Recombinant Actinobacillus succinogenes Ribosome-recycling factor (frr)

What is Actinobacillus succinogenes and why is it significant for research?

Actinobacillus succinogenes is a Gram-negative facultative anaerobic bacterium that has gained significant research attention due to its natural capacity to efficiently convert both pentose and hexose sugars to succinic acid (SA) with high yield as a tricarboxylic acid (TCA) cycle intermediate. This organism is capnophilic, meaning it incorporates CO₂ into succinic acid, making it an ideal candidate for the conversion of lignocellulosic sugars and CO₂ to commodity bioproducts from renewable feedstocks . A. succinogenes achieves among the highest reported succinic acid titers and yields, which explains its importance in metabolic engineering studies aimed at improving bioproduction systems .

What is the role of the ribosome-recycling factor (frr) in bacterial protein synthesis?

The ribosome-recycling factor (frr) plays a crucial role in the final stage of protein synthesis by disassembling the post-termination ribosomal complex. This recycling process is essential for making ribosomes available for new rounds of translation. In A. succinogenes, as in other bacteria, frr works in conjunction with elongation factor G (EF-G) and initiation factor 3 (IF3) to separate the ribosomal subunits, release the mRNA, and prepare the translation machinery for subsequent protein synthesis cycles. This process is particularly important in organisms like A. succinogenes that have been engineered for enhanced protein expression and metabolic flux toward specific products such as succinic acid.

How does the frr gene differ between A. succinogenes and other bacterial species?

While the search results don't provide specific sequence comparison data for the frr gene across bacterial species, it's important to note that ribosome-recycling factors are generally conserved across bacteria but show species-specific variations. In A. succinogenes, the frr gene would likely share homology with those from related organisms in the Pasteurellaceae family, such as Mannheimia succiniciproducens, which is also known for succinic acid production . Understanding these differences is crucial when designing recombinant expression systems, as codon optimization and signal sequence considerations may be necessary to ensure proper folding and function of the protein in heterologous hosts.

What are the optimal expression systems for producing recombinant A. succinogenes frr?

The optimal expression system for recombinant A. succinogenes frr depends on research objectives and downstream applications. For structural and functional studies, E. coli-based systems (particularly BL21(DE3) or its derivatives) often provide high yields with relatively simple protocols. For applications requiring post-translational modifications, yeast systems like Pichia pastoris may be more appropriate.

Expression parameters for recombinant bacterial proteins in E. coli typically include:

When expressing frr specifically, fusion tags (such as His₆, MBP, or SUMO) can significantly improve solubility and facilitate purification. Temperature optimization is particularly important as translation-related proteins can sometimes affect host cell viability when overexpressed.

What purification strategies yield the highest purity recombinant frr protein?

A multi-step purification approach typically yields the highest purity for recombinant frr protein:

• Initial capture: Affinity chromatography (IMAC for His-tagged constructs) serves as an effective first step, typically yielding 85-90% purity.

• Intermediate purification: Ion exchange chromatography (typically using a Q or SP column depending on the protein's pI) can remove host cell proteins and nucleic acid contaminants.

• Polishing step: Size exclusion chromatography (Superdex 75 or 200 depending on molecular weight) provides final purification and buffer exchange.

Typical purification yields for ribosomal proteins expressed in E. coli range from 5-15 mg/L of culture. Ensuring removal of nucleic acid contamination is particularly important for ribosome-associated proteins like frr, as they naturally bind RNA. Adding high salt (500 mM NaCl) during lysis and including a wash step with 50-100 mM imidazole for His-tagged constructs can significantly reduce nucleic acid contamination.

How can I confirm the proper folding and activity of recombinant frr protein?

Verifying proper folding and activity of recombinant frr protein requires several complementary approaches:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to evaluate secondary structure content

  • Thermal shift assays to determine protein stability

  • Limited proteolysis to assess compact folding

• Functional assays:

  • Ribosome-binding assays using purified ribosomes

  • Polysome dissociation assays measuring the release of mRNA and tRNA

  • In vitro translation termination efficiency tests

• Biophysical characterization:

  • Dynamic light scattering to assess monodispersity

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS) to determine oligomeric state

A particularly informative activity assay involves measuring the ability of purified frr to dissociate model post-termination complexes containing ribosomes, mRNA, and deacylated tRNA, which can be monitored using light scattering or fluorescently labeled components.

