Recombinant Saccharomyces cerevisiae 3-ketoacyl-CoA reductase (SCRG_02811)

What is SCRG_02811 and what is its function in Saccharomyces cerevisiae?

SCRG_02811 encodes a 3-ketoacyl-CoA reductase in Saccharomyces cerevisiae, a crucial enzyme involved in the β-oxidation pathway of fatty acid metabolism. This enzyme catalyzes the reduction of 3-ketoacyl-CoA to 3-hydroxyacyl-CoA during the β-oxidation cycle, which is the predominant pathway responsible for fatty acid catabolism in this organism . The full-length protein consists of 347 amino acids and plays a significant role in the peroxisomal fatty acid metabolism system of S. cerevisiae . As part of the multifunctional enzyme complex, it contributes to the organism's ability to utilize fatty acids as a carbon source and energy substrate.

What are the most effective expression systems for producing recombinant SCRG_02811?

For the production of recombinant SCRG_02811, E. coli has proven to be an effective heterologous expression system. The recombinant full-length Saccharomyces cerevisiae 3-Ketoacyl-CoA Reductase protein is commonly expressed with a His-tag to facilitate purification . When designing expression constructs, researchers should consider several factors:

• Codon optimization for the host organism to enhance expression levels

• Selection of appropriate promoters (e.g., T7 promoter for E. coli)

• Inclusion of fusion tags that don't interfere with enzyme activity

• Temperature and induction conditions that maximize soluble protein yield

It's worth noting that while E. coli is commonly used, expression in yeast systems such as Pichia pastoris may provide advantages for proper folding of this eukaryotic protein. Unlike some S. cerevisiae enzymes that cannot fold properly in cytoplasmic environments, proper localization signals and growth conditions must be optimized for functional expression of SCRG_02811 .

What purification challenges are specific to SCRG_02811 and how can they be addressed?

Purifying recombinant SCRG_02811 presents several challenges that researchers should address through optimized protocols:

• Protein solubility issues: Like many enzymes involved in lipid metabolism, SCRG_02811 may have hydrophobic regions that contribute to aggregation. These can be addressed by:

  • Using mild detergents during lysis and purification

  • Optimizing buffer composition with stabilizing agents

  • Employing lower expression temperatures (16-20°C)

• Maintaining enzyme activity: The catalytic function of 3-ketoacyl-CoA reductase is sensitive to oxidation and conformational changes. Consider:

  • Including reducing agents like DTT or β-mercaptoethanol in purification buffers

  • Adding glycerol (10-20%) to stabilize the protein structure

  • Minimizing freeze-thaw cycles

• Purification strategy: A multi-step approach is recommended:

  • Initial capture using immobilized metal affinity chromatography (IMAC) for His-tagged protein

  • Secondary purification via ion exchange or size exclusion chromatography

  • Activity-based validation of purified fractions

Researchers should note that, unlike cytoplasmic expression efforts with Fox2p that yielded no detectable activity, properly expressed and purified SCRG_02811 should maintain its catalytic function when appropriate conditions are maintained throughout the purification process .

What are the optimal conditions for assaying SCRG_02811 enzymatic activity?

The enzymatic activity of SCRG_02811 (3-ketoacyl-CoA reductase) can be assayed through spectrophotometric methods by monitoring the oxidation of NADPH or reduction of NAD+, depending on the direction of the reaction being measured. Optimal assay conditions include:

• Buffer composition and pH:

  • Phosphate buffer (50-100 mM) at pH 7.0-7.5

  • Addition of 0.1-0.5 mM EDTA to chelate metal ions that may inhibit activity

• Cofactor requirements:

  • NADPH or NADH as electron donors (typically 0.1-0.3 mM)

  • Optimal NADPH/NADH ratio may affect directionality of the reaction

• Substrate considerations:

  • Various chain length 3-ketoacyl-CoA substrates (C4-C18)

  • Substrate concentrations in the range of 10-100 μM

  • Inclusion of 0.01-0.05% Triton X-100 for improved substrate solubility

• Temperature and time course:

  • Temperature optimum typically between 25-30°C

  • Initial velocity measurements within linear range (first 1-5 minutes)

When designing activity assays, researchers should consider the substrate preferences observed in the S. cerevisiae β-oxidation pathway, as research has revealed distinct variations in β-oxidation among different fatty acids, primarily attributed to substrate preferences of key enzymes .

