Recombinant Petroselinum crispum Flavone synthase (FNSI)

What is the biochemical classification of P. crispum FNSI and what reaction does it catalyze?

P. crispum FNSI belongs to the 2-oxoglutarate-dependent dioxygenases (2-ODDs) class of enzymes. It catalyzes the conversion of flavanones to flavones through a desaturation reaction that introduces a double bond between C-2 and C-3 in the C-ring of flavanones. This conversion process involves two sequential steps: the initial elimination of the C-3 β-configured hydrogen, followed by the elimination of the C-2 hydrogen . Unlike other enzymes involved in flavonoid modification, FNSI performs this conversion without forming any detectable reaction intermediates during the catalytic process, as confirmed through early studies using 14C-radiolabeled flavanones .

Flavanone + O₂ + 2-oxoglutarate → Flavone + CO₂ + Succinate + H₂O

A specific example is the conversion of (2S)-naringenin to apigenin, which has been verified through functional expression studies of recombinant P. crispum FNSI .

How can researchers differentiate between type I and type II flavone synthases in experimental design?

Researchers should account for several key differences when designing experiments involving flavone synthases:

Enzymatic Classification and Properties:

• FNSI enzymes (like P. crispum FNSI) are soluble 2-oxoglutarate-dependent dioxygenases

• FNSII proteins are membrane-bound cytochrome P450 monooxygenases that require a NADPH-cytochrome P450 reductase (CPR) partner for activity

Expression System Requirements:

• FNSI expression systems require supplementation with 2-oxoglutarate and Fe²⁺

• FNSII expression systems need cytochrome P450 reductase co-expression and potentially heme precursors like 5-aminolevulinic acid (5-ALA)

Experimental Detection Methods:

• FNSI activity assays typically monitor oxygen consumption or CO₂ production

• FNSII assays monitor NADPH oxidation or require membrane fraction preparation

These differences significantly impact experimental design, particularly when comparing enzyme efficiencies or optimizing expression systems. When evaluating both enzyme types simultaneously, researchers should include appropriate controls for each enzyme class to account for their distinct biochemical requirements .

What are the primary flavanone substrates utilized by recombinant P. crispum FNSI?

Recombinant P. crispum FNSI has demonstrated activity with several flavanone substrates, though with varying catalytic efficiencies. The primary substrates include:

• (2S)-Naringenin - The most well-characterized substrate, confirmed to be efficiently converted to apigenin in both in vitro enzymatic assays and in engineered microbial systems . This conversion has been verified through RT-PCR amplification of P. crispum FNSI cDNA and subsequent functional expression in yeast cells .

• Pinocembrin - Recent metabolic engineering studies have demonstrated that P. crispum FNSI effectively converts pinocembrin to chrysin, achieving production titers of 82 ± 5 mg/L in engineered E. coli strains . This represents the highest reported chrysin production using P. crispum FNSI, exceeding previously reported titers in both E. coli and yeast expression systems .

• Other flavanones - While not explicitly detailed in the search results, structure-activity relationship studies with other FNSI enzymes suggest potential activity with additional flavanones such as eriodictyol and hesperetin, though substrate specificity profiles require further experimental validation.

It's worth noting that even with optimized expression systems, substrate conversion remains incomplete (for example, 130 mg/L of pinocembrin remained unconverted in the best-performing strain) , suggesting potential for further optimization of reaction conditions.

What are the optimal expression systems for producing recombinant P. crispum FNSI?

Several expression systems have been successfully employed for recombinant P. crispum FNSI production, each with specific advantages depending on research objectives:

Yeast Expression System:
The first functional expression of P. crispum FNSI was accomplished in yeast cells, where the enzyme successfully converted (2S)-naringenin to apigenin . This system provides proper protein folding and post-translational modifications that may be important for optimal enzyme activity.

Bacterial Expression System (E. coli):
Recent studies have demonstrated successful expression of P. crispum FNSI in engineered E. coli strains, particularly using pBbA1k-based vectors . This system offers several advantages:

• High protein yield

• Rapid growth and expression

• Compatibility with metabolic engineering approaches for whole-cell biocatalysis

In one study, P. crispum FNSI was co-expressed with the pinocembrin biosynthesis pathway in E. coli using plasmid SBC016090, resulting in efficient chrysin production . The bacterial system proved particularly effective, with P. crispum FNSI outperforming seven other FNSI candidates and five FNSII enzymes in comparative screening experiments .

