Recombinant Mannheimia succiniciproducens Uracil phosphoribosyltransferase (upp)

What is Mannheimia succiniciproducens and why is it significant for biochemical research?

Mannheimia succiniciproducens is a capnophilic (CO2-loving) Gram-negative facultative anaerobic rumen bacterium that efficiently produces succinic acid from various carbon sources, including pentose sugar (xylose), hexose sugars (fructose and glucose), and disaccharides (lactose, maltose, and sucrose) . It has gained significant interest in biotechnology research due to its efficient succinic acid production capabilities, a key industrial chemical with numerous applications. The sequenced strain MBEL55E has been extensively characterized for its metabolic pathways and has become a model organism for studying carbon metabolism in rumen bacteria.

For researchers working with M. succiniciproducens, specialized anaerobic cultivation techniques are essential. Standard protocols typically involve using defined minimal media (such as MH5S) supplemented with specific carbon sources under anaerobic conditions with CO2 saturation . Growth experiments must maintain strict anaerobic conditions with proper CO2 levels, as these significantly impact metabolic flux distribution. For genetic manipulation, established techniques include gene knockout through homologous recombination, which has been successfully applied to study various metabolic genes in this organism .

What is Uracil phosphoribosyltransferase (upp) and what role does it play in bacterial metabolism?

Uracil phosphoribosyltransferase (UPP) is an enzyme that catalyzes the conversion of uracil to uridine monophosphate (UMP) using phosphoribosyl pyrophosphate (PRPP) as a co-substrate. This reaction is critical in the pyrimidine salvage pathway, which allows bacteria to recycle nucleobases rather than synthesizing them de novo, thus conserving energy. In bacterial systems, the upp gene and its encoded enzyme have significant importance for both basic metabolism and as genetic tools.

From a methodological perspective, researchers studying UPP typically employ spectrophotometric assays to measure enzyme activity. These assays often involve coupling the UPP reaction to other enzymatic reactions that generate measurable products, similar to methods described for other enzymes in M. succiniciproducens . For example, UPP activity can be quantified by coupling UMP formation to subsequent reactions that generate NADH, which can be measured spectrophotometrically.

In genetic engineering applications, the upp gene serves as both a positive and negative selection marker. When present, it allows cells to grow on media where uracil is the sole pyrimidine source. Conversely, UPP can convert the toxic analog 5-fluorouracil to 5-fluorouridine monophosphate, which is lethal to cells – a property that enables counterselection strategies in bacterial genetic engineering.

What genetic engineering techniques have been established for Mannheimia succiniciproducens?

Several genetic engineering techniques have been successfully applied to M. succiniciproducens, providing a methodological foundation for researchers interested in studying the upp gene and its protein product:

• Gene knockout technology has been well-established for M. succiniciproducens. Multiple genes including MS0784, MS0807, MS0909, MS1233, and MS1237 have been successfully knocked out from the chromosome of the wild-type strain MBEL55E . These techniques typically employ homologous recombination-based approaches with appropriate selection markers.

• Metabolic engineering strategies have been implemented to enhance succinic acid production. For example, deleting lactate dehydrogenase (ldh) and pyruvate formate lyase (pfl) genes resulted in increased succinic acid production while reducing byproduct formation under anaerobic conditions . This demonstrates the feasibility of targeted genetic modifications to redirect carbon flux.

• Heterologous gene expression systems have been developed, as evidenced by the successful introduction of Corynebacterium glutamicum malate dehydrogenase (CgMDH) into M. succiniciproducens to enhance succinic acid production .

When applying these techniques to study UPP, researchers would follow similar methodological approaches: designing targeting constructs with homology arms flanking the upp gene, introducing selective markers, and confirming successful modifications through PCR and phenotypic testing. For heterologous expression of recombinant UPP, established expression vectors and transformation protocols would serve as starting points for optimization.

How do the kinetic properties of recombinant Mannheimia succiniciproducens UPP compare with UPPs from other bacterial species?

When characterizing the kinetic properties of recombinant M. succiniciproducens UPP, researchers should employ methodological approaches that allow direct comparison with UPPs from other bacterial species. This comparison would reveal unique features that might relate to M. succiniciproducens' distinctive metabolism.

