Recombinant Salmonella agona Nicotinate phosphoribosyltransferase (pncB)

What is nicotinate phosphoribosyltransferase (pncB) and what is its function in Salmonella agona?

Nicotinate phosphoribosyltransferase (pncB) is an enzyme encoded by the pncB gene that plays a crucial role in the NAD+ salvage pathway. In bacterial systems like Salmonella agona, pncB catalyzes the conversion of nicotinic acid to nicotinic acid mononucleotide (NaMN) using phosphoribosyl pyrophosphate (PRPP) as a co-substrate. This reaction represents a critical step in NAD+ biosynthesis, which serves as an essential cofactor for numerous metabolic reactions. The enzyme is particularly important for bacterial survival and metabolic activity during stress conditions and nutrient limitation. Based on studies in E. coli, pncB may play a significant role in the regulation of redox balance and energy metabolism in S. agona, especially during infection and persistence .

What are the common methods for isolating and identifying S. agona strains that express pncB?

Isolation and identification of S. agona strains expressing pncB typically employs a multi-faceted approach:

• Selective Media Culturing: Initial isolation on selective media such as XLD (Xylose Lysine Deoxycholate) or HE (Hektoen Enteric) agar.

• PCR-Based Detection: Amplification of the pncB gene using specific primers designed from conserved regions of the gene sequence.

• Whole Genome Sequencing: For comprehensive genetic characterization, using platforms such as Illumina or Oxford Nanopore for short and long-read sequencing, respectively.

• Enzymatic Activity Assay: Measurement of nicotinate phosphoribosyltransferase activity in cell lysates using spectrophotometric methods to detect NaMN production.

• Expression Analysis: RT-qPCR to quantify pncB mRNA levels under various growth conditions.

For more complete characterization, researchers often employ the BakCharak pipeline (version 3.0.4 or later) that integrates NCBI AMRFinderPlus, ABRicate, and other bioinformatics tools to analyze sequencing data and identify genetic elements including the pncB gene .

What are the optimal expression systems for producing recombinant S. agona pncB?

The optimal expression systems for recombinant S. agona pncB production balance yield, solubility, and activity considerations:

The choice of expression vector is equally important, with pET-series vectors (particularly pET-28a with an N-terminal His-tag) showing excellent results for bacterial NAD salvage pathway enzymes. Adding solubility tags such as SUMO or MBP can significantly improve protein solubility without compromising catalytic activity .

What purification strategies yield the highest purity and activity for recombinant S. agona pncB?

A multi-step purification approach typically yields the highest purity and activity for recombinant S. agona pncB:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-based resins for His-tagged protein, with elution using an imidazole gradient (50-300 mM).

• Intermediate Purification: Ion exchange chromatography (typically anion exchange using Q-Sepharose) at pH 8.0, which separates pncB from similarly sized contaminants.

• Polishing Step: Size exclusion chromatography using Superdex 75 or 200 columns to remove aggregates and achieve >95% purity.

• Tag Removal: If necessary, precision protease cleavage (TEV or HRV 3C) followed by subtractive IMAC.

Throughout purification, it's crucial to maintain buffer conditions that preserve enzyme activity:

• 50 mM Tris-HCl or phosphate buffer (pH 7.5-8.0)

• 100-150 mM NaCl

• 1-5 mM DTT or 2-mercaptoethanol

• 10% glycerol as a stabilizer

• 0.5-1 mM EDTA (after IMAC steps)

Enzyme activity should be monitored at each step using a standardized nicotinate phosphoribosyltransferase assay, and specific activity (μmol/min/mg) calculated to track purification efficiency .

How can researchers optimize the enzymatic activity assay for recombinant S. agona pncB?

The enzymatic activity assay for recombinant S. agona pncB can be optimized through several critical considerations:

Standard Spectrophotometric Assay Protocol:

• Reaction mixture components:

  • 50 mM Tris-HCl (pH 7.8)

  • 10 mM MgCl₂

  • 1 mM nicotinic acid

  • 1 mM PRPP

  • 1-5 μg purified enzyme

  • Total volume: 100 μL

• Incubation at 37°C for 10-15 minutes, followed by reaction termination with equal volume of 1M HCl.

• HPLC analysis of NaMN formation using C18 reverse-phase column with UV detection at 260 nm.

Key Optimization Parameters:

• pH optimization: Test activity across pH range 6.5-9.0 to determine optimal pH

• Temperature dependency: Assess activity from 25-50°C to establish temperature optima

• Metal ion requirements: Evaluate activity with different divalent cations (Mg²⁺, Mn²⁺, Ca²⁺)

• Substrate saturation curves: Determine Km and Vmax values for both nicotinic acid and PRPP

• Linearity verification: Ensure time and enzyme concentration linearity within working ranges

For high-throughput applications, a coupled enzyme assay can be developed where NaMN production is linked to NADH consumption, allowing continuous spectrophotometric monitoring at 340 nm. This approach requires careful optimization of coupling enzyme concentrations to ensure pncB activity remains rate-limiting .

How does pncB expression relate to antimicrobial resistance in S. agona?

The relationship between pncB expression and antimicrobial resistance in S. agona involves complex metabolic adaptations that affect bacterial survival under antibiotic stress:

• NAD⁺ Homeostasis and Stress Response: Altered pncB expression affects NAD⁺/NADH ratios, which can modulate cellular responses to oxidative stress generated by bactericidal antibiotics. Enhanced NAD⁺ metabolism through upregulated pncB may contribute to S. agona's ability to withstand antibiotic-induced stress.

• Plasmid-Mediated Co-Regulation: In multidrug-resistant (MDR) S. agona isolates, pncB may be co-regulated with other genes carried on large resistance plasmids. The 295,499 bp IncHI2 plasmid identified in S. agona isolates carries numerous antimicrobial resistance genes (ARGs) organized in distinct clusters, potentially creating metabolic networks where NAD⁺ metabolism intersects with resistance mechanisms .

• Biofilm Formation Connection: S. agona is recognized as a strong biofilm former, and NAD⁺ metabolism plays crucial roles in biofilm development. pncB upregulation may enhance biofilm formation, which in turn contributes to antibiotic tolerance through reduced penetration and metabolic dormancy of biofilm-embedded cells .

• Persistence and Viable but Non-Culturable State: S. agona can enter a viable but non-culturable state while remaining metabolically active, similar to strategies employed by S. Typhi. This physiological adaptation likely involves NAD⁺ metabolic reconfiguration mediated partly through pncB, contributing to both persistence during treatment and chronic carriage .

The metabolic flexibility conferred by modulated pncB expression may therefore represent an important adaptive mechanism that complements the more direct resistance determinants found in MDR S. agona strains.

What genomic contexts frequently contain the pncB gene in multiresistant S. agona strains?

The genomic contexts containing the pncB gene in multiresistant S. agona strains reveal important evolutionary and functional associations:

• Chromosomal Context: In S. agona, the pncB gene is typically located in the core genome region dedicated to nicotinamide metabolism. Analysis of S. agona isolates reveals that the chromosomal pncB gene exists in a conserved operon structure, often in proximity to genes involved in related metabolic pathways.

• Mobile Genetic Element Associations: While the pncB gene itself is not commonly found on mobile genetic elements, its expression and function appear to be metabolically linked to plasmid-borne resistance determinants. The large IncHI2 plasmid (pSE18-SA00377-1) identified in multidrug-resistant S. agona carries 16 antibiotic resistance genes organized in two distinct clusters, creating a complex regulatory network that may influence chromosomal gene expression, including pncB .

• Genomic Islands: Comparative genomic analyses of S. agona isolates have revealed structural variations and SNP patterns suggesting population expansion after acute infection. These genomic rearrangements likely affect metabolic gene regulation, potentially including NAD⁺ metabolism genes like pncB, as part of immune evasion mechanisms that enable persistent infection .

• Regulatory Elements: Upstream regions of pncB in resistant strains may contain modified promoter elements or transcription factor binding sites that allow for adaptive expression in response to antibiotic stress or host environments.

This complex genomic landscape underscores the importance of whole-genome sequencing approaches in understanding how metabolic genes like pncB interact with resistance determinants in the broader context of bacterial adaptation.

How does the metabolic function of pncB contribute to S. agona persistence in hosts?

The metabolic function of pncB contributes to S. agona persistence in hosts through several interconnected mechanisms:

• NAD⁺ Homeostasis During Nutrient Limitation: During host colonization, S. agona faces varying levels of nicotinamide precursor availability. The pncB-mediated salvage pathway provides metabolic flexibility by efficiently recycling available nicotinic acid into NAD⁺, supporting bacterial survival in nutrient-restricted host niches.

• Redox Balance Maintenance: pncB activity directly influences the NAD⁺/NADH ratio, which is critical for maintaining proper cellular redox balance during infection. This balance affects numerous cellular processes including energy generation, stress response, and virulence factor expression. Optimal redox homeostasis allows S. agona to respond appropriately to host immune defenses .

• Biofilm Matrix Production: NAD⁺ metabolism contributes to the production of extracellular polymeric substances (EPS) that form the biofilm matrix. S. agona has been identified as a strong biofilm former, and this property likely contributes to its persistence in both environmental and host settings. pncB-dependent NAD⁺ biosynthesis provides the reducing power necessary for EPS production .

• Metabolic Adaptation During Chronic Carriage: S. agona can transition from acute infection to chronic carriage, similar to S. Typhi. This transition involves complex metabolic adaptations, including the ability to enter a viable but non-culturable state while maintaining metabolic activity. pncB-mediated NAD⁺ metabolism likely plays a key role in sustaining basal metabolic functions during this reduced activity state .

• Response to Oxidative Stress: Host immune cells generate reactive oxygen species to combat bacterial pathogens. NAD⁺-dependent enzymes are critical components of bacterial defense against oxidative damage. By maintaining adequate NAD⁺ pools through pncB activity, S. agona can better withstand host-derived oxidative stress.

This metabolic versatility contributes to S. agona's ability to establish persistent infections and may partially explain its increasing recognition as a significant cause of foodborne gastroenteritis.

What are the most effective strategies for studying pncB regulation in S. agona under different environmental conditions?

Investigating pncB regulation in S. agona across diverse environmental conditions requires integrating multiple complementary approaches:

For comprehensive investigation, researchers should:

• First establish baseline pncB expression patterns during standard growth curves using qRT-PCR and western blotting to identify phases of maximal regulation.

• Conduct targeted experiments in models mimicking specific host environments (pH shifts, oxygen limitation, antimicrobial exposure, nutrient restriction).

• Apply system-wide approaches (RNA-Seq coupled with metabolomics) to place pncB regulation within broader metabolic networks.

• Validate findings with targeted genetic approaches, including promoter mutagenesis and regulatory protein deletions.

This multi-layered strategy enables the identification of both direct regulators of pncB expression and the metabolic signals that trigger regulatory responses .

How can researchers effectively engineer pncB variants with enhanced catalytic properties for biotechnological applications?

Engineering pncB variants with enhanced catalytic properties requires a systematic protein engineering approach:

• Structure-Guided Rational Design:

  • Homology modeling of S. agona pncB based on available crystal structures of homologous enzymes

  • Identification of catalytic residues and substrate-binding pockets

  • Computational prediction of mutations that could enhance substrate binding or catalytic efficiency

  • Site-directed mutagenesis targeting specific residues involved in:

    • Substrate specificity

    • Catalytic mechanism

    • Product release

    • Protein stability

• Directed Evolution Approaches:

  • Error-prone PCR to generate random mutation libraries

  • DNA shuffling with homologous pncB genes from other bacterial species

  • Creation of focused mutation libraries targeting substrate binding sites

  • High-throughput screening methods:

    • Colorimetric assays for nicotinamide mononucleotide production

    • Growth complementation in NAD⁺ biosynthesis-deficient strains

    • FACS-based screening using fluorescent indicators of NAD⁺ metabolism

• Semi-rational Approaches:

  • Consensus sequence analysis across multiple bacterial species

  • Ancestral sequence reconstruction to identify evolutionarily stable variants

  • Statistical coupling analysis to identify co-evolving residues

  • Combinatorial mutagenesis of hot spots identified through computational analysis

• Performance Evaluation and Optimization:

  • Comprehensive kinetic characterization (kcat, Km, catalytic efficiency)

  • Stability assessment (thermal shift assays, long-term activity retention)

  • pH and temperature optima determination

  • Tolerance to inhibitors and process conditions

  • Substrate scope expansion testing

• Protein Engineering Success Metrics:

  • Increased catalytic efficiency (kcat/Km)

  • Broader substrate specificity

  • Enhanced thermostability

  • Improved expression yields

  • Resistance to product inhibition

This systematic engineering workflow has been successfully applied to other NAD⁺ biosynthesis enzymes and can be adapted specifically for S. agona pncB, considering its unique structural features and catalytic properties .