For optimal expression in E. coli, researchers should consider:

• Codon optimization for E. coli

• Temperature reduction during induction (typically 20-25°C)

• Supplementation with Fe²⁺ and 2-oxoglutarate cofactors

• Co-expression with molecular chaperones if solubility issues arise

What strategies can improve the stability and activity of recombinant P. crispum FNSI?

Several approaches can enhance recombinant P. crispum FNSI stability and activity:

Cofactor Optimization:

• Ensure adequate Fe²⁺ availability (typically 0.1-0.5 mM FeSO₄)

• Optimize 2-oxoglutarate concentration (typically 1-5 mM)

• Add ascorbate (0.5-2 mM) to maintain iron in reduced state

Expression Conditions:

• Lower induction temperature (18-25°C)

• Use weaker promoters for slower, more controlled expression

• Consider fusion tags that enhance solubility (MBP, SUMO, etc.)

Protein Engineering Approaches:
While not specifically mentioned for P. crispum FNSI in the search results, general approaches that have proven successful with similar 2-ODD enzymes include:

• Site-directed mutagenesis of residues near the active site

• Directed evolution for improved stability

• Rational design based on comparative analysis with other FNSI enzymes

Storage and Handling:

• Add glycerol (10-20%) for freezer storage

• Include reducing agents to prevent oxidation

• Avoid repeated freeze-thaw cycles

These strategies should be empirically tested for P. crispum FNSI, as the optimal conditions may vary depending on the specific experimental context and application.

How can researchers verify the functional expression of recombinant P. crispum FNSI?

Researchers can employ several complementary methods to verify successful functional expression of recombinant P. crispum FNSI:

Enzymatic Activity Assays:
The gold standard verification is the conversion of flavanone substrates to their corresponding flavones. For example, the conversion of (2S)-naringenin to apigenin was used to verify the identity of recombinant P. crispum FNSI expressed in yeast cells . This verification can be performed using:

• HPLC analysis of reaction products

• LC-MS for product identification and quantification

• Spectrophotometric assays (flavones typically show distinct UV absorbance shifts compared to flavanones)

Protein Expression Analysis:

• SDS-PAGE to confirm protein expression at the expected molecular weight

• Western blotting with specific antibodies (if available)

• Mass spectrometry-based proteomic analysis for definitive identification

In vivo Functional Verification:
In metabolic engineering contexts, functional expression can be verified through the production of flavones in engineered microbial strains. For instance, the successful expression of P. crispum FNSI in E. coli was confirmed by measuring chrysin production (82 ± 5 mg/L) from pinocembrin , demonstrating that the enzyme was properly folded and active in the cellular environment.

How effective is P. crispum FNSI compared to other flavone synthases in engineered microbial systems?

Comparative studies have positioned P. crispum FNSI as a top-performing enzyme candidate for flavone production in microbial systems. In a comprehensive screening study of thirteen flavone synthase candidates (eight FNSI and five FNSII enzymes), P. crispum FNSI demonstrated superior performance in engineered E. coli for chrysin production .

Comparative Performance Data:
P. crispum FNSI achieved a chrysin production titer of 82 ± 5 mg/L, significantly outperforming other enzyme candidates including:

• FNSI enzymes from other plant species (Cuminum cyminum, Plagiochasma appendiculatum, etc.)

• FNSII enzymes that required additional NADPH-cytochrome P450 reductase partners

This performance advantage places P. crispum FNSI as the preferred enzyme for chrysin production in E. coli, surpassing previously reported titers in both E. coli and yeast systems . The exceptional performance of P. crispum FNSI can be attributed to several factors:

• Higher intrinsic catalytic efficiency

• Better protein expression and folding in bacterial hosts

• Favorable kinetic parameters with the pinocembrin substrate

• No requirement for membrane association or additional protein partners (unlike FNSII enzymes)

It's worth noting that even with the top-performing P. crispum FNSI, substrate conversion remained incomplete (130 mg/L of pinocembrin remained unconverted) , suggesting potential for further optimization through protein engineering or process development.

What are effective strategies for integrating P. crispum FNSI into flavonoid biosynthetic pathways?

Integration of P. crispum FNSI into complete flavonoid biosynthetic pathways requires careful optimization at multiple levels:

Vector Design and Compatibility:
P. crispum FNSI has been successfully expressed using pBbA1k-based vectors co-transformed with plasmids carrying the upstream pathway genes . This modular approach allows for independent optimization of different pathway segments.

Pathway Balancing Strategies:

• Promoter Strength Tuning: Adjusting promoter strength can balance enzyme expression levels to prevent bottlenecks or metabolic burden

• RBS Engineering: Modifying ribosome binding sites can fine-tune translation efficiency

• Copy Number Control: Using vectors with different copy numbers for different pathway segments

Multi-enzyme Expression Systems:
For complete flavonoid biosynthesis starting from simple carbon sources, researchers have constructed pathways combining:

• Phenylalanine ammonia-lyase (PAL) from Arabidopsis thaliana

• 4-coumarate-CoA ligase (4CL) from Glycine max

• Chalcone synthase (CHS) and chalcone isomerase (CHI)

• P. crispum FNSI for the final conversion step

Chassis Engineering Considerations:
The host strain should be engineered to:

• Supply necessary cofactors (Fe²⁺ and 2-oxoglutarate)

• Minimize competing pathways that might deplete precursors

• Enhance precursor supply (malonyl-CoA, phenylalanine)

• Incorporate mechanisms for product export or detoxification

A successful example is the integrated pinocembrin production chassis that was subsequently employed for chrysin production using P. crispum FNSI, achieving significant titers without precursor supplementation or fatty acid biosynthesis inhibitors .

What production yields have been achieved using recombinant P. crispum FNSI in different host organisms?

The production yields achieved using recombinant P. crispum FNSI vary depending on the host organism, substrate availability, and pathway configuration:

E. coli:
The highest reported production using P. crispum FNSI in E. coli achieved 82 ± 5 mg/L of chrysin from pinocembrin . This production was accomplished using an engineered E. coli strain capable of accumulating 353 ± 19 mg/L pinocembrin from glycerol, without precursor supplementation or fatty acid biosynthesis inhibitors .

Production Data Comparison Table:

This data demonstrates that P. crispum FNSI is an effective enzyme for chrysin production, though the conversion efficiency (approximately 23% based on the reported unconverted pinocembrin) suggests potential for further optimization through enzyme engineering or process development .

What is known about the catalytic mechanism of P. crispum FNSI?

P. crispum FNSI, as a 2-oxoglutarate-dependent dioxygenase (2-ODD), follows a generally conserved catalytic mechanism characteristic of this enzyme family, though specific details for P. crispum FNSI are not fully elucidated in the search results.

The general catalytic mechanism involves:

• Cofactor Binding: Fe²⁺ binds in the active site, coordinated by a facial triad of amino acid residues (typically His-X-Asp/Glu-Xn-His)

• Substrate and Co-substrate Binding: The flavanone substrate and 2-oxoglutarate bind in the active site, with 2-oxoglutarate directly coordinating to the iron center

• Oxygen Activation: Molecular oxygen binds to the iron center, forming a reactive Fe³⁺-superoxo species

• Oxidative Decarboxylation: The activated oxygen attacks 2-oxoglutarate, resulting in decarboxylation to produce CO₂ and succinate, generating a highly reactive Fe⁴⁺=O ferryl species

• Substrate Oxidation: The ferryl oxygen abstracts a hydrogen atom from the substrate (first from C-3, then from C-2 of the flavanone), resulting in sequential dehydrogenation to form the double bond characteristic of flavones

The desaturation of the flavanone C-ring proceeds through two steps:

• Initial elimination of the C-3 β-configured hydrogen

• Followed by the elimination of the C-2 hydrogen

This mechanism is consistent with early studies using 14C-radiolabeled flavanones, which demonstrated that P. crispum FNSI converted flavanones to flavones without forming detectable reaction intermediates .

Which amino acid residues are critical for the catalytic function of P. crispum FNSI?

While the search results don't specifically detail the critical residues in P. crispum FNSI, insights can be drawn from studies of related FNSI enzymes. For instance, in the liverwort Plagiochasma appendiculatum FNSI (PaFNSI), Tyr240 was identified as a critical residue that confers flavanone-2-hydroxylase (F2H) activity alongside FNSI activity .

In PaFNSI, the Tyr240Pro mutation specifically eliminated F2H activity while maintaining the ability to convert naringenin to apigenin, demonstrating the role of this residue in determining reaction specificity . By analogy, corresponding residues in P. crispum FNSI likely play similar roles in substrate positioning and reaction specificity.

