues to reduce slippage (uncoupled ATP hydrolysis)

    • Engineer the nucleotide binding pocket for improved affinity under fermentation conditions

    • Introduce mutations that modify the pH sensitivity of catalysis

    • Redesign subunit interfaces to enhance stability during stress conditions

  • Expected Outcomes:

    • 15-25% improvement in ATP synthesis coupling efficiency

    • Enhanced stability at the acidic pH typical during succinate fermentation

    • Reduced maintenance energy requirements during prolonged fermentation

• Directed Evolution Strategies:

  • Methodology:

    • Develop high-throughput screening systems based on growth under ATP-limiting conditions

    • Apply error-prone PCR with progressive selection under increasing stress

    • Implement PACE (Phage-Assisted Continuous Evolution) adapted for atpD

    • Combine shuffling of homologous atpD genes from different organisms

  • Target Parameters:

    • Select for variants with improved function at high CO₂ concentrations

    • Screen for mutants with enhanced activity during organic acid accumulation

    • Identify variants with improved thermostability for industrial application

• Heterologous atpD Substitution:

  • Source Selection:

    • Evaluate atpD homologs from extreme acidophiles for acid tolerance

    • Test variants from organisms with naturally efficient anaerobic metabolism

    • Consider chimeric constructs combining domains from different species

    • Screen atpD from other industrial production organisms (e.g., Mannheimia succiniciproducens)

  • Integration Strategy:

    • Replace native atpD while maintaining interactions with other ATP synthase subunits

    • Co-express heterologous atpD alongside native version with tunable ratios

    • Engineer compatibility with A. succinogenes-specific regulatory networks

• Systems Biology-Guided Modifications:

  • Approach:

    • Apply metabolic control analysis to identify optimal atpD activity levels

    • Design synthetic regulatory circuits that modulate atpD expression based on cellular energy state

    • Implement dynamic control of atpD activity coordinated with central metabolism

    • Develop biosensor-based feedback systems responding to ATP/ADP ratio

  • Expected Benefits:

    • Dynamic optimization of ATP synthase activity throughout fermentation phases

    • Coordinated balance between ATP generation and succinate production

    • Reduced metabolic burden during adaptation to changing conditions

• Post-translational Regulation Engineering:

  • Mechanism Modifications:

    • Alter phosphorylation sites to modify activity regulation

    • Engineer redox-sensitive switches for activity modulation under varying redox conditions

    • Modify allosteric regulatory sites to change response to inhibitors/activators

    • Implement synthetic protein scaffolds to optimize ATP synthase complex assembly

  • Implementation:

    • Introduce specific mutations at known regulatory residues

    • Create fusion proteins with engineered regulatory domains

    • Express modified regulatory factors that interact with atpD

These engineering approaches show promise for enhancing A. succinogenes performance during anaerobic fermentation, with preliminary studies indicating potential improvements in ATP efficiency of 20-30% and corresponding increases in succinate yield approaching theoretical maximum values .

How might CRISPR-Cas9 genome editing techniques be optimized for studying atpD function in A. succinogenes?

Optimizing CRISPR-Cas9 genome editing for studying atpD function in A. succinogenes requires addressing several specific challenges:

• Development of Efficient Transformation Protocols:

  • Critical Parameters:

    • Optimize electroporation conditions (1.8-2.2 kV/cm, 25 μF, 200 Ω)

    • Prepare cells at early-mid log phase (OD₆₀₀ 0.4-0.6)

    • Use glycine (1-1.5%) pretreatment to weaken cell wall

    • Include osmoprotectants (10% sucrose, 10% PEG 8000) in recovery media

    • Extend recovery time to 3-4 hours before selection

  • Expected Efficiency:

    • Target transformation efficiencies of 10⁵-10⁶ CFU/μg DNA

    • Validate using fluorescent protein reporters before attempting gene editing

• CRISPR-Cas9 System Adaptation:

  • Vector Design:

    • Develop shuttle vectors with compatible origins of replication

    • Use promoters functional in A. succinogenes (P₄₃, Pkan)

    • Employ codon-optimized Cas9 or alternative Cas variants (Cas12a)

    • Implement temperature-sensitive plasmids for transient expression

  • gRNA Optimization:

    • Design gRNAs with A. succinogenes-specific scoring algorithms

    • Target PAM sites unique to atpD without homology elsewhere

    • Validate gRNA efficiency using in vitro cleavage assays

    • Implement multiplexed gRNA expression for sequential editing

• Precise Genome Modification Strategies:

  • Editing Approaches:

    • Single nucleotide modifications using base editors (BE4, ABE8e)

    • Scarless editing via double-strand break repair with linear DNA templates

    • Precise domain swapping using homology-directed repair

    • Conditional expression systems integrated at the native locus

  • Template Design:

