Recombinant Clostridium kluyveri Triosephosphate isomerase (tpiA)

What is the function of triosephosphate isomerase in Clostridium kluyveri metabolism?

Triosephosphate isomerase (TPI) in C. kluyveri, as in other organisms, catalyzes a key reaction in glycolysis, converting dihydroxyacetone phosphate to glyceraldehyde-3-phosphate. This reaction is essential for central carbon metabolism and energy production. In C. kluyveri, which can grow autotrophically in co-culture conditions, TPI plays an important role in the organism's unique metabolism that enables chain elongation processes when grown with acetogens . The enzyme belongs to the triosephosphate isomerase (TIM) barrel domain metallolyase superfamily, a structural classification shared with several other metabolic enzymes .

How can the tpiA gene be identified and verified in Clostridium kluyveri isolates?

Species-specific internal fragments of the tpi gene can be targeted using PCR-based approaches for identification purposes. Based on techniques used for related Clostridium species, researchers should design primers targeting conserved regions of the tpiA gene . For verification of C. kluyveri specifically, molecular techniques comparing the sequence with characterized reference strains are recommended. A multiplex PCR approach similar to that used for C. difficile identification could be adapted, where primers target a species-specific internal fragment of the tpi gene alongside other identifying markers . The amplified fragments should then be sequenced and compared to database entries to confirm identity and detect any strain variations.

What genomic features distinguish C. kluyveri tpiA from other clostridial triosephosphate isomerases?

While the search results don't provide specific distinguishing features of C. kluyveri tpiA, comparative genomic analysis approaches would involve sequence alignment with other clostridial TPI genes. When analyzing the tpiA gene from C. kluyveri, researchers should examine sequence conservation patterns, particularly in catalytic domains. For experimental verification, species-specific internal fragments can be designed for PCR identification, similar to approaches used with C. difficile where the tpi gene provided reliable species identification when distinguished from 11 other Clostridium species . Analysis would include comparison of GC content, codon usage bias, and regulatory regions that might influence expression levels.

What expression systems are optimal for producing recombinant C. kluyveri triosephosphate isomerase?

Based on approaches used for related enzymes, E. coli expression systems using chaperone co-expression have proven effective for clostridial enzymes. For example, with Re-citrate synthase from C. kluyveri, researchers successfully cloned and overexpressed the gene in E. coli together with the genes encoding the chaperone GroEL . Similar approaches would be recommended for tpiA expression. The recombinant protein can be tagged (e.g., with a C-terminal Strep-tag) to facilitate purification . When designing expression constructs, codon optimization for E. coli may improve yields, and expression under control of strong inducible promoters such as T7 is advisable.

What purification challenges are specific to recombinant C. kluyveri TPI and how can they be overcome?

Purification of recombinant clostridial enzymes often faces challenges with solubility and proper folding. Based on approaches used for other recombinant clostridial proteins, affinity chromatography using tags like the Strep-tag system can be effective . For separation from chaperone proteins (which are often co-expressed to improve folding), techniques such as incubation with ATP, K⁺, and Mg²⁺ have been successful . Researchers should monitor enzyme activity throughout purification to ensure the protein remains functional. Additionally, maintaining anaerobic conditions during protein work may be important for preserving the native structure and function of enzymes from anaerobic organisms like C. kluyveri.

How can researchers verify the quality and activity of purified recombinant TPI?

Quality assessment should include multiple approaches:

• SDS-PAGE analysis to confirm size and purity

• Enzymatic activity assays measuring the conversion between dihydroxyacetone phosphate and glyceraldehyde-3-phosphate

• Structural verification through circular dichroism spectroscopy to confirm proper folding

• Mass spectrometry to verify the intact mass and potential post-translational modifications

For activity assays, researchers can measure the specific activity (units/mg protein) similar to approaches used for other recombinant enzymes . Metal dependency should be evaluated, as related enzymes from Clostridium species have shown enhanced activity in the presence of divalent metal ions such as Mn²⁺ or Co²⁺ .