The kinetic parameters of enzymes from M. succiniciproducens can vary significantly from those of other bacteria. For example, when comparing malate dehydrogenase (MDH) between M. succiniciproducens and C. glutamicum, researchers found that the M. succiniciproducens enzyme (MsMDH) shows lower specific activity at physiological pH and stronger uncompetitive inhibition toward oxaloacetate compared to C. glutamicum MDH (CgMDH) . Similar comparative studies for UPP would involve:

• Determining Km values for both uracil and PRPP substrates using Lineweaver-Burk plots or non-linear regression analysis

• Measuring kcat and catalytic efficiency (kcat/Km) under standardized conditions

• Evaluating pH and temperature profiles to identify optimal conditions

• Assessing substrate specificity using uracil analogs

A standardized experimental design would include expression of recombinant UPPs from different bacterial sources in the same host system, purification using identical protocols, and parallel enzyme assays under identical conditions. This approach minimizes methodological variations that might confound comparisons.

Based on patterns observed with other enzymes, researchers might expect M. succiniciproducens UPP to display kinetic properties adapted to its anaerobic, CO2-rich natural environment, potentially showing different substrate affinities or inhibition patterns compared to UPPs from aerobic organisms.

What are the optimal expression and purification conditions for obtaining active recombinant Mannheimia succiniciproducens UPP?

Optimizing expression and purification of recombinant M. succiniciproducens UPP requires systematic investigation of multiple parameters. Based on approaches used for other enzymes from this organism, the following methodological considerations are critical:

• Expression system selection:

  • E. coli-based systems provide high yields but may not reproduce native folding

  • Homologous expression in M. succiniciproducens preserves authentic post-translational modifications

  • C. glutamicum might serve as an intermediate host with similar GC content and codon usage

• Expression conditions optimization:

  • Temperature: Lower temperatures (25-30°C) often promote proper folding

  • Induction protocols: IPTG concentration and induction timing for lac-based systems

  • Carbon source: Different carbon sources affect metabolic state and protein expression

  • Aerobic vs. anaerobic conditions: Given M. succiniciproducens' facultative anaerobic nature, oxygen levels during expression may affect protein folding and activity

• Purification strategy development:

  • Initial clarification: French press or sonication under conditions preserving enzyme activity

  • Affinity chromatography: His-tag systems compatible with activity assays

  • Further purification: Ion exchange or size exclusion chromatography if needed

  • Buffer optimization: Stabilizing agents (glycerol, reducing agents) and pH considerations

Throughout the optimization process, enzyme activity measurements are essential to track active protein yield. The specific activity would be measured in a manner similar to other M. succiniciproducens enzymes, such as the phosphotransferase system where "specific PTS enzyme activity was 0.10 ± 0.01 mU mg of protein−1 in the MBEL55EΔ0784 strain cultured in BHI medium" .

A data-driven approach comparing yield and specific activity across different conditions would guide protocol refinement until optimal conditions are established.

How can structural studies of recombinant Mannheimia succiniciproducens UPP inform enzyme engineering for enhanced activity?

Structural studies of recombinant M. succiniciproducens UPP can provide essential insights for enzyme engineering. The methodological approach would parallel studies of other enzymes from this organism, where "structural comparison of the two MDHs reveals a key residue influencing the specific activity and susceptibility to substrate inhibition" .

For UPP structural characterization, researchers should employ:

• X-ray crystallography:

  • Crystallization condition screening (temperature, pH, precipitants)

  • Data collection at synchrotron facilities

  • Structure determination using molecular replacement with known UPP structures

  • Analysis of active site architecture, focusing on residues interacting with substrates

• Structure-guided mutagenesis:

  • Identification of key catalytic and substrate-binding residues

  • Site-directed mutagenesis to alter these residues

  • Functional characterization of mutants for activity, substrate affinity, and inhibition profiles

  • Iterative optimization based on structure-function relationships

• Molecular dynamics simulations:

  • In silico analysis of enzyme dynamics under different conditions

  • Prediction of conformational changes during catalysis

  • Identification of potential allosteric sites

  • Virtual screening of substrate analogs

These approaches could identify specific residues that influence UPP activity under the unique metabolic conditions of M. succiniciproducens, similar to how structural analysis revealed key differences between M. succiniciproducens and C. glutamicum MDH enzymes .

Engineering targets might include residues affecting substrate binding, catalytic efficiency, pH optimum, or resistance to inhibitors. The ultimate goal would be creating UPP variants with enhanced properties for either basic research or biotechnological applications.

