Recombinant Idiomarina loihiensis Orotidine 5'-phosphate decarboxylase (pyrF)

What is Idiomarina loihiensis and why is it significant in extremophile research?

Idiomarina loihiensis is a halophilic gamma-proteobacterium isolated from a hydrothermal vent at 1,300m depth on the Lōihi submarine volcano, Hawaii. It represents an important model organism for studying adaptations to deep-sea hydrothermal ecosystems . The bacterium is characterized as a Gram-negative rod, 0.35 μm wide and 0.7-1.8 μm in length, motile via a single polar or subpolar flagellum .

I. loihiensis demonstrates remarkable adaptability, surviving across a wide temperature range (4-46°C) and extreme salinity conditions (0.5-20% NaCl) . This broad adaptability makes it particularly valuable for studying stress response mechanisms in extremophiles. The organism's genome has been fully sequenced, consisting of 2,839,318 base pairs encoding 2,640 proteins, which provides extensive opportunities for comparative genomic and functional studies .

Unlike many other bacteria, I. loihiensis has evolved to rely primarily on amino acid catabolism rather than sugar fermentation for carbon and energy, suggesting specialized metabolic adaptations to its protein-rich deep-sea hydrothermal environment .

What is Orotidine 5'-phosphate decarboxylase (pyrF) and what is its metabolic function?

Orotidine 5'-phosphate decarboxylase (pyrF) is an essential enzyme in the de novo pyrimidine biosynthesis pathway. It catalyzes the decarboxylation of orotidine monophosphate (OMP) to form uridine monophosphate (UMP), a critical precursor for pyrimidine nucleotides used in DNA and RNA synthesis .

The enzyme is remarkable for its extraordinary catalytic efficiency, accelerating the uncatalyzed reaction rate by a factor of 10^17 . To put this in perspective, the uncatalyzed reaction would take approximately 78 million years to convert half the reactants into products, while the enzyme-catalyzed reaction completes the same transformation in just 18 milliseconds .

What makes this enzyme particularly interesting to researchers is that it achieves this remarkable rate enhancement without requiring any cofactors, metal ions, or prosthetic groups. Instead, pyrF relies solely on precise positioning of charged amino acid residues within its active site to destabilize the ground state relative to the transition state .

The pyrF enzyme functions as a dimer (E₂) under physiological conditions, with a dimer equilibrium dissociation constant of 0.25 μM in 0.01 M MOPS Na⁺ at pH 7.2 . The catalytically active dimeric form is stabilized by NaCl, remaining in the E₂ state at all tested enzyme concentrations in 100 mM NaCl .

How does the pyrF/5-FOA selection system work in genetic manipulation?

The pyrF/5-FOA system represents a powerful genetic selection and counter-selection method that has been successfully implemented in various organisms. The methodology operates on the following principles:

• Creation of pyrF-deleted (ΔpyrF) mutants: The first step involves deleting the endogenous pyrF gene, generating uracil auxotrophic host strains that cannot synthesize pyrimidines without supplementation .

• Selection mechanism: The ΔpyrF mutants require external uracil for growth. When transformed with a plasmid containing a functional pyrF gene, these cells regain the ability to grow on media lacking uracil, providing a positive selection marker .

• Counter-selection mechanism: 5-fluoroorotic acid (5-FOA) is converted to toxic 5-fluorouracil by cells expressing functional pyrF. Therefore, cells expressing pyrF cannot grow in the presence of 5-FOA, creating a powerful counter-selection tool .

• Application in gene manipulation: This dual-selection capability allows researchers to:

  • Select for initial transformants carrying pyrF (growth on uracil-free media)

  • Select for second crossover or plasmid loss events (growth on 5-FOA media)

Research has demonstrated that the pyrF/5-FOA system generates second-crossover colonies more efficiently than the traditional sacB/sucrose system, making it particularly valuable for marker-less gene editing in organisms like Acinetobacter baumannii .

A key advantage of this system is that it can be broadly applied across different species due to the conservation of the pyrF gene and its 500bp-flanking sequence, facilitating the rapid generation of ΔpyrF mutants in diverse organisms .

What expression systems are suitable for producing recombinant Idiomarina loihiensis pyrF?

Based on available research data, the following expression systems have proven effective for recombinant production of pyrF proteins, including those from extremophilic organisms like Idiomarina loihiensis:

Bacterial Expression Systems:

• pNYCOMPS-LIC-FH10T+ vector: This system has been specifically documented for expressing Idiomarina loihiensis L2TR proteins with N-terminal Flag and His tags, making it suitable for pyrF expression .

