Recombinant Debaryomyces hansenii Long chronological lifespan protein 2 (LCL2)

What is Debaryomyces hansenii and what makes it unique among yeasts?

Debaryomyces hansenii is a halophilic and osmotolerant non-conventional yeast species with remarkable abilities to withstand high osmotic pressure, high salinity (up to 4M NaCl), and low water activity . This yeast is particularly recognized for its involvement in cheese and meat product ripening processes and its role in synthesizing valuable biochemicals such as xylitol and riboflavin . D. hansenii is classified as an oleaginous yeast with the capacity to accumulate lipids exceeding 50% of its biomass, making it particularly interesting for biotechnological applications .

What are the optimal growth conditions for Debaryomyces hansenii cultures?

Optimal growth conditions for D. hansenii vary by strain, but most strains demonstrate enhanced growth in the presence of 1-2M NaCl . Research has shown that D. hansenii exhibits stable biomass formation when cultured with 1M NaCl present in the media . Temperature optimization studies indicate that while growth inhibition occurs at extreme temperatures, the presence of salt can stimulate growth even under these conditions . Typically, D. hansenii is grown at 25°C on standard yeast media (like YPD) supplemented with NaCl, with selection agents such as hygromycin B (25-50 mg/L), ClonNat (1.5-4 mg/L), or G418 disulphate (150-350 mg/L) used for transformed strains .

How does D. hansenii's tolerance to salt affect its cellular metabolism?

Salt tolerance in D. hansenii is directly linked to its cellular metabolism in several key ways:

• Enhanced respiratory activity: Studies have demonstrated increased respirative activity in D. hansenii in the presence of salts, especially sodium .

• ROS detoxification: High salt concentrations induce the expression of genes related to oxidative stress in D. hansenii, with sodium enabling better induction than potassium .

• Metabolic adaptation: The presence of salt (particularly 1M NaCl) has been shown to have protective and non-detrimental effects when D. hansenii is exposed to other stress factors, including extreme pH conditions, oxidative stress, or different temperatures .

This unique metabolic adaptation to salt helps explain why D. hansenii can thrive in environments that are hostile to other yeasts, making it valuable for specific biotechnological applications.

What transformation systems are available for D. hansenii?

Several transformation systems have been developed for D. hansenii:

• Histidine auxotrophy-based system: An efficient transformation system based on a histidine auxotrophic recipient strain (DBH9) generated by UV-induced random mutagenesis, with the DhHIS4 gene as the selectable marker .

• CRISPR-Cas9 system: A recently developed CRISPR-CUG/Cas9 toolbox enables efficient gene editing in D. hansenii .

• In vivo DNA assembly: This technique allows co-transformation of up to three different DNA fragments with 30-bp homologous overlapping overhangs, which are then fused in the correct order in a single step .

• Homologous recombination: This method has been employed for gene disruption in D. hansenii .

These transformation systems have significantly advanced genetic manipulation capabilities in D. hansenii, enabling more sophisticated research approaches.

How can I optimize the efficiency of genetic transformation in D. hansenii?

To optimize transformation efficiency in D. hansenii:

• Selection marker choice: For antibiotic selection, use appropriate concentrations: 25-50 mg/L hygromycin B, 1.5-4 mg/L ClonNat, or 150-350 mg/L G418 disulphate, with concentrations varying by strain .

• Plating density: For G418 selection specifically, plate cells at a relatively low density to minimize background growth, and replicate to a fresh selective plate after 24 hours of incubation .

• Promoter and terminator selection: When expressing recombinant proteins, the highest production has been achieved using the TEF1 promoter (from Arxula adeninivorans) and the CYC1 terminator .

• Homology arm length: For homologous recombination, use DNA fragments with 30-bp homologous overlapping overhangs for successful in vivo assembly .

• Growth conditions: Maintain cultures at 25°C, either on solid media or in liquid cultures with shaking at 200 rpm .

Following these optimization strategies can significantly improve transformation efficiency and expression of recombinant proteins in D. hansenii.

What expression systems are most effective for recombinant protein production in D. hansenii?

Based on recent research, the most effective expression systems for D. hansenii include:

• Promoter selection: The TEF1 promoter from Arxula adeninivorans has demonstrated the highest production of recombinant proteins (such as YFP used as a model) in D. hansenii .

• Terminator selection: The CYC1 terminator has shown good results for recombinant protein expression .

• Signal peptides: Various signal peptides have been screened to enhance D. hansenii's production of recombinant proteins, particularly in salt-rich by-products .

• Genomic safe landing sites: A genomic safe landing site has been identified for the expression of heterologous proteins in D. hansenii, which streamlines the development of expression strains .

• Culture media: D. hansenii can effectively produce recombinant proteins when grown in industrial by-products, particularly those rich in salt, without requiring nutritional supplements or freshwater .

This combination of genetic elements and culture conditions provides an optimized framework for recombinant protein expression in D. hansenii.

How can industrial by-products be utilized for recombinant protein production in D. hansenii?

D. hansenii can effectively utilize various industrial by-products for recombinant protein production:

• Salt-rich by-products: D. hansenii can grow in salt-rich industrial by-products from the dairy and pharmaceutical industries to produce recombinant proteins (such as YFP) without requiring any nutritional supplement or freshwater .

• Open cultivation advantages: The high salt concentration of these by-products favors D. hansenii's metabolism while hindering other non-halotolerant microorganisms, enabling successful open (non-sterile) cultivations at different laboratory scales (1.5 mL, 500 mL, and 1 L) .

• Scaling capabilities: Successful cultivation has been demonstrated at different laboratory scales, suggesting potential for industrial-scale applications .

• Lignocellulosic biomass: D. hansenii shows promise for industrial bioprocesses based on lignocellulosic biomass and other non-conventional feedstocks .

This ability to grow in by-products makes D. hansenii particularly valuable for sustainable biotechnology applications, as it avoids the use of freshwater and expensive carbon sources like glucose.

What methodologies are recommended for studying chronological lifespan proteins in D. hansenii?

For studying chronological lifespan proteins in D. hansenii, the following methodologies are recommended:

• Genetic manipulation techniques:

  • CRISPR-CUG/Cas9 system for precise gene editing

  • Homologous recombination for gene disruption

  • PCR-based methods for gene disruption

  • In vivo DNA assembly for efficient strain generation

• Cellular imaging approaches:

  • High-resolution live cell imaging to define ultrastructural and trafficking dynamics

  • Fluorescent labeling techniques such as using FM4-64 dye to temporally label endocytic trafficking to the lysosome/vacuole

• Metabolic analyses:

  • Characterization of NAD(H) homeostasis in peroxisomes, which can influence lifespan regulation

  • Evaluation of respiratory activity, which is enhanced in the presence of sodium

  • Assessment of ROS detoxification mechanisms, which are induced by high salt concentrations

• Stress resistance assays:

  • Testing survival under various stress conditions (temperature, pH, oxidative stress) with and without sodium chloride

  • Evaluating growth kinetics under different environmental conditions

These methodologies provide a comprehensive approach for investigating lifespan-related proteins in D. hansenii.

How does salt concentration affect the expression and function of lifespan-related proteins in D. hansenii?

Salt concentration significantly impacts the expression and function of proteins related to lifespan in D. hansenii through several mechanisms:

• Oxidative stress response: High salt concentrations induce the expression of genes related to oxidative stress response, with sodium enabling better induction than potassium . This is particularly relevant as oxidative stress management is a key determinant of cellular lifespan.

• Respiratory metabolism enhancement: Increased respirative activity has been observed in D. hansenii in the presence of salt, especially sodium . Respiratory metabolism is closely linked to chronological lifespan in yeasts.

• Protective effects: The presence of 1M NaCl has been shown to have protective effects when D. hansenii is exposed to other stress factors, including extreme pH conditions, oxidative stress, or temperature variations . These protective effects may extend to proteins involved in lifespan regulation.

• Stable biomass formation: Salt presence (1M NaCl) results in stable biomass formation in D. hansenii cultures over time , potentially affecting the expression stability of lifespan-related proteins.

• Metabolic shifts: Salt stress triggers metabolic adaptations that may alter NAD(H) homeostasis , which is known to influence chronological lifespan in yeasts.

Understanding these salt-dependent effects is crucial for optimizing the expression and studying the function of lifespan-related proteins in D. hansenii.

How does D. hansenii compare to S. cerevisiae as a model for studying lifespan-related proteins?

D. hansenii offers several distinct advantages and differences compared to S. cerevisiae for studying lifespan-related proteins:

This comparison highlights how D. hansenii can serve as a complementary model to S. cerevisiae, particularly for studying lifespan-related proteins under stress conditions that more closely resemble certain industrial or natural environments.

What are the unique cellular structures and physiological characteristics of D. hansenii that might impact lifespan studies?

D. hansenii possesses several unique cellular structures and physiological characteristics that could significantly impact lifespan studies:

• Endolysosomal system: Recent research has identified distinct endosomal trafficking patterns in D. hansenii. For example, studies using FM4-64 dye demonstrated that endocytic trafficking to the lysosome/vacuole follows a specific temporal pattern - from plasma membrane (0 min) to significant endosomal signal (2 min), then exclusively in endosomes (10 min), followed by successful trafficking to the vacuole (20 min) .

• Endosomal recycling route: D. hansenii has a unique endosomal recycling route that bypasses the trans-Golgi network, which differs from the pathway observed in S. cerevisiae .

• Peroxisomal NAD(H) homeostasis: D. hansenii exhibits specific mechanisms for NAD(H) homeostasis in peroxisomes, which could influence cellular lifespan as NAD+ metabolism is linked to aging processes .

• Halophilic adaptations: The cell membrane and wall structure of D. hansenii has evolved for optimal function in high-salt environments, which may affect cellular integrity maintenance during aging .

• ROS management systems: D. hansenii possesses enhanced mechanisms for managing reactive oxygen species, particularly when grown in sodium-rich environments . These systems are critical for preventing oxidative damage that accelerates aging.

These unique characteristics make D. hansenii a valuable model for studying lifespan-related proteins under conditions that may better represent certain environmental stresses than traditional model yeasts.

How can D. hansenii's antimycotic properties be harnessed in research contexts?

D. hansenii exhibits significant antimycotic properties that can be utilized in various research applications:

• Biocontrol mechanisms: D. hansenii strains produce both diffusible and volatile compounds that inhibit unwanted molds. Experimental results have shown inhibition rates surpassing 75% against various fungi, including Aspergillus, Penicillium, and Candida species . These compounds can be isolated and characterized for potential antimicrobial applications.

• Autochthonous strain screening: Research has established protocols for screening native D. hansenii strains isolated from food products to identify those with the strongest antifungal potential. Streak inhibition assays at various NaCl concentrations have been effective for selecting strains with the greatest inhibitory potential .

• Quantification methods: Radial inhibition and mouth-to-mouth approaches have been developed to evaluate the fungal antagonistic properties of diffusible and volatile compounds from D. hansenii .

• Practical applications: The antimycotic potential of D. hansenii has been demonstrated in real food systems, such as Iberian pork loins. D. hansenii LRC2 strain-inoculated products showed significantly fewer mold populations than non-inoculated ones, improving food safety without requiring high preservative concentrations .

• Morphology and sporulation effects: Beyond growth inhibition, D. hansenii can also inhibit sporulation in unwanted fungi, strengthening its biocontrol activity .

These antimycotic properties make D. hansenii valuable for developing natural preservation methods and studying fungal antagonism mechanisms.

What experimental design would you recommend for assessing the impact of recombinant LCL2 on chronological lifespan in yeast models?

A comprehensive experimental design for assessing the impact of recombinant LCL2 on chronological lifespan in yeast models would include:

• Strain construction and validation:

  • Generate D. hansenii strains expressing recombinant LCL2 using the TEF1 promoter from Arxula adeninivorans and the CYC1 terminator

  • Create control strains expressing a non-functional version of LCL2

  • Validate expression levels using Western blot and fluorescence microscopy techniques

  • Include S. cerevisiae transformants expressing the same constructs for comparative analysis

• Chronological lifespan assessment:

  • Culture strains in both standard and stress-inducing conditions (varying salt concentrations, pH levels, carbon sources)

  • Monitor cell viability over time using colony-forming unit (CFU) assays

  • Employ vital dyes to assess the proportion of metabolically active cells

  • Analyze survival curves and calculate mean and maximum lifespans

• Molecular and cellular analyses:

  • Measure ROS levels using fluorescent probes at different timepoints

  • Assess mitochondrial function using respiratory assays and membrane potential measurements

  • Evaluate NAD+/NADH ratios and peroxisomal NAD(H) homeostasis

  • Monitor endosomal trafficking patterns using FM4-64 dye labeling

• Genetic interaction studies:

  • Perform gene knockout or knockdown studies of known lifespan regulators

  • Assess epistatic relationships between LCL2 and other longevity genes

  • Conduct transcriptome analysis to identify genes differentially expressed in response to LCL2 overexpression

• Cross-species validation:

  • Express D. hansenii LCL2 in S. cerevisiae and assess phenotypic effects

  • Compare chronological lifespan effects between multiple yeast species

This experimental design provides a comprehensive approach to characterizing the impact of LCL2 on chronological lifespan while leveraging D. hansenii's unique physiological characteristics.

What are the most common challenges in working with D. hansenii and how can they be addressed?

Researchers working with D. hansenii frequently encounter specific challenges that can be addressed through targeted approaches:

Addressing these challenges will improve research outcomes and facilitate more effective work with D. hansenii in laboratory settings.

How can inconsistent results in lifespan studies with D. hansenii be resolved?

Inconsistent results in lifespan studies with D. hansenii can be resolved through several methodological approaches:

• Standardize strain background and verification:

  • Verify strain identity using MALDI-TOF biotyping to ensure values higher than two, confirming D. hansenii identification

  • Document the exact origin and lineage of strains used, as D. hansenii strains exhibit diverse properties

  • Maintain proper strain storage practices to prevent genetic drift

• Control environmental variables:

  • Standardize media composition precisely, including salt concentration which significantly affects D. hansenii metabolism

  • Maintain consistent temperature (25°C) and agitation (200 rpm) for liquid cultures

  • Control relative humidity in experimental chambers, as this affects D. hansenii growth kinetics

• Implement robust experimental protocols:

  • Establish consistent cell density measurements for initiating experiments

  • Use multiple, complementary methods to assess viability (e.g., CFU counts, vital dyes, metabolic activity assays)

  • Include appropriate positive and negative controls in each experimental setup

• Account for strain-specific variations:

  • Acknowledge that different D. hansenii strains may have very diverse properties that could lead to different experimental outcomes

  • Use multiple strains when possible to identify strain-independent effects

  • Document strain-specific growth characteristics under experimental conditions

• Apply statistical rigor:

  • Ensure sufficient biological and technical replicates (minimum three independent biological replicates)

  • Use appropriate statistical methods that account for the non-normal distribution often observed in lifespan data

  • Conduct power analyses to determine adequate sample sizes for detecting expected effect sizes

By implementing these approaches, researchers can minimize variability and produce more consistent, reproducible results in lifespan studies with D. hansenii.

What are the most promising areas for future research involving D. hansenii lifespan proteins?

The most promising areas for future research involving D. hansenii lifespan proteins include:

• Stress resistance mechanisms: Further investigation into how D. hansenii's exceptional stress tolerance contributes to its longevity, particularly focusing on how halotolerance pathways intersect with known longevity mechanisms .

• Cross-species lifespan regulation: Comparative studies of lifespan regulatory pathways between D. hansenii and other yeast models like S. cerevisiae could reveal evolutionarily conserved and divergent aspects of aging regulation .

• Peroxisomal NAD(H) homeostasis: Deeper exploration of NAD+ metabolism in D. hansenii peroxisomes and its impact on cellular lifespan, as NAD+ levels are known regulators of aging across species .

• Endolysosomal dynamics: Investigation of the unique endosomal recycling routes in D. hansenii that bypass the trans-Golgi network and how these may contribute to cellular maintenance during aging .

• Proteostasis under salt stress: Examination of how protein quality control mechanisms function under high salt conditions and their role in maintaining cellular integrity during aging.

• Metabolic adaptations and longevity: Research into how D. hansenii's metabolic flexibility and ability to utilize diverse carbon sources impacts its chronological lifespan.

• Industrial applications of lifespan extension: Development of D. hansenii strains with enhanced longevity for improved industrial applications, particularly in continuous fermentation processes.

• Environmental adaptation and longevity: Studies exploring the relationship between D. hansenii's ability to thrive in extreme environments and its lifespan regulation mechanisms.

These research directions could significantly advance our understanding of lifespan regulation in non-conventional yeasts and potentially reveal novel longevity factors relevant to other organisms.

How might findings from D. hansenii lifespan research translate to other biological systems?

Findings from D. hansenii lifespan research have significant potential to translate to other biological systems in several key ways:

• Stress resistance mechanisms: D. hansenii's exceptional ability to thrive under salt stress has parallels to stress resistance in extremophile organisms and could provide insights into how stress resistance pathways contribute to longevity across diverse species .

• Metabolic adaptation principles: The metabolic flexibility that allows D. hansenii to utilize various carbon sources and grow in industrial by-products may reveal fundamental principles of metabolic adaptation that contribute to longevity in other organisms .

• Conserved longevity pathways: Identification of novel longevity genes in D. hansenii could lead to the discovery of previously uncharacterized longevity factors that are conserved in higher eukaryotes, including humans.

• Industrial and biotechnological applications: Understanding lifespan regulation in D. hansenii could inform the development of other yeast strains with enhanced longevity for industrial applications, particularly for continuous fermentation processes .

• Environmental adaptation models: D. hansenii's adaptations to extreme environments provide models for understanding how organisms adapt to challenging ecological niches, with potential applications in environmental remediation and climate change adaptation strategies .

• Food preservation technologies: Insights from D. hansenii's antimycotic properties could inform the development of novel food preservation approaches that are natural and less dependent on chemical preservatives .

• Probiotic applications: Understanding the molecular basis of D. hansenii's potential probiotic properties could inform the development of novel probiotics for both human and animal health applications .