How do substrate specificity patterns of SCRG_02811 compare with similar enzymes from other organisms?

The substrate specificity of SCRG_02811 (3-ketoacyl-CoA reductase) from S. cerevisiae shows distinctive patterns compared to similar enzymes from other organisms:

• Chain length preferences:

  • S. cerevisiae SCRG_02811 shows activity toward medium to long-chain (C8-C18) 3-ketoacyl-CoA substrates

  • Unlike bacterial homologs that may prefer shorter chain substrates

• Saturation state preferences:

  • Research indicates differential handling of saturated versus unsaturated fatty acid derivatives

  • The enzyme shows activity toward both saturated and unsaturated substrates, with Faa1p and Faa4p playing major roles in activating intracellular C14 and C16 saturated fatty acids upstream of this enzyme

• Comparative kinetic parameters:

  Organism	Enzyme	Preferred Substrates	Km Range (μM)	kcat/Km (M⁻¹s⁻¹)
  S. cerevisiae	SCRG_02811	C14-C18 3-ketoacyl-CoA	10-50	10³-10⁵
  Y. lipolytica	3-ketoacyl-CoA reductase	C16-C18 3-ketoacyl-CoA	5-30	10⁴-10⁶
  C. albicans	3-ketoacyl-CoA reductase	C14-C18 3-ketoacyl-CoA	15-60	10³-10⁴

• Regulatory differences:

  • S. cerevisiae SCRG_02811 activity is integrated with peroxisomal functions

  • In contrast to Y. lipolytica, which shows distinct fatty acid activation patterns with ACS I activating long-chain fatty acids in the cytoplasm and ACS II activating medium and short-chain fatty acids directly in peroxisomes

These comparative analyses reveal that while the fundamental catalytic function remains conserved, the substrate preferences and regulatory mechanisms have evolved to suit the metabolic needs of different organisms.

How can SCRG_02811 expression be regulated in S. cerevisiae for enhanced fatty acid metabolism studies?

Regulation of SCRG_02811 expression in S. cerevisiae can be achieved through several genetic and environmental strategies:

• Promoter manipulation:

  • Replacement of native promoter with carbon source-responsive promoters

  • Similar to FOX1 gene regulation, where transcription is strictly regulated by carbon source. FOX1 transcription is undetectable when glucose concentration exceeds 0.1%, minimally expressed with ethanol as carbon source, and significantly upregulated (25x and 10x higher than glucose and ethanol conditions, respectively) when oleic acid is the sole carbon source

• Environmental regulation:

  • Carbon source selection (glucose repression, oleic acid induction)

  • Growth phase considerations (expression increases during stationary phase)

  • Oxygen availability (aerobic conditions favor β-oxidation pathway expression)

• Genetic background optimization:

  • Deletion or overexpression of upstream regulatory factors

  • Co-expression with other β-oxidation pathway components

  • Similar to approaches used with FAA1/FAA4 where their knockout significantly impacts fatty acid activation and consumption

• Integration with redox balancing:

  • Address NADH/NAD+ ratio imbalances that affect enzymatic function

  • Similar to approaches used in FFA-producing strains where deletion of GPD1/GPD2 and overexpression of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GapN) helped balance redox factors

These regulatory approaches should be carefully designed based on the specific research objectives, whether focused on basic understanding of enzyme function or metabolic engineering for enhanced fatty acid utilization.

What are the most effective knockout and overexpression strategies for studying SCRG_02811 function?

For comprehensive functional analysis of SCRG_02811, researchers should consider these effective genetic manipulation strategies:

• Knockout methodologies:

  • CRISPR-Cas9 genome editing for precise gene deletion

  • Homologous recombination-based approaches using selectable markers

  • Conditional knockout systems (e.g., tetracycline-regulated expression) for essential genes

• Overexpression approaches:

  • Genomic integration vs. episomal expression considerations

  • Selection of appropriate promoters (constitutive vs. inducible)

  • Codon optimization and fusion tag design for enhanced expression and detection

• Complementation studies:

  • Expression of wild-type SCRG_02811 in knockout strains to verify phenotype rescue

  • Cross-species complementation with homologs from Y. lipolytica or C. albicans to study functional conservation