    • Include 1-1.5 kb homology arms for efficient recombination

    • Incorporate silent mutations in PAM sites to prevent re-cutting

    • Design counterselection markers for plasmid curing

    • Consider integration of landing pads for subsequent modifications

• Screening and Verification Protocols:

  • Selection Strategies:

    • Implement MAGE (Multiplex Automated Genome Engineering) for subtle modifications

    • Develop high-throughput phenotypic screens relevant to ATP synthase function

    • Create reporter systems linked to ATP synthase activity

    • Design PCR screens with high specificity for modified regions

  • Validation Methods:

    • Whole-genome sequencing to verify on-target edits and detect off-target effects

    • RT-qPCR to confirm expression levels of modified atpD

    • Western blotting with specific antibodies to verify protein production

    • Enzyme activity assays to confirm functional consequences

• Complementation Systems:

  • Rescue Strategies:

    • Establish plasmid-based complementation with wild-type atpD

    • Develop inducible systems for controlled expression during editing

    • Create merodiploid strains for essential gene modifications

    • Implement CRISPR interference for partial knockdowns when full knockouts are lethal

These optimized CRISPR-Cas9 techniques can achieve editing efficiencies of 10-15% for simple modifications and 1-5% for complex edits in A. succinogenes, enabling precise study of atpD function through targeted mutations, domain swapping, or regulatory element engineering .

What potential exists for atpD as a target for enhancing A. succinogenes stress tolerance during high-density fermentation?

The ATP synthase beta subunit (atpD) represents a promising target for enhancing stress tolerance in A. succinogenes during high-density fermentation:

• Acid Stress Tolerance Enhancement:

  • Mechanisms:

    • ATP-dependent maintenance of cytoplasmic pH homeostasis requires efficient ATP synthase function

    • Modified atpD can maintain functionality at lower pH, supporting continued ATP generation during acid accumulation

    • Improved coupling efficiency reduces proton leakage across the membrane

  • Engineering Approaches:

    • Target residues at the catalytic site to optimize function at low pH (pH 5.0-5.5)

    • Introduce mutations observed in acid-tolerant microorganisms

    • Modify regulatory regions to maintain expression under acid stress

    • Engineer allosteric regulation to respond to cytoplasmic acidification

• Osmotic Stress Adaptation:

  • Physiological Relevance:

    • High-density fermentation creates significant osmotic pressure from media components and product accumulation

    • ATP-dependent compatible solute transport and synthesis require robust energy metabolism

    • atpD optimization can support energy needs during osmoadaptation

  • Target Modifications:

    • Engineer protein stability under high ionic strength conditions

    • Modify regulatory mechanisms to maintain activity during osmotic shifts

    • Enhance interaction with other ATP synthase subunits under stress conditions

    • Develop variants that maintain activity despite membrane fluidity changes

• Redox Stress Management:

  • Mechanistic Connection:

    • Maintaining redox balance during succinate production requires coordinated energy metabolism

    • ATP synthase activity influences the cell's capacity to manage electron flow

    • Engineered atpD can support optimal energetic coupling during redox perturbations

  • Design Strategies:

    • Incorporate redox-sensing domains to modulate activity based on cellular redox state

    • Engineer variants less susceptible to oxidative damage

    • Modify regions involved in proton translocation for improved efficiency

    • Design versions with altered inhibitory response to high NADH/NAD+ ratios

• Carbon-utilization Efficiency Under Stress:

  • Metabolic Integration:

    • ATP availability influences carbon flux distribution during stress

    • Optimized atpD can maintain adequate ATP supply for anaplerotic reactions

    • Enhanced energy efficiency reduces carbon diversion to maintenance requirements

  • Performance Metrics:

    • 15-25% improvement in succinate yield under osmotic stress

    • Extended productive fermentation time during acid accumulation

    • Reduced lag phase after environmental perturbations

    • Increased cell viability maintaining productivity at high product titers

• Integrated Stress Response Coordination:

  • Regulatory Engineering:

    • Link atpD expression or activity to cellular stress response networks

    • Develop synthetic regulatory circuits connecting stress sensors to ATP synthase regulation

    • Fine-tune energy allocation between growth and stress tolerance

    • Implement dynamic control systems responding to changing fermentation conditions

Preliminary studies with engineered atpD variants have demonstrated promising results, including:

• Maintenance of 60-70% ATP synthase activity at pH 4.8 compared to 20-30% in wild-type

• Extended fermentation time by 15-20 hours before productivity decline

• Increased final succinate titers by 15-30% under high-osmolarity conditions

• Improved recovery from transient stresses during fed-batch operation

These improvements suggest significant potential for atpD engineering as a strategy to enhance A. succinogenes performance in industrial fermentation settings .