What are the current challenges in studying the role of pncB in S. agona pathogenesis and host adaptation?

Researchers investigating pncB's role in S. agona pathogenesis face several significant challenges:

• Genetic Manipulation Limitations:

  • Development of reliable genetic tools specifically optimized for S. agona remains incomplete

  • Construction of clean pncB deletion mutants without polar effects on adjacent genes

  • Challenges in complementation studies due to copy number effects and expression level differences

  • Limited availability of S. agona-specific inducible promoter systems for controlled expression studies

• Physiological Complexity:

  • Redundancy in NAD⁺ biosynthetic pathways complicating interpretation of single-gene knockout studies

  • Interconnection between NAD⁺ metabolism and numerous other metabolic pathways

  • Difficulty in distinguishing direct from indirect effects of pncB manipulation on virulence

  • Context-dependent gene expression across different infection stages and host environments

• Host Interaction Dynamics:

  • Limited understanding of nicotinamide availability within host microenvironments

  • Complex interplay between bacterial NAD⁺ metabolism and host NAD⁺-dependent immune functions

  • Challenges in real-time monitoring of NAD⁺/NADH ratios during host-pathogen interactions

  • Lack of suitable animal models that fully recapitulate S. agona chronic carriage

• Technical Barriers:

  • Difficulty in studying viable but non-culturable states relevant to persistence

  • Challenges in correlating genome-scale SNP variations with metabolic phenotypes

  • Limited spatial resolution in analyzing pncB expression in different microenvironments during infection

  • Integration of multi-omics data (transcriptomics, proteomics, metabolomics) to build comprehensive models

• Translation to Clinical Applications:

  • Establishing direct links between NAD⁺ metabolism modulation and treatment outcomes

  • Development of pncB inhibitors with selectivity for bacterial over human NAD⁺ salvage pathways

  • Understanding how pncB function intersects with existing antibiotic resistance mechanisms

  • Translating basic research findings into therapeutic approaches against persistent S. agona infections

Overcoming these challenges requires innovative approaches combining advanced genetic tools, host-relevant infection models, and systems biology perspectives to decipher the complex role of pncB in S. agona's lifecycle from initial infection to persistent carriage .

What are the most promising approaches for developing inhibitors targeting S. agona pncB for antimicrobial development?

The development of S. agona pncB inhibitors represents a promising antimicrobial strategy, with several complementary approaches showing potential:

• Structure-Based Drug Design:

  • Homology modeling of S. agona pncB based on crystal structures of bacterial homologs

  • Virtual screening of compound libraries against the enzyme's active site

  • Fragment-based approaches to identify scaffolds with high binding efficiency

  • Molecular dynamics simulations to identify transient binding pockets and allosteric sites

  • Rational design of transition state analogs specifically targeting the phosphoribosyltransferase reaction mechanism

• High-Throughput Screening Platforms:

  • Biochemical assays measuring inhibition of purified recombinant enzyme

  • Cell-based assays in NAD⁺ auxotrophic strains complemented with S. agona pncB

  • Phenotypic screens for compounds that specifically impair bacterial growth under conditions requiring pncB function

  • Thermal shift assays to identify compounds that bind and stabilize/destabilize the enzyme

• Mechanistic Inhibitor Classes to Explore:

  • Substrate competitive inhibitors (nicotinic acid or PRPP analogs)

  • Product mimics (NaMN analogs with modifications preventing further metabolism)

  • Transition state analogs

  • Covalent inhibitors targeting catalytic residues

  • Allosteric inhibitors affecting enzyme oligomerization or conformational changes

• Selectivity Considerations:

  • Structural differences between bacterial and human nicotinate phosphoribosyltransferases

  • Differential inhibitor uptake leveraging bacterial-specific transport systems

  • Prodrug approaches with activation by bacterial-specific enzymes

  • Targeting bacterial-specific regulatory mechanisms controlling pncB expression

• Combination Approaches:

  • pncB inhibitors as antibiotic adjuvants to enhance efficacy of existing drugs

  • Dual targeting of multiple NAD⁺ biosynthesis enzymes to overcome pathway redundancy

  • Combination with efflux pump inhibitors to enhance intracellular accumulation

  • Integration with biofilm dispersal agents to target persistent populations

• Validation Strategies:

  • Confirmation of on-target activity through resistant mutant generation and characterization

  • Metabolomic profiling to verify NAD⁺ depletion mechanism

  • Efficacy testing in infection models that recapitulate S. agona persistence

  • Assessment of resistance development frequency and mechanisms

This multi-faceted approach to inhibitor development takes advantage of the essential nature of NAD⁺ metabolism while addressing the challenges of target selectivity and bacterial persistence mechanisms .

What are best practices for ensuring reproducibility in recombinant S. agona pncB expression and characterization studies?

Ensuring reproducibility in recombinant S. agona pncB studies requires systematic attention to multiple experimental variables:

• Genetic Material Standardization:

  • Use verified reference sequences with accession numbers for all gene constructs

  • Document and validate all cloning junctions and vector features by sequencing

  • Establish seed stocks of expression constructs with detailed passage history

  • Consider codon optimization strategies and document algorithms used

  • Deposit final constructs in public repositories with complete annotation

• Expression System Standardization:

  • Fully document bacterial strain genotypes and their source

  • Create standardized protocols for competent cell preparation

  • Establish defined criteria for expression strain selection

  • Control for batch variation in media components with detailed composition reporting

  • Monitor and report growth curves with standardized metrics (OD600, cell density)

• Expression Parameter Control:

  • Calibrated induction protocols with precisely defined timing and inducer concentration

  • Temperature control within ±0.5°C during expression phases

  • Standardized cell harvest criteria (OD, time post-induction)

  • Detailed documentation of all buffer compositions with measured final pH

  • Consistent cell disruption methods with validated efficiency metrics

• Purification Method Validation:

  • Column performance qualification before each purification

  • Inclusion of standard quality control samples

  • Use of calibrated chromatography systems with regular performance verification

  • Documentation of elution profiles with reproducibility metrics

  • Application of consistent protein quantification methods with standard curves

• Enzyme Characterization Controls:

  • Inclusion of activity standards with known specific activity

  • Temperature and pH monitoring during activity assays

  • Substrate quality verification before use

  • Multi-lot testing for critical reagents

  • Statistical power analysis to determine appropriate replicate numbers

• Data Reporting Standards:

  • Raw data availability in public repositories

  • Detailed methods sections with no omitted steps

  • Explicit reporting of failed approaches and optimization attempts

  • Comprehensive reporting of statistical analyses including outlier criteria

  • Use of reporting checklists specific to enzyme characterization studies

Implementation of these practices significantly improves study reproducibility and facilitates translation of findings between different research groups working on NAD⁺ metabolism in pathogenic bacteria .

How can researchers effectively integrate multiple omics approaches to study pncB function in S. agona?

Effective integration of multiple omics approaches to elucidate pncB function in S. agona requires strategic experimental design and sophisticated data integration:

• Experimental Design Considerations:

  • Synchronized sample collection across all omics platforms

  • Inclusion of genetically modified strains (pncB knockout, overexpression, point mutants)

  • Carefully selected environmental conditions relevant to S. agona lifecycle

  • Time-course sampling to capture dynamic responses

  • Biological and technical replicates appropriate for each platform

• Multi-omics Data Generation:

  • Genomics: Whole genome sequencing to identify genetic variants in pncB and regulatory regions

  • Transcriptomics: RNA-seq to measure pncB expression and global transcriptional responses

  • Proteomics: LC-MS/MS to quantify PncB protein levels and post-translational modifications

  • Metabolomics: Targeted and untargeted approaches focusing on NAD⁺ metabolism intermediates

  • Fluxomics: 13C-labeled tracer experiments to measure metabolic flux through NAD⁺-related pathways

• Analytical Integration Approaches:

  • Correlation Networks: Identify relationships between pncB expression, protein levels, and metabolite abundances

  • Pathway Enrichment Analysis: Map multi-omics data to metabolic pathways to identify functional impacts

  • Genome-Scale Metabolic Modeling: Integrate omics data into metabolic models to predict flux distributions

  • Machine Learning Approaches: Supervised and unsupervised methods to identify patterns across datasets

  • Causal Network Inference: Establish causality between pncB perturbation and downstream effects

• Validation Strategies:

  • Targeted Gene Manipulations: Confirm key findings through specific genetic interventions

  • Enzyme Assays: Measure PncB activity in conditions highlighted by multi-omics analysis

  • Reporter Systems: Develop fluorescent or luminescent reporters for real-time monitoring

  • In vivo Relevance Testing: Translate findings to infection models to confirm physiological significance

• Data Management and Integration Tools:

This comprehensive integration approach enables researchers to move beyond correlative observations to mechanistic understanding of how pncB functions within the complex metabolic network of S. agona under various environmental conditions .

What are the emerging technologies that could advance our understanding of S. agona pncB function in antimicrobial resistance?

Several cutting-edge technologies show promise for elucidating the complex role of pncB in S. agona antimicrobial resistance:

• Single-Cell Technologies:

  • Single-cell RNA-seq to reveal heterogeneity in pncB expression across bacterial populations

  • Single-cell metabolomics to measure NAD⁺/NADH ratios in individual bacteria

  • Microfluidic platforms for real-time monitoring of metabolic adaptations at single-cell resolution

  • Time-lapse microscopy combined with fluorescent biosensors to track NAD⁺ metabolism during antibiotic exposure

• Advanced Genetic Manipulation Tools:

  • CRISPR interference (CRISPRi) for precise temporal control of pncB expression

  • Base editing for introducing specific point mutations without selection markers

  • Inducible degradation systems for rapid protein depletion studies

  • Multiplex genome engineering to study pncB in different genetic backgrounds

• Structural Biology Innovations:

  • Cryo-EM analysis of PncB protein complexes in native conditions

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes

  • Time-resolved crystallography to capture enzyme intermediates

  • In-cell NMR to monitor protein-metabolite interactions in live bacteria

• Systems-Level Approaches:

  • Transposon-sequencing (Tn-seq) to identify genetic interactions with pncB

  • Multi-strain comparative proteogenomics to link genomic variations to protein function

  • Machine learning approaches to predict resistance phenotypes from omics data

  • Metabolic flux analysis under antibiotic stress conditions

• Host-Pathogen Interaction Technologies:

  • Organoid infection models to study pncB function in tissue-specific environments

  • Dual RNA-seq of host-pathogen interfaces during various infection stages

  • In vivo biosensors to monitor NAD⁺ dynamics during infection

  • MALDI-imaging mass spectrometry to map metabolite distributions in infected tissues

Implementation of these technologies will provide unprecedented insights into how pncB function contributes to both direct and indirect mechanisms of antimicrobial resistance in S. agona, potentially revealing novel intervention strategies that target metabolic vulnerabilities rather than conventional antibiotic targets .

What are the potential applications of engineered S. agona pncB variants in biotechnology and medicine?

Engineered S. agona pncB variants offer diverse applications across biotechnology and medicine:

• Biocatalytic Applications:

  • Production of NAD⁺ and derivatives for pharmaceutical and nutraceutical industries

  • Cofactor regeneration systems for biocatalytic processes

  • Development of enzyme cascades for synthesis of complex nicotinamide-containing compounds

  • Immobilized enzyme systems for continuous-flow biocatalysis

• Biosensing Platforms:

  • Development of pncB-based biosensors for nicotinic acid detection in environmental and clinical samples

  • Engineering of allosteric pncB variants with altered substrate specificity for detecting related compounds

  • Integration into whole-cell biosensors for monitoring metabolic states

  • Creation of bioreporter systems to study NAD⁺ metabolism in various microenvironments

• Therapeutic Applications:

  • Design of recombinant pncB variants as enzyme replacement therapy for specific metabolic disorders

  • Development of pncB-based drug delivery systems targeting NAD⁺-dependent processes

  • Engineering of attenuated S. agona strains with modified pncB for vaccine development

  • Creation of probiotics with enhanced NAD⁺ production capabilities for gut health applications

• Metabolic Engineering Tools:

  • Optimized pncB variants for enhancing NAD⁺ availability in industrial microorganisms

  • Development of regulatory tools based on pncB-dependent switches

  • Creation of tunable NAD⁺/NADH ratio control systems for optimizing production pathways

  • Engineering of synthetic NAD⁺ metabolism modules for introduction into heterologous hosts

• Diagnostic Applications:

  • Development of pncB-based assays for detecting S. agona in clinical and food samples

  • Creation of activity-based probes for studying NAD⁺ metabolism in complex biological samples

  • Use of engineered pncB variants in multiplexed detection systems for bacterial identification

  • Integration into point-of-care diagnostic devices for rapid antimicrobial resistance profiling

These applications leverage the unique catalytic properties and substrate specificity of S. agona pncB while addressing challenges in bioprocessing, diagnostics, and therapeutic intervention through protein engineering and synthetic biology approaches .