Recombinant Scheffersomyces stipitis 3-ketoacyl-CoA reductase (PICST_79198)

What is PICST_79198 and what is its basic structure?

PICST_79198 is a 3-ketoacyl-CoA reductase enzyme from the yeast Scheffersomyces stipitis (formerly Pichia stipitis). It is a full-length protein consisting of 346 amino acids (residues 1-346) . The protein can be recombinantly expressed with histidine tags to facilitate purification while maintaining its enzymatic activity . This enzyme belongs to the short-chain dehydrogenase/reductase (SDR) family and plays a role in fatty acid metabolism and potentially in redox balance mechanisms in S. stipitis.

What are the primary physiological roles of 3-ketoacyl-CoA reductase in Scheffersomyces stipitis?

3-Ketoacyl-CoA reductase (PICST_79198) primarily functions in the fatty acid biosynthesis pathway, where it catalyzes the reduction of 3-ketoacyl-CoA to 3-hydroxyacyl-CoA using NADPH as a cofactor . In S. stipitis, this enzyme may have additional significance beyond fatty acid metabolism, particularly in maintaining redox balance during xylose fermentation. Unlike Saccharomyces cerevisiae, S. stipitis can naturally ferment xylose to ethanol, and the redox metabolism differences between these two yeasts likely contribute to this phenotypic distinction . The enzyme might be involved in NADPH regeneration pathways that are crucial for xylose metabolism in anaerobic conditions.

How does the PICST_79198 protein sequence compare to homologs in other organisms?

Sequence comparison analyses reveal that PICST_79198 has relatively low sequence identity with proteins from distantly related organisms. BLAST search results show only 28% identity (23/81 amino acids) in aligned regions with certain proteins from other species . This moderate sequence conservation suggests functional conservation of the catalytic domains while allowing for species-specific adaptations. Within the Scheffersomyces genus, higher conservation is expected, though exact figures weren't provided in the search results. This divergence makes PICST_79198 an interesting subject for comparative studies of enzyme evolution and specialization.

What are the optimal expression systems for producing recombinant PICST_79198?

For recombinant expression of PICST_79198, Escherichia coli has been successfully employed as a host organism . The protein can be expressed with histidine tags to facilitate purification through affinity chromatography. Based on similar proteins, the following methodological approach is recommended:

• Clone the full-length gene (without native signal peptide if present) into an expression vector with an appropriate promoter and His-tag

• Transform into an E. coli expression strain (such as BL21)

• Induce protein expression with IPTG or other inducers depending on the vector system

• Lyse cells and purify using nickel affinity chromatography

• Verify protein identity and purity using SDS-PAGE and Western blotting

• Assess enzymatic activity through reduction assays with 3-ketoacyl-CoA substrates

Alternative expression systems, such as Komagataella phaffii (formerly Pichia pastoris), might be considered for eukaryotic expression, especially when post-translational modifications are critical for function .

What are the most reliable methods for assessing PICST_79198 enzymatic activity?

To assess the enzymatic activity of PICST_79198, researchers should design assays that monitor the reduction of 3-ketoacyl-CoA substrates to 3-hydroxyacyl-CoA while tracking NADPH consumption. A methodological approach includes:

• Spectrophotometric assays: Monitor the decrease in NADPH absorbance at 340 nm as it is oxidized to NADP+ during the reaction

• HPLC analysis: Separate and quantify substrate and product to determine conversion rates

• Coupled enzyme assays: For situations where direct measurement is challenging

• Controls should include:

  • Reactions without enzyme (negative control)

  • Reactions with known 3-ketoacyl-CoA reductases (positive control)

  • Substrate specificity tests using various chain lengths of 3-ketoacyl-CoA

  • Cofactor specificity tests comparing NADPH vs. NADH preference

Temperature, pH, and buffer composition optimization should be performed to determine optimal reaction conditions for the enzyme from S. stipitis.

How can researchers design experiments to study PICST_79198's role in redox balance during xylose fermentation?

Designing experiments to elucidate PICST_79198's potential role in redox balance requires a multi-faceted approach:

• Gene knockout/knockdown studies:

  • Create PICST_79198 deletion mutants in S. stipitis

  • Compare growth rates and fermentation capacity on xylose vs. glucose media

  • Monitor intracellular NAD(P)H/NAD(P)+ ratios using fluorescence-based assays

• Metabolic flux analysis:

  • Use 13C-labeled xylose to trace carbon flow through different pathways

  • Compare wild-type and PICST_79198 mutant strains

  • Apply flux balance analysis models to predict metabolic adjustments

• Heterologous expression studies:

  • Express PICST_79198 in S. cerevisiae (which cannot naturally ferment xylose)

  • Assess whether expression enhances xylose fermentation capacity

  • Pair with expression of other components like XR and XDH from S. stipitis

• Cofactor engineering experiments:

  • Test whether PICST_79198 interacts with NAD(P)H regeneration systems

  • Examine effects of overexpressing NADH kinase with PICST_79198

This approach follows established experimental design principles for functional characterization of metabolic enzymes in the context of specific physiological processes.

How does PICST_79198 potentially interact with the xylose reductase-xylitol dehydrogenase (XR-XDH) pathway in S. stipitis?

The xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway is central to xylose fermentation in S. stipitis. The potential interaction with PICST_79198 involves complex redox balance mechanisms:

• The XR enzyme from S. stipitis can use both NADPH (60% preference) and NADH as cofactors, whereas XDH specifically requires NAD+

• This cofactor imbalance can result in xylitol accumulation when NADPH is preferentially consumed but NAD+ regeneration is limited

• PICST_79198, as a 3-ketoacyl-CoA reductase, typically uses NADPH. Its activity might be coordinated with NADPase and NADH kinase to maintain appropriate cofactor pools

Research indicates that S. stipitis has evolved mechanisms to balance redox cofactors in the absence of oxygen, potentially including NADPase and NADH kinase activities . Experimental approaches to investigate PICST_79198's interaction with this system should:

• Examine co-expression patterns of PICST_79198 with XR-XDH genes under xylose fermentation conditions

• Test whether PICST_79198 activity affects xylitol accumulation in metabolic engineering contexts

• Investigate potential protein-protein interactions between PICST_79198 and components of the XR-XDH pathway

How can researchers apply flux balance analysis to study the metabolic context of PICST_79198?

Flux balance analysis (FBA) is a powerful computational approach for studying metabolic networks and predicting phenotypes. To apply FBA to understand PICST_79198's metabolic context:

• Use genome-scale metabolic models of S. stipitis, such as iBB814, iSS884, iTL885, or iPL912, or create a consensus model

• Incorporate accurate constraints for PICST_79198 reactions, including:

  • Stoichiometry of the reaction

  • Cofactor preferences (NADPH vs. NADH)

  • Reversibility constraints

  • Gene-protein-reaction associations

• Simulate growth and fermentation with different carbon sources (glucose vs. xylose)

• Perform in silico gene deletion studies to predict the systemic effects of PICST_79198 absence

• Use alternative optima analysis to identify potential metabolic solutions that might involve PICST_79198

This approach can help identify the most critical roles of PICST_79198 in the context of the entire metabolic network of S. stipitis, particularly during xylose fermentation.

What are the critical considerations when investigating evolutionary conservation of PICST_79198 function across yeast species?

Investigating the evolutionary conservation of PICST_79198 requires careful experimental design and analytical approaches:

• Phylogenetic analysis:

  • Construct phylogenetic trees based on sequence homology

  • Focus on the Scheffersomyces-Spathaspora clade, which includes xylose-fermenting yeasts

  • Correlate enzyme conservation with metabolic capabilities across species

• Functional complementation:

  • Express PICST_79198 homologs from different yeasts in S. stipitis knockout strains

  • Assess restoration of phenotypes related to fatty acid metabolism and xylose fermentation

  • Compare enzyme kinetics and substrate specificity across homologs

• Structural biology approaches:

  • Determine protein structures through crystallography or cryo-EM

  • Compare active sites and substrate binding pockets across species

  • Identify conserved residues that may be critical for function

• Ecological and evolutionary context:

  • Correlate PICST_79198 sequence variations with ecological niches of different yeasts

  • Consider horizontal gene transfer events that might have influenced enzyme evolution

  • Examine selective pressures on PICST_79198 across lineages

This multi-faceted approach can provide insights into how PICST_79198 function has been conserved or altered throughout yeast evolution, particularly in relation to xylose metabolism capabilities.

What are common challenges in purifying active PICST_79198 and how can they be addressed?

Purifying active recombinant PICST_79198 can present several challenges:

• Protein solubility issues:

  • Use solubility tags (e.g., SUMO, MBP) if His-tag alone results in inclusion bodies

  • Optimize expression temperature (often lower temperatures improve solubility)

  • Test different lysis buffers with various detergents and stabilizing agents

• Cofactor retention:

  • Include NADPH at low concentrations in purification buffers

  • Avoid harsh elution conditions that might displace bound cofactors

  • Consider dialysis protocols that maintain cofactor association

• Activity preservation:

  • Add reducing agents (DTT, β-mercaptoethanol) to prevent oxidation of active site residues

  • Include glycerol (10-20%) to stabilize the protein during storage

  • Perform activity assays immediately after purification and after storage to assess stability

• Protein yield optimization:

  • Test multiple expression systems (E. coli, K. phaffii, etc.)

  • Optimize codon usage for the expression host

  • Evaluate different induction conditions (temperature, inducer concentration, duration)

Implementing these strategies can help overcome common technical challenges in obtaining functionally active PICST_79198 for biochemical and structural studies.

How can contradictory experimental results regarding PICST_79198 function be resolved?

When faced with contradictory results regarding PICST_79198 function, researchers should consider a systematic approach to resolution:

• Experimental conditions analysis:

  • Carefully compare buffer compositions, pH, temperature, and other reaction conditions

  • Assess purity and integrity of enzyme preparations using multiple methods

  • Consider post-translational modifications that might vary between preparations

• Alternative hypothesis testing:

  • Design experiments that can distinguish between competing hypotheses

  • Use multiple independent methods to measure the same parameter

  • Perform time-course studies to capture dynamic behavior

• Biological context consideration:

  • Test function in both in vitro reconstituted systems and in vivo contexts

  • Consider metabolic state differences that might influence enzyme behavior

  • Examine enzyme regulation under different growth conditions

• Technical validation:

  • Implement rigorous controls for each experiment

  • Use reference standards when appropriate

  • Consider blind experimental design to reduce bias

• Collaborative cross-validation:

  • Engage multiple laboratories to independently verify key findings

  • Use standardized protocols to facilitate comparison

When analyzing contradictory results, it's essential to apply fundamental principles of experimental design, including proper controls, technical replicates, and statistical analysis .

What novel approaches could advance our understanding of PICST_79198's role in xylose fermentation?

Several innovative approaches could significantly advance our understanding of PICST_79198:

• Systems biology integration:

  • Multi-omics studies combining transcriptomics, proteomics, and metabolomics

  • Network analysis to identify regulatory interactions affecting PICST_79198

  • Mathematical modeling of dynamic responses to changing carbon sources

• Synthetic biology applications:

  • Design minimal synthetic pathways containing PICST_79198 to test specific hypotheses

  • Create chimeric enzymes by domain swapping with related reductases

  • Employ directed evolution to alter cofactor specificity or catalytic efficiency

• Advanced microscopy techniques:

  • Use fluorescent protein tagging to track PICST_79198 localization under different conditions

  • Apply super-resolution microscopy to examine potential enzyme clustering

  • Implement FRET-based sensors to monitor enzyme-substrate interactions in real-time

• CRISPR-based approaches:

  • Apply CRISPRi for tunable repression of PICST_79198 expression

  • Use CRISPR activation to upregulate expression at native loci

  • Implement base editing for precise amino acid substitutions

• Structural dynamics studies:

  • Apply hydrogen-deuterium exchange mass spectrometry to examine conformational changes

  • Use molecular dynamics simulations to predict functional motions

  • Implement time-resolved crystallography to capture catalytic intermediates

These approaches could provide unprecedented insights into PICST_79198's functional role and regulatory mechanisms in the context of xylose fermentation.

How might PICST_79198 be leveraged in metabolic engineering for improved biofuel production?

The potential applications of PICST_79198 in metabolic engineering include:

• Cofactor balance optimization:

  • Express modified versions of PICST_79198 with altered NADPH/NADH preferences

  • Co-express with complementary enzymes that regenerate preferred cofactors

  • Fine-tune expression levels to match cofactor utilization with regeneration rates

• Pathway integration strategies:

  • Incorporate PICST_79198 into synthetic pathways designed for optimal carbon flux

  • Create fusion proteins with interacting enzymes to enhance substrate channeling

  • Coordinate expression with xylose transporters and downstream glycolytic enzymes

• Chassis organism development:

  • Introduce PICST_79198 along with other S. stipitis genes into industrial production strains

  • Evaluate performance in different host backgrounds (S. cerevisiae, E. coli, etc.)

  • Test robustness under industrial fermentation conditions

• Regulatory circuit design:

  • Develop dynamic regulatory systems that modulate PICST_79198 expression in response to metabolic state

  • Create feedback loops that maintain optimal redox balance during fermentation

  • Implement genetic switches for conditional expression

These strategies could help overcome current limitations in lignocellulosic biofuel production, particularly by addressing redox imbalance issues that often limit xylose fermentation efficiency in engineered organisms .