What kinetic parameters characterize the catalytic efficiency of recombinant C. kluyveri TPI?

A comprehensive kinetic characterization would include determination of:

• Km values for both substrates (dihydroxyacetone phosphate and glyceraldehyde-3-phosphate)

• kcat (turnover number)

• kcat/Km (catalytic efficiency)

• Optimal pH and temperature ranges

• Effects of potential inhibitors

While specific values for C. kluyveri TPI are not provided in the search results, approaches similar to those used for other TPIs would be appropriate. For example, with recombinant S. japonicum TPI, researchers determined a Km value of 406.7 μM using glyceraldehyde-3-phosphate as substrate . Comparable methodologies could be applied to characterize C. kluyveri TPI, with enzyme assays conducted at varying substrate concentrations to generate Michaelis-Menten plots.

How does metal ion dependency affect C. kluyveri TPI activity and stability?

Based on findings with other clostridial enzymes, researchers should investigate the effects of various divalent metal ions on TPI activity. For example, Re-citrate synthase from C. kluyveri contained stoichiometric amounts of Ca²⁺ as isolated but achieved higher specific activities in the presence of Mn²⁺ (1.2 U/mg) or Co²⁺ (2.0 U/mg) . For TPI characterization, activity assays should be conducted with various concentrations of different metal ions (Ca²⁺, Mg²⁺, Mn²⁺, Co²⁺, Zn²⁺) to determine optimal metal cofactor requirements. Metal content analysis using atomic absorption spectroscopy or inductively coupled plasma mass spectrometry can determine metal stoichiometry in the purified enzyme.

What methods are most effective for monitoring TPI activity in complex biological systems?

Effective methods include:

• Coupled enzyme assays that link TPI activity to NAD(P)H oxidation/reduction, allowing spectrophotometric monitoring

• Metabolic flux analysis using isotope labeling (e.g., 13C) to track carbon flow through the pathway

• In situ activity staining following native PAGE separation

• Real-time monitoring of substrate/product levels using mass spectrometry or NMR

For co-culture systems (e.g., C. kluyveri with other Clostridium species), enzyme activity can be correlated with species-specific cell counts obtained through techniques like fluorescence in situ hybridization followed by flow cytometry (FISH-FC), as demonstrated with C. kluyveri and C. carboxidivorans co-cultures . This allows researchers to distinguish metabolic contributions of individual species in mixed cultures.

What structural features define the TIM barrel domain in C. kluyveri TPI?

The TIM barrel is a highly conserved protein fold consisting of eight α-helices and eight parallel β-strands that alternate along the protein backbone. In C. kluyveri TPI, as with other TPI enzymes, this domain is expected to form the core catalytic structure. While specific structural data for C. kluyveri TPI is not provided in the search results, the enzyme belongs to the triosephosphate isomerase (TIM) barrel domain metallolyase superfamily . Detailed structural characterization would require X-ray crystallography or cryo-electron microscopy studies of the purified recombinant enzyme. Computational homology modeling based on closely related TPI structures could provide preliminary structural insights before experimental determination.

How can researchers determine the quaternary structure of recombinant C. kluyveri TPI?

Methods for quaternary structure determination include:

• Size exclusion chromatography to estimate native molecular weight

• Analytical ultracentrifugation to determine sedimentation coefficients

• Native gel electrophoresis comparing migration with known standards

• Cross-linking studies followed by SDS-PAGE analysis

• Multi-angle light scattering for precise molecular weight determination

Based on related enzymes, TPI typically forms homodimers, but the specific quaternary structure of C. kluyveri TPI should be experimentally verified. For comparison, some recombinant clostridial enzymes form homotetramers, as seen with Re-citrate synthase from C. kluyveri (4 × 72,892 Da) .

How can recombinant C. kluyveri TPI be used to enhance product yields in synthetic microbial co-cultures?