• E. coli-based systems: These represent the most common approach due to:

  • Rapid growth and high protein yields

  • Compatibility with halophilic proteins (when properly optimized)

  • Availability of specialized strains for toxic or problematic proteins

Key optimization parameters for extremophilic protein expression:

When expressing recombinant pyrF from extremophiles like I. loihiensis, researchers should consider the unique characteristics of the source organism, including its growth temperature range (4-46°C) and high salt tolerance (up to 20% NaCl) , as these factors may influence optimal expression conditions.

What are the unique structural and functional characteristics of Idiomarina loihiensis pyrF?

Idiomarina loihiensis pyrF, like other orotidine 5'-phosphate decarboxylases, likely adopts the conserved TIM-barrel fold with the ligand binding site positioned near the open end of the barrel, based on structural data from related organisms . While the specific crystal structure of I. loihiensis pyrF has not been determined in the search results, comparative analysis with related pyrF enzymes reveals several expected key features:

Predicted structural elements:

• TIM-barrel core structure with eight parallel β-strands surrounded by α-helices

• Active site located at the C-terminal end of the β-barrel

• Conserved protein loops that likely envelop the substrate upon binding

Functional characteristics:

• Adaptation to the extremophilic nature of I. loihiensis, functioning across the organism's broad temperature range (4-46°C)

• Possible tolerance to high salt concentrations, reflecting the halophilic nature of the source organism (0.5-20% NaCl)

• Conservation of critical active site residues involved in catalysis, including:

  • Lysine residue anchored to optimize electrostatic interactions with developing negative charge at C-6 of the pyrimidine ring

  • Hydrogen bonds from the active site to O-2 and O-4 that help delocalize negative charge in the transition state

  • Interactions with the phosphoribosyl group to anchor the pyrimidine within the active site

The extremophilic origin of I. loihiensis pyrF suggests potential unique adaptations that could confer enhanced thermal stability and salt tolerance compared to mesophilic homologs, making it particularly interesting for comparative enzymology studies and potential biotechnological applications.

How can I optimize the heterologous expression and purification of Idiomarina loihiensis pyrF?

Optimizing the heterologous expression and purification of Idiomarina loihiensis pyrF requires careful consideration of its extremophilic origin. Based on research with similar proteins, the following methodological approach is recommended:

Expression optimization strategy:

• Vector selection:

  • Use pNYCOMPS-LIC-FH10T+ (N-terminal Flag and His-tagged) vector, which has been specifically used for I. loihiensis proteins

  • Consider vectors with strong, inducible promoters (T7, tac) and fusion tags that enhance solubility (MBP, SUMO)

• Expression conditions optimization:

• Purification strategy:

  • Initial IMAC (immobilized metal affinity chromatography) using His-tag

  • Secondary purification by ion exchange chromatography

  • Final polishing step with size exclusion chromatography

• Buffer optimization for halophilic protein:

  • Include 0.5-1.0 M NaCl in all purification buffers

  • Test stability in different salt types (NaCl, KCl) and concentrations

  • Evaluate the effect of osmolytes (glycerol, betaine) on stability

• Activity validation:

  • Develop a spectrophotometric assay to monitor OMP decarboxylation

  • Validate enzyme activity under different temperature and salt conditions

  • Determine kinetic parameters (Km, kcat, kcat/Km) under optimal conditions

• Storage conditions:

  • Test stability at 4°C, -20°C, and -80°C

  • Evaluate the effect of glycerol (10-50%) as a cryoprotectant

  • Consider lyophilization for long-term storage

By systematically optimizing these parameters, researchers can achieve higher yields of active I. loihiensis pyrF for subsequent structural and functional characterization.

What are the challenges in expressing genes from extremophiles like Idiomarina loihiensis in standard laboratory hosts?

Expression of extremophile genes in conventional laboratory hosts presents several unique challenges that must be addressed through careful experimental design:

Major challenges and methodological solutions:

• Codon usage bias:

  • Challenge: Extremophiles often have distinct codon preferences compared to mesophilic expression hosts like E. coli.

  • Solution: Utilize codon-optimized synthetic genes or express in Rosetta strains containing rare tRNAs. Analyze the codon adaptation index (CAI) of the native sequence to determine optimization needs.

• Protein folding and stability:

  • Challenge: Proteins from I. loihiensis (temperature range 4-46°C, high salt tolerance up to 20% NaCl) may fold incorrectly in mesophilic hosts under standard conditions.

  • Solution: Express at lower temperatures (16-25°C), supplement growth media with osmolytes, and consider co-expression with chaperones (GroEL/ES, DnaK/J).

• Post-translational modifications:

  • Challenge: Required modifications may differ between source and host organisms.

  • Solution: Characterize potential modifications in native I. loihiensis pyrF and select expression systems capable of producing required modifications.

• Toxicity to host cells:

  • Challenge: Heterologous expression of pyrF may be toxic to host cells lacking the native regulatory mechanisms.

  • Solution: Use tightly regulated expression systems, lower induction levels, or employ specialized strains designed for toxic protein expression.

• Halophilic adaptation:

  • Challenge: I. loihiensis proteins may require high salt concentrations for proper folding and activity.

  • Solution: Include appropriate salt concentrations in growth media and all purification buffers.

Comparative performance of expression systems for extremophilic proteins:

Understanding these challenges and employing appropriate strategies is essential for successful recombinant expression of I. loihiensis pyrF and similar extremophilic proteins.

How does the catalytic mechanism of Orotidine 5'-phosphate decarboxylase differ between mesophilic and extremophilic organisms?

The catalytic mechanism of Orotidine 5'-phosphate decarboxylase (OMP decarboxylase) involves several key steps that may exhibit adaptations in extremophiles like Idiomarina loihiensis compared to mesophilic counterparts:

Core catalytic mechanism conserved across species:

• Destabilization of the ground state by electrostatic repulsion between an aspartate residue in the enzyme's active site and the substrate's carboxyl group

• Protonation at C-5 of the orotate ring concurrent with decarboxylation

• Formation of a vinyl carbanion intermediate stabilized by active site interactions

• Proton donation to C-6 to complete the reaction

Comparative adaptations in extremophilic OMP decarboxylases:

Research data on other enzymes from extremophiles suggests that while the fundamental catalytic mechanism would be conserved in I. loihiensis pyrF, specific adaptations likely exist to maintain optimal function under the extreme conditions of temperature and salinity faced by this organism .

The mesophilic yeast OMP decarboxylase demonstrates kinetics governed by two ionizations (pK₁ = 6.1 and pK₂ = 7.7) , and it would be valuable to determine whether these ionization constants differ in the I. loihiensis enzyme as an adaptation to its unique environment.

How can Idiomarina loihiensis pyrF be engineered for improved thermostability or catalytic efficiency?

Engineering I. loihiensis pyrF for enhanced properties requires a structured approach combining computational analysis and experimental validation. The following methodology represents the current best practices for engineering extremozymes like pyrF:

Strategic approach to pyrF engineering:

• Computational analysis and design:

  • Homology modeling based on available crystal structures of OMP decarboxylase (PDB: 1DQX)

  • Molecular dynamics simulations under varying temperature/salt conditions

  • Identification of flexible regions that may limit thermostability

  • Computational prediction of stabilizing mutations using algorithms like FRESCO, FireProt, or Rosetta

• Rational design strategies for enhanced thermostability:

• Directed evolution methodologies:

  • Error-prone PCR to generate diversity

  • Screening using thermal challenge followed by activity assays

  • DNA shuffling with homologous pyrF genes from other thermophiles

  • Implementation of PACE (Phage-Assisted Continuous Evolution) for continuous selection

• High-throughput screening strategy:

  • Development of a colorimetric or fluorescent assay for pyrF activity

  • Thermal challenge at temperatures exceeding I. loihiensis optimal growth (>46°C)

  • Microplate-based screening of variant libraries

  • Deep sequencing to identify enriched mutations after selection

• Validation and iterative improvement:

  • Detailed characterization of promising variants

  • Determination of melting temperatures (Tm) using differential scanning calorimetry

  • Measurement of kinetic parameters at elevated temperatures

  • Structural analysis of successful variants

  • Combination of beneficial mutations and testing for additivity or synergy

• Semi-rational approaches:

  • B-factor analysis to identify regions of high mobility

  • Back-to-consensus mutagenesis targeting flexible regions

  • Ancestral sequence reconstruction to identify historically conserved residues

The combination of these approaches has proven successful for engineering thermostable variants of other enzymes and could be effectively applied to I. loihiensis pyrF, potentially yielding variants with improved properties for biotechnological applications.

How does environmental adaptation influence the structure-function relationship of Idiomarina loihiensis pyrF?

The extreme environmental conditions of deep-sea hydrothermal vents have likely shaped the structure-function relationship of I. loihiensis pyrF through adaptations that balance catalytic efficiency with stability under fluctuating conditions:

Environmental factors at the Lōihi hydrothermal vent and their molecular impacts:

• Temperature fluctuations (4-46°C growth range) :

  • Presumed molecular adaptation: Balanced conformational rigidity that maintains structural integrity at higher temperatures while preserving necessary flexibility for catalysis at lower temperatures

  • Functional consequence: Broader temperature optimum compared to mesophilic homologs

  • Potential structural features: Increased number of salt bridges and hydrophobic interactions, but fewer thermally-labile residues (Asn, Gln) in surface loops

• High salinity tolerance (0.5-20% NaCl) :

  • Presumed molecular adaptation: Increased proportion of acidic residues on protein surface, reduced hydrophobic surface patches

  • Functional consequence: Requirement for higher salt concentrations for optimal folding and activity

  • Potential structural features: Negative surface charge distribution, bound water molecules forming hydration networks

• Oxygen concentration gradients:

  • Presumed molecular adaptation: Enhanced stability against oxidative damage to maintain pyrimidine synthesis capability

  • Functional consequence: Potential differences in redox sensitivity compared to mesophilic homologs

  • Potential structural features: Strategic positioning of oxidation-resistant residues near the active site

• Pressure effects:

  • Presumed molecular adaptation: Volume-minimizing protein packing

  • Functional consequence: Potential pressure-dependent activity profile

  • Potential structural features: Reduced internal cavities, tighter packing of side chains

Comparative analysis with pyrF from other environmental niches:

Understanding these environment-specific adaptations provides valuable insights for both fundamental enzymology and the potential biotechnological applications of I. loihiensis pyrF, particularly for processes requiring stability under fluctuating conditions.

What are the comparative benefits and limitations of pyrF-based genetic systems versus other selection markers for marine extremophiles?

A systematic comparison of genetic selection systems reveals the unique advantages and potential limitations of pyrF-based approaches for marine extremophiles like Idiomarina loihiensis:

Comparative analysis of selection systems for marine extremophiles:

*Success rates estimated from comparable studies; actual rates may vary by specific organism and laboratory conditions.

Methodological considerations for implementing pyrF-based systems in marine extremophiles:

• Host preparation:

  • Utilize the conserved pyrF and its 500bp-flanking sequences to rapidly generate ΔpyrF hosts

  • Confirm uracil auxotrophy on minimal media with and without uracil supplementation

  • Verify 5-FOA resistance (typically 50-100 μg/ml effective concentration)

• Vector design:

  • Include the pyrF gene under native or constitutive promoter control

  • Design homologous regions (≥500bp) flanking the target modification site

  • Incorporate origin of replication compatible with the target organism

• Optimization parameters:

  • Determine minimal inhibitory concentration of 5-FOA specific to your organism

  • Optimize uracil concentration for auxotrophic growth (typically 40-50 mg/L)

  • Establish appropriate transformation methods (electroporation protocols may require salt-optimization for halophiles)

• Validation strategy:

  • PCR verification of gene modifications

  • Sequencing to confirm precise edits

  • Phenotypic assessment of edited strains

The pyrF system has demonstrated particular value for extremophiles, with research showing it was "very effective and enabled us to obtain marker-free mutants in a very short period of time" , making it an excellent choice for genetic manipulation of marine extremophiles like I. loihiensis.

How can the structural and catalytic insights from Idiomarina loihiensis pyrF contribute to understanding evolutionary adaptation in deep-sea microorganisms?

Idiomarina loihiensis pyrF represents a valuable model for investigating evolutionary adaptation principles in deep-sea environments, offering insights that extend beyond a single enzyme to broader ecological and evolutionary processes:

Conceptual framework for evolutionary analysis using I. loihiensis pyrF:

• Molecular signatures of environmental adaptation:

  • Comparative sequence analysis between I. loihiensis pyrF and homologs from different thermal/pressure regimes

  • Identification of positively selected residues using methods like dN/dS ratio analysis

  • Correlation of adaptive substitutions with specific environmental parameters (temperature, pressure, salinity)

• Reconstruction of adaptive pathways:

  • Ancestral sequence reconstruction to determine the evolutionary trajectory of pyrF

  • Synthesis and characterization of ancestral enzymes to map functional transitions

  • Identification of epistatic interactions that constrained or facilitated adaptation

• Enzymatic properties as environmental biosensors:

  • Analysis of kinetic parameters (kcat, Km) across temperature/pressure gradients

  • Development of mathematical models correlating enzyme properties with historical environmental fluctuations

  • Investigation of substrate specificity as an indicator of metabolic adaptation

• Structural insights into adaptation mechanisms:

  • Comparative structural analysis of pyrF across environmental gradients

  • Molecular dynamics simulations under varying conditions

  • Identification of conserved vs. variable regions in relation to function

Research methodology for investigating evolutionary adaptation:

Broader ecological implications:

I. loihiensis relies primarily on amino acid catabolism rather than sugar fermentation for carbon and energy , suggesting a specialized ecological niche. The enzyme's characteristics may reflect adaptation to this specialized metabolism, providing insights into how deep-sea vent ecosystems function at the molecular level.

The organism's genome contains a cluster of 32 genes for exopolysaccharide synthesis , suggesting adaptation for biofilm formation. Investigating how pyrF activity connects to broader cellular processes like biofilm formation could reveal metabolic integration strategies in extreme environments.

By systematically investigating these aspects, researchers can use I. loihiensis pyrF as a model to understand fundamental principles of protein evolution and adaptation in extreme environments, with implications extending beyond this specific enzyme to broader questions of microbial ecology and evolution.

What are the optimal conditions for assaying recombinant Idiomarina loihiensis pyrF activity?

Establishing reliable assay conditions is critical for accurately characterizing the activity of recombinant I. loihiensis pyrF. Based on studies of pyrF enzymes and the known properties of I. loihiensis, the following methodological approach is recommended:

Standard pyrF activity assay protocol:

• Spectrophotometric assay principle:

  • Monitoring the conversion of OMP to UMP at 285nm (Δε₂₈₅ = 1660 M⁻¹cm⁻¹)

  • Alternative: Coupled assay with detection of released CO₂ using carbonic anhydrase

• Buffer composition optimization:

• Temperature considerations:

  • Test activity across 20-50°C range (I. loihiensis grows from 4-46°C)

  • Pre-incubate enzyme in assay buffer at test temperature for thermal equilibration

  • Allow substrate solution to reach identical temperature before initiating reaction

• Reaction conditions:

  • Enzyme concentration: Initially 50-500nM, optimize based on activity

  • Substrate concentration: 0.1-10 × estimated Km value (typically 1-100μM for pyrF enzymes)

  • Reaction volumes: 200-500μL for standard cuvettes; 50-100μL for microplates

• Data collection and analysis:

  • Monitor continuous absorbance changes for 2-10 minutes

  • Calculate initial rates from linear portion of progress curves

  • Determine kinetic parameters using Michaelis-Menten regression analysis

Specialized assay considerations for I. loihiensis pyrF:

• Salt effects characterization:

  • Test activity in NaCl gradient (0-2M) to determine halophilic profile

  • Compare effects of different salts (NaCl, KCl, MgCl₂) on activity

• Temperature-activity relationship:

  • Generate temperature-activity profile between 4-60°C

  • Determine temperature optimum and activation energy (Ea) from Arrhenius plot

• Stability assays:

  • Heat inactivation studies: Preincubate enzyme at different temperatures (30-80°C) before assaying remaining activity

  • Long-term stability: Monitor activity retention at 4°C, 25°C over several days

• Alternative substrate testing:

  • Evaluate activity with modified OMP analogs to probe substrate specificity

  • Test potential inhibitors to characterize active site properties

By systematically optimizing these parameters, researchers can establish reliable assay conditions that accurately reflect the true catalytic capabilities of I. loihiensis pyrF while accounting for its adaptation to extreme environmental conditions.

What troubleshooting strategies are effective when encountering challenges with recombinant pyrF expression and purification?

Recombinant expression of pyrF from extremophiles like Idiomarina loihiensis can present several challenges. The following troubleshooting decision tree addresses common issues and their methodological solutions:

1. Low expression yield:

2. Insoluble protein formation:

3. Purification challenges:

4. Activity problems:

5. Special considerations for I. loihiensis pyrF:

• Halophilic adaptation: Given I. loihiensis grows in 0.5-20% NaCl , ensure adequate salt concentration throughout purification

• Temperature sensitivity: Since I. loihiensis grows between 4-46°C , the enzyme may have specific temperature requirements for proper folding

• Oxygen sensitivity: Consider whether anaerobic purification conditions improve yield or activity

Implementation of these troubleshooting strategies in a systematic manner can resolve most challenges encountered during recombinant expression and purification of I. loihiensis pyrF.

What emerging technologies could advance our understanding of pyrF enzymes from extremophiles like Idiomarina loihiensis?

Several cutting-edge technologies are poised to revolutionize our understanding of extremophilic enzymes like I. loihiensis pyrF in the coming years:

1. Advanced structural biology approaches:

• Cryo-electron microscopy (Cryo-EM): Enables visualization of protein structures in near-native conditions without crystallization, potentially revealing conformational states relevant to extremophilic adaptations

• Time-resolved X-ray crystallography: Captures structural snapshots during catalysis, illuminating the dynamic aspects of the reaction mechanism

• Neutron crystallography: Provides hydrogen atom positions, critical for understanding proton transfer in the catalytic mechanism of pyrF

• Serial femtosecond crystallography: Uses X-ray free electron lasers to obtain room-temperature structures without radiation damage, revealing physiologically relevant conformations

2. Computational and simulation techniques:

• Deep learning structure prediction: Methods like AlphaFold2 and RoseTTAFold allow accurate prediction of protein structures without experimental data

• Enhanced sampling molecular dynamics: Captures rare conformational transitions relevant to catalysis at extremes of temperature and pressure

• Quantum mechanics/molecular mechanics (QM/MM): Provides detailed insights into electronic changes during catalysis under extreme conditions

• Metadynamics simulations: Explores free energy landscapes under varying conditions (temperature, pressure, salt)

3. Systems biology and synthetic approaches:

• Single-cell proteomics: Examines cell-to-cell variation in pyrF expression under environmental stress

• In situ cryo-electron tomography: Visualizes pyrF within native cellular contexts

• Cell-free synthetic biology: Enables rapid prototyping and testing of pyrF variants

• Minimally altered laboratory evolution (MAGE): Facilitates targeted evolutionary studies of pyrF adaptation

4. Emerging analytical methods:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Probes protein dynamics and solvent accessibility under extreme conditions

• Single-molecule enzymology: Reveals heterogeneity in catalytic behavior masked in ensemble measurements

• Microfluidic platforms: Enables high-throughput screening of enzyme variants under precisely controlled conditions

• Native mass spectrometry: Analyzes protein-ligand interactions and oligomerization states in near-native conditions

Methodological integration for comprehensive understanding:

These emerging technologies will provide unprecedented insights into how extremophilic enzymes like I. loihiensis pyrF function under challenging conditions, potentially informing the design of industrial enzymes with enhanced stability and activity.

What are the potential biotechnological applications of engineered Idiomarina loihiensis pyrF?

Engineered variants of Idiomarina loihiensis pyrF offer promising biotechnological potential due to the enzyme's remarkable catalytic properties and adaptation to extreme conditions:

1. Biocatalysis and Green Chemistry:

• Selective decarboxylation reactions: Engineered pyrF variants could catalyze decarboxylation of non-natural substrates under environmentally friendly conditions

• Halotolerant biocatalysis: Catalytic processes in high salt concentrations that would denature conventional enzymes

• Temperature-flexible processes: Enzymes functional across broad temperature ranges (4-46°C) enable energy-efficient bioprocesses

2. Genetic Tools and Synthetic Biology:

• Selection marker system: Development of pyrF-based marker systems for marine and extremophilic organisms beyond current applications in A. baumannii

• Gene editing tools: Counterselection systems for CRISPR-based genetic manipulation in non-model organisms

• Biosensors: Development of pyrF-based biosensors for environmental monitoring

3. Pharmaceutical Applications:

• Drug discovery platforms: Use of pyrF structural insights to design inhibitors targeting pathogen-specific OMP decarboxylases

• Pyrimidine analog synthesis: Enzymatic modification of nucleotide analogs for antiviral and anticancer applications

• Thermostable diagnostic enzymes: Components in nucleic acid amplification diagnostic kits

4. Industrial Process Enhancements:

5. Research methodology development:

Development of a unified pyrF-based genetic manipulation system applicable across diverse bacterial species would represent a significant advance in molecular biology tools. The system would build upon successful implementations in organisms like A. baumannii , leveraging the highly conserved nature of pyrF and its flanking sequences.

Engineered properties needed for specific applications:

To realize these applications, systematic protein engineering approaches combining computational design, directed evolution, and structure-guided mutagenesis would be required to enhance the enzyme's natural properties while maintaining its exceptional catalytic efficiency and environmental adaptability.