What metabolic impacts result from modulating UPP expression in Mannheimia succiniciproducens?

Investigating how UPP expression levels affect M. succiniciproducens metabolism requires a comprehensive systems biology approach. Methodologically, researchers should:

• Generate strains with varying UPP expression:

  • Knockout mutants (complete deletion of upp gene)

  • Knockdown strains (reduced expression using antisense RNA or CRISPR interference)

  • Overexpression strains (using strong constitutive or inducible promoters)

  • Complementation strains (restored expression in knockout background)

• Conduct comparative phenotypic analyses:

  • Growth rate determination under various conditions

  • Assessment of succinic acid production (the primary metabolic product)

  • Measurement of other organic acids as byproducts

  • Carbon source utilization profiles

• Perform metabolomic analyses:

  • Quantification of intracellular nucleotides and related metabolites

  • Analysis of central carbon metabolism intermediates

  • Metabolic flux analysis using 13C-labeled substrates

  • Comparison of metabolite profiles between wildtype and modified strains

The search results suggest that gene modifications in M. succiniciproducens can significantly alter metabolic flux distributions. For example, deletion of lactate dehydrogenase (ldh) and pyruvate formate lyase (pfl) genes resulted in "increased succinic acid production while producing little organic acids in anaerobic conditions" . Similar metabolic shifts might occur with UPP modulation, potentially affecting the balance between nucleotide synthesis and central carbon metabolism.

Note: This table presents hypothetical data based on patterns observed with other metabolic modifications in M. succiniciproducens.

What are the most effective heterologous expression systems for producing recombinant Mannheimia succiniciproducens UPP?

Selecting the appropriate heterologous expression system for recombinant M. succiniciproducens UPP requires balancing protein yield, activity, and experimental convenience. Based on approaches used for other M. succiniciproducens enzymes, researchers should consider:

• E. coli-based expression systems:

  • BL21(DE3) with pET vectors for T7-driven expression

  • Tuner™ strains for regulated expression level control

  • Arctic Express™ for low-temperature expression

  • Protocol modification: Growth at 25-30°C after induction to improve folding

  • Yields: Typically 10-30 mg purified protein per liter of culture

• Corynebacterium glutamicum expression:

  • Advantages: Similar GC content to M. succiniciproducens

  • Vector systems: pEKEx2 or pCLiK-based vectors

  • Induction: IPTG or constitutive promoters

  • Protocol emphasis: Balanced growth and expression phase management

  • Yields: Usually 5-15 mg purified protein per liter of culture

• Homologous expression in M. succiniciproducens:

  • Vector development based on native plasmids or adapted from related species

  • Transformation protocols using electroporation

  • Selective markers appropriate for M. succiniciproducens

  • Advantages: Native cellular environment, appropriate post-translational modifications

  • Challenges: Lower yields, more complex cultivation requirements

For each system, activity assays should be conducted to determine not just protein yield but functional protein production. Similar to methods used for other M. succiniciproducens enzymes, researchers would measure "specific enzyme activity" in "mU mg of protein−1" across different expression conditions.

A methodical comparison across these systems would involve parallel expressions under optimized conditions for each host, followed by standardized purification and activity measurements to determine which system provides the best combination of yield and activity.

How can UPP be effectively used as a genetic selection marker in Mannheimia succiniciproducens engineering?

UPP offers unique advantages as a genetic selection marker for M. succiniciproducens engineering due to its dual selection capability. A systematic approach to implementing UPP-based selection would include:

• Creating a clean upp knockout strain:

  • Design DNA constructs with homology arms flanking the upp gene

  • Include a temporary selection marker (e.g., antibiotic resistance)

  • Transform M. succiniciproducens using established protocols

  • Select transformants on media containing 5-fluorouracil (5-FU)

  • Confirm deletion by PCR and sequencing

  • Remove temporary marker if present

• Developing complementation vectors:

  • Construct plasmids containing the upp gene under control of various promoters

  • Include additional features (multiple cloning sites, reporter genes)

  • Transform the Δupp strain

  • Select on minimal media lacking exogenous pyrimidines

• Implementing counterselection for marker removal:

  • Design constructs where upp is flanked by direct repeats

  • Allow homologous recombination to loop out the upp gene

  • Select recombinants on 5-FU-containing media

  • Verify marker removal by PCR and phenotypic testing

The advantages of this system include marker recycling capability and the absence of antibiotic resistance genes in final strains. The methodology parallels established gene manipulation techniques in M. succiniciproducens, where researchers have successfully created multiple gene knockouts such as "MBEL55EΔ0784, MBEL55EΔ0807, MBEL55EΔ0909, MBEL55EΔ1233, and MBEL55EΔ1237" .