Recombinant TPI could be employed to optimize glycolytic flux in synthetic co-cultures involving C. kluyveri. When designing such systems, researchers should consider:

• Expression levels - balancing TPI activity with other pathway enzymes to prevent metabolic bottlenecks

• Compatibility with partner organisms - ensuring metabolic integration when pairing C. kluyveri with partners like C. carboxidivorans in syngas fermentation systems

• Growth conditions - optimizing parameters like pH and substrate availability that affect both TPI activity and co-culture stability

In synthetic co-cultures of C. kluyveri with C. carboxidivorans, manipulating TPI expression could potentially enhance chain elongation processes that lead to production of longer-chain alcohols like butanol and hexanol . The ability to monitor individual species' contributions using techniques like FISH-FC would be essential for optimizing such systems.

What role does TPI play in C. kluyveri's unique metabolism during syntrophic growth?

In syntrophic growth conditions, C. kluyveri demonstrates specialized metabolic capabilities for chain elongation. While the search results don't directly address TPI's specific role in syntrophy, studies of C. kluyveri in co-culture with C. carboxidivorans show that C. kluyveri participates in converting primary fermentation products (acetate and ethanol) to butyrate and caproate, which can then be reduced to butanol and hexanol by C. carboxidivorans .

The metabolic flux through glycolysis, where TPI operates, likely influences the availability of reducing equivalents needed for these reactions. Researchers investigating this aspect should examine TPI expression levels under different co-culture conditions using techniques like qRT-PCR, similar to approaches used for detecting transcripts of other metabolic genes in axenic versus syntrophic cultures .

How does C. kluyveri TPI compare functionally with TPI enzymes from other industrially relevant Clostridium species?

A comprehensive comparative analysis would include:

• Sequence alignments to identify conserved and variable regions

• Kinetic parameter comparison (Km, kcat, substrate specificity)

• Stability under various conditions (temperature, pH, oxidative stress)

• Regulatory mechanisms controlling expression

While the search results don't provide direct comparisons of TPI across Clostridium species, approaches used for other enzymes can be applied. For instance, when studying C. difficile, researchers were able to use tpi gene amplification to distinguish it from 11 other Clostridium species , suggesting sufficient sequence divergence for species discrimination while maintaining core catalytic function. For industrial applications, comparing the stability and activity of TPI from different clostridia under process-relevant conditions would be valuable.

What differential expression patterns of tpiA are observed when comparing axenic versus syntrophic growth of C. kluyveri?

To investigate differential expression:

• Quantitative reverse transcriptase PCR (qRT-PCR) can detect transcripts of the tpiA gene under different growth conditions, similar to approaches used for other metabolic genes in C. kluyveri

• RNA-seq analysis would provide genome-wide context for tpiA expression changes

• Proteomics approaches could confirm if transcript-level changes translate to protein abundance differences

Results for other metabolic genes have shown that C. kluyveri can express different enzyme profiles when grown axenically versus in syntrophic co-cultures . Researchers should correlate tpiA expression with metabolic shifts, particularly in carbon flux through glycolysis versus alternative pathways that might be favored under different growth conditions.

How can CRISPR-Cas9 techniques be optimized for genetic manipulation of the tpiA gene in C. kluyveri?

Implementing CRISPR-Cas9 for tpiA modification in C. kluyveri would require:

• Optimization of transformation protocols, addressing the challenge of low transformation efficiency often observed in Clostridium species

• Design of specific guide RNAs targeting tpiA with minimal off-target effects

• Selection of appropriate promoters for Cas9 and guide RNA expression that function efficiently in C. kluyveri

• Development of effective homology-directed repair templates for precise gene editing

While the search results don't provide specific CRISPR protocols for C. kluyveri tpiA, forward and reverse genetics approaches have been developed for industrially important Clostridia . Researchers should adapt these techniques, potentially incorporating improvements in transformation methods to overcome the "low frequencies of plasmid transfer by electroporation" noted as a challenge in clostridial genetics .

What are the most effective strategies for expressing recombinant C. kluyveri TPI as a potential vaccine candidate?

