Recombinant Lodderomyces elongisporus 3-ketoacyl-CoA reductase (LELG_03198)

What is Lodderomyces elongisporus 3-ketoacyl-CoA reductase and what is its role in fatty acid biosynthesis?

Lodderomyces elongisporus 3-ketoacyl-CoA reductase is an enzyme that catalyzes the second step in the very long-chain fatty acid (VLCFA) elongation process. Within the microsomal fatty acid elongation (FAE) complex, this enzyme specifically reduces the 3-ketoacyl-CoA intermediate generated by 3-ketoacyl-CoA synthase (KCS). The reaction involves the reduction of a carbonyl group in the 3-ketoacyl-CoA to produce a 3-hydroxyacyl-CoA intermediate. This step is crucial in the four-enzyme cycle of fatty acid elongation, which also includes subsequent dehydration and final reduction steps to yield a fatty acyl-CoA extended by two carbon atoms .

What distinguishes Lodderomyces elongisporus from other yeast species and why is it valuable for recombinant protein studies?

Lodderomyces elongisporus is distinguished from other yeast species by several key characteristics. Microscopically, it exhibits a significantly higher proportion of elongated budding yeast cells compared to other species, with conidia typically measuring approximately 2–6 × 4–7 μm . L. elongisporus can be identified using chromogenic agar, where it displays a distinctive blue-turquoise coloration, contrasting with the green color of Candida albicans and the metallic dark blue of Candida tropicalis .

A defining metabolic characteristic is its inability to utilize L-arabinose and D-xylose as carbon sources, which forms the basis of the specialized Loddy test for identification . This yeast also demonstrates significant bioreduction activity on various ketones, showing optimal activity in the pH range of 7-10 with conversions of 60-80% for certain substrates . These unique properties make L. elongisporus valuable for recombinant protein studies, particularly for enzymes involved in redox reactions.

How does the structure of Lodderomyces elongisporus 3-ketoacyl-CoA reductase compare to similar enzymes in other organisms?

While specific structural information about L. elongisporus 3-ketoacyl-CoA reductase is not detailed in the provided search results, we can draw some inferences from related enzymes. Based on homology with other 3-ketoacyl reductases, it likely adopts a structure with a Rossmann fold typical of short-chain dehydrogenase/reductase family proteins, containing a nucleotide-binding domain for the cofactor (typically NADPH) and a substrate-binding domain.

Similar enzymes in the fatty acid elongation pathway, such as those in the Type III PKS family, display a characteristic five-layer ⍺β⍺β⍺ fold that is conserved across related enzymes . The active site would contain a catalytic triad essential for the reduction reaction. Substrate specificity would be determined by the shape and properties of the binding pocket, similar to how the binding tunnel in KCS enzymes influences their substrate preferences .

What are the optimal conditions for expressing recombinant Lodderomyces elongisporus 3-ketoacyl-CoA reductase in heterologous systems?

The optimal expression conditions for recombinant L. elongisporus 3-ketoacyl-CoA reductase should consider several key parameters based on enzymatic characteristics. First, select an expression system capable of proper protein folding and post-translational modifications. While E. coli might offer high yields, yeast expression systems like Pichia pastoris or Saccharomyces cerevisiae often provide better functionality for eukaryotic enzymes.

For expression in yeast systems, use strong inducible promoters (such as AOX1 for P. pastoris or GAL1 for S. cerevisiae) with temperatures between 25-30°C to minimize protein aggregation. Maintain pH between 7-10, as L. elongisporus demonstrates optimal enzymatic activity in this range . Include a histidine or similar tag for purification, positioned to avoid interfering with the active site.

Expression should be monitored through time-course sampling at 24, 48, and 72 hours post-induction. Supplement the media with zinc ions, as ketoreductases often require metal cofactors for proper folding. Following expression, perform purification under mild conditions (pH 7.5, 4°C) to preserve enzyme activity.

How can the enzymatic activity of recombinant Lodderomyces elongisporus 3-ketoacyl-CoA reductase be accurately measured?

The enzymatic activity of recombinant L. elongisporus 3-ketoacyl-CoA reductase can be accurately measured through several complementary approaches:

Spectrophotometric Assay: Monitor the oxidation of NADPH to NADP+ at 340 nm, as the reduction of 3-ketoacyl-CoA consumes NADPH. The reaction mixture should contain:

• Purified enzyme (0.1-1 μg)

• 3-ketoacyl-CoA substrate (50-200 μM)

• NADPH (100-200 μM)

• Buffer (typically 50-100 mM phosphate or Tris at pH 7.0-8.0)

HPLC Analysis: Measure the conversion of substrate to product directly using reverse-phase HPLC, which allows quantification of reaction kinetics.

Gas Chromatography: For volatile substrates like phenylacetone, use GC to measure substrate depletion and product formation.

Activity should be expressed as μmol substrate converted per gram of enzyme per minute. As demonstrated with various yeast strains, productivity can vary significantly based on substrate concentration, with L. elongisporus showing productivity of up to 40 μmol g⁻¹ min⁻¹ for certain substrates .

What immobilization techniques are most effective for enhancing the stability and reusability of Lodderomyces elongisporus 3-ketoacyl-CoA reductase?

Based on successful approaches with L. elongisporus, sol-gel matrix immobilization has proven particularly effective for enhancing enzyme stability and reusability . The following protocol is recommended:

• Sol-gel Matrix Preparation:

  • Mix tetramethoxysilane (TMOS) or tetraethoxysilane (TEOS) with water

  • Add the enzyme solution (0.5-2 mg/mL) in phosphate buffer (pH 7.5)

  • Allow gelation at 4°C for 24 hours

  • Wash with buffer to remove unbound enzyme

• Cross-linked Enzyme Aggregates (CLEAs):

  • Precipitate the enzyme using ammonium sulfate (60-80% saturation)

  • Cross-link with glutaraldehyde (0.5-2% v/v)

  • Wash and recover the CLEAs

• Enzyme Performance Analysis:

  • Evaluate activity retention after immobilization (typically 60-80%)

  • Test reusability through multiple reaction cycles (minimum 10 cycles)

  • Assess thermal stability at 30-50°C

  • Determine pH stability across the range of 5-10

L. elongisporus immobilized in sol-gel matrices has demonstrated excellent stability, maintaining over 70% of initial activity after 5 cycles of use. Additionally, immobilization expands the operational pH range and enhances resistance to substrate inhibition, allowing higher substrate concentrations (up to 10 mM) before observing productivity decline .

How can directed evolution strategies be applied to enhance the catalytic properties of Lodderomyces elongisporus 3-ketoacyl-CoA reductase?

Directed evolution of L. elongisporus 3-ketoacyl-CoA reductase requires a systematic approach to generate and select improved variants:

• Library Generation Methods:

  • Error-prone PCR: Use skewed dNTP ratios (0.2-0.7 mM) and MnCl₂ (0.05-0.5 mM) to introduce random mutations with a target mutation rate of 2-5 nucleotides per gene

  • Site-saturation mutagenesis: Focus on residues lining the substrate binding pocket and those involved in cofactor binding

  • DNA shuffling: Recombine gene fragments from related ketoreductases from various yeast species

• Screening Strategy:

  • Develop a high-throughput colorimetric assay using redox indicators (such as tetrazolium salts) that change color upon NADPH oxidation

  • Implement microplate-based screening for initial rounds, examining 10³-10⁴ variants

  • Screen for multiple parameters simultaneously: activity, thermostability, pH tolerance, and substrate specificity

• Iterative Improvement:

  • Combine beneficial mutations through site-directed mutagenesis

  • Validate improvements through detailed kinetic analysis (k_cat, K_M)

  • Compare enzyme performance under conditions mimicking the fatty acid elongation pathway

The mutations targeting residues in the hydrophobic substrate binding tunnel would be particularly valuable, as these regions determine substrate specificity in related enzymes . Focus especially on residues equivalent to those in the kinked regions of substrate binding tunnels, which influence preference for saturated versus unsaturated substrates.

What are the effects of cofactor engineering on the substrate specificity and catalytic efficiency of Lodderomyces elongisporus 3-ketoacyl-CoA reductase?

Cofactor engineering can significantly alter the catalytic properties of L. elongisporus 3-ketoacyl-CoA reductase through several strategic modifications:

How can Lodderomyces elongisporus 3-ketoacyl-CoA reductase be integrated into synthetic metabolic pathways for the production of specialized fatty acid derivatives?

Integration of L. elongisporus 3-ketoacyl-CoA reductase into synthetic metabolic pathways requires careful pathway design and optimization:

• Pathway Design Considerations:

  • Ensure balanced expression of all pathway enzymes using promoters of appropriate strength

  • Co-localize enzymes through scaffolding proteins or fusion constructs to enhance substrate channeling

  • Include regulatory elements responsive to product accumulation or substrate depletion

• Implementation Strategies:

  • Build a modular pathway with standardized genetic parts for easy optimization

  • Express the complete fatty acid elongation complex (KCS, KCR, HCD, ECR) rather than KCR alone

  • Balance NADPH availability through concurrent expression of NADPH-generating enzymes

• Performance Optimization:

  • Use adaptive laboratory evolution to enhance pathway flux

  • Implement dynamic regulation to respond to changing cellular conditions

  • Knock out competing pathways that might divert intermediates

The integration would be particularly effective for producing specialized fatty acid derivatives like hydroxylated fatty acids or α,ω-dicarboxylic acids. Since L. elongisporus demonstrates high productivity in the bioreduction of various ketones (up to 40 μmol g⁻¹ min⁻¹) , it could be effectively coupled with KCS enzymes with different substrate specificities to produce diverse elongated products.

Monitor pathway performance through metabolite profiling using LC-MS/MS, analyze flux distribution with ¹³C metabolic flux analysis, and use biosensors to provide real-time feedback on pathway intermediates.

What strategies can overcome substrate inhibition observed at high concentrations with Lodderomyces elongisporus 3-ketoacyl-CoA reductase?

Substrate inhibition has been observed with L. elongisporus at substrate concentrations above 10 mM, showing significant productivity decline . Several approaches can effectively address this challenge:

• Fed-batch Substrate Addition:

  • Maintain substrate concentration below inhibitory levels (4-8 mM) through controlled feeding

  • Implement an automated system that adds substrate based on consumption rates

  • Optimize feeding profile through design of experiments methodology

• Biphasic Reaction Systems:

  • Create a two-phase system using an organic solvent (e.g., n-hexane, toluene, or MTBE)

  • The organic phase serves as a substrate reservoir, releasing substrate into the aqueous phase gradually

  • Test multiple solvent systems to identify optimal partition coefficients

• Protein Engineering Approaches:

  • Target residues in the substrate binding pocket that influence substrate affinity

  • Introduce mutations that reduce binding affinity slightly but improve turnover rate

  • Screen for variants with higher K_i values for substrate inhibition

• Enzyme Immobilization Benefits:

  • Immobilize the enzyme in sol-gel matrix to create a microenvironment that buffers against high substrate concentrations

  • Use porous materials that control substrate diffusion rates

  • Combine immobilization with protein engineering for additive improvements

• Process Optimization:

  • Implement in situ product removal strategies to shift reaction equilibrium

  • Optimize reaction parameters (temperature, pH, ionic strength) to minimize inhibition effects

  • Use mathematical modeling to predict optimal operational conditions

The implementation of these strategies has shown that with proper immobilization techniques and process design, L. elongisporus biocatalysts can maintain high productivity (>60% of maximum) even at elevated substrate concentrations .

How can the stability of recombinant Lodderomyces elongisporus 3-ketoacyl-CoA reductase be improved for industrial applications?

Improving stability of recombinant L. elongisporus 3-ketoacyl-CoA reductase requires a multi-faceted approach:

• Protein Engineering for Enhanced Stability:

  • Introduce disulfide bridges at positions identified through computational analysis

  • Replace surface-exposed hydrophobic residues with polar residues

  • Identify and mutate residues in flexible regions to reduce unfolding

  • Use consensus design approaches based on alignment with thermostable homologs

• Formulation Strategies:

  • Add stabilizing additives (10-20% glycerol, trehalose, or arginine)

  • Optimize buffer compositions to include stabilizing ions

  • Control pH within the enzyme's stability range (pH 7-10)

  • Include reducing agents to prevent oxidation of cysteine residues

• Immobilization Techniques:

  • Covalent attachment to epoxy-activated supports

  • Cross-linked enzyme aggregates with polyethylenimine

  • Entrapment in silica-based sol-gel matrices as demonstrated for L. elongisporus

  • Multi-point attachment strategies to rigid supports

• Storage and Operational Stability:

  • Lyophilization with appropriate cryoprotectants

  • Immobilization in hydrophobic supports for operation in organic solvents

  • Controlled dehydration to enhance thermostability

  • Co-immobilization with stabilizing proteins or cofactors

Implementation of these strategies has shown significant improvements in enzyme stability. For example, lyophilized forms of related yeast enzymes have demonstrated efficient biocatalytic activity in the production of enantiopure alcohols on preparative scale , indicating that properly stabilized L. elongisporus 3-ketoacyl-CoA reductase could achieve similar industrial performance.

What are the common analytical challenges in studying Lodderomyces elongisporus 3-ketoacyl-CoA reductase activity and how can they be addressed?

Researchers face several analytical challenges when studying L. elongisporus 3-ketoacyl-CoA reductase:

• Challenge: Distinguishing Enzyme Activity from Background Cell Metabolism
  Solution:

  • Use cell-free extract controls from non-expressing host cells

  • Implement specific inhibitors for related enzymes

  • Develop selective assays using structurally unique substrates

  • Employ isotope-labeled substrates to track specific conversions

• Challenge: Accurately Quantifying Cofactor Consumption in Complex Mixtures
  Solution:

  • Implement HPLC methods for direct NADPH/NADP⁺ quantification

  • Use coupled enzyme assays with specific detection systems

  • Develop fluorescence-based assays for enhanced sensitivity

  • Account for non-enzymatic NADPH oxidation through proper controls

• Challenge: Measuring Kinetics with Hydrophobic Substrates
  Solution:

  • Use co-solvents (5-10% DMSO, ethanol) that don't impact enzyme activity

  • Employ cyclodextrins to improve substrate solubility

  • Develop biphasic reaction systems with proper mixing

  • Use substrate analogs with improved water solubility for initial characterization

• Challenge: Determining True Substrate Specificity Profiles
  Solution:

  • Create a standardized substrate panel with diverse chain lengths and functional groups

  • Normalize activity data to account for differences in substrate solubility

  • Use competition assays to determine relative substrate preferences

  • Implement LC-MS/MS methods to detect all potential products

• Challenge: Correlating In Vitro Activity with In Vivo Function
  Solution:

  • Develop cell-based assays measuring fatty acid profile changes

  • Use metabolic labeling with stable isotopes

  • Implement liposome-based systems mimicking natural membrane environment

  • Correlate enzyme kinetics with physiological substrate concentrations

Implementing these solutions allows for more accurate characterization of enzyme activity. For example, researchers have successfully used standardized testing conditions to characterize the pH profiles and substrate specificity of various yeast strains, revealing that L. elongisporus exhibits optimal bioreduction activity between pH 7-10 with conversions in the 60-80% range for specific substrates .

How does the genetic diversity of natural Lodderomyces elongisporus isolates impact the functional properties of 3-ketoacyl-CoA reductase enzymes?

The genetic diversity among natural L. elongisporus isolates can significantly influence the functional properties of their 3-ketoacyl-CoA reductases. Research in this area should address:

• Population Genetics Analysis:

  • Sequence the 3-ketoacyl-CoA reductase gene from geographically diverse L. elongisporus isolates

  • Identify natural variants and polymorphic sites through comparative genomics

  • Analyze selection pressure on different domains of the enzyme

  • Correlate genetic variation with ecological niches of the isolates

• Functional Characterization of Natural Variants:

  • Express and purify enzymes from diverse isolates

  • Compare kinetic parameters (k_cat, K_M) across substrates

  • Assess thermal stability and pH optima variations

  • Evaluate cofactor preference and efficiency

• Structure-Function Relationship Analysis:

  • Model the structural differences between variants

  • Identify key residues responsible for functional differences

  • Use site-directed mutagenesis to confirm the role of specific variations

  • Develop predictive models relating sequence variations to functional properties

These analyses would provide valuable insights into evolutionary adaptations of L. elongisporus 3-ketoacyl-CoA reductase and identify naturally optimized variants for specific applications. The approach is similar to how researchers have analyzed different yeast strains for their bioreduction capabilities, revealing significant variations in productivity (ranging from 10-60 μmol g⁻¹ min⁻¹) and substrate preferences .

What potential exists for utilizing Lodderomyces elongisporus 3-ketoacyl-CoA reductase in the biosynthesis of novel fatty acid-derived compounds?

The potential for L. elongisporus 3-ketoacyl-CoA reductase in novel fatty acid-derived compound biosynthesis is substantial and can be explored through several approaches:

• Designer Fatty Acid Production:

  • Engineer pathways incorporating the reductase for omega-3/omega-6 fatty acid production

  • Create branched-chain fatty acids through modified elongation cycles

  • Produce fatty acids with precisely positioned functional groups

  • Generate cyclopropane or cyclopropene fatty acids with unique properties

• Therapeutic Compound Development:

  • Synthesize fatty acid-derived signaling molecules like prostaglandins

  • Produce specialized hydroxy fatty acids with anti-inflammatory properties

  • Generate precursors for lipid-based drug delivery systems

  • Create fatty acid-peptide conjugates with enhanced bioavailability

• Biofuel and Biomaterial Applications:

  • Engineer pathways for medium-chain fatty acid production optimized for biofuels

  • Create precursors for bioplastics with tailored properties

  • Produce wax esters with specific melting points for industrial applications

  • Generate fatty alcohols and aldehydes for fragrance and cosmetic applications

• Proof-of-Concept Studies:

  • Reconstruct complete fatty acid elongation systems with L. elongisporus 3-ketoacyl-CoA reductase

  • Test compatibility with various 3-ketoacyl-CoA synthases for diverse substrate incorporation

  • Evaluate the reductase's ability to process non-natural substrates

  • Optimize systems for the production of commercially valuable compounds

The high efficiency of L. elongisporus in bioreduction reactions (with productivity of up to 40 μmol g⁻¹ min⁻¹ for certain substrates) suggests strong potential for applications requiring stereoselective reduction steps in complex biosynthetic pathways.

How can systems biology approaches improve our understanding of Lodderomyces elongisporus 3-ketoacyl-CoA reductase in the context of cellular metabolism?

Systems biology approaches can provide comprehensive insights into L. elongisporus 3-ketoacyl-CoA reductase function within cellular metabolism:

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics data from L. elongisporus under various growth conditions

  • Identify co-regulated genes and proteins associated with 3-ketoacyl-CoA reductase

  • Map metabolite changes associated with altered reductase expression

  • Develop regulatory network models for fatty acid metabolism

• Flux Analysis and Modeling:

  • Perform ¹³C metabolic flux analysis to quantify carbon flow through fatty acid elongation

  • Develop genome-scale metabolic models of L. elongisporus

  • Simulate the effects of reductase overexpression or knockout

  • Identify limiting steps and bottlenecks in fatty acid elongation

• Comparative Systems Analysis:

  • Compare metabolic networks across yeast species with different fatty acid profiles

  • Identify unique regulatory features of L. elongisporus fatty acid metabolism

  • Evaluate the impact of different growth conditions on pathway regulation

  • Assess the integration of reductase function with cellular redox balance

• Advanced Visualization and Analysis:

  • Develop interactive metabolic maps highlighting reductase activity

  • Implement machine learning to identify non-obvious relationships in multi-omics data

  • Use time-resolved analysis to capture dynamic responses

  • Create predictive models for metabolic engineering applications

This systems-level understanding would complement the existing knowledge about L. elongisporus enzymatic capabilities, which has already revealed significant bioreduction activity across various pH ranges (optimal between pH 7-10) and substrate types .

What are the best practices for optimizing gene expression systems for high-yield production of recombinant Lodderomyces elongisporus 3-ketoacyl-CoA reductase?

Optimizing gene expression systems for high-yield production of recombinant L. elongisporus 3-ketoacyl-CoA reductase requires attention to multiple factors:

• Codon Optimization Strategy:

  • Analyze the codon usage bias of the expression host

  • Optimize the coding sequence while preserving mRNA secondary structure

  • Consider harmonization rather than maximization of codon adaptation index

  • Validate optimization through predictive algorithms before synthesis

• Expression Vector Design:

  • Select appropriate promoter strength based on protein solubility concerns

  • Include optimal ribosome binding sites or Kozak sequences

  • Consider adding introns for enhanced expression in eukaryotic hosts

  • Include appropriate secretion signals if extracellular production is desired

• Host Cell Engineering:

  • Select hosts with appropriate post-translational modification capabilities

  • Consider chaperone co-expression for improved folding

  • Engineer redox environment for optimal disulfide bond formation

  • Modify central carbon metabolism to enhance precursor and energy supply

• Fermentation Process Development:

  • Implement fed-batch strategies to maintain optimal growth rates

  • Control dissolved oxygen to support proper protein folding

  • Optimize induction timing and inducer concentration

  • Monitor and adjust pH within the enzyme's stability range (pH 7-10)

• Experimental Design Approach:

  • Use factorial design to identify critical parameters

  • Implement response surface methodology for optimization

  • Apply statistical process control during production

  • Develop scale-down models for process characterization

The successful implementation of these practices can significantly improve yield and quality of the recombinant enzyme, similar to how optimization enabled efficient biocatalyst production for various yeast strains used in preparative-scale bioreduction reactions .

How can crystallography and structural biology techniques be applied to elucidate the catalytic mechanism of Lodderomyces elongisporus 3-ketoacyl-CoA reductase?

Elucidating the catalytic mechanism of L. elongisporus 3-ketoacyl-CoA reductase through crystallography and structural biology requires a systematic approach:

• Protein Crystallization Strategy:

  • Screen various construct designs with different N/C-terminal truncations

  • Test multiple crystallization conditions (>1000) using sparse matrix screens

  • Optimize promising conditions through fine gradient screening

  • Consider surface entropy reduction mutations to promote crystal contacts

  • Explore co-crystallization with substrates, products, and cofactors

  • Use microseeding techniques to improve crystal quality

• Structural Determination Methods:

  • Collect high-resolution X-ray diffraction data (aiming for <2.0 Å)

  • Use molecular replacement with related structures as search models

  • Consider selenomethionine labeling for experimental phasing if necessary

  • Perform careful model building and refinement

• Mechanistic Studies:

  • Obtain structures with bound substrate analogs and cofactors

  • Capture reaction intermediates through cryo-trapping techniques

  • Perform time-resolved crystallography when possible

  • Generate structures of catalytically important mutants

• Complementary Techniques:

  • Use hydrogen-deuterium exchange mass spectrometry to map dynamics

  • Apply nuclear magnetic resonance for solution-state analysis

  • Implement molecular dynamics simulations to model catalytic steps

  • Perform quantum mechanics/molecular mechanics calculations to model transition states

These approaches would provide insights similar to those obtained for related enzymes, where structural analysis revealed binding tunnels with distinct shapes influencing substrate specificity . Understanding these structural features would explain L. elongisporus 3-ketoacyl-CoA reductase's performance in the bioreduction of various ketones, including its optimal pH range (7-10) and substrate preferences .

What high-throughput screening methods are most effective for identifying improved variants of Lodderomyces elongisporus 3-ketoacyl-CoA reductase?

Effective high-throughput screening for improved L. elongisporus 3-ketoacyl-CoA reductase variants requires specialized methods tailored to the enzyme's characteristics:

• Colorimetric Activity Assays:

  • Develop NAD(P)H-coupled assays using tetrazolium salts (NBT, INT)

  • Implement pH indicators for proton-consuming/producing reactions

  • Use chromogenic substrate analogs that change color upon conversion

  • Optimize signal-to-noise ratio through reaction condition tuning

• Fluorescence-Based Methods:

  • Develop assays using fluorogenic substrate analogs

  • Implement FRET-based sensors for conformational changes

  • Use fluorescence polarization to detect product formation

  • Apply flow cytometry for single-cell analysis with cell-surface displayed variants

• Microfluidic Approaches:

  • Develop droplet microfluidics for ultrahigh-throughput screening (10⁶-10⁸ variants)

  • Create gradient microfluidic devices for simultaneous condition optimization

  • Implement microarray-based screening for immobilized enzyme variants

  • Use microchamber arrays for parallel reaction monitoring

• Automation and Data Analysis:

  • Implement robotic systems for assay miniaturization to 384 or 1536-well formats

  • Develop image analysis algorithms for colony-based screens

  • Use machine learning to identify patterns in screening data

  • Implement design of experiments for efficient parameter optimization

The effectiveness of these screening methods can be evaluated by comparing the improvement factors achieved. For reference, optimization studies of different yeast strains have demonstrated productivity variations ranging from 10 to 60 μmol g⁻¹ min⁻¹ for bioreduction reactions , suggesting significant potential for improvement through directed evolution and high-throughput screening.