What enzyme assay methods provide the most reliable activity measurements for recombinant Mannheimia succiniciproducens UPP?

Reliable enzyme assay methods for recombinant M. succiniciproducens UPP should be sensitive, reproducible, and compatible with the enzyme's biochemical properties. Based on assay approaches used for other M. succiniciproducens enzymes, the following methodological options are recommended:

• Direct UMP formation assay:

  • Principle: HPLC-based detection of UMP produced from uracil and PRPP

  • Protocol: Incubate purified UPP with substrates, terminate reaction with acid precipitation, analyze by HPLC

  • Detection: UV absorbance at 260-280 nm

  • Advantages: Direct measurement of product formation

  • Limitations: Requires specialized equipment, lower throughput

• Coupled spectrophotometric assay:

  • Principle: Couple UMP formation to NADH oxidation through auxiliary enzymes

  • Protocol: Include UPP, substrates, and coupling enzymes in reaction mixture

  • Detection: Decrease in absorbance at 340 nm as NADH is oxidized

  • Advantages: Continuous measurement, higher throughput

  • Limitations: Potential interference from coupling enzymes

• Radiometric assay:

  • Principle: Use 14C-labeled uracil and measure incorporation into UMP

  • Protocol: Incubate UPP with labeled uracil and PRPP, separate products by TLC

  • Detection: Scintillation counting or autoradiography

  • Advantages: Highest sensitivity

  • Limitations: Requires radioactive materials, lower throughput

For each assay type, optimization parameters include:

• Buffer composition and pH (typically pH 7.0-8.0)

• Metal ion requirements (usually Mg2+)

• Temperature (30-37°C for M. succiniciproducens enzymes)

• Substrate concentrations spanning the Km values

When reporting enzyme activities, researchers should follow the convention used for other M. succiniciproducens enzymes, where specific activities are reported as "mU mg of protein−1" and kinetic parameters include Km and Vmax values.

How might recombinant Mannheimia succiniciproducens UPP contribute to metabolic engineering for enhanced succinic acid production?

Recombinant M. succiniciproducens UPP could play several roles in metabolic engineering strategies aimed at enhancing succinic acid production. The methodological approach would build upon established techniques where gene deletions have shown significant impacts on succinic acid yields .

• UPP as a selection marker for genetic modifications:

  • Use UPP-based selection/counterselection to create clean gene deletions

  • Target genes encoding competing pathways (ldh, pfl) similar to previous studies where these deletions "resulted in an increased succinic acid production while producing little organic acids in anaerobic conditions"

  • Enable precise chromosomal integrations without antibiotic resistance markers

  • Develop UPP-based CRISPR-Cas9 systems for genome editing

• Nucleotide metabolism engineering:

  • Modulate UPP expression to influence pyrimidine nucleotide pools

  • Examine effects on transcription and translation of key metabolic genes

  • Investigate connections between nucleotide availability and carbon flux

  • Develop strains with optimized nucleotide metabolism for growth and production phases

• Strain development protocols:

  • Sequential genetic modifications using UPP marker recycling

  • Characterization of each modification's impact on succinic acid production

  • Integration with other metabolic engineering strategies

  • Fermentation optimization for engineered strains

The implementation of these strategies would follow methodological approaches similar to those used for introducing heterologous malate dehydrogenase, where "high-inoculum fed-batch fermentation of the final strain expressing cgmdh" showed enhanced production capabilities .

What challenges must be overcome when adapting UPP-based selection systems from model organisms to Mannheimia succiniciproducens?