• Protein localization considerations:

  • Proper peroxisomal localization signals must be maintained

  • Unlike Fox2p, which cannot fold correctly in the cytoplasm, careful design of expression constructs is needed to ensure proper subcellular targeting and folding

• Multi-gene modification strategies:

  • Simultaneous manipulation of related pathway genes

  • For example, research has shown that knockout of FAA1 or FAA4 alone did not effectively reduce activation and consumption of intracellular fatty acids, whereas their combined knockout significantly reduced activation of cytoplasmic fatty acids

These genetic approaches provide powerful tools for dissecting the role of SCRG_02811 in fatty acid metabolism and its interactions with other pathway components.

How does SCRG_02811 interact with other enzymes in the β-oxidation pathway?

SCRG_02811 (3-ketoacyl-CoA reductase) functions as part of a coordinated enzymatic cascade in the β-oxidation pathway of S. cerevisiae, with specific interactions:

• Sequential pathway interactions:

  • Receives 3-ketoacyl-CoA substrates generated by Fox2p's 3-hydroxyacyl-CoA dehydrogenase activity

  • Fox2p is a multifunctional enzyme with both enoyl-CoA hydratase and hydroxyl-CoA dehydrogenase activity, catalyzing the transformation of trans-2,3-enoyl-CoA to 3-ketoacyl-CoA

  • Products of SCRG_02811 are further processed by thiolase enzymes (Fox3p/Pot1p)

• Regulatory interactions:

  • Expression is coordinated with other β-oxidation enzymes

  • Responds to similar regulatory factors as Fox1p, whose transcription is regulated by intracellular glucose, ethanol, and long-chain fatty acid levels

• Physical complex formation:

  • Evidence suggests potential protein-protein interactions within the pathway

  • May function within multienzyme complexes for efficient substrate channeling

• Cofactor competition and sharing:

  • Competes for or shares NAD+/NADH with other dehydrogenases in the pathway

  • Integration with cellular redox balance mechanisms

• Peroxisomal localization interactions:

  • Requires proper targeting to peroxisomes via PTS1/PTS2 signals

  • Interacts with peroxisomal import machinery encoded by PEX genes

  • Functions downstream of the Pxa1p/Pxa2p transporter complex that imports activated long-chain fatty acids into peroxisomes

These interactions highlight the integrated nature of β-oxidation and the critical role of SCRG_02811 within this metabolic network.

What role does SCRG_02811 play in alternative metabolic pathways beyond fatty acid oxidation?

Beyond its primary role in β-oxidation, SCRG_02811 (3-ketoacyl-CoA reductase) influences several interconnected metabolic pathways:

• Fatty acid synthesis crosstalk:

  • Provides metabolic intermediates that may feed into fatty acid synthesis

  • Changes in SCRG_02811 activity can affect the balance between degradation and synthesis pathways

  • Similar to observations where deletion of FAA1/FAA4 and expression of thioesterase ACOT5s increased expression levels of fatty acid synthesis genes ACC1, FAS1, FAS2, and OLE1

• Central carbon metabolism connections:

  • Acetyl-CoA generated from β-oxidation feeds into the TCA cycle and glyoxylate cycle

  • SCRG_02811 activity indirectly influences carbon flux through these pathways

• Redox balance mechanisms:

  • NADH generated or consumed by SCRG_02811 affects cellular redox state

  • Similar to observations in engineered strains where GPD1/GPD2 deletion and GapN overexpression balanced NAD/NADH redox factors to improve fatty acid production

• Lipid membrane homeostasis:

  • Influences phospholipid composition through fatty acid availability

  • Affects membrane fluidity and composition, particularly regarding the saturation state of fatty acids

• Peroxisome proliferation pathways:

  • β-oxidation activity links to peroxisome biogenesis and abundance

  • SCRG_02811 function may influence peroxisomal protein import via the Pex11p pathway, which participates in medium-chain fatty acid transport

These interconnections demonstrate that SCRG_02811 serves not just as a β-oxidation enzyme but as a node in the broader metabolic network of S. cerevisiae.

How can SCRG_02811 be leveraged for enhancing fatty acid-derived bioproduct synthesis?

