Recombinant Mannheimia succiniciproducens [Protein-PII] uridylyltransferase (glnD), partial

What is [Protein-PII] uridylyltransferase (glnD) and what is its function in Mannheimia succiniciproducens?

[Protein-PII] uridylyltransferase (glnD) is a key enzyme involved in nitrogen metabolism regulation in bacteria including M. succiniciproducens. It functions by post-translationally modifying PII proteins through uridylylation/deuridylylation in response to nitrogen availability. The enzyme catalyzes the transfer of UMP groups to PII proteins when nitrogen is limited and removes these groups when nitrogen is abundant. This mechanism enables bacteria to adjust their metabolic pathways according to environmental nitrogen conditions, which is particularly important for organisms used in metabolic engineering applications for succinic acid production .

How should recombinant [Protein-PII] uridylyltransferase be stored and handled in laboratory settings?

For optimal stability and activity retention, recombinant [Protein-PII] uridylyltransferase should be stored according to its formulation:

• Liquid formulations maintain stability for approximately 6 months at -20°C/-80°C

• Lyophilized formulations remain stable for up to 12 months at -20°C/-80°C

Working aliquots can be maintained at 4°C for up to one week, but repeated freeze-thaw cycles should be strictly avoided as they significantly diminish enzyme activity. For reconstitution of lyophilized protein, use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL, followed by addition of glycerol to a final concentration of 5-50% (optimally 50%) before aliquoting for long-term storage .

What expression systems are suitable for producing recombinant M. succiniciproducens proteins?

Recombinant M. succiniciproducens proteins, including [Protein-PII] uridylyltransferase, can be successfully expressed in several systems with distinct advantages:

Yeast expression systems have been successfully employed for producing recombinant [Protein-PII] uridylyltransferase with purity levels exceeding 85% as determined by SDS-PAGE analysis . When developing expression systems, optimization of experimental conditions through Design of Experiments (DoE) approaches rather than one-factor-at-a-time methods significantly improves production efficiency .

How can Design of Experiments (DoE) be applied to optimize recombinant [Protein-PII] uridylyltransferase production?

DoE methodology provides a systematic approach to optimize recombinant [Protein-PII] uridylyltransferase production by:

• Defining critical factors affecting protein expression (temperature, inducer concentration, media composition, pH)

• Designing a minimal set of experiments that captures factor interactions

• Developing statistical models that predict optimal conditions

Unlike the inefficient one-factor-at-a-time approach, DoE simultaneously evaluates multiple variables and their interactions, enabling researchers to identify optimal production conditions with fewer experiments. For recombinant protein production, response surface methodology (RSM) is particularly valuable for identifying the optimal balance of factors affecting expression level, solubility, and activity.

Implementation involves:

• Initial screening designs (Plackett-Burman) to identify significant factors

• Optimization designs (central composite, Box-Behnken) to determine optimal settings

• Verification experiments to confirm model predictions

Available software packages facilitate DoE implementation, from experimental design through statistical analysis, making this approach accessible to researchers without extensive statistical backgrounds .

What purification strategies yield high-purity recombinant [Protein-PII] uridylyltransferase?

Purification of recombinant [Protein-PII] uridylyltransferase to high purity (>85%) involves a strategic multi-step approach:

• Initial clarification: Cell lysis followed by centrifugation to remove cellular debris

• Capture phase: Affinity chromatography utilizing appropriate tags determined during the manufacturing process

• Intermediate purification: Ion exchange chromatography based on the protein's theoretical pI

• Polishing phase: Size exclusion chromatography to remove aggregates and achieve final purity

For quality control, SDS-PAGE analysis confirms purity levels exceeding 85%, which is standard for research-grade recombinant proteins . The purification strategy must be optimized for each specific construct, considering the protein's unique physicochemical properties and the presence of any affinity tags.

How does M. succiniciproducens [Protein-PII] uridylyltransferase activity relate to metabolic engineering for succinic acid production?

The relationship between [Protein-PII] uridylyltransferase activity and succinic acid production in M. succiniciproducens involves complex nitrogen-carbon metabolism integration:

• Nitrogen sensing: [Protein-PII] uridylyltransferase acts as a nitrogen sensor, modifying PII proteins based on intracellular glutamine levels

• Metabolic flux regulation: Modified PII proteins influence key enzymes in central carbon metabolism, including those in succinic acid production pathways

• Integration with gene knockouts: In metabolically engineered M. succiniciproducens strains optimized for succinic acid production (such as the LPK7 strain), nitrogen metabolism must be balanced with carbon flux

The LPK7 strain, with disruptions in the ldhA, pflB, pta, and ackA genes, produces 13.4 g/liter of succinic acid from 20 g/liter glucose with minimal by-product formation, achieving a yield of 0.97 mol succinic acid per mol glucose . Understanding the role of [Protein-PII] uridylyltransferase in this context may provide opportunities for further optimization of nitrogen utilization efficiency during succinic acid production.

What structural characteristics distinguish M. succiniciproducens [Protein-PII] uridylyltransferase from homologs in other bacterial species?

While detailed structural information specific to M. succiniciproducens [Protein-PII] uridylyltransferase is limited in the provided sources, we can infer likely structural characteristics based on homologous proteins:

• Domain organization: Typically contains an N-terminal nucleotidyltransferase domain, central sensor domain, and C-terminal ACT domain

• Active site architecture: Conserved catalytic residues coordinating ATP and facilitating UMP transfer

• Species-specific variations: Likely contains unique surface residues that affect substrate specificity and regulatory interactions

Similar to how structural comparisons between M. succiniciproducens MDH and C. glutamicum MDH revealed key residues influencing enzyme activity and substrate inhibition , comparative structural analysis of [Protein-PII] uridylyltransferase from different species could identify residues responsible for species-specific regulatory properties. Identification of these distinctive features would enable targeted protein engineering to enhance desired characteristics.

How can protein engineering approaches improve the catalytic efficiency of M. succiniciproducens [Protein-PII] uridylyltransferase?

Protein engineering strategies to enhance [Protein-PII] uridylyltransferase catalytic properties can be approached through:

• Rational design: Based on structural insights and sequence analysis

  • Targeting active site residues to modify substrate affinity

  • Modifying allosteric sites to alter regulatory properties

  • Engineering surface residues to improve protein stability

• Directed evolution: Creating and screening libraries for improved variants

  • Error-prone PCR to generate random mutations

  • DNA shuffling to recombine beneficial mutations

  • Site-saturation mutagenesis of key residues

As demonstrated with malate dehydrogenase in M. succiniciproducens, even single amino acid substitutions can dramatically impact enzyme performance. For example, the G11Q variant of M. succiniciproducens MDH showed significantly improved catalytic properties, leading to enhanced succinic acid production when expressed in engineered strains . Similar approaches could identify critical residues in [Protein-PII] uridylyltransferase that, when modified, improve its regulatory function in nitrogen metabolism.

How does [Protein-PII] uridylyltransferase activity interact with the knockout strategy used for succinic acid production in M. succiniciproducens?

The integration of [Protein-PII] uridylyltransferase activity with the gene knockout strategy for enhanced succinic acid production involves complex metabolic interconnections:

• Redox balance effects: The deletion of ldhA, pflB, pta, and ackA genes in strains like LPK7 alters redox balance (NAD+/NADH ratio), which indirectly impacts nitrogen metabolism regulated by [Protein-PII] uridylyltransferase

• Precursor availability: Modified carbon flux resulting from gene knockouts changes the availability of α-ketoglutarate, a key intermediate connecting carbon and nitrogen metabolism

• Regulatory crosstalk: Signals processed by [Protein-PII] uridylyltransferase influence expression of genes involved in carbon metabolism

In the LPK7 strain, disruption of ldhA, pflB, pta, and ackA genes redirects carbon flux toward succinic acid production, achieving yields of 0.97 mol succinic acid per mol glucose with minimal by-product formation . Understanding how [Protein-PII] uridylyltransferase activity responds to these altered metabolic conditions could reveal strategies to further optimize nitrogen utilization efficiency during high-yield succinic acid production.

What analytical methods are most effective for measuring [Protein-PII] uridylyltransferase activity in metabolically engineered M. succiniciproducens?

Comprehensive analysis of [Protein-PII] uridylyltransferase activity in metabolically engineered strains requires multiple complementary approaches:

For in vivo studies monitoring [Protein-PII] uridylyltransferase activity during fermentation, techniques that can distinguish between modified and unmodified PII proteins are essential. This information, when correlated with carbon flux measurements and succinic acid production rates, provides insights into the dynamic relationship between nitrogen sensing and carbon metabolism under production conditions.

How might systems biology approaches integrate [Protein-PII] uridylyltransferase regulation with metabolic flux optimization?

Future integration of [Protein-PII] uridylyltransferase regulation with metabolic flux optimization will likely employ multi-omics approaches:

• Genome-scale metabolic models: Incorporating nitrogen regulatory networks into existing carbon metabolism models

• Dynamic flux balance analysis: Predicting how [Protein-PII] uridylyltransferase activity affects metabolic flux distribution under changing conditions

• Regulatory-metabolic network integration: Developing computational tools that predict how nitrogen signaling influences carbon metabolism during fermentation

Such integrated approaches could guide the development of M. succiniciproducens strains with coordinated carbon and nitrogen metabolism optimization. Current metabolically engineered strains like LPK7 achieve high succinic acid yields (0.97 mol/mol glucose) , while more advanced strains expressing optimized enzymes like C. glutamicum MDH reach productivities of 21.3 g/L/h and titers of 134.25 g/L . Integration of nitrogen metabolism optimization could potentially push these metrics even higher.

What is the potential for using CRISPR-Cas9 genome editing to optimize [Protein-PII] uridylyltransferase expression or activity in M. succiniciproducens?

CRISPR-Cas9 genome editing offers precise approaches for optimizing [Protein-PII] uridylyltransferase function in M. succiniciproducens:

• Promoter engineering: Modifying native promoter elements to fine-tune expression levels

• Single nucleotide modifications: Introducing specific amino acid changes identified through structural studies

• Regulatory element engineering: Modifying regulatory sequences that control expression in response to environmental conditions

• Multiplex editing: Simultaneously modifying [Protein-PII] uridylyltransferase and related components of the nitrogen regulatory network

The advantages of CRISPR-Cas9 over traditional methods include higher efficiency, ability to make marker-free modifications, and capacity for multiplexed editing. Learning from previous metabolic engineering success in M. succiniciproducens, where enzyme optimization of malate dehydrogenase significantly improved succinic acid production , similar precision engineering of [Protein-PII] uridylyltransferase could enhance nitrogen metabolism regulation in production strains.