Recombinant Clostridium phytofermentans Triosephosphate isomerase (tpiA)

What is the biological function of tpiA in Clostridium phytofermentans metabolism? (Basic)

Triosephosphate isomerase (tpiA) in C. phytofermentans plays a crucial role in glycolysis by catalyzing the reversible interconversion of dihydroxyacetone phosphate (DHAP) to glyceraldehyde 3-phosphate (G3P). This reaction is essential for energy metabolism, particularly during fermentation of plant biomass. As C. phytofermentans is known for its ability to ferment lignocellulosic material primarily to ethanol, tpiA functions as a key metabolic enzyme that supports the conversion of plant-derived sugars into fermentation products . The enzyme maintains a balance between these two triose phosphates, ensuring efficient carbon flow through the glycolytic pathway. Without functional tpiA, the organism would accumulate DHAP, resulting in reduced ATP production and compromised fermentation capability.

What expression systems are most effective for recombinant C. phytofermentans tpiA production? (Basic)

Several expression systems have been validated for recombinant C. phytofermentans tpiA production:

E. coli has been successfully used to express C. phytofermentans tpiA as indicated in several product datasheets . For functional studies, researchers typically employ a pET expression system with a C-terminal His6-tag to facilitate purification. When expressing in E. coli, optimal conditions include induction with 50-100 μM IPTG at 16°C overnight to prevent inclusion body formation, similar to protocols used for other C. phytofermentans proteins .

What genetic transformation methods are available for C. phytofermentans and how might they be applied to tpiA studies? (Advanced)

Several genetic transformation approaches have been developed specifically for C. phytofermentans:

• Electroporation method: A simple benchtop electroporation protocol has been developed that doesn't require an anaerobic glovebox. This involves preparing competent cells with 0.5M sucrose and applying a specific electric pulse followed by immediate recovery in rich medium. This method yields approximately 10^3 transformants/μg DNA .

• Interspecific conjugation: Conjugation with E. coli can be used to transfer plasmids containing resistance markers and replication origins that function in C. phytofermentans. This approach has been successfully used to disrupt genes including the GH9 gene, showing it could be applied to tpiA modification .

• Group II intron-based gene disruption: For targeted chromosomal insertions without selection, a designed group II intron system has been demonstrated. This system can be adapted for tpiA disruption to study its metabolic significance .

Specifically for tpiA studies, researchers have determined that plasmids with pBP1, pCB102, and pAMβ1 origins are efficiently maintained in C. phytofermentans and can be selected using erythromycin (ermB), spectinomycin (aad9), or thiamphenicol (catP) resistance markers. The tetracycline resistance gene (tetA) is not functional in this organism .

How can CRISPRi systems be designed for modulating tpiA expression in C. phytofermentans? (Advanced)

Development of a CRISPRi system for tpiA expression modulation requires careful design considerations:

• CRISPR effector selection: LbCas12a from Lachnospiraceae bacterium ND2006 has been validated in C. phytofermentans, chosen because it originates from bacteria in the same family. This system appears less toxic than Cas9-based systems reported in other Clostridia .

• PAM site identification: For targeting tpiA, researchers must identify the LbCas12a PAM (5'-TTTV-3') sites within the promoter or early coding region. Studies show that while the Cas12a PAM is less common across the C. phytofermentans genome compared to Cas9 PAM sites, it is more abundant in highly expressed promoter regions .

• Inducible expression system: To mitigate potential toxicity, an anhydrotetracycline (aTc)-inducible system has been engineered using:

  • TetR repressor driven by the miniPthl promoter

  • aTc-inducible dLbCas12a

  • gRNA targeting the gene of interest

The system allows for tunable repression with aTc concentrations between 5-200 ng/mL providing a range of expression levels without affecting bacterial growth . For tpiA targeting, guide RNA design should focus on the promoter region or early in the coding sequence for maximum repression efficiency.

What purification strategies yield the highest purity and activity for recombinant C. phytofermentans tpiA? (Basic)

The following purification workflow has been demonstrated to yield >85% pure and active recombinant C. phytofermentans tpiA:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using nickel-nitrilotriacetic acid resin equilibrated in 30 mM Tris-HCl, pH 8.0, 1 mM CaCl₂. Elution with 100 mM imidazole in the same buffer .

• Secondary purification options:
  a. Ion exchange chromatography: Using Q-Sepharose fast flow equilibrated in 30 mM Tris-HCl, pH 8.0, 1 mM CaCl₂ with elution by linear gradient of 0-500 mM NaCl.
  b. Size exclusion chromatography: For highest purity, concentrate the IMAC fraction to 2 mL and apply to Superdex 200 10/300 GL resin equilibrated in 30 mM Tris-HCl, pH 8.0, 1 mM CaCl₂, 150 mM NaCl .

• Final conditioning: Dialysis by ultrafiltration against 10 mM Tris-HCl, pH 8.0, 1 mM CaCl₂ .

• Storage stability: Addition of 50% glycerol allows storage at -80°C for up to 12 months with minimal activity loss .

Protein concentration can be estimated by absorbance at 280 nm using extinction coefficients calculated from the amino acid sequence via ProtParam. Activity assays should monitor the conversion of DHAP to G3P spectrophotometrically at 340 nm using coupling enzymes such as glycerol-3-phosphate dehydrogenase.

How does methylome analysis improve transformation efficiency in C. phytofermentans and how might this affect tpiA studies? (Advanced)

The methylome analysis of C. phytofermentans has revealed critical epigenetic barriers that must be overcome for efficient transformation:

• DNA modification patterns: Two complementary sequencing methods have identified specific methylation patterns in C. phytofermentans:

  • RIMS-seq revealed m5C modification at 5′-GATC-3′ sites (p = 1.23 × 10^-4061)

  • SMRT sequencing detected m6A modification preferentially at 5′-CTKCAG-3′ motifs

• Methylation impact: These modifications represent restriction-modification systems that protect C. phytofermentans from foreign DNA but also create barriers for introducing recombinant constructs.