How should I design gene knockout experiments to study the role of frr in A. succinogenes metabolism?

Designing gene knockout experiments for frr in A. succinogenes requires careful consideration as ribosome recycling is typically essential for cell viability. Consider these approaches:

• Conditional knockout system: Implement an inducible promoter (such as a tetracycline-responsive system) to control frr expression, allowing gradual depletion rather than complete knockout.

• Partial knockdown: Use antisense RNA or CRISPR interference (CRISPRi) to reduce but not eliminate frr expression, then monitor effects on growth rate, protein synthesis, and succinic acid production.

• Complementation testing: Create a strain where chromosomal frr is deleted but complemented by a plasmid-borne copy, then use plasmid loss experiments to confirm essentiality.

When studying the metabolic impacts of frr modification, it's crucial to monitor:

• Growth rate in different carbon sources

• Protein synthesis rates using metabolic labeling

• Ribosome profiles using sucrose gradient fractionation

• Metabolic flux distribution using 13C-labeled substrates

Similar approaches have been successfully applied in metabolic engineering studies of A. succinogenes targeting other genes involved in central metabolism , providing a methodological foundation for frr-focused experiments.

What are the key considerations when designing site-directed mutagenesis experiments for frr functional analysis?

When designing site-directed mutagenesis experiments for frr functional analysis, consider:

• Target selection based on structural information:

  • Active site residues involved in ribosome binding

  • Residues at the interface with EF-G

  • Conserved residues across bacterial species

  • Species-specific residues that might confer unique properties

• Mutation strategy:

  • Conservative substitutions to probe specific interactions

  • Charge reversals to disrupt electrostatic interactions

  • Alanine scanning to identify critical residues

• Phenotypic analysis:

  • Growth rate measurements under various conditions

  • In vitro translational activity assays

  • Ribosome binding affinity measurements

  • Protein synthesis error rates

• Controls:

  • Wild-type frr expressed from the same vector

  • Structurally characterized mutations from related organisms

  • Mutations in non-conserved, surface-exposed residues

Analysis should focus on correlating biochemical properties with physiological outcomes, particularly how mutations affect A. succinogenes' ability to produce succinic acid under fermentation conditions. This approach allows mapping of structure-function relationships specific to this industrially relevant organism.

How can I design experiments to study the impact of frr expression levels on succinic acid production in A. succinogenes?

To investigate how frr expression levels affect succinic acid production, design experiments that carefully modulate frr expression while monitoring metabolic outputs:

• Expression modulation approaches:

  • Construct a series of promoters with different strengths to drive frr expression

  • Develop an inducible system with tunable expression levels

  • Implement ribosome binding site (RBS) variants with different translation efficiencies

• Fermentation setup:

  • Batch fermentation in controlled bioreactors

  • Monitoring pH, CO₂ availability, and substrate consumption

  • Analysis of both growth phase and stationary phase metabolism

• Key parameters to measure:

  • Succinic acid titer, yield, and productivity

  • Expression levels of frr (via RT-qPCR and Western blotting)

  • Ribosome profiles and polysome distribution

  • Formation of competing products (formate, acetate, lactate)

  • Global protein synthesis rates

Previous metabolic engineering studies in A. succinogenes have demonstrated that modifying pathways involved in competing acid production can significantly impact succinic acid yields . By extending this approach to translation-related factors like frr, researchers can explore how protein synthesis regulation might influence metabolic flux distribution toward desired products.

How can multi-omics approaches enhance our understanding of frr function in A. succinogenes?

Multi-omics approaches provide powerful tools for understanding the systemic role of frr in A. succinogenes metabolism:

• Transcriptomics: RNA-seq analysis comparing wild-type and frr-modified strains can reveal gene expression changes, particularly focusing on:

  • Translational machinery components

  • Stress response pathways

  • Central carbon metabolism genes

  • Changes in expression patterns under different fermentation conditions

• Proteomics: Quantitative proteomic analysis using techniques like iTRAQ or TMT labeling can identify:

  • Changes in protein abundance across metabolic pathways

  • Post-translational modifications affecting enzyme activity

  • Protein turnover rates and stability differences

• Metabolomics: Targeted and untargeted metabolomic approaches can measure:

  • Flux through succinic acid biosynthetic pathways

  • Accumulation of intermediates suggesting bottlenecks

  • Redistribution of carbon flux in response to translation machinery modifications

• Integration of multi-omics data:

  • Correlation analysis between transcripts, proteins, and metabolites

  • Pathway enrichment analysis to identify systems-level effects

  • Flux balance analysis incorporating experimental data

Previous studies have employed systems biology approaches to understand succinic acid production in A. succinogenes , providing a framework for integrating frr-specific investigations into broader metabolic contexts.

What computational approaches are most effective for analyzing the impact of frr mutations on protein structure and function?

Effective computational approaches for analyzing frr mutations include:

• Homology modeling and structure prediction:

  • Building A. succinogenes frr models based on crystal structures from related organisms

  • Refinement using molecular dynamics simulations

  • Validation through Ramachandran analysis and quality assessment tools

• Molecular dynamics simulations:

  • Analysis of protein stability and conformational changes

  • Evaluation of mutation effects on flexible regions

  • Identification of altered interaction networks

• Molecular docking and binding analysis:

  • Modeling frr interactions with ribosomal components

  • Calculating binding free energy changes due to mutations

  • Identifying key interaction residues

• Sequence-based predictions:

  • Coevolution analysis to identify functionally linked residue networks

  • Conservation analysis across bacterial species

  • Prediction of mutation effects using tools like SIFT and PolyPhen

• Integration with experimental data:

  • Correlating computational predictions with biochemical assays

  • Refining models based on mutagenesis results

  • Generating testable hypotheses for further experimental validation

These computational approaches can guide experimental design by prioritizing mutations likely to affect specific aspects of frr function, particularly those relevant to A. succinogenes metabolism and succinic acid production.

How can I accurately quantify the effects of frr modification on translation efficiency and metabolic flux in A. succinogenes?

Accurately quantifying the effects of frr modification requires combining several specialized techniques:

• Translation efficiency measurement:

  • Polysome profiling to assess ribosome loading on mRNAs

  • Ribosome profiling (Ribo-seq) to determine ribosome positioning and translation rates

  • Pulse-chase labeling with radioactive or stable isotope-labeled amino acids

  • Reporter systems (luciferase or fluorescent proteins) driven by various promoters

• Metabolic flux analysis:

  • 13C metabolic flux analysis using isotopically labeled glucose or other carbon sources

  • Measurement of extracellular metabolite concentrations over time

  • Enzymatic activity assays for key metabolic enzymes

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

• Experimental design considerations:

  • Use of chemostats to maintain steady-state conditions

  • Carefully controlled batch fermentations

  • Sampling across multiple growth phases

  • Technical and biological replicates to ensure statistical significance

• Data integration framework:

  • Correlation analysis between translation metrics and metabolic outputs

  • Mathematical modeling relating ribosome recycling efficiency to metabolic flux

  • Statistical analysis to establish causality vs. correlation

Previous metabolic engineering studies in A. succinogenes have shown that modifying key enzymes in the reductive branch of the TCA cycle enhances flux to succinic acid . Extending this approach to include translation factors like frr provides an opportunity to understand how protein synthesis regulation might be leveraged for metabolic engineering applications.

How can frr engineering be integrated into broader metabolic engineering strategies for enhanced succinic acid production?

