Recombinant Aspergillus oryzae 3-ketoacyl-CoA reductase (AO090026000492)

What is 3-ketoacyl-CoA reductase (AO090026000492) and what is its biological function?

3-ketoacyl-CoA reductase (AO090026000492) from Aspergillus oryzae is an enzyme involved in fatty acid biosynthesis, specifically in the reduction of 3-ketoacyl-CoA to 3-hydroxyacyl-CoA using NADPH as a cofactor. It is also known by several synonyms including Very-long-chain 3-oxoacyl-CoA reductase, 3-ketoreductase (KAR), and Microsomal beta-keto-reductase. The enzyme is encoded by the AO090026000492 gene in A. oryzae and has a UniProt ID of Q2UET3. The protein consists of 346 amino acids and functions within the fatty acid elongation pathway, catalyzing the second step in the four-reaction cycle of fatty acid elongation .

What expression systems are commonly used for producing recombinant AO090026000492?

The most common expression system for recombinant production of AO090026000492 is Escherichia coli. The full-length protein (amino acids 1-346) can be expressed with an N-terminal His tag to facilitate purification. E. coli provides several advantages for recombinant protein production, including rapid growth, high protein yields, and well-established protocols for transformation and protein expression . For researchers interested in studying the native function or interactions in a fungal system, expression in Aspergillus species may be preferable. A. oryzae itself can be used as a host for homologous expression, leveraging various synthetic biology tools including DNA assembly technologies and genome editing systems. Transformation methods such as protoplast-mediated transformation (PMT) and Agrobacterium tumefaciens-mediated transformation (ATMT) have been successfully employed in A. oryzae .

What are the optimal storage and handling conditions for recombinant AO090026000492?

Recombinant AO090026000492 protein is typically supplied as a lyophilized powder. For optimal stability and activity, the protein should be stored at -20°C to -80°C upon receipt, with aliquoting recommended for multiple use to avoid repeated freeze-thaw cycles. Working aliquots can be stored at 4°C for up to one week. For reconstitution, the protein should be briefly centrifuged prior to opening to bring contents to the bottom, then reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Addition of glycerol to a final concentration of 5-50% (with 50% being standard) is recommended for long-term storage at -20°C or -80°C . The storage buffer typically contains a Tris/PBS-based solution with 6% Trehalose at pH 8.0. To maintain enzyme activity, it is critical to avoid repeated freeze-thaw cycles as this can lead to protein denaturation and activity loss .

How can I design experiments to characterize the kinetic properties of AO090026000492?

Designing experiments to characterize the kinetic properties of AO090026000492 requires a systematic approach focusing on substrate specificity, reaction rates, and cofactor requirements. Begin by establishing a reliable activity assay, typically monitoring the oxidation of NADPH at 340 nm as the enzyme reduces 3-ketoacyl-CoA substrates. For comprehensive kinetic characterization:

• Prepare a range of substrate concentrations (typically 0.1-10× the estimated Km) with various chain-length 3-ketoacyl-CoA substrates to determine substrate specificity.

• Maintain constant enzyme concentration (typically 10-100 nM).

• Use optimal buffer conditions (usually Tris-HCl buffer pH 7.5-8.0) containing necessary cofactors.

• Measure initial reaction velocities at various substrate concentrations.

• Plot data using Michaelis-Menten, Lineweaver-Burk, or Eadie-Hofstee plots to determine Km, Vmax, and kcat.

• Analyze the effect of pH (range 6.0-9.0), temperature (25-45°C), and ionic strength on enzyme activity.

• Examine cofactor specificity by comparing activity with NADPH versus NADH.

For inhibition studies, perform similar assays in the presence of potential inhibitors, varying both substrate and inhibitor concentrations to determine the type of inhibition (competitive, non-competitive, or uncompetitive) and Ki values . These characterizations are essential for understanding the enzymatic mechanism and comparing the properties with orthologous enzymes from other organisms.

What are the challenges and strategies for heterologous expression of AO090026000492 in different host systems?

Heterologous expression of AO090026000492 presents several challenges depending on the host system selected. When expressing in E. coli, common issues include protein misfolding, formation of inclusion bodies, and lack of post-translational modifications. To overcome these challenges:

• Optimize codon usage for the host organism to improve translation efficiency.

• Use fusion tags (His, GST, MBP) to enhance solubility and facilitate purification.

• Express at lower temperatures (16-25°C) to reduce inclusion body formation.

• Co-express with molecular chaperones to assist proper folding.