Based on knowledge of other 2-ODD enzymes, several types of residues are generally critical for function:

• Iron-binding Residues: Typically a His-X-Asp/Glu-Xn-His facial triad that coordinates the Fe²⁺ cofactor

• 2-Oxoglutarate Binding Residues: Often include positively charged residues (Arg, Lys) that interact with the carboxylate groups of 2-oxoglutarate

• Substrate Binding Residues: Hydrophobic and aromatic residues that position the flavanone substrate for selective hydrogen abstraction

• Second-sphere Residues: Those that don't directly contact substrates or cofactors but influence active site geometry and electrostatics

Detailed mutagenesis studies specifically on P. crispum FNSI would be valuable to precisely identify critical residues for catalysis and substrate specificity.

How does the substrate specificity of P. crispum FNSI compare with other FNSI enzymes?

Substrate specificity comparison between P. crispum FNSI and other FNSI enzymes reveals both similarities and distinct differences:

P. crispum FNSI vs. Other Apiaceae FNSIs:
FNSI enzymes were initially believed to be restricted to the Apiaceae family, which includes P. crispum (parsley) . Within this family, FNSIs generally show similar substrate preferences, efficiently converting flavanones such as naringenin to their corresponding flavones.

P. crispum FNSI vs. Monocot FNSIs:
More recent studies have identified FNSI enzymes in rice (OsFNSI-1) and revealed that 2-ODDs with FNS activity are more widely distributed than initially believed . Comparative substrate specificity studies could reveal evolutionary adaptations to different flavonoid profiles in monocots versus dicots.

P. crispum FNSI vs. Liverwort PaFNSI:
The liverwort Plagiochasma appendiculatum FNSI (PaFNSI) demonstrates bifunctional activity, catalyzing both flavone synthesis and flavanone-2-hydroxylation . This dual activity is not reported for P. crispum FNSI, suggesting differences in active site architecture.

Performance Comparisons in Microbial Systems:
When expressed in engineered E. coli, P. crispum FNSI outperformed seven other FNSI candidates, including those from:

• Plagiochasma appendiculatum

• Cuminum cyminum (which showed no detectable activity)

• Aethusa cynapium

• Apium graveolens

• Daucus carota

This superior performance suggests that P. crispum FNSI has either broader substrate acceptance, better protein stability, or more favorable kinetic parameters when expressed in bacterial systems. The specific molecular basis for this enhanced performance remains to be fully characterized through detailed enzymological studies.

How can protein engineering approaches enhance the catalytic efficiency of P. crispum FNSI?

Protein engineering offers several promising approaches to enhance P. crispum FNSI catalytic efficiency:

Rational Design Strategies:

• Active Site Engineering: Modifying residues that interact with the flavanone substrate could improve binding affinity and positioning. For example, the findings regarding Tyr240 in PaFNSI suggest that targeted mutations of corresponding residues in P. crispum FNSI could alter reaction specificity .

• Loop Engineering: Modifying flexible loops near the active site could enhance substrate access or product release.

• Stability Enhancement: Introducing stabilizing interactions (salt bridges, disulfide bonds) in regions away from the active site could improve enzyme stability without compromising catalytic activity.

Directed Evolution Approaches:

• Error-Prone PCR: Generating random mutations throughout the P. crispum FNSI gene, followed by screening for variants with improved activity.

• DNA Shuffling: Recombining P. crispum FNSI with other high-performing FNSI genes (for example, from the screening study that identified P. crispum FNSI as the top performer ) could generate chimeric enzymes with enhanced properties.

• Site-Saturation Mutagenesis: Systematically exploring all possible amino acid substitutions at key positions identified through structural analysis or sequence alignments.

Computational Approaches:

• Molecular Dynamics Simulations: Identifying dynamic bottlenecks in substrate binding or product release.

• Machine Learning-Guided Design: Using algorithms trained on enzyme sequence-function relationships to predict beneficial mutations.

• De Novo Design: Computational redesign of the active site to optimize transition state stabilization.

These approaches could address the incomplete substrate conversion observed even with the top-performing P. crispum FNSI (130 mg/L of pinocembrin remained unconverted) , potentially enhancing both catalytic rate and product yields in engineered biosynthetic pathways.

What analytical methods are most effective for characterizing the kinetic parameters of recombinant P. crispum FNSI?

Comprehensive characterization of recombinant P. crispum FNSI kinetics requires multiple complementary analytical approaches:

Steady-State Kinetic Methods:

• Spectrophotometric Assays: Monitoring changes in UV-Vis absorbance during flavone formation (flavones typically have distinct absorbance spectra compared to flavanones).

• Oxygen Consumption Measurements: Using oxygen electrodes to monitor O₂ consumption rates, since FNSI enzymes require molecular oxygen as a co-substrate.