Integrating frr engineering into metabolic engineering strategies requires a coordinated approach targeting multiple cellular systems:

• Coupling frr modifications with pathway engineering:

  • Optimize frr expression in conjunction with overexpression of key enzymes in the reductive TCA cycle

  • Coordinate frr modifications with knockout of competing pathways (acetate and formate production)

  • Balance translation efficiency with metabolic flux capacity

• Stress response considerations:

  • Engineer frr variants with enhanced function under acidic conditions

  • Couple frr optimization with acid tolerance improvements achieved through genome shuffling

  • Design frr modifications that maintain function during biofilm formation

• Integrated strain engineering strategy:

  • Stage 1: Optimize central carbon metabolism (as demonstrated in previous studies )

  • Stage 2: Enhance stress tolerance through genome shuffling or targeted modifications

  • Stage 3: Fine-tune translation machinery components including frr

  • Stage 4: Optimize fermentation conditions for the engineered strain

Previous studies have demonstrated that overexpression of succinic acid biosynthetic machinery, particularly malate dehydrogenase, enhances flux to succinic acid . Combining these approaches with translation optimization through frr engineering could potentially address bottlenecks in protein synthesis for key metabolic enzymes.

What are the most promising approaches for engineering frr to enhance its function under industrial fermentation conditions?

Several promising approaches can enhance frr function under industrial fermentation conditions:

• Directed evolution for acid tolerance:

  • Error-prone PCR to generate frr variant libraries

  • Selection under increasingly acidic conditions

  • High-throughput screening for variants supporting growth and SA production at low pH

• Rational design based on structure-function relationships:

  • Engineering surface charge distribution to maintain function at low pH

  • Stabilizing key interaction interfaces with ribosomal components

  • Modifying regulatory sites to optimize activity under stress

• Hybrid approaches:

  • Semi-rational design focusing on flexible regions identified from molecular dynamics

  • Structure-guided recombination with frr from acid-tolerant organisms

  • Targeted randomization of specific residue clusters

• Expression optimization:

  • Developing acid-inducible promoters for frr expression

  • Engineering translation efficiency through codon optimization

  • Designing synthetic ribosome binding sites for optimal expression

Previous work has shown that A. succinogenes can be engineered for improved acid tolerance through genome shuffling, with modified strains able to grow at pH as low as 3.5 and showing significant improvements in succinic acid yields . Applying similar approaches specifically to frr could yield variants that support translation efficiency under industrial fermentation conditions.

How can I design experiments to evaluate the industrial relevance of frr-engineered A. succinogenes strains?

To evaluate industrial relevance of frr-engineered strains, design experiments that simulate industrial conditions while measuring relevant performance metrics:

• Scale-up fermentation studies:

  • Bench-scale bioreactors (5-10L) simulating industrial parameters

  • Fed-batch processes with defined feeding strategies

  • Extended fermentation times (>72 hours)

  • Evaluation of performance consistency across replicate runs

• Key performance indicators to measure:

  • Succinic acid titer (g/L), with industrial relevance typically requiring >50 g/L

  • Yield (g succinic acid/g substrate), targeting >0.8 g/g

  • Productivity (g/L/h), with industrial targets typically >1 g/L/h

  • Product purity (percentage of succinic acid relative to other organic acids)

  • Strain stability over multiple generations

• Substrate flexibility assessment:

  • Performance on industrial carbon sources (corn stover hydrolysates, molasses)

  • Tolerance to inhibitors present in lignocellulosic feedstocks

  • Ability to utilize mixed sugar streams

• Comparative analysis framework:

  • Direct comparison with parent strains under identical conditions

  • Benchmarking against reported performance of commercial strains

  • Economic analysis of improvements (cost per kg of product)

Previous studies have evaluated A. succinogenes strains in 5L bioreactors, maintaining pH at 4.8 with 7.0M NH₄OH, achieving succinic acid yields of 31.2 g/L . This provides a baseline for comparison when evaluating frr-engineered strains, with improvements in titer, yield, or productivity directly demonstrating industrial relevance.

What are the most common challenges when working with recombinant frr and how can they be addressed?

Common challenges when working with recombinant frr include:

• Protein solubility issues:

  • Challenge: frr often forms inclusion bodies when overexpressed

  • Solutions:

    • Lower induction temperature (16-20°C)

    • Use solubility-enhancing fusion tags (SUMO, MBP)

    • Co-express with chaperones (GroEL/GroES)

    • Optimize induction conditions (lower IPTG, later induction)

• Protein stability problems:

  • Challenge: Purified frr may aggregate or lose activity during storage

  • Solutions:

    • Include stabilizing agents (10% glycerol, 150-300 mM NaCl)

    • Optimize buffer pH to 7.0-8.0

    • Store at -80°C in small aliquots

    • Add reducing agents (1-5 mM DTT or TCEP)

• Activity assay limitations:

  • Challenge: Measuring frr activity often requires complex reconstituted systems

  • Solutions:

    • Develop simplified assays focusing on specific aspects (e.g., ribosome binding)

    • Use coupled enzyme assays that produce measurable signals

    • Implement fluorescence-based assays for higher sensitivity

• Expression toxicity:

  • Challenge: High-level expression of translation factors can inhibit host growth

  • Solutions:

    • Use tight expression control (glucose repression, lower temperature)

    • Express in specialized strains (C41/C43 for toxic proteins)

    • Balance expression levels with cell growth using auto-induction media

Each of these challenges requires systematic optimization, often specific to the particular construct and expression system being used.

How can I troubleshoot unexpected metabolic changes in frr-modified A. succinogenes strains?

When encountering unexpected metabolic changes in frr-modified strains, implement a systematic troubleshooting approach:

• Verify frr expression and function:

  • Confirm frr expression levels by RT-qPCR and Western blotting

  • Assess ribosome profiles using sucrose gradient centrifugation

  • Measure global protein synthesis rates using metabolic labeling

• Investigate metabolic pathway activities:

  • Measure key enzyme activities in central carbon metabolism

  • Quantify metabolic intermediates to identify accumulation points

  • Monitor redox balance (NAD+/NADH ratio)

  • Assess energy charge (ATP/ADP/AMP levels)

• Examine stress responses:

  • Check for upregulation of stress-response genes

  • Assess membrane integrity and potential changes

  • Measure reactive oxygen species (ROS) levels

  • Evaluate cell morphology using microscopy

• Consider indirect effects:

  • Examine translation of specific mRNAs (especially metabolic enzymes)

  • Look for changes in protein turnover rates

  • Investigate potential regulatory RNA effects

  • Consider changes in growth phase timing

Previous studies have shown that removal of competing carbon pathways in A. succinogenes can trigger unexpected by-product formation (e.g., lactic acid) . Similar compensatory mechanisms might be activated in response to translation machinery modifications, requiring careful analysis to uncover the underlying regulatory connections.

What controls and validation experiments are essential when publishing research on recombinant A. succinogenes frr?

Essential controls and validation experiments for publishing research on recombinant A. succinogenes frr include:

• Expression and purification validation:

  • SDS-PAGE and Western blot confirmation of protein identity

  • Mass spectrometry verification of protein sequence

  • Size exclusion chromatography to confirm oligomeric state

  • Circular dichroism to verify secondary structure elements

• Functional validation:

  • In vitro activity assays with appropriate controls

  • Complementation tests in conditional knockout strains

  • Dose-response relationships for activity measurements

  • Comparative analysis with well-characterized frr proteins

• Phenotypic characterization controls:

  • Empty vector controls for all expression studies

  • Wild-type frr expressed at similar levels

  • Growth curves under multiple conditions

  • Controls for metabolic analysis without frr modification

• Reproducibility and statistical validation:

  • Minimum of three biological replicates for all experiments

  • Appropriate statistical tests with reported p-values

  • Technical replicates for all analytical measurements

  • Independent validation of key findings using alternative methods

• Strain authentication:

  • Whole genome sequencing to confirm strain identity and detect any secondary mutations

  • Strain deposition in a public culture collection

  • Detailed description of strain construction and maintenance

These controls ensure that observed effects can be confidently attributed to frr modifications rather than experimental artifacts or secondary effects, providing a solid foundation for published research in this field.