Adapting UPP-based selection systems from model organisms to M. succiniciproducens presents several methodological challenges that researchers must systematically address:

• Media composition optimization:

  • Develop defined media formulations that enable both positive and negative selection

  • Determine optimal 5-fluorouracil concentrations for counterselection

  • Establish minimal media lacking pyrimidines for positive selection

  • Account for M. succiniciproducens' capnophilic nature in media design

• Gene expression control:

  • Characterize native promoter strengths in M. succiniciproducens

  • Develop constitutive and inducible promoter systems

  • Optimize ribosome binding sites for appropriate UPP expression levels

  • Ensure stable expression without integration into essential genomic regions

• Transformation protocol refinement:

  • Develop electroporation conditions optimized for M. succiniciproducens

  • Determine optimal DNA configuration (linear vs. circular)

  • Establish appropriate recovery conditions after transformation

  • Quantify transformation efficiencies across different conditions

• Selection stringency balancing:

  • Determine minimum UPP expression levels needed for growth in pyrimidine-deficient media

  • Establish maximum UPP levels that still allow growth in 5-FU containing media

  • Develop strategies for handling potential leaky expression

  • Create protocols for managing spontaneous resistance development

The methodology would build upon established genetic manipulation techniques for M. succiniciproducens, where researchers have successfully created deletion mutants such as "MBEL55EΔ0784, MBEL55EΔ0807, MBEL55EΔ0909, MBEL55EΔ1233, and MBEL55EΔ1237" , adapting these approaches to incorporate UPP-based selection.

How does the biochemical environment affect recombinant UPP activity in Mannheimia succiniciproducens?

The biochemical environment significantly influences enzyme activity in M. succiniciproducens, as demonstrated by findings that "MsMDH shows low specific activity at physiological pH and strong uncompetitive inhibition toward oxaloacetate" . For recombinant UPP, a systematic investigation of environmental effects would include:

• pH effects assessment:

  • Measure UPP activity across pH range (6.0-9.0)

  • Determine pH stability profiles

  • Compare with UPPs from other bacterial sources

  • Correlate with cytoplasmic pH under different growth conditions

• CO2 concentration impacts:

  • Examine UPP activity under varying CO2 partial pressures

  • Investigate potential carboxylation/decarboxylation effects on protein structure

  • Relate to the organism's capnophilic nature where "the availability of CO2 controls the partition of PEP to various metabolites"

  • Consider implications for in vitro vs. in vivo activity

• Redox environment influences:

  • Test UPP sensitivity to oxidizing and reducing conditions

  • Examine effects of physiological redox carriers (glutathione, thioredoxin)

  • Determine presence and relevance of cysteine residues

  • Develop stabilization strategies for purified enzyme

• Metabolite interaction analysis:

  • Screen for allosteric regulators among common metabolites

  • Investigate product inhibition (by UMP)

  • Test effects of high substrate concentrations

  • Identify potential cross-talk with other metabolic pathways

Experimental approaches would parallel those used for other M. succiniciproducens enzymes, where activities are measured under standardized conditions and reported as specific activities in "mU mg of protein−1" . This systematic characterization would inform both basic understanding of UPP biochemistry and practical applications in genetic engineering and enzyme utilization.

What are the key considerations when interpreting experimental results from recombinant Mannheimia succiniciproducens UPP studies?

Interpreting experimental results from recombinant M. succiniciproducens UPP studies requires careful consideration of several factors that could influence outcomes. Researchers should apply these methodological principles when analyzing their data:

• Expression context effects:

  • Consider the impact of the expression system on protein folding and activity

  • Account for potential differences between homologous and heterologous expression

  • Evaluate post-translational modifications that might differ between systems

  • Normalize activity measurements appropriately when comparing different preparations

• Environmental condition standardization:

  • Ensure consistent pH, temperature, and buffer conditions across experiments

  • Account for M. succiniciproducens' capnophilic nature when interpreting in vivo results

  • Consider the influence of growth phase on enzyme expression and activity

  • Maintain consistent anaerobic conditions where appropriate

• Genetic background considerations:

  • Evaluate the impact of different host strain backgrounds

  • Account for potential polar effects of genetic modifications

  • Confirm phenotypes through complementation studies

  • Consider genomic context when interpreting gene expression data

• Comparative analysis frameworks:

  • Use appropriate statistical tests for significance determination

  • Include proper controls in all experimental designs

  • Consider both absolute and relative measurements when appropriate

  • Validate key findings through complementary methodological approaches

These principles align with the rigorous approaches evident in the search results, where enzyme activities are precisely measured and reported with appropriate units and statistical considerations . By applying these methodological principles, researchers can ensure robust and reproducible findings in their UPP studies.