• Use specialized E. coli strains designed for expression of eukaryotic proteins.

For expression in fungal hosts like Aspergillus species, considerations include:

• Selection of appropriate promoters (e.g., amyB promoter) and terminators for efficient transcription.

• Use of suitable selection markers - either dominant selectable markers (ptrA, AosdhB, Blmb) or auxotrophic markers (pyrG, sC, argB, niaD, adeA, amdS, adeB) depending on the host strain.

• Choice of transformation method - protoplast-mediated transformation (PMT) or Agrobacterium tumefaciens-mediated transformation (ATMT), with each having specific advantages .

• Integration strategy - either random integration through non-homologous end joining (NHEJ) or site-specific integration through homologous recombination (HR).

For fungal expression, host strain selection is critical. Strains like NSAR1 (niaD−, sC−, ΔargB, adeA−) provide multiple auxotrophic markers allowing for successive transformations when expressing multiple genes . Gateway cloning technology has been successfully employed for heterologous expression in A. oryzae, allowing site-specific recombination between attL and attR sites for efficient integration of target genes .

How does AO090026000492 compare structurally and functionally to 3-ketoacyl-CoA reductases from other organisms?

AO090026000492 from A. oryzae belongs to the short-chain dehydrogenase/reductase (SDR) superfamily, sharing the characteristic Rossmann fold for nucleotide binding. Structural comparison with 3-ketoacyl-CoA reductases from other organisms reveals:

• Conserved catalytic triad (Ser-Tyr-Lys) essential for the reaction mechanism.

• NADPH binding motif (typically TGxxxGxG) in the N-terminal region.

• Substrate binding pocket variations that influence chain-length specificity.

Functionally, the enzyme catalyzes the same reduction reaction across species, but with different substrate preferences and kinetic parameters:

Note: The values presented are typical ranges based on similar enzymes; exact values for AO090026000492 should be determined experimentally.

Sequence alignment of AO090026000492 with its homologs reveals higher similarity to fungal counterparts (70-80% identity) compared to bacterial (30-40%) or mammalian (25-35%) orthologs. The A. oryzae enzyme contains unique membrane-spanning regions indicated by the hydrophobic amino acid stretches in its sequence (e.g., "LFLLAAGSLFVASRALTFVRVLLSLFVLPGKSL") , suggesting it may be associated with the endoplasmic reticulum membrane, similar to mammalian 3-ketoacyl-CoA reductases in the fatty acid elongation system.

What transformation methods are most efficient for genetic manipulation of AO090026000492 in A. oryzae?

Two primary transformation methods have proven effective for genetic manipulation of genes in A. oryzae, including AO090026000492:

• Protoplast-Mediated Transformation (PMT):

  • This traditional method involves enzymatic removal of the fungal cell wall to create protoplasts.

  • Heterologous genes are loaded onto vectors containing appropriate selection markers.

  • Transformation occurs through PEG/CaCl₂-mediated uptake under high osmotic pressure conditions.

  • Integration occurs via either non-homologous end joining (NHEJ) or homologous recombination (HR).

  • Advantages: Simple procedure, ability to co-transform multiple DNA fragments.

  • Limitations: Difficult protoplast culture, low regeneration frequency, high reagent requirements .

• Agrobacterium tumefaciens-Mediated Transformation (ATMT):

  • Utilizes A. tumefaciens to insert T-DNA containing the target gene into the fungal genome.

  • Requires construction of a binary vector system with the gene of interest between T-DNA borders.

  • Success has been demonstrated in A. oryzae using pyrG as a selection marker fused with GFP.

  • Advantages: Higher transformation efficiency, simpler operation procedure.

  • This method was first applied to A. oryzae in 2016 .

For gene editing specifically targeting AO090026000492, CRISPR-Cas9 systems adapted for filamentous fungi can be employed. The selection of appropriate markers is crucial, with options including:

• Dominant selectable markers: ptrA (pyrithiamine resistance), AosdhB/cxr (carboxin resistance), Blmb (bleomycin resistance)

• Auxotrophic selectable markers: pyrG, sC, argB, niaD, adeA, amdS, adeB

For multiple genetic manipulations, marker recycling systems can be employed:

• Short repeat sequence-based excision of pyrG using 5-FOA counter-selection

• Cre/loxP recombinase systems for marker removal after successful transformation

The choice between these methods depends on the specific experimental requirements, with ATMT generally recommended for higher efficiency and PMT when co-transformation of multiple constructs is needed.

What are the optimal conditions for assaying AO090026000492 enzymatic activity?

Establishing optimal conditions for assaying AO090026000492 enzymatic activity requires careful consideration of reaction components and environmental parameters:

• Buffer System and pH:

  • Optimal buffer: 50-100 mM Tris-HCl or phosphate buffer

  • pH range: 7.5-8.0 (with activity profiling recommended across pH 6.0-9.0)

  • Addition of 1-5 mM DTT or β-mercaptoethanol to maintain reducing environment

• Cofactor Requirements:

  • NADPH as primary cofactor (typically 0.1-0.5 mM)

  • MgCl₂ (1-5 mM) to enhance activity

  • BSA (0.1-0.5 mg/ml) to stabilize enzyme and prevent non-specific binding

• Substrate Preparation:

  • Various chain-length 3-ketoacyl-CoA substrates (C4-C20)

  • Substrate concentration range for kinetic studies: 1-200 μM

  • Use of substrate analogues for structure-function studies

• Reaction Monitoring:

  • Spectrophotometric assay: Measure NADPH oxidation at 340 nm (Δε = 6,220 M⁻¹cm⁻¹)

  • HPLC analysis: Monitor substrate depletion and product formation

  • Coupled assay systems for continuous monitoring

• Enzyme Concentration and Stability:

  • Use protein at 10-100 nM for initial rate measurements

  • Pre-incubation periods and thermal stability assessment

  • Addition of glycerol (5-10%) to stabilize enzyme during assay

• Reaction Conditions:

  • Temperature: 25-30°C (with temperature profiling from 20-45°C)

  • Reaction time: 5-30 minutes (ensuring linear response)

  • Agitation: Gentle mixing to prevent protein denaturation

• Controls and Validations:

  • Heat-inactivated enzyme as negative control

  • Known KAR enzymes as positive controls

  • Inhibitor studies (e.g., using NAD+ analogs)

For accurate results, it's essential to determine the linear range of the assay with respect to both time and enzyme concentration before proceeding with detailed kinetic characterization .

How can I design experiments to study the role of AO090026000492 in fatty acid metabolism?

Designing experiments to elucidate the role of AO090026000492 in fatty acid metabolism requires a multi-faceted approach combining genetic, biochemical, and analytical techniques:

• Genetic Manipulation Strategies:

  • Gene knockout using CRISPR-Cas9 or homologous recombination in A. oryzae

  • Gene overexpression using strong promoters (e.g., amyB promoter)

  • Site-directed mutagenesis of catalytic residues to create inactive variants

  • Creation of fluorescently tagged versions for localization studies

• Fatty Acid Profile Analysis:

  • Extract total lipids from wild-type and mutant strains

  • Perform gas chromatography-mass spectrometry (GC-MS) to quantify fatty acid profiles

  • Focus on changes in chain-length distribution and saturation levels

  • Isotope labeling experiments using ¹³C-labeled acetate to track fatty acid synthesis

• Metabolic Flux Analysis:

  • Use metabolic tracers to follow carbon flow through fatty acid pathways

  • Quantify intermediate metabolites using liquid chromatography-mass spectrometry (LC-MS)

  • Develop a computational model of fatty acid metabolism incorporating enzyme kinetics

  • Identify rate-limiting steps and regulatory nodes in the pathway

• Protein Interaction Studies:

  • Perform co-immunoprecipitation to identify interacting proteins

  • Use yeast two-hybrid or split-GFP assays to confirm direct interactions

  • Conduct pull-down assays with recombinant His-tagged AO090026000492

  • Map interaction domains through truncation and mutation analysis

• Transcriptional Response Analysis:

  • RNA-seq to identify genes co-regulated with AO090026000492

  • qRT-PCR validation of key pathway components

  • ChIP-seq to identify transcription factors regulating AO090026000492 expression

  • Compare transcriptional responses under various growth conditions

• Physiological Studies:

  • Growth phenotyping under different carbon sources and stress conditions

  • Microscopic analysis of lipid droplet formation using lipophilic dyes

  • Membrane composition analysis in response to environmental changes

  • Comparative analysis between wild-type and mutant strains

• Comparative Studies with Other Organisms:

  • Heterologous expression of AO090026000492 in model organisms (yeast, E. coli)

  • Complementation assays with known 3-ketoacyl-CoA reductase mutants

  • Cross-species activity assays with substrates from different organisms

Experimental controls should include parallel analysis of known fatty acid metabolism genes (e.g., fatty acid synthases, desaturases, elongases) to establish pathway context and validate experimental approaches .