Based on approaches used for other TPI enzymes as vaccine candidates, researchers should consider:

• Expression system selection - bacterial systems like E. coli have shown success for producing recombinant TPI with high enzymatic activity

• Purification under non-denaturing conditions to maintain native conformation and activity

• Quality control assessing both enzymatic activity and absence of contaminants

• Formulation with appropriate adjuvants based on the target immune response

For example, recombinant S. japonicum TPI (re-SjcTPI) was successfully expressed in bacteria and purified to >98% homogeneity under non-denaturing conditions, maintaining high enzymatic activity (7687 units/mg protein) . Similar approaches could be applied to C. kluyveri TPI if being developed as a potential vaccine antigen, though the search results don't specifically address vaccines targeting C. kluyveri.

How can isotope labeling experiments with recombinant TPI illuminate C. kluyveri's metabolic pathways in complex fermentation processes?

Isotope labeling experiments could include:

• Use of 13C-labeled substrates to track carbon flux through TPI-catalyzed reactions

• Metabolic flux analysis comparing wild-type versus TPI-overexpressing strains

• Position-specific labeling to determine stereospecificity of the enzyme similar to approaches used for citrate synthase

For instance, researchers studying Re-citrate synthase in Syntrophus aciditrophicus used 14C-labeled substrates to determine enzyme stereospecificity by tracking the labeled atoms through the reaction pathway . Similar approaches could be applied to study C. kluyveri TPI, particularly in co-culture conditions where distinguishing metabolic contributions between species is challenging. These experiments would be particularly valuable for understanding C. kluyveri's role in syntrophic relationships that enable chain elongation processes for producing higher alcohols .

What approaches can resolve low expression yields of recombinant C. kluyveri TPI in heterologous hosts?

To address low expression yields:

• Co-express molecular chaperones (e.g., GroEL/GroES) to aid proper protein folding

• Optimize codon usage for the expression host

• Evaluate different fusion tags and their positions (N- versus C-terminal)

• Test various induction conditions (temperature, inducer concentration, induction time)

• Consider alternative expression hosts beyond E. coli, such as Bacillus or yeast systems

The successful approach used for Re-citrate synthase from C. kluyveri involved co-expression with the chaperone GroEL in E. coli, followed by purification using a C-terminal Strep-tag . Similar strategies could be applied to overcome expression challenges with recombinant TPI from C. kluyveri.

How can researchers address enzyme instability issues with purified recombinant C. kluyveri TPI?

Stability enhancement strategies include:

• Screening buffer compositions with various stabilizing agents (glycerol, trehalose, specific metal ions)

• Identifying optimal pH and temperature storage conditions

• Evaluating the effect of reducing agents to prevent oxidative damage

• Testing protein engineering approaches to enhance stability while maintaining activity

Metal ion dependency should be thoroughly investigated, as related clostridial enzymes have shown enhanced activity in the presence of specific divalent cations. For example, Re-citrate synthase from C. kluyveri contained Ca2+ but showed higher activity with Mn2+ or Co2+ , suggesting that metal cofactor optimization could also enhance stability.

Note: This table presents a hypothetical framework for investigating metal ion effects on C. kluyveri TPI based on patterns observed with other clostridial enzymes.

What are the most promising applications of engineered C. kluyveri TPI variants in synthetic biology?

Engineered TPI variants could enable:

• Enhanced thermal stability for industrial bioprocesses

• Modified substrate specificity to accommodate non-native metabolic pathways

• Altered allosteric regulation to optimize flux through glycolysis

• Immobilization-compatible variants for continuous bioprocessing

In the context of synthetic co-cultures for biofuel production, engineered TPI variants could help optimize the carbon flux when C. kluyveri is paired with partners like C. carboxidivorans . This could potentially enhance the production of valuable products like butanol and hexanol through more efficient chain elongation processes.

How might systems biology approaches integrate TPI function into genome-scale metabolic models of C. kluyveri?

Systems biology approaches would involve: