Recombinant Debaryomyces hansenii Polyadenylate-binding protein, cytoplasmic and nuclear (PAB1), partial

What is the functional role of PAB1 in Debaryomyces hansenii?

PAB1 (Polyadenylate-binding protein) in yeasts primarily binds to the poly(A) tail of mRNA and serves as an important mediator of multiple roles of the poly(A) tail in mRNA biogenesis, stability, and translation. While specific characterization of PAB1 in D. hansenii is still developing, studies in related yeasts show that PAB1 functions in both nuclear and cytoplasmic compartments . In the nucleus, it interacts with cleavage factors required for mRNA processing and polyadenylation, while in the cytoplasm, it affects both translation and mRNA decay .

Notably, D. hansenii exhibits a unique polyadenylation pattern compared to other yeasts like Saccharomyces cerevisiae and Kluyveromyces lactis, with a higher focus on a single dominant polyadenylation point closer to the ORF terminus . This suggests that PAB1 might have evolved specific functional adaptations in D. hansenii related to its halotolerant lifestyle.

How does D. hansenii differ from other yeast species as a host for recombinant protein production?

D. hansenii possesses several distinctive characteristics that make it an attractive alternative host system:

These characteristics make D. hansenii particularly suitable for expression of recombinant proteins under conditions that would be detrimental to conventional expression hosts . The yeast's ability to metabolize lactic and citric acids also provides flexibility in culture media options .

What are the current genetic modification tools available for D. hansenii?

Recent advances have significantly improved the genetic toolbox for D. hansenii:

• PCR-based gene targeting using homologous recombination with 50 bp flanking sequences (>75% efficiency in wild-type isolates)

• Selectable marker cassettes using Hygromycin B (hygromycin B phosphotransferase gene) or G418 (kanamycin resistance gene)

• CRISPR-CUG/Cas9 system adapted specifically for D. hansenii

• In vivo DNA assembly technique for co-transformation of up to three DNA fragments with 30-bp homologous overlapping overhangs

• Promoter options including TEF1 promoter from Arxula adeninivorans for high-level expression

The selection of appropriate tools depends on the specific experimental goals. For simple gene disruptions, the PCR-based approach with selectable markers is efficient, while for more complex modifications, the CRISPR-Cas9 system offers greater precision .

How can homologous recombination efficiency be optimized for PAB1 modification in D. hansenii?

Optimizing homologous recombination in D. hansenii requires addressing several factors that influence recombination efficiency:

• NHEJ pathway inhibition: Unlike S. cerevisiae, D. hansenii prefers the non-homologous end joining (NHEJ) pathway for DNA repair. Creating NHEJ-deficient mutants significantly improves homologous recombination efficiency and reduces random genome integration .

• Length of homologous regions: While the PCR-based method using 50 bp homology arms has shown high efficiency (>75% for gene disruption) , increasing the length of homologous sequences to 500-1000 bp can further improve targeting efficiency for complex modifications of essential genes like PAB1.

• Selection marker optimization: Using heterologous selectable markers that have been codon-optimized for D. hansenii, particularly regarding CTG codon usage, improves expression. Both Hygromycin B resistance (hph gene) and G418 resistance (kanr gene) markers have been successfully employed .

• Target site selection: The genomic context of the insertion site affects recombination efficiency. For PAB1 modification, considering chromatin structure and transcriptional activity at the target site is crucial.

• DNA delivery method: Electroporation protocols specifically optimized for D. hansenii yield higher transformation efficiencies than standard lithium acetate methods used for S. cerevisiae .

• Recovery conditions: Given D. hansenii's halotolerance, including 0.5-1.0M NaCl in recovery media can improve cell viability post-transformation and increase recombination efficiency .

The combination of these approaches can significantly enhance homologous recombination efficiency for PAB1 manipulation in D. hansenii.

What are the implications of salt stress on PAB1 expression and function in D. hansenii?

Salt stress significantly impacts gene expression and protein function in D. hansenii, with particular relevance to PAB1:

• Transcriptional regulation: Integrated multi-omics analysis of D. hansenii growing at high salt concentrations (1M NaCl or KCl) revealed distinct transcriptomic profiles compared to normal conditions . PAB1, being involved in post-transcriptional regulation, may be differentially expressed under salt stress.

• Post-translational modifications: Phosphoproteomic analysis has identified unique phosphorylation patterns in D. hansenii under high salt conditions . As PAB1 function is regulated by phosphorylation in other yeasts, salt-stress induced modifications likely affect its activity in D. hansenii.

• Protein-protein interactions: Salt stress alters the interaction landscape of RNA-binding proteins. PAB1 interactions with translation initiation factors (like eIF4G homologs) may be modified under high salt conditions, affecting translation efficiency .

• mRNA stability regulation: PAB1's role in mRNA stability becomes particularly crucial under stress conditions. In D. hansenii, PAB1 may be involved in stabilizing specific transcripts required for salt adaptation .

• Subcellular localization: Studies in other yeasts show that stress conditions cause redistribution of PAB1 between the nucleus, cytoplasm, and stress granules . In D. hansenii, salt-specific stress granule formation mechanisms may uniquely involve PAB1.

Research examining recombinant PAB1 function should account for these salt-dependent alterations, particularly when studying its role in stress adaptation mechanisms.

How does the polyadenylation pattern in D. hansenii influence recombinant PAB1 expression and function?

The unique polyadenylation characteristics of D. hansenii have significant implications for recombinant PAB1 expression:

• Higher focus on dominant poly(A) sites: Unlike S. cerevisiae and K. lactis that exhibit extensive heterogeneity in polyadenylation sites, D. hansenii shows a stronger preference for a single dominant poly(A) site closer to the ORF terminus . This may result in more uniform mRNA populations for recombinant PAB1, potentially enhancing translation efficiency.

• Species-specific factors in poly(A) site selection: When D. hansenii genes are expressed in S. cerevisiae, they adopt the S. cerevisiae polyadenylation profile, indicating that species-specific factors primarily determine the polyadenylation pattern . This suggests that optimal expression of recombinant PAB1 requires the native D. hansenii cellular machinery.

• Secondary structure influence: Many dominant poly(A) sites adopt a common secondary structure recognized by the cleavage/polyadenylation machinery . Engineering these structural elements into expression constructs can enhance proper processing of recombinant PAB1 mRNA.

• Sequence context dependencies: Polyadenylation in D. hansenii relies on a highly degenerate sequence over a broad region and a local sequence that depends on A residues after the cleavage point . These sequence requirements should be preserved in expression constructs.

• Terminator selection importance: Given the species-specific nature of polyadenylation, using native D. hansenii terminators rather than those from other organisms can significantly improve recombinant PAB1 expression levels and mRNA stability .

These considerations highlight the importance of preserving native polyadenylation signals when designing expression constructs for recombinant PAB1 in D. hansenii.

What CRISPR-Cas9 strategy is most effective for PAB1 modification in D. hansenii?

An effective CRISPR-Cas9 strategy for PAB1 modification in D. hansenii should incorporate these key considerations:

• CRISPR-CUG/Cas9 adaptation: Using a CRISPR system specifically adapted for D. hansenii's unique genetic code, accounting for the nonstandard usage of the CUG codon .

• Guide RNA design: For PAB1 modification, design guide RNAs targeting non-essential regions of the gene or its regulatory elements. Multiple guides increase the chances of successful editing but may lead to unwanted modifications.

• Repair template construction:

  • For gene disruption: Design a repair template with 50 bp homology arms flanking a selectable marker

  • For precise modifications: Include longer homology arms (500+ bp) with the desired mutations

  • For domain modifications: Target specific functional domains based on PAB1 structural information from related yeasts

• NHEJ inhibition: To improve HDR (homology-directed repair) efficiency, consider temporarily inhibiting the NHEJ pathway during transformation . This can be achieved through targeting key NHEJ components like Ku70/80.

• Multiplexing considerations: When targeting multiple PAB1 domains simultaneously, use a single CRISPR-Cas9 vector expressing multiple guide RNAs rather than co-transforming multiple vectors.

• Off-target analysis: Due to limited genomic information for D. hansenii compared to model yeasts, conduct thorough bioinformatic analysis to minimize off-target effects, particularly in genes sharing sequence similarity with PAB1.

This approach maximizes editing efficiency while minimizing unwanted effects on D. hansenii cellular function.

What purification strategy yields highest purity and activity for recombinant PAB1 from D. hansenii?

A comprehensive purification strategy for recombinant PAB1 from D. hansenii:

• Affinity tag selection: For PAB1 purification, a C-terminal 6×His tag is recommended as it minimally interferes with RNA-binding activity. Alternative tags include FLAG or Strep-II tag when antibody-based detection is preferred .

• Cell lysis optimization:

  • Buffer composition: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol

  • Protease inhibitors: Complete EDTA-free cocktail

  • Phosphatase inhibitors: Critical to preserve native phosphorylation state

  • RNase inhibitors: Include to maintain RNA-binding capabilities

  • Lysis method: Glass bead disruption in high-salt conditions (0.5-1.0M NaCl) leverages D. hansenii's halotolerance while reducing contaminant protein solubility

• Multi-step purification protocol:

  Step 1: Affinity chromatography

  • For His-tagged PAB1: Ni-NTA resin

  • Binding: 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 20 mM imidazole

  • Washing: Increase imidazole to 50 mM

  • Elution: Linear gradient to 300 mM imidazole

  Step 2: Ion exchange chromatography

  • Cation exchange (SP-Sepharose) at pH 6.5

  • Elution: Linear gradient from 150 mM to 1M NaCl

  Step 3: Size exclusion chromatography

  • Superdex 200 column

  • Buffer: 20 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM DTT, 5% glycerol

• Quality control assessment:

  • Purity: >95% by SDS-PAGE and silver staining

  • Identity: Western blotting and mass spectrometry

  • Activity: Poly(A) binding assay using fluorescently labeled oligo(A) substrates

  • Structural integrity: Circular dichroism to verify proper folding

• Storage conditions:

  • Short-term: 4°C in 20 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM DTT, 10% glycerol

  • Long-term: -80°C in small aliquots with 20% glycerol or lyophilized

This multi-step strategy accounts for D. hansenii's specific characteristics and preserves PAB1's functional properties.

How can functional assays be designed to characterize recombinant D. hansenii PAB1 activity?

Comprehensive functional characterization of recombinant D. hansenii PAB1 requires multiple complementary assays:

• RNA binding assays:

  • Electrophoretic Mobility Shift Assay (EMSA): Using fluorescently labeled poly(A) RNA oligonucleotides to determine binding constants and specificity

  • Fluorescence Anisotropy: For quantitative measurement of binding affinities to different RNA substrates

  • UV Cross-linking: To identify specific RNA sequence preferences unique to D. hansenii PAB1

  • Filter Binding Assay: For rapid screening of binding conditions and RNA substrate preferences

• Protein-protein interaction studies:

  • Co-immunoprecipitation: To identify interaction partners, particularly focusing on translation initiation factors like eIF4G homologs

  • Yeast Two-Hybrid: Using D. hansenii PAB1 as bait against a D. hansenii cDNA library

  • Surface Plasmon Resonance: For quantitative measurement of binding kinetics with putative partners

  • Pull-down assays with Tif4631p homologs: To compare interaction patterns with those observed in S. cerevisiae

• Functional complementation:

  • Heterologous expression in S. cerevisiae pab1Δ mutants: To test functional conservation

  • Domain swapping experiments: Between D. hansenii PAB1 and S. cerevisiae PAB1 to identify functional domains

  • Growth assays under stress conditions: Particularly in high salt media to assess stress-specific functions

• mRNA stability and translation assays:

  • In vitro deadenylation assays: To measure the effect of PAB1 on poly(A) tail shortening

  • mRNA half-life measurements: Using reporter constructs in the presence/absence of PAB1

  • Polysome profiling: To assess effects on translation efficiency

  • In vitro translation systems: Using D. hansenii extracts supplemented with recombinant PAB1

• Structural characterization:

  • Limited proteolysis: To identify domain boundaries and stable fragments

  • Circular dichroism: To assess secondary structure content

  • NMR spectroscopy: For detailed structural analysis of the RRM domains

  • Crystallography: To determine high-resolution structure

• Salt-dependent functionality:

  • All above assays should be performed under varying salt concentrations (0.5-2.0M NaCl) to assess how D. hansenii PAB1's unique adaptations respond to salt stress

These assays collectively provide a comprehensive functional profile of recombinant D. hansenii PAB1, highlighting its unique properties compared to homologs from other yeast species.

How might PAB1 interact with the halotolerance mechanisms in D. hansenii?

Recent evidence suggests several potential mechanisms by which PAB1 may contribute to halotolerance in D. hansenii:

• Stress granule dynamics: Under high salt conditions, PAB1 may participate in the formation of stress granules that sequester specific mRNAs, protecting them from degradation while temporarily halting translation . D. hansenii's adaptation to salt stress might involve unique PAB1-mediated regulation of these structures.

• mRNA stability regulation: Integrated multi-omics analysis of D. hansenii under salt stress revealed differential expression of numerous genes . PAB1 likely plays a critical role in stabilizing mRNAs encoding salt-stress response proteins through its interaction with the poly(A) tail.

• Selective translation promotion: In S. cerevisiae, PAB1 stimulates translation through interaction with eIF4G . In D. hansenii, this mechanism might be specialized to preferentially translate transcripts required for salt adaptation even under high salt conditions that normally inhibit translation.

• Interaction with ion transporters: The phosphoproteomic analysis of D. hansenii under salt stress implicated a novel uncharacterized cation transporter in the response to high sodium concentrations . PAB1 might regulate the expression of this and other transporters at the post-transcriptional level.

• Nuclear export of stress-response transcripts: PAB1's role in mRNA export from the nucleus might be particularly important for rapidly responding to changing salt concentrations, ensuring that stress-response transcripts are efficiently exported for translation.

Research in this area would benefit from comparative studies examining PAB1 binding targets under normal versus high salt conditions, potentially revealing unique adaptations that contribute to D. hansenii's remarkable halotolerance.

What are the biotechnological applications of engineered variants of D. hansenii PAB1?

Engineered PAB1 variants offer several promising biotechnological applications:

• Enhanced recombinant protein production:

  • PAB1 variants with increased binding affinity to poly(A) tails could enhance mRNA stability and translation efficiency of recombinant transcripts

  • Domain-optimized variants could improve translation of specific recombinant proteins in high-salt industrial environments

• Stress-resistant strains for biotechnology:

  • Overexpression of engineered PAB1 could generate D. hansenii strains with enhanced tolerance to combined stresses (salt, temperature, pH)

  • Such strains would be valuable for bioprocessing of high-salt industrial waste streams

• Antimicrobial applications:

  • D. hansenii produces killer toxins active against pathogenic Candida species

  • PAB1-modified strains could potentially enhance production of these antimicrobial compounds

• Biosensor development:

  • PAB1-reporter fusions could serve as sensitive biosensors for environmental stresses

  • Applications in monitoring industrial bioprocesses or environmental contamination

• RNA-targeting therapeutics platform:

  • Engineered PAB1 RRM domains could serve as scaffolds for developing RNA-binding proteins with novel specificities

  • Potential applications in targeting pathogenic RNA or modulating gene expression

These applications leverage D. hansenii's natural capabilities while enhancing them through targeted engineering of the PAB1 protein, potentially opening new avenues in industrial biotechnology and biomedical applications.

How can low transformation efficiency be addressed when working with D. hansenii?

When facing low transformation efficiency with D. hansenii, consider these evidence-based approaches:

• Optimize competent cell preparation:

  • Harvest cells in early-mid logarithmic phase (OD600 0.6-0.8)

  • Pre-condition cells in media containing 0.5-1.0M NaCl to activate stress response mechanisms

  • Use freshly prepared competent cells rather than frozen stocks

• Modify transformation protocols:

  • Increase DNA concentration to 5-10 μg for complex constructs

  • Extend heat shock duration to 40-45 minutes at 42°C

  • Include recovery phase in YPD with 0.5M NaCl before selective plating

  • Consider electroporation (1.5 kV, 200 Ω, 25 μF) as an alternative to chemical transformation

• Address homologous recombination efficiency:

  • Use longer homology arms (500+ bp) for complex genetic modifications

  • Consider generating NHEJ-deficient strains to improve homologous recombination

  • Include carrier DNA (salmon sperm DNA, 100 μg) to improve transformation efficiency

• Optimize selective conditions:

  • Use lower antibiotic concentrations initially (50-70% of standard concentration)

  • Gradually increase selective pressure in subsequent passages

  • Consider the influence of media salt concentration on antibiotic efficacy

• Vector design considerations:

  • Ensure codon optimization, particularly CTG codons

  • Use strong promoters suitable for D. hansenii (TEF1, ACT1)

  • Confirm that selective markers are functional in D. hansenii

By systematically addressing these factors, transformation efficiency can be significantly improved, facilitating genetic manipulation of D. hansenii for PAB1 studies.

What approaches can resolve protein expression and solubility issues with recombinant PAB1?

When encountering expression or solubility challenges with recombinant PAB1 in D. hansenii, implement these strategies:

• Expression optimization:

  • Promoter screening: Test multiple promoters (TEF1, ACT1, PGK1) to identify optimal expression levels

  • Codon optimization: Ensure comprehensive CTG codon adaptation for D. hansenii

  • Growth conditions: Optimize temperature (20-30°C), media composition, and induction timing

  • Expression kinetics: Monitor expression over time to identify optimal harvest point

• Solubility enhancement:

  • Domain-based approach: Express individual RRM domains rather than full-length protein

  • Fusion partners: Test solubility-enhancing tags (MBP, SUMO, TrxA) at N-terminus

  • Buffer optimization: Screen buffers containing various salt concentrations (0.3-1.0M NaCl)

  • Additives: Include stabilizers like glycerol (10-20%), low concentrations of non-ionic detergents, or arginine (50-100 mM)

• Structural considerations:

  • N-terminal modifications: The N-terminal helix in RRM domains may affect solubility; consider truncation variants

  • Disulfide engineering: Strategic introduction or removal of cysteine residues

  • Surface charge manipulation: Modify surface residues to enhance solubility while preserving function

• Expression temperature:

  • Lower temperature (16-20°C) often improves folding and solubility

  • For D. hansenii, due to its cryotolerance, expression at lower temperatures is particularly feasible

• Co-expression strategies:

  • Co-express with binding partners (e.g., eIF4G fragments)

  • Co-express with molecular chaperones to improve folding

• Extraction optimization:

  • Cell lysis under native salt concentration for D. hansenii (0.5-1.0M NaCl)

  • Include RNase inhibitors to preserve RNA-binding capabilities

  • Test various lysis methods (sonication, enzymatic, high-pressure homogenization)

By methodically testing these approaches, researchers can overcome expression and solubility challenges for recombinant PAB1 from D. hansenii.

What are the most promising research directions for understanding PAB1's role in D. hansenii's unique adaptations?

Several high-potential research avenues deserve exploration:

• Comparative structural biology:

  • High-resolution structures of D. hansenii PAB1 compared to homologs from non-halotolerant yeasts

  • Focus on unique structural adaptations that enable function in high-salt environments

  • Investigation of salt-dependent conformational changes using techniques like SAXS or cryo-EM

• System-wide RNA-protein interaction studies:

  • CLIP-seq to identify PAB1 binding targets genome-wide under various salt conditions

  • Comparative analysis with PAB1 targets in S. cerevisiae to identify D. hansenii-specific interactions

  • Integration with transcriptome and translatome data to build comprehensive models of post-transcriptional regulation

• Genetic interaction mapping:

  • Synthetic genetic array analysis with PAB1 mutations

  • Identification of genetic interactions unique to high-salt conditions

  • Mapping the functional relationship between PAB1 and known halotolerance factors

• Role in stress granule dynamics:

  • Characterization of stress granule composition and dynamics in D. hansenii

  • Investigation of PAB1's role in recruiting specific mRNAs to stress granules under salt stress

  • Comparison with stress granule properties in non-halotolerant yeasts

• Integration with phosphoproteome studies:

  • Identification of salt-dependent phosphorylation patterns on PAB1

  • Characterization of kinases and phosphatases regulating PAB1 activity

  • Functional consequences of phosphorylation on RNA binding and protein interactions

• Evolutionary analysis:

  • Comprehensive phylogenetic analysis of PAB1 across yeasts with varying halotolerance

  • Identification of signatures of adaptive evolution in D. hansenii PAB1

  • Reconstruction of ancestral PAB1 sequences to trace the evolution of halotolerance

These research directions would significantly advance our understanding of how PAB1 contributes to D. hansenii's remarkable environmental adaptations and could inform biotechnological applications leveraging these unique properties.

How might advances in synthetic biology enable new applications for engineered D. hansenii PAB1 variants?

Emerging synthetic biology approaches offer exciting possibilities for PAB1 engineering:

• Designer RNA regulatory circuits:

  • Engineered PAB1 variants with altered RNA binding specificities could serve as regulators in synthetic gene circuits

  • Applications in controlling gene expression in high-salt industrial processes

  • Creation of salt-responsive genetic switches based on PAB1 conformational changes

• Orthogonal translation systems:

  • PAB1 variants that selectively enhance translation of specific mRNA subsets

  • Development of orthogonal translation systems for expressing toxic proteins

  • Creation of synthetic genetic codes optimized for high-salt environments

• Engineered stress response networks:

  • Synthetic stress response pathways incorporating modified PAB1 proteins

  • Fine-tuned sensing and response to environmental conditions

  • Applications in biocontainment systems for engineered organisms

• Biomolecular condensate engineering:

  • Designed PAB1 variants that form specific RNA-protein condensates under controlled conditions

  • Applications in creating subcellular microenvironments for specialized metabolic processes

  • Potential for creating synthetic organelles with specialized functions

• Cell-free expression systems:

  • Development of D. hansenii-based cell-free protein synthesis systems for high-salt environments

  • Incorporation of engineered PAB1 variants to enhance translation efficiency

  • Applications in producing proteins that are typically difficult to express in conventional systems