SCRG_02811 presents several strategic opportunities for metabolic engineering to enhance fatty acid-derived bioproduct synthesis:

• Modification of enzyme properties:

  • Protein engineering to alter substrate specificity

  • Directed evolution to enhance catalytic efficiency

  • Structure-guided mutations to reduce product inhibition

• Pathway flux optimization:

  • Controlled expression to balance β-oxidation with product synthesis

  • Similar to approaches where FFA-producing strains with gene deletions (Δfaa1, Δfaa4, Δpox1, Δfaa4, ΔHFD1) were used as chassis cells for metabolic engineering

• Integration with synthetic pathways:

  • Coupling modified SCRG_02811 activity with heterologous biosynthetic modules

  • Redirection of 3-hydroxyacyl-CoA intermediates toward valuable products

  • Engineering strategies similar to those where overexpression of the bacterial pyruvate dehydrogenase PDH complex increased acetyl-CoA supply, resulting in 840.5 mg/L FFA production, 2.08% higher than control strains

• Redox engineering approaches:

  • Balancing NADH/NAD+ ratios to favor desired reaction directionality

  • Co-expression with complementary redox-balancing enzymes

  • Implementation of strategies similar to those using NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GapN) overexpression to balance redox factors

• Regulatory circuit design:

  • Development of synthetic regulatory systems for dynamic control

  • Biosensor-based feedback mechanisms responsive to product or intermediate levels

These approaches represent promising strategies for leveraging SCRG_02811 in the development of yeast-based platforms for sustainable production of fatty acid-derived chemicals and biofuels.

What are the most promising targets for improvement when engineering SCRG_02811 for biotechnological applications?

When engineering SCRG_02811 for biotechnological applications, researchers should focus on these high-priority targets for improvement:

• Catalytic efficiency enhancement:

  • Rational design based on structural modeling and homology with characterized reductases

  • Active site modifications to improve substrate binding (Km) and turnover rate (kcat)

  • Laboratory evolution to identify variants with enhanced performance under industrial conditions

• Substrate specificity engineering:

  • Broadening or narrowing substrate range based on desired products

  • Targeting the accommodation of non-native or synthetic substrates

  • Engineering to preferentially process specific fatty acid chain lengths or saturation states

• Stability improvement:

  • Thermostability enhancement for robustness in industrial processes

  • pH tolerance expansion for versatility in different process conditions

  • Tolerance to organic solvents for biphasic fermentation systems

• Cofactor preference manipulation:

  • Engineering NADH vs. NADPH preference to align with cellular availability

  • Creating variants with altered cofactor binding to influence reaction directionality

  • Similar to strategies addressing redox imbalances in FFA production, where the NADH/NAD ratio was optimized

• Protein-protein interaction engineering:

  • Optimizing interactions with other β-oxidation enzymes for efficient substrate channeling

  • Creating synthetic scaffolds to co-localize pathway enzymes

  • Designing enzyme fusions to improve pathway efficiency

These engineering targets offer promising avenues for developing SCRG_02811 variants with improved properties for various biotechnological applications.

What are the most effective techniques for analyzing SCRG_02811 structure-function relationships?

Comprehensive analysis of SCRG_02811 structure-function relationships requires a multi-faceted approach combining several analytical techniques:

• Crystallography and structural determination:

  • X-ray crystallography of purified protein (native and with substrates/inhibitors)

  • Cryo-electron microscopy for structural analysis in different functional states

  • NMR spectroscopy for solution-state dynamics and ligand interactions

• Computational modeling approaches:

  • Homology modeling based on related reductases with known structures

  • Molecular dynamics simulations to predict substrate binding and catalytic mechanisms

  • Quantum mechanics/molecular mechanics (QM/MM) calculations for reaction mechanism elucidation

• Mutagenesis and functional assays:

  • Alanine scanning mutagenesis of conserved residues

  • Site-directed mutagenesis guided by structural predictions

  • Activity assays with various substrates to correlate structural changes with functional impacts

• Biophysical characterization:

  • Circular dichroism (CD) spectroscopy for secondary structure analysis

  • Differential scanning calorimetry (DSC) for thermal stability assessment

  • Isothermal titration calorimetry (ITC) for substrate and cofactor binding kinetics

• Proteomic approaches:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational dynamics

  • Cross-linking mass spectrometry for protein-protein interaction mapping

  • Limited proteolysis to identify flexible regions and domains

These methodologies, when applied in combination, provide comprehensive insights into how SCRG_02811 structure relates to its catalytic function, substrate specificity, and integration within the β-oxidation pathway.

How can researchers accurately quantify SCRG_02811 activity in complex cellular environments?

Accurately quantifying SCRG_02811 activity in complex cellular environments presents several challenges that can be addressed through specialized analytical approaches:

• Activity-based protein profiling:

  • Development of selective activity-based probes for 3-ketoacyl-CoA reductase

  • In-gel fluorescence analysis of labeled enzyme

  • Mass spectrometry quantification of probe-labeled enzyme

• Metabolomic approaches:

  • Targeted LC-MS/MS analysis of 3-ketoacyl-CoA and 3-hydroxyacyl-CoA intermediates

  • Stable isotope labeling to track specific substrate conversions

  • Flux analysis using 13C-labeled fatty acids to measure pathway activity

• Reporter systems:

  • Development of biosensors responsive to substrate/product ratios

  • Fluorescent or luminescent reporter constructs linked to pathway activity

  • Split-protein complementation assays for enzyme-substrate interactions

• In situ activity measurements:

  • Permeabilized cell assays maintaining cellular compartmentalization

  • Subcellular fractionation to isolate peroxisomes for direct activity measurement

  • Development of cell-penetrating fluorogenic substrates

• Proteomics-integrated approaches:

  • Correlation of enzyme abundance (via targeted proteomics) with metabolite levels

  • Post-translational modification analysis affecting enzyme activity

  • Protein turnover studies to account for degradation effects

These methods provide researchers with powerful tools to quantify SCRG_02811 activity within its native cellular context, accounting for the complexities of compartmentalization, regulation, and interaction with other pathway components that may not be captured in reconstituted in vitro systems.

How do 3-ketoacyl-CoA reductases from different yeast species compare in terms of structure and function?

3-Ketoacyl-CoA reductases from different yeast species exhibit notable variations in structure and function that reflect their evolutionary adaptations to different ecological niches and metabolic requirements:

• Sequence conservation and divergence:

  • Core catalytic domains show high conservation across species

  • Regulatory regions and targeting sequences display greater divergence

  • S. cerevisiae SCRG_02811 shares significant homology with counterparts in other yeasts, while maintaining species-specific features

• Subcellular localization patterns:

  • S. cerevisiae 3-ketoacyl-CoA reductase localizes to peroxisomes for β-oxidation

  • Y. lipolytica shows distinct fatty acid activation patterns with transport functions in addition to activation roles, with long-chain fatty acids activated by ACS I in the cytoplasm and medium/short-chain fatty acids activated directly in peroxisomes by ACS II

  • C. albicans exhibits patterns similar to S. cerevisiae but with unique regulatory aspects

• Enzyme kinetics and substrate preferences:

  • S. cerevisiae enzymes show distinct substrate preferences compared to other yeasts

  • C. albicans has three 3-ketoacyl-CoA thiolases (Pot1p, Fox3p, and Pot13p) with Pot1p and Fox3p sharing high homology with S. cerevisiae Pot1p

  • Y. lipolytica enzymes often demonstrate broader substrate ranges reflecting its oleaginous nature

• Regulatory mechanisms:

  • Carbon source responsiveness varies between species

  • Transcriptional regulation shows species-specific patterns

  • Post-translational modifications differ among homologs

• Functional roles in metabolism:

  • Core β-oxidation function is conserved across species

  • Integration with other metabolic pathways shows species-specific patterns

  • Contribution to lipid homeostasis varies based on species-specific lipid metabolism

These comparative analyses provide valuable insights into the evolution of fatty acid metabolism in fungi and can guide rational engineering approaches based on advantageous features from different species.

What insights from other organisms' 3-ketoacyl-CoA reductases can be applied to understanding or improving SCRG_02811?

Comparative analysis of 3-ketoacyl-CoA reductases across diverse organisms offers valuable insights that can be applied to understanding and engineering SCRG_02811:

• Bacterial enzyme insights:

  • Bacterial FabG (3-ketoacyl-ACP reductase) structures provide templates for modeling SCRG_02811

  • Bacterial enzymes often show higher thermostability, informing stability engineering

  • Insights from bacterial directed evolution studies can guide mutagenesis approaches

• Mammalian system comparisons:

  • Mammalian 3-ketoacyl-CoA reductases exhibit specialized regulatory mechanisms

  • Structural features enabling broader substrate ranges could be transferred to SCRG_02811

  • Mammalian enzymes like those from M. musculus provide insights, as seen with the successful expression of truncated acyl-CoA thioesterase ACOT5 (Acot5s) from M. musculus in engineered yeast strains

• Plant enzyme characteristics:

  • Plant 3-ketoacyl-ACP reductases show unique substrate specificities

  • Regulation by plant-specific factors reveals alternative control mechanisms

  • Adaptation to different cellular compartments offers insights for subcellular targeting

• Other yeast species applications:

  • Y. lipolytica's dual transport and activation functions could be engineered into SCRG_02811

  • C. albicans enzymes offer insights into pathogen-specific adaptations

  • Analysis of oleaginous yeasts provides strategies for enhancing fatty acid metabolism

• Extremophile enzyme lessons:

  • Thermophilic archaea provide templates for thermostable variants

  • Halophilic adaptations offer strategies for salt tolerance

  • Psychrophilic features could inspire cold-active variants for low-temperature applications

By integrating insights from these diverse systems, researchers can develop comprehensive models of 3-ketoacyl-CoA reductase function and engineer SCRG_02811 variants with novel or enhanced properties suited to specific research or biotechnological applications.

What are common challenges in working with SCRG_02811 and how can they be overcome?

Researchers working with SCRG_02811 frequently encounter several technical challenges that can be addressed through optimized approaches:

• Expression and solubility issues:

  • Challenge: Low expression levels or inclusion body formation

  • Solution: Optimize codon usage, lower induction temperature (16-20°C), use solubility-enhancing fusion tags (MBP, SUMO), or employ specialized expression strains designed for difficult proteins

• Enzymatic activity instability:

  • Challenge: Loss of activity during purification or storage

  • Solution: Include stabilizing agents (glycerol 10-20%, reducing agents), optimize buffer composition, minimize freeze-thaw cycles, and consider activity-preserving immobilization techniques

• Substrate availability limitations:

  • Challenge: Commercial unavailability of 3-ketoacyl-CoA substrates

  • Solution: Develop enzymatic synthesis methods, collaborate with specialized chemical synthesis laboratories, or implement coupled enzyme assays to generate substrates in situ

• Assay interference in complex samples:

  • Challenge: Background signal or competing activities in cell lysates

  • Solution: Develop specific inhibitors for competing enzymes, optimize extraction protocols to preserve compartmentalization, or implement selective activity-based probes

• Localization challenges:

  • Challenge: Improper subcellular targeting affecting function

  • Solution: Verify and optimize peroxisomal targeting sequences, ensure proper folding conditions, and incorporate lessons from Fox2p which cannot fold correctly in the cytoplasm of S. cerevisiae

These solutions represent proven strategies for overcoming common technical hurdles in SCRG_02811 research, enabling more efficient and reliable experimental outcomes.

How can researchers optimize assay conditions to accurately measure SCRG_02811 activity in different experimental contexts?

Optimizing assay conditions for SCRG_02811 requires systematic adjustment across multiple parameters to ensure accurate activity measurements in various experimental contexts:

• Spectrophotometric assay optimization:

  • Buffer selection: Test multiple buffer systems (phosphate, HEPES, Tris) at 50-100 mM across pH range 6.5-8.0

  • Cofactor concentration: Titrate NADPH/NADH (0.05-0.5 mM) to determine optimal concentration

  • Substrate concentration range: Develop Michaelis-Menten kinetics across substrate range (5-200 μM)

  • Temperature optimization: Determine activity profile across 20-40°C

  • Additives screening: Test effects of metal ions, stabilizers, and detergents

• High-throughput screening adaptations:

  • Miniaturization to microtiter plate format (96/384-well)

  • Development of fluorescent or colorimetric endpoint assays

  • Automation-compatible protocol modifications

  • Statistical validation for Z-factor and signal-to-noise optimization

• In vivo activity monitoring:

  • Development of whole-cell assays with permeabilization

  • Reporter system construction linked to pathway activity

  • Metabolite profiling to track product formation

  • Growth-based phenotypic screens in defined media

• Inhibitor and activator profiling:

  • Systematic screening of potential inhibitors and activators

  • Determination of IC50/EC50 values and inhibition mechanisms

  • Specificity testing against related enzymes

  • Structure-activity relationship development

• Coupled enzyme assay development:

  • Design of linked enzyme systems for continuous monitoring

  • Optimization of coupling enzyme ratios

  • Validation of rate-limiting step determination

  • Implementation of internal standards and controls

By systematically addressing these parameters, researchers can develop robust, reproducible assays for accurately measuring SCRG_02811 activity across diverse experimental contexts, from basic enzymatic characterization to high-throughput screening applications.

What are the most promising research frontiers for understanding SCRG_02811 function in S. cerevisiae metabolism?

Several cutting-edge research directions hold significant promise for advancing our understanding of SCRG_02811's role in S. cerevisiae metabolism:

• Systems biology integration:

  • Multi-omics profiling (transcriptomics, proteomics, metabolomics) to map SCRG_02811's influence on global metabolic networks

  • Flux balance analysis to quantify contributions to fatty acid metabolism

  • Development of comprehensive computational models integrating enzyme kinetics with whole-cell metabolism

• Single-cell analysis approaches:

  • Investigation of cell-to-cell variability in SCRG_02811 expression and activity

  • Correlation of peroxisome number and distribution with enzyme function

  • Time-lapse microscopy to track dynamic responses to changing fatty acid availability

• Structural biology advancements:

  • Cryo-EM studies of SCRG_02811 within native peroxisomal complexes

  • Time-resolved crystallography to capture catalytic intermediates

  • Integrative structural modeling incorporating diverse experimental data

• Synthetic biology applications:

  • Development of SCRG_02811-based biosensors for fatty acid metabolism

  • Creation of orthogonal β-oxidation pathways for specialized functions

  • Integration with non-native metabolic modules for novel product synthesis

• Evolutionary and comparative studies:

  • Reconstruction of ancestral 3-ketoacyl-CoA reductases to trace functional evolution

  • Horizontal gene transfer analysis to identify advantageous enzyme variants

  • Comparative studies with oleaginous yeasts to identify optimization targets

These research frontiers represent promising avenues for elucidating the fundamental biology of SCRG_02811 while simultaneously establishing foundations for biotechnological applications.

How might new technological advances impact our ability to study and utilize SCRG_02811?

Emerging technologies are poised to revolutionize SCRG_02811 research and applications through several transformative approaches:

• CRISPR-based technologies:

  • Base editing for precise modification of catalytic residues without double-strand breaks

  • CRISPRi/CRISPRa for tunable regulation of expression levels

  • Prime editing for sophisticated gene modifications without donor templates

  • High-throughput CRISPR screening to identify genetic interactions

• Advanced imaging techniques:

  • Super-resolution microscopy for visualizing peroxisomal enzyme organization

  • Correlative light and electron microscopy (CLEM) to link enzyme localization with ultrastructure

  • Live-cell metabolic imaging with genetically encoded sensors

  • Label-free techniques for monitoring enzymatic activity in situ

• Artificial intelligence applications:

  • Machine learning for protein structure prediction and function annotation

  • Deep learning models to predict enzyme specificity from sequence

  • AI-driven design of improved enzyme variants with desired properties

  • Automated laboratory systems for high-throughput enzyme characterization

• Synthetic biology platforms:

  • Cell-free systems for rapid prototyping of engineered enzymes

  • Genome-reduced chassis strains for simplified pathway optimization

  • DNA assembly methods for combinatorial pathway engineering

  • Microdroplet platforms for ultrahigh-throughput enzyme evolution

• Multi-scale modeling approaches:

  • Integrated models spanning quantum mechanics to whole-cell physiology

  • Spatiotemporal modeling of peroxisomal reactions and metabolite transport

  • Enzyme design algorithms incorporating machine learning and molecular dynamics

  • Constraint-based modeling to predict metabolic responses to enzyme modifications

These technological advances will enable unprecedented insights into SCRG_02811 structure, function, and regulation while accelerating the development of engineered variants for biotechnological applications, similar to progress made with other enzymes in the fatty acid metabolic pathway .