Recombinant Coprinopsis cinerea Acetyl-coenzyme A synthetase (ACS-1), partial

What is Acetyl-coenzyme A synthetase (ACS-1) and what is its function in Coprinopsis cinerea?

Acetyl-coenzyme A synthetase (ACS-1) is an enzyme that catalyzes the activation of acetate to acetyl-CoA through an ATP-dependent reaction. In Coprinopsis cinerea, this enzyme plays a critical role in central carbon metabolism, particularly in acetate utilization pathways. While specific research on C. cinerea ACS-1 is limited in the provided search results, the enzyme family is known to be involved in various metabolic processes including lipid biosynthesis, protein acetylation, and energy production through the TCA cycle.

The reaction catalyzed follows the general mechanism:
Acetate + ATP + CoA → Acetyl-CoA + AMP + PPi

Research methodologies for functional characterization typically include enzyme assays measuring either CoA consumption or pyrophosphate/AMP production using spectrophotometric methods.

What expression systems are commonly used for recombinant production of fungal enzymes like C. cinerea ACS-1?

Several expression systems can be employed for the recombinant production of fungal enzymes from C. cinerea, with selection depending on research goals and required protein characteristics:

• Fungal hosts: Heterologous expression in industrial fungal hosts has been successfully used for C. cinerea enzymes, as demonstrated with recombinant peroxygenase .

• Pichia pastoris (Komagataella phaffis): This methylotrophic yeast is particularly valuable for fungal enzyme expression due to its ability to perform post-translational modifications similar to those in native fungal systems. The PichiaPink system and GlycoSwitch strains offer enhanced capabilities for controlled glycosylation patterns .

• C. cinerea itself: Self-expression systems using C. cinerea as both gene source and expression host have been developed, especially for laccases, allowing proper folding and post-translational modifications .

For optimal expression, consider that temperature and medium composition significantly affect enzyme production rates and yields. Studies with C. cinerea laccases showed differential expression based on temperature conditions and nutrient availability .

What are the optimal growth conditions for recombinant C. cinerea enzyme production?

Based on studies with recombinant laccase production in C. cinerea, the following parameters are critical for optimizing enzyme production:

Temperature Effects:
Temperature significantly impacts enzyme secretion and activity. Recombinant laccase production in C. cinerea showed optimal activity at specific temperature ranges, with notable differences in enzyme stability and production rates at different temperatures .

Medium Composition:
Different media formulations support varying levels of enzyme production:

• Carbon source type and concentration directly affect enzyme expression levels

• Nitrogen sources impact production efficiency

• Inducers may be necessary for optimal expression

• Trace elements and cofactors often enhance enzyme activity and stability

Growth Morphology:
Pellet formation characteristics impact enzyme production, with most pellets in the optimal range of 3-5 mm² yielding better enzyme production for some C. cinerea transformants .

pH Considerations:
Initial medium pH and pH control during fermentation significantly impact enzyme production and stability.

A methodological approach requires systematic optimization of these parameters for each specific recombinant enzyme, typically using design of experiments (DoE) approaches to identify optimal conditions.

What strategies can improve heterologous expression yields of C. cinerea ACS-1?

Several advanced strategies can be employed to enhance the expression yields of recombinant C. cinerea ACS-1:

Codon Optimization:
Adjusting the coding sequence to match the codon usage bias of the expression host can significantly improve translation efficiency. This is particularly important when expressing fungal genes in bacterial or yeast systems.

Promoter Engineering:
Selection or modification of promoters affects transcription levels. For example, methanol-inducible promoters like AOX1 in Pichia pastoris can be modified for methanol-free induction, reducing toxicity while maintaining high expression levels .

Signal Peptide Optimization:
The choice of secretion signal can dramatically affect secretion efficiency. Testing various signal sequences (native or host-specific) is a methodological approach to identify optimal secretion.

Host Cell Engineering:
Modifying the expression host through:

• Glycosylation pathway engineering for proper post-translational modifications

• Secretion pathway enhancement to reduce bottlenecks

• Protease deletion strains to minimize degradation

Fermentation Optimization:
Developing fed-batch or continuous culture strategies with controlled nutrient delivery can enhance yields. Temperature shifts during induction phase have shown improved production for C. cinerea laccases .

Metabolic Flux Analysis:
Identifying and alleviating metabolic bottlenecks through precursor supplementation or pathway engineering can enhance energy availability for protein production.

Implementation requires systematic testing of these strategies, often in combination, with quantitative analysis of yield improvements at each step.

How can researchers troubleshoot low activity of recombinant C. cinerea ACS-1?

When confronting low enzymatic activity of recombinant ACS-1, a systematic troubleshooting approach is essential:

Protein Folding Assessment:

• Analyze protein structure using circular dichroism spectroscopy

• Evaluate thermal stability profiles

• Compare with native enzyme where available

Post-translational Modification Analysis:

• Assess glycosylation patterns using glycan-specific staining or mass spectrometry

• Investigate other modifications like phosphorylation or acetylation that may affect activity

Enzyme Assay Optimization:

• Develop robust activity assays with appropriate controls

• Test various buffer systems, pH ranges, and ionic strengths

• Evaluate cofactor requirements and concentration optimization

Stability Considerations:

• Test stabilizers such as glycerol, BSA, or specific ions

• Evaluate storage conditions (temperature, buffer composition)

• Measure half-life under various conditions

Expression System Evaluation:
If activity issues persist, consider alternative expression systems. Research with C. cinerea enzymes has shown that enzyme characteristics can vary significantly depending on the expression host .

Methodological Approach to Troubleshooting:
Create a decision tree with systematic testing of each variable while maintaining controls. Document all conditions tested and results obtained to identify patterns that may reveal the underlying cause of low activity.

What are the key considerations for designing kinetic studies of recombinant ACS-1?

Designing robust kinetic studies for recombinant ACS-1 requires careful attention to several factors:

Assay Development:

• Select appropriate detection methods (spectrophotometric, HPLC, coupled enzyme assays)

• Ensure linearity within the concentration ranges tested

• Validate assay reproducibility with statistical analysis

Reaction Conditions Optimization:

• Determine optimal pH through pH-activity profiles (typically pH 4-9 range)

• Establish temperature optima and stability profiles

• Identify buffer components that may influence activity

Substrate Specificity Analysis:

• Test various potential substrates beyond acetate (propionate, butyrate, etc.)

• Develop structure-activity relationships

• Compare specificity constants (kcat/Km) across substrates

Kinetic Parameter Determination:

• Use appropriate models (Michaelis-Menten, allosteric, etc.)

• Determine Km, Vmax, kcat values under standardized conditions

• Evaluate product inhibition effects

Data Analysis Approaches:

• Apply multiple plotting methods (Lineweaver-Burk, Eadie-Hofstee, Hanes-Woolf)

• Use non-linear regression for direct fitting to rate equations

• Assess goodness of fit and statistical significance

Based on research with other C. cinerea enzymes, incorporation of stability testing against potential inhibitors and in the presence of organic solvents can provide valuable insights into enzyme robustness and potential applications .

What purification strategies are most effective for recombinant C. cinerea enzymes?

Effective purification of recombinant C. cinerea enzymes requires a multi-step approach, typically following these methodological guidelines:

Initial Clarification:

• Centrifugation to remove cellular debris (10,000-15,000g, 15-30 minutes)

• Filtration through 0.45-0.22 μm filters

• Ammonium sulfate precipitation for initial concentration when appropriate

Chromatographic Separation Sequence:
Based on successful purification of C. cinerea laccases, a typical sequence includes :

• Ion Exchange Chromatography:

  • Anion exchange (Q-Sepharose) for initial capture

  • Optimal salt gradient elution (typically 0-1M NaCl)

• Hydrophobic Interaction Chromatography:

  • Phenyl-Sepharose or similar matrices

  • Decreasing ammonium sulfate gradients

• Size Exclusion Chromatography:

  • Final polishing step

  • Separation based on molecular size

  • Buffer exchange capability

Affinity-Based Approaches:
When applicable, affinity tags (His-tag, GST) can simplify purification, though tag removal may be necessary if it affects enzyme activity.

Purification Monitoring:
Track purification progress through:

• Activity assays at each step

• SDS-PAGE analysis

• Protein concentration determination

• Calculation of specific activity and purification fold

Quality Control Criteria:

• Homogeneity assessment via IEF and SDS-PAGE

• Mass spectrometry confirmation

• N-terminal sequencing when necessary

• Stability testing of the purified enzyme

This methodological approach has successfully yielded pure C. cinerea enzymes, as demonstrated in the purification of laccases to homogeneity with high specific activity .

How can researchers accurately characterize the biochemical properties of recombinant ACS-1?

Comprehensive biochemical characterization of recombinant ACS-1 requires a systematic approach examining multiple enzyme properties:

pH and Temperature Profiles:

• Determine pH optima across a broad range (pH 3-10)

• Establish temperature optima (typically 25-80°C)

• Measure pH and thermal stability over time

Substrate Specificity Assessment:

• Test various potential substrates with different chain lengths and structures

• Determine kinetic parameters for each viable substrate

• Create substrate specificity profiles based on relative activity

Cofactor Requirements:

• Evaluate dependence on metal ions (Mg²⁺, Mn²⁺, Zn²⁺, etc.)

• Test effect of chelating agents (EDTA, EGTA)

• Determine optimal ATP concentrations and potential alternatives

Inhibitor Sensitivity:
Following approaches used for other C. cinerea enzymes :

• Test common enzyme inhibitors (product inhibition, competitive inhibitors)

• Evaluate stability in presence of organic solvents

• Determine IC₅₀ values for significant inhibitors

Structural Characterization:

• Molecular weight determination via SDS-PAGE and mass spectrometry

• Isoelectric point determination through IEF

• Glycosylation analysis if relevant

• Secondary structure assessment via circular dichroism

Methodological Considerations:
For each parameter, establish standard conditions, include appropriate controls, ensure reproducibility through replicate measurements, and apply statistical analysis to determine significance of observed differences.

What analytical techniques are recommended for monitoring ACS-1 activity and product formation?

Multiple analytical approaches can be employed to reliably monitor ACS-1 activity and product formation:

Spectrophotometric Assays:

• Coupled Enzyme Assays:

  • Link acetyl-CoA formation to reactions producing measurable chromophores

  • Malate dehydrogenase/citrate synthase coupling for NADH oxidation monitoring

• Direct Assays:

  • Measure CoA consumption through thiol-reactive reagents (DTNB/Ellman's reagent)

  • Monitor ATP consumption via luciferase-based assays

Chromatographic Methods:

• HPLC Analysis:

  • Reverse-phase separation of reaction components

  • UV detection of acetyl-CoA (260 nm)

  • Gradient elution systems for optimal separation

• Ion Exchange Chromatography:

  • Separation of charged intermediates and products

  • Particularly useful for separating AMP from ATP

Mass Spectrometry Approaches:

• LC-MS/MS for direct detection of acetyl-CoA formation

• Isotope labeling studies to track carbon flux

• High-resolution MS for detailed reaction mechanism studies

Radiochemical Methods:

• ¹⁴C-labeled acetate incorporation into acetyl-CoA

• Thin-layer chromatography with radiometric detection

Methodological Workflow:

• Begin with higher-throughput spectrophotometric assays for initial screening

• Confirm key findings with more specific chromatographic methods

• Apply MS-based approaches for detailed mechanism studies or complex sample analysis

Based on approaches used for other C. cinerea enzymes, method validation should include linearity assessment, reproducibility testing, and sensitivity determination for each analytical technique employed .

How should researchers approach the comparative analysis of recombinant versus native ACS-1?

Comparative analysis between recombinant and native ACS-1 requires comprehensive characterization across multiple parameters:

Enzyme Kinetics Comparison:

• Determine and compare kinetic parameters (Km, kcat, Vmax)

• Create a comparative table like:

Structural Comparison:

• Molecular weight verification via SDS-PAGE and mass spectrometry

• Glycosylation pattern analysis using glycoprotein staining techniques

• Peptide mapping through protease digestion and MS analysis

• Secondary structure comparison via circular dichroism

Stability Profiles:

• Thermal stability assessment (half-life at various temperatures)

• pH stability ranges

• Storage stability under various conditions

• Resistance to inhibitors and denaturants

Methodological Approach:

• Ensure both enzymes are analyzed under identical conditions

• Use statistical methods (t-tests, ANOVA) to determine significance of differences

• Consider multiple biological and technical replicates

• Control for buffer components and other environmental factors

Drawing from the experience with C. cinerea laccases, where recombinant enzyme characteristics varied depending on expression conditions, researchers should thoroughly document all expression and purification steps to aid in interpreting observed differences .

What statistical approaches are recommended for analyzing enzyme kinetic data of recombinant ACS-1?

Robust statistical analysis of enzyme kinetic data requires multiple complementary approaches:

Parameter Estimation Methods:

• Non-linear Regression Analysis:

  • Direct fitting to Michaelis-Menten equation

  • Weighted regression when variance is heteroscedastic

  • Robust regression methods for outlier resistance

• Linear Transformation Analysis:

  • Lineweaver-Burk plots (1/v vs 1/[S])

  • Eadie-Hofstee plots (v vs v/[S])

  • Hanes-Woolf plots ([S]/v vs [S])

Goodness of Fit Assessment:

• Residual analysis (randomness and distribution)

• R² values and adjusted R² for model comparison

• Akaike Information Criterion (AIC) for model selection

Statistical Comparison of Parameters:

• Extra sum-of-squares F-test for comparing models

• Confidence intervals for parameter estimates

• Bootstrap resampling for parameter distribution analysis

Methodological Recommendations:

• Collect sufficient data points across the substrate concentration range (minimum 7-8 points)

• Include concentrations below and above Km (ideally 0.2-5× Km)

• Perform multiple independent experiments (n≥3)

• Report standard errors or confidence intervals for all parameter estimates

Software Tools:

• GraphPad Prism for comprehensive enzyme kinetics analysis

• R with specialized packages (drc, nlstools)

• Python with scipy.optimize for custom model fitting

Following the detailed characterization approaches used for other C. cinerea enzymes, researchers should document all statistical methods employed and provide clear justification for model selection .

How can researchers effectively interpret differences in substrate specificity and catalytic efficiency between ACS-1 variants?

Interpreting differences in substrate specificity and catalytic efficiency between ACS-1 variants requires a structured analytical framework:

Specificity Constant Analysis:

• Calculate kcat/Km values for each substrate-enzyme combination

• Create relative specificity profiles using a reference substrate

• Apply log transformations for clearer visualization of large specificity ranges

Comparative Visualization Methods:

• Radar charts for multi-substrate comparison across variants

• Heat maps for visualizing specificity patterns

• Bar graphs with error bars for direct statistical comparison

Structure-Function Correlation:

• Map variations to predicted structural elements

• Analyze potential interactions with substrates based on molecular modeling

• Identify key residues responsible for specificity differences

Example Specificity Profile Table:

Methodological Integration:

• Combine kinetic data with structural information

• Consider evolutionary relationships between variants

• Perform molecular dynamics simulations for insight into substrate binding differences

Statistical Considerations:

• Apply ANOVA with post-hoc tests for multi-variant comparison

• Use multiple comparison corrections (Bonferroni, Tukey HSD)

• Report effect sizes in addition to p-values

Drawing from approaches used with other C. cinerea enzymes, researchers should ensure that all experimental conditions are standardized across variants to enable meaningful comparisons of catalytic parameters .

How can recombinant C. cinerea ACS-1 be applied in metabolic engineering studies?

Recombinant C. cinerea ACS-1 offers several valuable applications in metabolic engineering:

Acetate Utilization Enhancement:

• Overexpression in host organisms to improve acetate assimilation

• Integration into acetyl-CoA dependent pathways for bioproduct synthesis

• Balancing acetate metabolism in industrial fermentation processes

Biofuel and Biochemical Production:

• Engineering acetyl-CoA pools for fatty acid-derived biofuels

• Enhancing precursor availability for isoprenoid biosynthesis

• Improving carbon flux through the TCA cycle for organic acid production

Methodological Approaches:

• Gene Integration Strategies:

  • Genomic integration using homologous recombination

  • Plasmid-based expression with controlled induction

  • Copy number optimization for balanced expression

• Pathway Design Considerations:

  • Cofactor balance (ATP, CoA) assessment

  • Feedback inhibition management

  • Flux balancing with connected pathways

• Performance Evaluation:

  • Metabolic flux analysis using isotope labeling

  • Growth characterization under various carbon sources

  • Product yield and productivity measurements

Case Study Framework:
Based on approaches with other fungal systems, researchers could design experiments evaluating:

• Impact of ACS-1 overexpression on acetate consumption rates

• Effect on product yields from acetate-supplemented media

• Changes in central carbon metabolism flux distribution

The recombinant enzyme approach allows for variant testing and optimization without extensive genetic modification of the production organism, similar to the strategies used with other C. cinerea enzymes in biotransformation applications .

What considerations are important when using recombinant ACS-1 for in vitro biochemical studies?

When utilizing recombinant ACS-1 for in vitro biochemical studies, several methodological considerations are critical:

Enzyme Preparation and Quality:

• Ensure consistent purification protocol across batches

• Verify enzyme homogeneity through SDS-PAGE and activity assays

• Determine specific activity for standardization of enzyme amounts

• Consider storage stability and optimize preservation conditions

Reaction System Design:

• Buffer Composition:

  • Optimize pH and ionic strength

  • Select buffers without interfering components

  • Consider physiological relevance of conditions

• Cofactor Management:

  • Ensure sufficient ATP, CoA, and Mg²⁺ availability

  • Monitor potential product inhibition

  • Consider regeneration systems for expensive cofactors

• Substrate Delivery:

  • Account for solubility limitations of hydrophobic substrates

  • Consider using organic solvent co-solvents when necessary (with appropriate controls)

  • Design concentration gradients that span below and above Km values

Analytical Considerations:

• Select detection methods with appropriate sensitivity

• Ensure linearity of signal response across the concentration range

• Include proper controls for background reactions

• Consider time-course measurements for reaction progress monitoring

Methodological Controls:

• Include enzyme-free controls

• Perform substrate-free controls

• Test heat-inactivated enzyme controls

• Use known inhibitors as positive controls for specificity

Drawing from experience with other C. cinerea enzymes, researchers should document all reaction conditions in detail and validate assay reproducibility before complex experimental designs .

How can structural studies of recombinant ACS-1 inform enzyme engineering efforts?

Structural studies provide critical insights for rational enzyme engineering of ACS-1:

Structural Characterization Approaches:

• X-ray Crystallography:

  • Determine high-resolution structures of ACS-1

  • Co-crystallization with substrates, products, or inhibitors

  • Analysis of key catalytic residues and binding pockets

• Homology Modeling:

  • Prediction of structure based on related enzymes

  • Molecular docking of substrates and cofactors

  • Identification of potential engineering targets

• Hydrogen-Deuterium Exchange MS:

  • Analyze protein dynamics and conformational changes

  • Identify flexible regions and substrate-induced conformational shifts

  • Map solvent accessibility of different regions

Engineering Target Identification:

• Active site residues for altered substrate specificity

• Stability-determining regions for enhanced thermostability

• Surface residues for improved solubility or reduced aggregation

• Dynamic loops for altered catalytic rates

Rational Design Strategies:

• Site-Directed Mutagenesis:

  • Single point mutations of catalytic residues

  • Conservative substitutions for specificity alteration

  • Non-conservative changes for novel activities

• Domain Swapping:

  • Exchange of functional domains with related enzymes

  • Creation of chimeric enzymes with hybrid properties

  • Integration of regulatory domains

• Loop Engineering:

  • Modification of substrate-binding loops

  • Rigidification of flexible regions for stability

  • Introduction of disulfide bridges

Validation Methodologies:

• Activity assays comparing wild-type and variant enzymes

• Stability testing under various conditions

• Kinetic parameter determination for altered specificity

• Structural confirmation of engineered variants

Drawing from approaches used with other fungal enzymes, researchers should employ iterative cycles of design, construction, testing, and analysis, with each cycle informed by the results of previous rounds .

What are common challenges in expression and purification of recombinant ACS-1 and how can they be addressed?

Researchers commonly encounter several challenges when working with recombinant ACS-1, each requiring specific troubleshooting approaches:

Low Expression Yields:

Protein Solubility Issues:

Purification Difficulties:

Methodological Approach to Troubleshooting:

• Systematically isolate variables and test one at a time

• Document all conditions and outcomes thoroughly

• Implement small-scale tests before scaling up

• Consider parallel approaches for critical bottlenecks

Based on experience with other C. cinerea enzymes, temperature effects and media composition particularly impact recombinant enzyme production and should be prioritized in optimization efforts .

How can researchers optimize the functional characterization assays for recombinant ACS-1?

Optimizing functional characterization assays for recombinant ACS-1 requires systematic refinement across multiple parameters:

Assay Sensitivity Enhancement:

• Selection of optimal detection methods based on signal-to-noise ratios

• Buffer optimization to minimize background reactions

• Signal amplification through coupled enzyme systems when appropriate

• Instrument optimization (PMT voltage, integration time, etc.)

Reproducibility Improvement:

• Standardization of enzyme preparation and storage protocols

• Preparation of master mixes to reduce pipetting variations

• Temperature control during all assay steps

• Inclusion of internal standards for normalization

High-Throughput Adaptation:

• Miniaturization to microplate format with optimized volumes

• Automation of reagent addition and mixing steps

• Development of endpoint assays when possible

• Data processing automation with quality control metrics

Methodological Validation Approach:

• Linearity Assessment:

  • Determine linear range for enzyme concentration

  • Establish linear time course range

  • Verify substrate concentration linearity

• Precision Evaluation:

  • Calculate intra-assay coefficient of variation (CV) from replicates

  • Determine inter-assay CV across multiple days

  • Establish minimum acceptable CV thresholds

• Accuracy Confirmation:

  • Recovery testing with spiked samples

  • Comparison with orthogonal methods

  • Standard addition experiments

Optimization Table Example:

Based on experiences with other C. cinerea enzymes, careful optimization of reaction conditions can significantly improve assay performance and reliability .

What strategies can address enzyme instability issues with recombinant ACS-1?

Addressing stability challenges with recombinant ACS-1 requires a comprehensive approach:

Physical Stabilization Methods:

• Buffer Optimization:

  • Screen various buffer systems (phosphate, HEPES, MOPS)

  • Test pH ranges for optimal stability (typically pH 6-8)

  • Evaluate ionic strength effects (50-500 mM salt ranges)

• Additive Screening:

  • Polyols (glycerol, sorbitol) for hydration layer stabilization

  • Sugars (trehalose, sucrose) to prevent denaturation

  • Surfactants (Tween-20, Triton X-100) at low concentrations

  • Protein stabilizers (BSA, gelatin) to prevent adsorption losses

• Storage Condition Optimization:

  • Temperature evaluation (-80°C, -20°C, 4°C)

  • Freeze-thaw stability testing

  • Lyophilization with appropriate cryoprotectants

Chemical Stabilization Approaches:

• Cofactor Management:

  • Maintain critical cofactors (Mg²⁺, ATP, CoA) at appropriate levels

  • Add reducing agents (DTT, β-mercaptoethanol) for thiol protection

  • Include metal chelators (EDTA) to prevent metal-catalyzed oxidation

• Cross-linking Strategies:

  • Glutaraldehyde treatment for thermostability enhancement

  • Chemical modification of surface residues

  • Polymer conjugation (PEGylation) for increased solubility

Protein Engineering for Stability:

• Disulfide bridge introduction at strategic positions

• Surface charge optimization to enhance solubility

• Flexible loop rigidification based on B-factor analysis

• Consensus approach using alignment of homologous sequences

Methodological Stability Assessment:

• Thermal inactivation kinetics at various temperatures

• Long-term storage stability monitoring

• Activity retention under various stress conditions

• Conformational stability analysis via circular dichroism

Drawing from experiences with C. cinerea laccases, which showed variable stability profiles depending on purification and storage conditions, researchers should systematically evaluate multiple stabilization strategies in combination for optimal results .