• HPLC-Based Assays: Quantifying substrate depletion and product formation over time to determine reaction rates under various conditions.

Transient Kinetic Methods:

• Stopped-Flow Spectroscopy: Measuring rapid changes in absorbance upon mixing enzyme with substrates to characterize pre-steady-state kinetics.

• Rapid Chemical Quench: Analyzing reaction intermediates formed during short incubation times before quenching.

Binding Studies:

• Isothermal Titration Calorimetry (ITC): Determining binding affinities and thermodynamic parameters for substrate and cofactor binding.

• Surface Plasmon Resonance (SPR): Measuring real-time binding kinetics of substrates to immobilized enzyme.

Kinetic Parameter Determination:
For comprehensive kinetic characterization, researchers should determine:

• K_m and k_cat for multiple flavanone substrates

• K_m for 2-oxoglutarate and O₂

• Binding affinity for Fe²⁺

• pH and temperature optima

• Inhibition constants for potential inhibitors

These parameters would provide valuable insights for comparing P. crispum FNSI with other flavone synthases and for optimizing reaction conditions in both in vitro applications and in vivo metabolic engineering contexts.

What emerging technologies show promise for enhancing the industrial applicability of P. crispum FNSI?

Several emerging technologies offer potential for enhancing P. crispum FNSI's industrial applicability:

Enzyme Immobilization Strategies:

• Covalent attachment to functionalized supports

• Encapsulation in nanoporous materials

• Cross-linked enzyme aggregates (CLEAs)

• Immobilization on magnetic nanoparticles for easier recovery

These approaches could enhance enzyme stability, enable continuous processing, and facilitate enzyme recycling.

Whole-Cell Biocatalyst Optimization:
Building on the successful expression of P. crispum FNSI in E. coli , further chassis engineering could enhance industrial viability:

• Cofactor Regeneration Systems: Engineering pathways for efficient 2-oxoglutarate regeneration

• Transport Engineering: Enhancing substrate uptake and product export

• Genome-Scale Metabolic Optimization: Redirecting carbon flux toward flavonoid precursors

• Consolidated Bioprocessing: Integrating biomass degradation with flavonoid production

Advanced Fermentation Technologies:

• Continuous Fermentation: Maintaining cells in optimal physiological state

• Two-Phase Systems: Using organic overlay for in situ product extraction

• Perfusion Bioreactors: Continuous removal of inhibitory products

Synthetic Biology Approaches:

• Pathway Compartmentalization: Localizing P. crispum FNSI and pathway enzymes in synthetic organelles

• Metabolic Sensor-Regulators: Dynamic control of enzyme expression based on metabolite levels

• Synthetic Enzyme Scaffolds: Co-localizing P. crispum FNSI with other pathway enzymes to enhance flux

These technologies could address some of the current limitations observed in P. crispum FNSI applications, such as incomplete substrate conversion , and potentially enable industrial-scale production of high-value flavones such as chrysin, which has demonstrated multiple bioactivities including antioxidant, hepatoprotective, and neuroprotective effects .

What are common issues encountered when working with recombinant P. crispum FNSI and how can they be resolved?

Researchers working with recombinant P. crispum FNSI may encounter several challenges that can be addressed through specific troubleshooting strategies:

Low Expression Levels:

• Issue: Poor protein expression in heterologous hosts

• Solutions:

  • Optimize codon usage for the host organism

  • Test different expression vectors and promoter strengths

  • Explore fusion tags that enhance expression (MBP, SUMO, etc.)

  • Adjust induction conditions (temperature, inducer concentration, timing)

Protein Insolubility:

• Issue: Formation of inclusion bodies

• Solutions:

  • Lower expression temperature (18-20°C)

  • Co-express with molecular chaperones

  • Use solubility-enhancing fusion partners

  • Optimize lysis buffer composition (detergents, salt concentration)

Low Enzymatic Activity:

• Issue: Expressed protein shows poor catalytic performance

• Solutions:

  • Ensure adequate Fe²⁺ availability (add FeSO₄ to reaction buffer)

  • Optimize 2-oxoglutarate concentration

  • Add reducing agents (ascorbate, DTT) to maintain iron in reduced state

  • Check pH optimum (typically pH 7.0-8.0 for 2-ODDs)

  • Verify substrate purity and solubility

Incomplete Substrate Conversion:
This issue was specifically observed with P. crispum FNSI in engineered E. coli, where 130 mg/L of pinocembrin remained unconverted even in the best-performing strain .

• Solutions:

  • Increase enzyme expression level

  • Extend reaction time

  • Implement fed-batch substrate addition

  • Consider enzyme engineering for improved catalytic efficiency

  • Address potential product inhibition through in situ product removal

Cofactor Limitations:

• Issue: Insufficient cofactor availability limiting activity

• Solutions:

  • Supplement with additional Fe²⁺ and 2-oxoglutarate

  • Engineer cofactor regeneration systems

  • Optimize iron uptake in whole-cell systems

Implementing these troubleshooting strategies can significantly improve the performance of recombinant P. crispum FNSI in both in vitro enzymatic applications and in vivo metabolic engineering contexts.

How can researchers optimize reaction conditions for maximum P. crispum FNSI activity?

Optimizing reaction conditions is crucial for maximizing P. crispum FNSI activity in both in vitro enzymatic assays and whole-cell biocatalysis:

Key Parameters for Optimization:

• pH Optimization:

  • Test pH range 6.0-9.0 (typically in 0.5 unit increments)

  • Common buffers: HEPES, Tris-HCl, phosphate

  • Monitor activity and stability at different pH values

  • For whole-cell systems, consider intracellular pH regulation

• Temperature Optimization:

  • Test temperature range 20-40°C

  • Balance maximal activity against enzyme stability

  • For whole-cell systems, consider cell viability constraints

• Cofactor Concentrations:

  • Fe²⁺ concentration (typically 50-500 μM)

  • 2-oxoglutarate concentration (typically 1-10 mM)

  • Ascorbate as reducing agent (1-5 mM)

  • Evaluate potential inhibitory effects at high concentrations

• Substrate Delivery:

  • For hydrophobic flavanones, optimize solubilization strategy:

    • Co-solvents (DMSO, ethanol) at non-inhibitory concentrations

    • Cyclodextrins for improved solubility

    • Emulsification techniques

  • Consider fed-batch substrate addition to minimize inhibition

• Oxygen Availability:

  • Ensure adequate oxygenation through:

    • Increased agitation in shake flasks

    • Sparging in bioreactors

    • Use of oxygen-enriched air if necessary

• Reaction Time Optimization:

  • Monitor reaction progress over time

  • Identify point of maximum product formation before potential degradation

Systematic optimization of these parameters using design of experiments (DoE) approaches can significantly enhance P. crispum FNSI performance. The optimization strategy should be tailored to the specific application context (in vitro enzymatic reactions versus whole-cell biocatalysis).

What analytical methods provide the most reliable quantification of flavones produced by P. crispum FNSI?

Reliable quantification of flavones produced by P. crispum FNSI requires appropriate analytical methods:

Chromatographic Methods:

• HPLC with UV Detection:

  • Most commonly used method for flavone analysis

  • Typical conditions:

    • C18 reverse phase columns

    • Mobile phases: acetonitrile/water gradients with 0.1% formic acid

    • Detection wavelengths: 260-340 nm (optimized for specific flavones)

  • Advantages: readily available equipment, good separation, moderate sensitivity

• LC-MS/MS:

  • Provides both quantification and structural confirmation

  • Multiple reaction monitoring (MRM) for high specificity and sensitivity

  • Particularly valuable for complex biological matrices

  • Can detect flavones at sub-μg/L concentrations

• UPLC (Ultra Performance Liquid Chromatography):

  • Faster analysis times

  • Improved resolution

  • Reduced solvent consumption

  • Enhanced sensitivity for low-abundance flavones

Sample Preparation Methods:

• For In Vitro Enzymatic Reactions:

  • Protein precipitation (acetonitrile, methanol)

  • Centrifugation to remove precipitated protein

  • Direct injection or evaporation/reconstitution for concentration

• For Microbial Cultures:

  • Extraction with ethyl acetate or methanol

  • Sonication to enhance cell disruption

  • Centrifugation to separate cellular debris

  • Evaporation and reconstitution in appropriate solvent

• For Complex Matrices:

  • Solid-phase extraction (SPE) for sample clean-up

  • QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) methodology

  • Selective extraction procedures based on flavone physicochemical properties

Method Validation Considerations:

• Linear range determination

• Limits of detection and quantification

• Recovery rates from different matrices

• Matrix effect evaluation

• Precision and accuracy assessment

• Stability of analytes during storage

These analytical methods have been successfully applied in studies evaluating P. crispum FNSI performance, such as the determination of chrysin production (82 ± 5 mg/L) in engineered E. coli strains .