• Improving transformation efficiency: For tpiA studies, researchers can:

  • Express C. phytofermentans methyltransferases in E. coli strains used for plasmid preparation

  • Use E. coli strains lacking methyl-specific restriction systems (such as dcm⁻/dam⁻ strains)

  • Prepare plasmids from C. phytofermentans itself for subsequent transformations

Transformation efficiency improvements of 10-100 fold have been observed when plasmid DNA is appropriately methylated prior to electroporation . For tpiA expression or knockout studies, this approach significantly increases the likelihood of successful genetic manipulation and enables more complex genetic engineering strategies.

What are the implications of tpiA activity on biofuel production by C. phytofermentans? (Advanced)

The activity of tpiA has profound implications for biofuel production in C. phytofermentans:

• Carbon flux regulation: As a glycolytic enzyme, tpiA influences the flow of carbon from plant biomass-derived sugars toward ethanol production. Modeling suggests tpiA may be a rate-limiting step during high cellulose conversion rates .

• Redox balance: The activity of tpiA affects NADH/NAD⁺ ratios through its position in glycolysis, which subsequently impacts the distribution of fermentation products (ethanol vs. acetate and hydrogen).

• Engineering potential: Modulating tpiA expression levels could redirect carbon flux:

  • Increased tpiA expression may accelerate glycolysis, potentially increasing ethanol yields

  • Careful downregulation might create a bottleneck that could increase flux through alternative pathways that may produce valuable products

Studies on amino acid catabolism-directed biofuel production in the related Clostridium sticklandii have established mathematical models that could be adapted to predict the effects of tpiA modulation in C. phytofermentans metabolic engineering .

How does C. phytofermentans tpiA structure compare to tpiA from other Clostridium species? (Basic)

Structural comparison of C. phytofermentans tpiA with other Clostridium species reveals significant conservation with specific variations:

The amino acid sequence analysis reveals that all Clostridial tpiA enzymes share the canonical TIM barrel fold with eight alternating α-helices and β-strands. The active site residues (including the catalytic glutamate) are highly conserved across all species, explaining the preserved enzymatic function.

Key functional regions found in all Clostridial tpiA proteins include:

• The conserved IAGNW motif near the N-terminus, crucial for maintaining the structural integrity of the active site

• A flexible loop region that closes over the active site during catalysis

• The catalytic glutamate residue essential for proton transfer during catalysis

These structural comparisons provide insights for experiments involving heterologous expression or protein engineering studies of tpiA .

What assays can be used to measure recombinant C. phytofermentans tpiA enzyme activity? (Basic)

Several assays have been validated for measuring the activity of recombinant C. phytofermentans tpiA:

• Coupled spectrophotometric assay: The most common method involves coupling tpiA activity to glycerol-3-phosphate dehydrogenase and monitoring NADH oxidation at 340 nm.

  Reaction setup:

  • 50 mM Tris-HCl buffer (pH 7.5)

  • 10 mM MgCl₂

  • 0.5 mM NADH

  • 1 mM DHAP (substrate)

  • 1-5 U/mL α-glycerophosphate dehydrogenase (coupling enzyme)

  • 0.1-1 μg purified tpiA

  • Monitor decrease in A₃₄₀ at 25°C

• Direct assay by NMR: For detailed kinetic studies, ¹³C or ³¹P NMR can be used to directly track the interconversion between DHAP and G3P without coupling enzymes.

• High-performance anion exchange chromatography (HPAEC): Similar to methods used for analyzing C. phytofermentans enzymes, HPAEC can be employed to quantify substrate and product :

  • Dionex CarboPac PA1 column (4 × 250 mm)

  • Elution with 0.1 M NaOH and 0.5 M sodium acetate gradient

  • Detection by pulsed amperometric detection

  • Analysis against standards of known concentration

• Enzyme kinetics determination: Comprehensive kinetic analysis can be performed using Michaelis-Menten and Lineweaver-Burk plots, determining parameters including:

  • K_m for DHAP and G3P (typically in the range of 0.1-0.5 mM)

  • k_cat (catalytic constant)

  • Enzyme efficiency (k_cat/K_m)

  • pH optimum (typically pH 7.0-8.0 for Clostridial tpiA)

  • Temperature optimum (typically 30-37°C)

How can site-directed mutagenesis of tpiA be used to study C. phytofermentans metabolism? (Advanced)

Site-directed mutagenesis of tpiA provides a powerful approach to understanding C. phytofermentans metabolism:

• Key residues for targeted mutagenesis:

  • Catalytic residues: The glutamate that acts as a proton acceptor/donor

  • Substrate binding loop residues: Modifications can alter substrate specificity or catalytic efficiency

  • Dimer interface residues: Changes may affect protein stability and activity regulation

• Methodological approach:
  a. Design and create mutants: Using PCR-based site-directed mutagenesis with the tpiA gene cloned in pET28a(+) vector at NcoI/XhoI sites with C-terminal His-tag .
  b. Express in E. coli: Transform BL21(DE3), induce with 50-100 μM IPTG at 16°C overnight to prevent inclusion body formation.
  c. Purify and characterize: Using IMAC and ion exchange chromatography, followed by enzyme activity assays and structural characterization (CD spectroscopy).
  d. In vivo analysis: Introduce mutations into C. phytofermentans chromosome using group II intron or CRISPR-Cas systems .

• Metabolic insights from tpiA mutations: