Recombinant Kluyveromyces lactis 3-ketoacyl-CoA reductase (KLLA0B09812g)

What is Kluyveromyces lactis 3-ketoacyl-CoA reductase and what is its function?

Kluyveromyces lactis 3-ketoacyl-CoA reductase (KLLA0B09812g) is an enzyme that catalyzes the NADPH-dependent reduction of 3-ketoacyl intermediates to (R)-3-hydroxyacyl isomers in the fatty acid biosynthesis pathway. The enzyme is also known as Very-long-chain 3-oxoacyl-CoA reductase, 3-ketoreductase, KAR, or Microsomal beta-keto-reductase . It plays a crucial role in lipid metabolism, particularly in the elongation of very long chain fatty acids (VLCFAs).

Similar to other 3-ketoacyl-CoA reductases, such as those found in E. coli and Pseudomonas species, this enzyme is involved in the conversion of 3-ketoacyl-CoA to 3-hydroxyacyl-CoA, which can be important for various metabolic processes including synthesis of cellular lipids and membrane components .

Why is Kluyveromyces lactis an important expression system for recombinant proteins?

Kluyveromyces lactis has emerged as one of the most important yeast species for research and industrial biotechnology due to several advantageous characteristics:

• It is a Crabtree-negative yeast, which means it does not produce ethanol under aerobic conditions with excess glucose, allowing for higher biomass yields .

• K. lactis has exceptional protein secretion capabilities, making it particularly attractive for heterologous protein production .

• Since 1991, almost 100 recombinant proteins have been successfully expressed in K. lactis, with 20% of those produced in recent years (as of 2016), demonstrating its growing importance .

• It is considered "food-safe" and suitable for applications in food and pharmaceutical industries, offering advantages for downstream applications requiring safety compliance .

• Well-established genetic modification techniques, including CRISPR/Cas9 systems, are available for K. lactis, facilitating strain design and optimization .

How does substrate specificity of K. lactis 3-ketoacyl-CoA reductase compare to other homologous enzymes?

The substrate specificity of K. lactis 3-ketoacyl-CoA reductase can be compared to other homologous enzymes by examining its ability to process various chain-length substrates. While specific data for K. lactis 3-ketoacyl-CoA reductase is limited in the provided search results, insights can be drawn from studies of similar enzymes:

Studies with 3-ketoacyl-CoA reductases from other organisms, such as E. coli FabG and Pseudomonas sp. 61-3 FabG, have shown these enzymes can process substrates of different chain lengths (C4 to C12) . Testing substrate specificity typically involves measuring NADPH formation at 340 nm when the enzyme is provided with different chain-length substrates .

In Arabidopsis, KCS enzymes (similar in function) demonstrated specific elongation patterns, where some can elongate fatty acids up to C24 while others push further to C26 and beyond . This suggests that different 3-ketoacyl-CoA reductases may have evolved distinct substrate preferences based on their biological roles.

For rigorous characterization of K. lactis 3-ketoacyl-CoA reductase specificity, researchers should perform comparative enzyme assays using:

What are the optimal expression conditions for recombinant K. lactis 3-ketoacyl-CoA reductase?

The optimal expression conditions for recombinant K. lactis 3-ketoacyl-CoA reductase depend on the host system being used. Based on available information and general principles for recombinant protein expression:

When expressed in E. coli:

• The full-length protein (1-346 amino acids) has been successfully expressed with an N-terminal His-tag .

• The protein is typically obtained as a lyophilized powder after purification and can be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• For long-term storage, addition of 5-50% glycerol and storage at -20°C/-80°C is recommended to maintain enzyme activity .

When expressed in K. lactis itself:

• K. lactis has demonstrated high-level secretion of recombinant proteins, making it potentially suitable for homologous expression of its own enzymes .

• Genetic modifications used for heterologous gene expression in K. lactis can be applied, including integration into the genome or use of expression vectors .

• The choice between intracellular retention and secretion would depend on the research objectives and downstream applications.

Optimization experiments should systematically evaluate:

• Promoter strength and induction conditions

• Growth media composition

• Temperature and pH during growth and induction

• Cell density at induction

• Duration of expression

• Codon optimization for the host organism

How can structural analysis of K. lactis 3-ketoacyl-CoA reductase inform enzyme engineering efforts?

Structural analysis of K. lactis 3-ketoacyl-CoA reductase can provide critical insights for enzyme engineering through the following approaches:

• Cofactor binding site analysis: 3-ketoacyl-CoA reductases utilize NADPH as a cofactor. Identifying and characterizing the NADPH binding domain through structural studies can help in engineering efforts to alter cofactor specificity (NADPH vs. NADH) or improve binding affinity.

• Substrate binding pocket characterization: Detailed structural information about the substrate binding pocket would reveal amino acid residues that interact with the acyl chain. This knowledge is crucial for engineering substrate specificity, particularly for applications targeting specific chain-length fatty acids.

• Catalytic residue identification: Identifying the catalytic residues involved in the reduction reaction would allow for targeted mutagenesis to enhance catalytic efficiency or alter reaction mechanisms.

• Structural comparisons with homologs: Comparative analysis with structurally characterized homologs, such as those from E. coli or Pseudomonas species that have been studied for substrate specificity in processing different chain lengths (C4-C12) , could reveal evolutionary adaptations that confer specific functional properties.

• Protein stability assessment: Structural data can identify regions that contribute to protein stability, which is particularly important for industrial applications requiring robust enzymes that can withstand harsh reaction conditions.

What is the recommended protocol for purifying recombinant K. lactis 3-ketoacyl-CoA reductase?

A comprehensive purification protocol for recombinant K. lactis 3-ketoacyl-CoA reductase (KLLA0B09812g) with an N-terminal His-tag should include the following steps:

• Cell Lysis:

  • Harvest cells expressing the recombinant protein

  • Resuspend in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, 1 mM PMSF, and appropriate protease inhibitors

  • Lyse cells using sonication or mechanical disruption

  • Centrifuge at 15,000 × g for 30 minutes at 4°C to remove cell debris

• Immobilized Metal Affinity Chromatography (IMAC):

  • Load cleared lysate onto a Ni-NTA column pre-equilibrated with binding buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole)

  • Wash extensively with wash buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole)

  • Elute the protein with elution buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 250 mM imidazole)

• Buffer Exchange and Concentration:

  • Pool elution fractions containing the target protein

  • Dialyze against storage buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl)

  • Concentrate using a centrifugal concentrator with appropriate molecular weight cut-off

• Size Exclusion Chromatography (optional):

  • For higher purity, apply the concentrated protein to a size exclusion column

  • Collect fractions containing the purified protein

• Quality Control:

  • Verify purity by SDS-PAGE (should be >90%)

  • Confirm identity by Western blotting or mass spectrometry

  • Assess activity using an enzyme assay measuring NADPH formation at 340 nm

• Storage:

  • Add glycerol to a final concentration of 50%

  • Aliquot to avoid freeze-thaw cycles

  • Store at -20°C/-80°C for long-term storage

For reconstitution, the lyophilized protein should be briefly centrifuged prior to opening and then reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

How can enzymatic activity of K. lactis 3-ketoacyl-CoA reductase be accurately measured?

The enzymatic activity of K. lactis 3-ketoacyl-CoA reductase can be accurately measured using spectrophotometric assays that monitor the NADPH-dependent reduction of 3-ketoacyl-CoA substrates. Based on methodologies used for similar enzymes , the following protocol is recommended:

Standard Spectrophotometric Assay:

• Reaction Mixture Preparation:

  • 50 mM Tris-HCl buffer (pH 8.0)

  • 0.25 mM 3-ketoacyl-CoA substrate (various chain lengths: C4, C6, C8, C10, C12)

  • 0.5 mM NADPH

  • Purified K. lactis 3-ketoacyl-CoA reductase (1-5 μg)

  • Total reaction volume: 400 μl

• Assay Procedure:

  • Pre-warm all components to room temperature

  • Add all components except the enzyme to a quartz cuvette

  • Record baseline at 340 nm

  • Initiate reaction by adding the enzyme

  • Monitor the decrease in absorbance at 340 nm for 2-5 minutes

  • Calculate activity based on the rate of NADPH consumption (ε₃₄₀ = 6,220 M⁻¹cm⁻¹)

• Data Analysis:

  • Calculate the initial reaction rate (ΔA₃₄₀/min)

  • Convert to enzyme activity units: 1 Unit = amount of enzyme that catalyzes the oxidation of 1 μmol of NADPH per minute

  • Determine specific activity (Units/mg protein)

• Kinetic Parameters Determination:

  • Perform assays at varying substrate concentrations

  • Plot data using Michaelis-Menten, Lineweaver-Burk, or Eadie-Hofstee methods

  • Calculate Km, Vmax, and kcat values

  • Determine substrate specificity by comparing kinetic parameters across different chain-length substrates

This methodology is similar to the approach used to evaluate substrate specificity of E. coli FabG and Pseudomonas sp. 61-3 FabG proteins, where reactions were monitored by the increase in absorbance at 340 nm due to NADPH formation .

What are the key considerations for coexpressing K. lactis 3-ketoacyl-CoA reductase with other enzymes of the fatty acid synthesis pathway?

Coexpression of K. lactis 3-ketoacyl-CoA reductase with other enzymes of the fatty acid synthesis pathway requires careful consideration of several factors to ensure functional integration and productive metabolic flux:

• Vector Design and Compatibility:

  • For prokaryotic expression: Use compatible plasmids with different origins of replication and selection markers

  • For eukaryotic expression: Consider polycistronic constructs or multiple integration sites

  • Balance expression levels using different promoter strengths to avoid metabolic burden

• Stoichiometric Balance:

  • Previous studies have shown that coexpression of fabG genes with type II PHA synthase genes enables recombinant E. coli to accumulate MCL PHA copolymers

  • The relative expression levels of pathway enzymes should be optimized to prevent bottlenecks

  • Consider using promoters with similar strengths or tunable induction systems

• Subcellular Localization:

  • Ensure proper targeting of enzymes to the same cellular compartment

  • For K. lactis expression, consider that homologous proteins like KCS family members in plants are typically localized to the endoplasmic reticulum

  • If needed, include targeting sequences to direct enzymes to the appropriate compartment

• Cofactor Availability:

  • 3-Ketoacyl-CoA reductase requires NADPH as a cofactor

  • Ensure sufficient regeneration of NADPH, possibly by coexpressing cofactor regeneration systems

  • Consider the metabolic state of the host and its ability to provide necessary cofactors

• Substrate Channeling:

  • Investigate fusion protein strategies to enhance substrate channeling between sequential enzymes

  • Consider spatial organization of pathway enzymes to minimize diffusion of intermediates

• Optimization Table for Coexpression Systems:

How can researchers address issues of protein insolubility when expressing K. lactis 3-ketoacyl-CoA reductase?

When facing protein insolubility issues with K. lactis 3-ketoacyl-CoA reductase expression, researchers should implement the following systematic troubleshooting approaches:

• Expression Condition Optimization:

  • Lower induction temperature (16-20°C) to slow protein folding and reduce inclusion body formation

  • Reduce inducer concentration to decrease expression rate

  • Use rich media supplemented with osmolytes like sorbitol or glycine betaine

  • Consider auto-induction media for gradual protein expression

• Protein Engineering Approaches:

  • Fusion partners: Add solubility-enhancing tags such as MBP, SUMO, or Thioredoxin

  • Domain truncation: Express functional domains separately if full-length protein is problematic

  • Surface charge modification: Introduce mutations to increase surface hydrophilicity

• Solubilization and Refolding Strategies:

  • If the protein remains insoluble, solubilize inclusion bodies using 8M urea or 6M guanidinium chloride

  • Implement step-wise dialysis to gradually remove denaturant

  • Use additives like L-arginine, glycerol, or non-detergent sulfobetaines during refolding

  • Consider on-column refolding during affinity purification

• Host System Considerations:

  • If expressing in E. coli, try specialized strains like Rosetta (for rare codons), Origami (for disulfide bonds), or Arctic Express (with cold-adapted chaperones)

  • Consider expression in K. lactis itself, as homologous expression may improve folding

  • Coexpress with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

• Buffer Optimization:

  • Test various pH conditions (pH 6.0-9.0) during lysis and purification

  • Include stabilizing additives such as glycerol (10-20%)

  • Add reducing agents (DTT, β-mercaptoethanol) if the protein contains cysteine residues

  • Consider detergents (0.1% Triton X-100, 0.5% CHAPS) for membrane-associated proteins

The storage conditions recommended for purified K. lactis 3-ketoacyl-CoA reductase (Tris/PBS-based buffer with 6% Trehalose, pH 8.0) suggest that the protein benefits from stabilizing agents, which could be incorporated earlier in the expression and purification process to improve solubility.

What approaches can be used to analyze the role of K. lactis 3-ketoacyl-CoA reductase in lipid metabolism?

To comprehensively analyze the role of K. lactis 3-ketoacyl-CoA reductase in lipid metabolism, researchers should employ a multi-faceted approach combining genetic, biochemical, and analytical techniques:

• Genetic Manipulation Strategies:

  • Generate knockout strains (ΔKLLA0B09812g) in K. lactis using CRISPR/Cas9 technology

  • Create overexpression strains with the native gene under strong, inducible promoters

  • Develop complementation systems in knockout strains using wild-type and mutant variants

  • Construct chimeric enzymes with domains from related reductases to probe structure-function relationships

• Lipidomic Analysis:

  • Employ liquid chromatography-mass spectrometry (LC-MS) to profile changes in:

    • Very long chain fatty acids (VLCFAs)

    • Triacylglycerols (TAGs), particularly polyunsaturated TAGs (puTAGs)

    • Membrane lipids and their fatty acid composition

  • Apply multivariate analysis techniques like Principal Component Analysis (PCA) to identify patterns in lipidomic data

  • Calculate Pearson correlations between different lipid species to identify metabolically linked compounds

• Metabolic Flux Analysis:

  • Use isotope labeling experiments with 13C-labeled substrates

  • Track carbon flow through fatty acid synthesis and elongation pathways

  • Quantify flux changes in knockout vs. wild-type strains

• Heterologous Expression Studies:

  • Express K. lactis 3-ketoacyl-CoA reductase in model organisms like yeast (S. cerevisiae) or plants

  • Assess complementation of mutant phenotypes in these systems

  • Similar to studies with Arabidopsis KCS4, analyze the effect on VLCFA production in heterologous systems

• Growth and Phenotypic Analysis under Different Conditions:

  • Compare growth rates of wild-type and mutant strains under different carbon sources

  • Assess cold sensitivity, heat tolerance, and membrane stress responses

  • Examine changes in lipid droplet formation using fluorescent microscopy

• Data Integration Framework:

This integrated approach would provide comprehensive insights into the role of K. lactis 3-ketoacyl-CoA reductase in lipid metabolism, similar to the multifaceted analysis performed for Arabidopsis KCS4 in triacylglycerol synthesis regulation .

How can researchers distinguish between the activity of K. lactis 3-ketoacyl-CoA reductase and other similar enzymes in complex biological samples?

Distinguishing the activity of K. lactis 3-ketoacyl-CoA reductase from other similar enzymes in complex biological samples requires specific analytical approaches that exploit unique characteristics of the enzyme:

• Immunological Methods:

  • Develop specific antibodies against K. lactis 3-ketoacyl-CoA reductase

  • Use immunoprecipitation to isolate the enzyme from complex samples

  • Perform Western blotting with these antibodies to specifically detect the protein

  • Employ immunohistochemistry to determine subcellular localization

• Activity-Based Protein Profiling:

  • Design activity-based probes that covalently bind to the active site of 3-ketoacyl-CoA reductases

  • Incorporate features that allow selective labeling of K. lactis 3-ketoacyl-CoA reductase

  • Use click chemistry to attach reporter tags for visualization or affinity purification

  • Analyze labeled proteins by SDS-PAGE and mass spectrometry

• Differential Inhibition Studies:

  • Identify selective inhibitors that affect K. lactis 3-ketoacyl-CoA reductase differently than homologous enzymes

  • Perform activity assays in the presence of various inhibitors

  • Analyze inhibition patterns to distinguish between different reductases

• Substrate Specificity Analysis:

  • Exploit potential differences in substrate chain-length preferences

  • Similar to studies with E. coli and Pseudomonas FabG proteins , test activity with substrates ranging from C4 to C26

  • Compare activity profiles across chain lengths to identify unique signatures

• Recombinant Expression and Kinetic Comparison:

  • Express recombinant K. lactis 3-ketoacyl-CoA reductase with a unique tag (e.g., His-tag)

  • Express other potentially interfering reductases with different tags

  • Compare kinetic parameters (Km, Vmax, kcat) across various substrates

  • Identify unique kinetic signatures that can be used for discrimination

• Mass Spectrometry-Based Approaches:

  • Use targeted proteomics (MRM/PRM) to quantify specific peptides unique to K. lactis 3-ketoacyl-CoA reductase

  • Analyze post-translational modifications that might be enzyme-specific

  • Employ top-down proteomics to analyze intact protein masses and distinguish between closely related proteins

• Genetic Approaches in K. lactis:

  • Generate knockout strains and assess the residual 3-ketoacyl-CoA reductase activity

  • Complement with specific point mutants to verify activity restoration

  • Use cell-free extracts from these strains as controls for activity assays

What are the potential applications of engineered K. lactis 3-ketoacyl-CoA reductase in synthetic biology?

Engineered K. lactis 3-ketoacyl-CoA reductase holds significant potential for various synthetic biology applications, particularly in metabolic engineering for specialized lipid production:

• Biofuel Production:

  • Engineer the enzyme for altered chain-length specificity to produce medium-chain fatty acids (MCFAs) that are ideal precursors for biodiesel and jet fuels

  • Integrate with other enzymes to create synthetic pathways for advanced biofuel production

  • Similar to how FabG enzymes have been shown to intercept 3-ketoacyl-CoA intermediates from β-oxidation , engineered variants could be designed to efficiently channel carbon toward fuel molecule synthesis

• Polyhydroxyalkanoate (PHA) Production:

  • Co-express with PHA synthase genes to enable production of biodegradable bioplastics

  • Studies have demonstrated that coexpression of fabG genes with type II PHA synthase genes enables accumulation of medium-chain-length PHA copolymers

  • Engineer substrate specificity to produce PHAs with novel monomer compositions and improved material properties

• Specialty Lipid Production:

  • Modify the enzyme to alter stereoselectivity or regioselectivity in fatty acid modification

  • Engineer pathways for production of omega-3 fatty acids, structured lipids, or other high-value lipid products

  • Create synthetic metabolons by fusing K. lactis 3-ketoacyl-CoA reductase with other enzymes in the pathway to enhance flux

• Biosensors for Metabolic Engineering:

  • Develop biosensors based on K. lactis 3-ketoacyl-CoA reductase activity that respond to specific fatty acid intermediates

  • Use these biosensors in high-throughput screening of strain libraries

  • Implement dynamic pathway regulation using these sensing modules

• Cell-Free Biocatalysis:

  • Develop robust cell-free systems incorporating engineered K. lactis 3-ketoacyl-CoA reductase for in vitro synthesis of specialized lipids

  • Optimize enzyme stability and cofactor regeneration for industrial applications

  • The demonstrated ability to express and purify the enzyme with high stability makes it a good candidate for cell-free systems

These applications leverage the food-grade status of K. lactis and its established role in biotechnology , providing advantages for applications requiring regulatory approval or consumer acceptance.

How might comparative studies between K. lactis 3-ketoacyl-CoA reductase and homologs from other organisms advance our understanding of evolutionary adaptations in lipid metabolism?

Comparative studies between K. lactis 3-ketoacyl-CoA reductase and homologs from other organisms can provide valuable insights into evolutionary adaptations in lipid metabolism through several investigative approaches:

• Phylogenetic Analysis and Structural Comparisons:

  • Construct comprehensive phylogenetic trees of 3-ketoacyl-CoA reductases across the tree of life

  • Correlate evolutionary relationships with environmental niches and metabolic requirements

  • Compare critical structural features across different organisms to identify conserved motifs and organism-specific adaptations

  • Similar to how Arabidopsis KCS family members show functional diversity , identify how 3-ketoacyl-CoA reductases have diversified

• Substrate Specificity Variations:

  • Compare substrate range and catalytic efficiency across homologs from different organisms

  • Identify how substrate preferences correlate with the lipid composition requirements of different organisms

  • E. coli and Pseudomonas FabG proteins have been assayed for substrate specificity with chain lengths from C4 to C12 , providing a basis for comparison with K. lactis 3-ketoacyl-CoA reductase

• Environmental Adaptation Analysis:

  • Examine 3-ketoacyl-CoA reductases from organisms adapted to extreme environments (thermophiles, psychrophiles, halophiles)

  • Identify molecular adaptations that enable function under these conditions

  • Correlate with the lipid composition changes required for membrane function in extreme environments

• Functional Complementation Studies:

  • Express K. lactis 3-ketoacyl-CoA reductase in organisms with disrupted endogenous reductases

  • Assess the degree of functional complementation

  • Identify organism-specific requirements for integration into metabolic networks

• Cofactor Preference Evolution:

  • Analyze variations in NADPH versus NADH preference across different organisms

  • Correlate cofactor preference with cellular redox balance strategies

  • Identify key residues that determine cofactor specificity through site-directed mutagenesis

• Regulatory Network Integration:

  • Compare how 3-ketoacyl-CoA reductases are regulated in different organisms

  • Identify conserved and divergent regulatory mechanisms

  • Similar to how KCS4 in Arabidopsis acts as a branch point in the regulation of triacylglycerol synthesis , determine if K. lactis 3-ketoacyl-CoA reductase plays comparable regulatory roles

• Comparative Framework Example:

What are the key considerations for researchers beginning work with recombinant K. lactis 3-ketoacyl-CoA reductase?

Researchers beginning work with recombinant Kluyveromyces lactis 3-ketoacyl-CoA reductase (KLLA0B09812g) should consider several key factors to ensure successful experimental outcomes:

• Expression System Selection:

  • For high-purity protein production: E. coli expression with N-terminal His-tag has been demonstrated to be effective

  • For metabolic engineering: K. lactis itself may be preferable as a host due to its "food-grade" status and natural compatibility with the enzyme

  • For functional studies: Consider the cofactor availability (NADPH) in the chosen expression system

• Protein Handling Considerations:

  • The purified protein should be stored with stabilizing agents (6% Trehalose has been used)

  • Addition of glycerol (5-50%) is recommended for long-term storage at -20°C/-80°C

  • Avoid repeated freeze-thaw cycles to maintain enzymatic activity

• Experimental Design:

  • Include appropriate controls when assessing enzymatic activity

  • Consider the chain-length specificity when selecting substrates for activity assays

  • Design experiments to distinguish between native activities in the host and the recombinant enzyme

• Integration with Other Systems:

  • When coexpressing with other enzymes, consider the stoichiometric balance and metabolic burden

  • For production of polyhydroxyalkanoates or other specialized lipids, coexpression with appropriate synthases may be required

  • Consider the cellular localization of the enzyme and its partners in the expression system

• Technical Expertise Required:

  • Protein biochemistry skills for expression, purification, and enzyme assays

  • Analytical chemistry expertise for product analysis (GC-MS, LC-MS)

  • Molecular biology techniques for genetic manipulation and strain development

By addressing these considerations, researchers can establish robust experimental systems for studying K. lactis 3-ketoacyl-CoA reductase and leverage its potential for both basic research and biotechnological applications.

How can researchers effectively integrate computational approaches with experimental methods when studying K. lactis 3-ketoacyl-CoA reductase?

Researchers can effectively integrate computational approaches with experimental methods when studying K. lactis 3-ketoacyl-CoA reductase through a multi-tiered strategy that enhances both experimental design and data interpretation:

• Homology Modeling and Structural Prediction:

  • Generate structural models of K. lactis 3-ketoacyl-CoA reductase based on crystal structures of homologous enzymes

  • Use these models to identify critical residues for substrate binding and catalysis

  • Guide site-directed mutagenesis experiments to test computational predictions

  • Validate models through experimental approaches such as circular dichroism or limited proteolysis

• Molecular Dynamics Simulations:

  • Simulate enzyme-substrate interactions with various chain-length substrates

  • Predict conformational changes during catalysis

  • Identify potential allosteric sites that could be targeted for enzyme engineering

  • Use simulation results to design experiments testing substrate preference

• Metabolic Modeling Integration:

  • Incorporate K. lactis 3-ketoacyl-CoA reductase into genome-scale metabolic models of K. lactis

  • Simulate the effects of enzyme modifications on metabolic flux

  • Predict optimal conditions for desired product formation

  • Use flux balance analysis to identify potential bottlenecks in engineered pathways

• Machine Learning for Data Analysis:

  • Apply clustering algorithms to lipidomic data to identify patterns in fatty acid profiles

  • Use supervised learning to correlate enzyme variants with specific activity profiles

  • Implement neural networks to predict enzyme-substrate compatibility

  • Similar to multivariate analysis techniques used in lipidomic studies , develop predictive models for lipid metabolism

• Automated High-Throughput Experimental Design:

  • Use algorithms to design optimal mutation libraries for directed evolution

  • Implement design of experiments (DOE) approaches for optimization of expression conditions

  • Develop computational pipelines for automated analysis of high-throughput enzyme assays

  • Create feedback loops between computational predictions and experimental validations

• Integration Framework: