Recombinant Debaryomyces hansenii Polarized growth chromatin-associated controller 1 (PCC1)

What is Debaryomyces hansenii and why is it significant for PCC1 research?

D. hansenii is a halophilic non-conventional yeast found in natural environments with high salt concentrations and osmotic pressure (e.g., seawater, soil, glaciers, and salty food). Its significance for PCC1 research stems from its unique ability to withstand high osmotic pressure, high salinity, and low water activity . As an osmotolerant yeast that can grow in the presence of high concentrations of salt or ethanol, it offers a distinctive cellular environment for studying polarized growth and chromatin-associated proteins like PCC1 .

How does one initiate D. hansenii culture for experimental work with recombinant proteins?

For initiating D. hansenii cultures, the following methodological approach is recommended:

• Media selection: YPD medium (1% yeast extract, 2% peptone, 2% glucose) or YM DEB medium are suitable for routine cultivation .

• Growth conditions: Optimal growth occurs at temperatures below 30°C (23-28°C is ideal) .

• Adaptation procedure:

  • Start with standard YM DEB medium

  • Gradually adapt cells to experimental conditions through sequential subculturing

  • For starvation experiments, follow the adaptation process shown below:

Adaptation Process for D. hansenii Cultures:

This adaptation process is particularly important when studying stress responses or preparing cells for recombinant protein expression .

What are the key considerations for designing primers for PCC1 amplification in D. hansenii?

When designing primers for PCC1 amplification in D. hansenii, researchers should consider:

• Codon usage bias: D. hansenii belongs to the CTG clade, which encodes serine instead of leucine in CTG codons . This necessitates codon optimization of heterologous genes.

• Primer design parameters:

  • For targeted gene integration, include 50 bp flanking regions identical to the target site in the genome .

  • For higher efficiency integration, extend homology arms to 90-100 bp .

  • Include restriction sites for subsequent cloning steps.

• Special considerations for PCC1:

  • Check for CTG codons in PCC1 sequence and modify if necessary.

  • If expressing in another host, consider that CTG codons may need to be changed to serine codons (TCA) for compatibility .

Using this approach, targeted integration efficiency of >75% can be achieved through homologous recombination .

What are the most effective selectable markers for recombinant PCC1 expression in D. hansenii?

The selection of appropriate markers is crucial for successful recombinant protein expression. Based on current research, several effective markers for D. hansenii have been established:

• Auxotrophic markers:

  • DhHIS4 and DhARG1 genes - suitable for laboratory strains with corresponding auxotrophy

  • Limited to specific laboratory strains with auxotrophic mutations

• Heterologous dominant selectable markers (applicable to wild-type isolates):

  • Hygromycin B resistance (HygR)

  • G418/Geneticin resistance (KanR)

  • Nourseothricin resistance (NatR) - using the sat-1 gene from bacterial transposon Tn1825

Selection marker efficiency comparison:

For PCC1 expression, heterologous markers are generally recommended as they allow work with wild-type D. hansenii strains that may exhibit better growth and stress tolerance characteristics .

How can we improve the efficiency of homologous recombination for PCC1 integration in D. hansenii?

Despite previous reports of inefficient homologous recombination in D. hansenii, recent studies demonstrate that high efficiency can be achieved with these methodological improvements:

• Flank length optimization:

  • 50 bp homology flanks can achieve >75% integration efficiency

  • Extending flanks to 90-100 bp provides higher efficiency for targeted integration and expression

• Selection marker design:

  • Use heterologous selectable markers (HygR, KanR, NatR) for wild-type strains

  • Ensure the marker cassette consists exclusively of heterologous DNA sequences

• Transformation protocol optimization:

  • Electroporation has proven most effective for D. hansenii transformation

  • Cell wall weakening treatments can improve transformation efficiency

  • Addition of sorbitol as an osmotic stabilizer significantly improves transformation efficiency (>1.5×10⁵ transformants/μg)

• Safe harbor site targeting:

  • The DhARG1 locus has been established as a safe genomic harbor site for the integrated expression of heterologous genes

  • This site allows stable expression without disrupting essential cellular functions

This optimized approach enables efficient targeted genomic modification through homologous recombination in D. hansenii isolates .

What promoters and terminators are most effective for PCC1 expression in D. hansenii?

Selection of appropriate promoter and terminator sequences significantly impacts recombinant protein expression levels. The following have proven effective in D. hansenii:

• Promoters:

  • Heterologous TEF1 promoter from Arxula adeninivorans - highest production of reporter proteins

  • Meyerozyma guilliermondii ACT1 promoter (MgACT1pr) - demonstrated to direct GFP expression in D. hansenii

  • Saccharomyces stipitis GPD1 promoter (500 bp upstream of GPD1 ORF)

  • D. hansenii TEF1 promoter (DhTEF) - successful for reporter gene expression

• Terminators:

  • S. cerevisiae PGK1 terminator - proven functionality in D. hansenii

  • D. hansenii native terminators - provide proper transcription termination signals

For optimal expression, in vivo DNA assembly techniques can be used to screen potential promoters and terminators to enhance D. hansenii's production of recombinant proteins in specific growth conditions .

What are the key methodological considerations when applying CRISPR/Cas9 for PCC1 editing in D. hansenii?

CRISPR/Cas9 technology has recently been adapted for D. hansenii, providing powerful options for precise gene editing. Key methodological considerations include:

• CRISPR system optimization:

  • A CRISPR/Cas9 method facilitating efficient oligo-mediated gene editing has been developed

  • CRISPR-CUG/Cas9 toolbox specifically developed for D. hansenii

• PAM site selection:

  • Target PAM sites that avoid CTG codons to prevent off-target effects

  • Consider the chromatin structure around the PCC1 locus for accessibility

• Repair template design:

  • For precise editing, design repair templates with extended homology arms (>100 bp)

  • Include selectable markers for efficient screening of edited cells

  • Consider the tendency of D. hansenii to prefer non-homologous end joining over homology-directed repair

• Delivery method:

  • Ribonucleoprotein (RNP) complex delivery may improve efficiency

  • Plasmid-based expression systems using ARS elements for transient Cas9 expression

• Strain selection:

  • Different D. hansenii isolates show heterogeneity in editing efficiency

  • Some isolates may retain wild-type copies alongside disrupted gene copies

The CRISPR/Cas9 method allows for precise gene editing, which is essential for the development of D. hansenii as a cell factory for various biotechnological applications, including controlled expression of proteins like PCC1 .

How can researchers address the challenge of genetic heterogeneity and ploidy variation when studying PCC1 function in D. hansenii?

D. hansenii exhibits significant genetic heterogeneity and potential ploidy variations that complicate genetic studies. To address these challenges:

• Strain characterization:

  • Perform pulsed-field gel electrophoresis (PFGE) to determine chromosome polymorphism

  • Use cluster analysis with dice coefficient similarity calculation to establish genetic relationships

  • Confirm ploidy through flow cytometry analysis

• Multi-strain approach:

  • Study PCC1 function across multiple isolates to account for strain variability

  • Include reference strains like CBS 767 and well-characterized strains NCYC102 and NCYC3363

• Confirmation of gene disruption:

  • Screen all potential gene deletion mutants for both the presence of gene deletion and absence of wild-type copy

  • Some isolates may retain wild-type copies alongside disrupted gene copies, requiring thorough screening

• Strain-specific optimization:

  • Adjust transformation and selection protocols for each strain

  • Consider that homologous recombination efficiency varies between strains

Genetic heterogeneity analysis example:

This approach accounts for the significant genetic heterogeneity among D. hansenii strains, which can be reflected in intra-species variation at the phenotypic level .

What advanced imaging techniques can be applied to study PCC1 localization and dynamics in D. hansenii?

Understanding the subcellular localization and dynamics of PCC1 requires sophisticated imaging techniques. Recent advances applicable to D. hansenii include:

• High-resolution live cell fluorescent imaging:

  • Fluorescent protein tagging strategies have been optimized for D. hansenii

  • N-terminal GFP tagging using the MgACT1 promoter with (Gly-Ala)3 linker

  • C-terminal tagging strategies for proteins like DhGpd1

• Label-free holotomography:

  • Recently optimized for yeast cells including D. hansenii

  • Allows visualization of subcellular features like membranes and vacuoles without fluorescent labels

  • Physical parameters defined for D. hansenii cells: sphericity values (>0.75 for single cells, <0.72 for dividing cells)

• Combined approaches:

  • Integrate fluorescent imaging with holotomography for comprehensive analysis

  • Correlative light and electron microscopy (CLEM) to combine fluorescence imaging with ultrastructural details

• Protein detection methods:

  • Some antibodies raised against S. cerevisiae proteins can recognize D. hansenii orthologs

  • Western blot analysis confirms detection of orthologs at predicted molecular weights

This combinatorial approach serves as a template for studying cell biological systems like D. hansenii that are not amenable to standard genetic procedures .

How should researchers design experiments to study the role of PCC1 in D. hansenii's response to osmotic stress?

To effectively study PCC1's role in osmotic stress response, a comprehensive experimental design approach is recommended:

• Strain construction:

  • Generate PCC1 deletion mutants using PCR-based gene targeting with 50-100 bp homology flanks

  • Create PCC1-GFP fusion strains to track protein localization during stress

  • Develop conditional PCC1 expression strains using controllable promoters

• Stress conditions setup:

  • Establish chemostat cultivations in controlled bioreactors with defined salt concentrations

  • Compare responses to different osmotic agents (NaCl vs. KCl at 1M concentration)

  • Include time-course sampling to capture immediate and adaptive responses

• Multi-omics analysis:

  • Transcriptomic analysis to identify PCC1-dependent gene expression changes

  • Proteomic and phosphoproteomic analyses to detect post-translational modifications

  • Metabolomic profiling to understand metabolic adaptations

• Experimental matrix design:

• Key parameters to measure:

  • Growth rates and biomass production

  • PCC1 expression levels and localization

  • Global transcriptional changes

  • Chromatin structure changes using ChIP-seq

  • Metabolite profiles, especially polyol production and utilization

This experimental design will provide comprehensive insights into PCC1's role in D. hansenii's remarkable osmotolerance and stress adaptation capabilities .

What is the optimal experimental design for studying PCC1's interaction with chromatin in D. hansenii?

To effectively study PCC1's interaction with chromatin in D. hansenii, researchers should implement this methodological framework:

• Chromatin immunoprecipitation (ChIP) protocol optimization:

  • Generate epitope-tagged PCC1 strains (e.g., PCC1-FLAG, PCC1-HA)

  • Optimize cell fixation conditions for D. hansenii's robust cell wall

  • Develop chromatin fragmentation protocols specific to D. hansenii's chromatin structure

  • Validate antibody specificity and efficiency in D. hansenii context

• Chromatin interaction mapping:

  • Perform ChIP-seq to identify genome-wide binding sites of PCC1

  • Implement ChIP-exo or CUT&RUN for higher resolution binding site mapping

  • Correlate binding sites with gene expression data to identify direct regulatory targets

• Protein-protein interaction analysis:

  • Perform co-immunoprecipitation to identify PCC1 interaction partners

  • Validate interactions through reciprocal pull-downs

  • Use proximity labeling approaches (BioID, APEX) to identify chromatin-associated interaction partners

• Experimental conditions matrix:

• Controls and validations:

  • Include non-tagged strains as negative controls

  • Use known chromatin-associated proteins as positive controls

  • Validate binding sites through reporter assays or mutagenesis

This comprehensive approach will provide insights into how PCC1 interacts with chromatin under different physiological conditions and contributes to D. hansenii's unique stress adaptation capabilities.

How can researchers apply high-throughput screening to identify conditions that modulate PCC1 function in D. hansenii?

High-throughput screening approaches can efficiently identify conditions affecting PCC1 function. An optimal methodological framework includes:

• Reporter system development:

  • Create PCC1-fluorescent protein fusions to monitor expression and localization

  • Develop promoter-reporter constructs for PCC1-regulated genes

  • Establish growth-based readouts for PCC1 functionality

• Micro-cultivation screening setup:

  • Implement micro-fermentation screening analysis that allows for a semi-controlled environment

  • Use scattered light signal as an indicator for biomass formation

  • Monitor growth in real-time to determine maximum specific growth rates

• Experimental condition matrix:

  • Test varying salt concentrations (0-2M NaCl, KCl)

  • Evaluate pH range effects (pH 4-8)

  • Assess carbon source variations (glucose, xylose, glycerol)

  • Examine temperature effects (15-37°C)

  • Test industrial by-products or complex feedstocks

• Data analysis and integration:

  • Apply experimental design principles for big data analysis

  • Implement Algorithm 1 from Wang et al. for optimal subset selection

  • Use the following utility function for selecting optimal design:
    s = |I(θ, d)| where I is the observed information matrix

• Validation in controlled bioreactors:

  • Confirm high-throughput findings in controlled chemostat cultivations

  • Perform transcriptomic and proteomic analyses of validated conditions

  • Examine PCC1 localization and activity under validated conditions

Example screening matrix:

This high-throughput approach allows for systematic identification of conditions that modulate PCC1 function while efficiently managing experimental resources .

How can researchers troubleshoot low expression of recombinant PCC1 in D. hansenii?

When facing low expression of recombinant PCC1, implement this systematic troubleshooting approach:

• Expression construct optimization:

  • Verify codon optimization for D. hansenii's CTG clade context

  • Test alternative promoters (TEF1 from A. adeninivorans shows highest expression)

  • Optimize the Kozak sequence for D. hansenii

  • Evaluate different signal peptides if secretion is intended

• Integration site assessment:

  • Confirm integration at the intended genomic locus

  • Consider using the DhARG1 locus as a safe chromosomal harbor site

  • Test multiple integration sites to identify optimal expression loci

• Host strain selection:

  • Evaluate expression in multiple D. hansenii strains due to genetic heterogeneity

  • Consider using strains with characterized proteolytic profiles

  • Test expression in protease-deficient strains if available

• Culture condition optimization:

  • Adjust cultivation temperature (optimal growth below 30°C)

  • Optimize media composition and pH

  • Test expression in the presence of varying salt concentrations

  • Monitor growth phase-dependent expression

• Protein stability assessment:

  • Check for proteolytic degradation of expressed PCC1

  • Consider fusion tags to enhance stability

  • Implement pulse-chase experiments to determine protein half-life

Systematic troubleshooting matrix:

This systematic approach will help identify and address the specific factors limiting PCC1 expression in D. hansenii .

What strategies can be employed to resolve contradictory experimental results when studying PCC1 function in D. hansenii?

When facing contradictory experimental results in PCC1 research, implement this methodological framework to resolve discrepancies:

• Genetic heterogeneity assessment:

  • Verify strain identity through ribosomal DNA sequencing

  • Perform pulsed-field gel electrophoresis (PFGE) to detect chromosome polymorphism

  • Use UPGMA method with Dice coefficient to analyze genetic relationships between strains

  • Sequence the PCC1 locus across strains to identify potential variations

• Experimental condition standardization:

  • Document precise cultivation conditions (media composition, pH, temperature)

  • Standardize growth phase for sampling (e.g., mid-log phase at specific OD)

  • Control environmental factors like salt concentration and osmotic pressure

  • Implement chemostat cultivation for precise physiological control

• Methodological validation:

  • Perform cross-laboratory validation of key protocols

  • Include multiple technical and biological replicates

  • Use complementary methods to confirm findings

  • Implement appropriate statistical analyses for result validation

• Strain-specific effects investigation:

  • Test PCC1 function across multiple D. hansenii strains

  • Create isogenic strains differing only in PCC1 alleles

  • Perform complementation tests with different PCC1 alleles

• Systematic literature review and meta-analysis:

  • Compare experimental conditions across published studies

  • Identify potential sources of variation between studies

  • Implement the PCC research question framework to structure analysis

Resolution framework matrix:

This systematic approach acknowledges the significant strain variation in D. hansenii and implements rigorous controls to resolve contradictory results .