Recombinant Zygosaccharomyces rouxii Altered inheritance of mitochondria protein 39, mitochondrial (AIM39)

How does Z. rouxii AIM39 compare to similar proteins in other yeast species?

Z. rouxii AIM39 belongs to a conserved family of mitochondrial proteins found across various yeast species, including:

• Ashbya gossypii (AGOS_AGL215W)

• Lachancea thermotolerans (KLTH0C04708g)

• Candida glabrata (CAGL0I09966g)

While these proteins likely share core functions in mitochondrial inheritance, Z. rouxii AIM39 may possess unique adaptations related to extreme osmotolerance. Comparative genomic analysis suggests these homologs maintain similar structural features but with species-specific variations that likely reflect their ecological niches. To study these differences, researchers should:

• Perform multiple sequence alignments to identify conserved domains

• Use phylogenetic analysis to understand evolutionary relationships

• Compare expression patterns under stress conditions across species

• Conduct complementation studies to test functional conservation

Z. rouxii's ability to thrive in environments with up to 60% glucose suggests its AIM39 may have evolved distinctive properties that support mitochondrial function under extreme osmotic pressure.

What are the optimal growth conditions for Z. rouxii when studying AIM39 expression?

For optimal Z. rouxii culture when studying AIM39, researchers should consider:

Growth Media Compositions:

Culture Conditions:

• Temperature: 28-30°C for optimal growth ; 40°C for heat stress studies

• Agitation: 180 rpm for aerobic conditions

• Culture Duration: 3-4 days for reaching stationary phase (~2×10⁸ CFU/mL)

• Secondary Culture: 5% inoculum, 30-35 hours to reach 10⁸ CFU/mL

When specifically studying AIM39 expression patterns, researchers should monitor protein levels across growth phases and stress conditions. Z. rouxii exhibits unique adaptations to osmotic stress, so comparing AIM39 expression under normal versus high sugar/salt conditions can provide valuable insights into its potential role in stress adaptation mechanisms .

What are the most effective systems for recombinant expression of Z. rouxii AIM39?

Several expression systems have been successfully employed for Z. rouxii AIM39 production, each with distinct advantages:

E. coli Expression System:
The most commonly utilized approach involves expressing AIM39 with an N-terminal His-tag in E. coli . For optimal results:

• Use BL21(DE3) or Rosetta strains to address potential codon bias

• Express at lower temperatures (16-20°C) after induction to enhance solubility

• Employ autoinduction media for gentler protein expression

• Include 0.5-1% glucose during initial growth to suppress basal expression

Yeast Expression Systems:
For native-like post-translational modifications:

• Saccharomyces cerevisiae can be used with GAL1/GAL10 promoters as employed for similar Z. rouxii proteins

• Z. rouxii itself can serve as an expression host, particularly when studying functional aspects in the native environment

• The pECS-URA vector system has been successfully used for Z. rouxii protein expression

Advanced Expression Systems:

• Baculovirus expression may be beneficial for larger-scale production

• Cell-free expression systems can circumvent toxicity issues

The choice of system should be guided by the research question. Structural studies may benefit from E. coli expression for higher yields, while functional studies might require yeast-based systems that preserve native modifications and interactions.

What purification protocol yields the highest purity and activity for recombinant AIM39?

A robust purification strategy for recombinant AIM39 involves multiple chromatographic steps:

Step 1: Affinity Chromatography

• Use Ni-NTA resin for His-tagged AIM39

• Equilibrate column with 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 20 mM imidazole

• Elute with imidazole gradient (50-500 mM)

• Add 10% glycerol and 1 mM DTT to all buffers to enhance stability

Step 2: Size Exclusion Chromatography

• Apply concentrated affinity-purified protein to a Superdex 200 column

• Use buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5% glycerol

• Collect monomeric protein fractions based on molecular weight (~35 kDa)

Step 3: Ion Exchange Chromatography (Optional)

• For >95% purity, apply pooled fractions to an anion exchange column

• Use a gradient of 0-500 mM NaCl in 20 mM Tris-HCl (pH 8.0)

Storage Conditions:
For optimal stability, store purified AIM39 in Tris/PBS-based buffer with 6% trehalose at pH 8.0 . Aliquot and flash-freeze in liquid nitrogen before storing at -80°C to prevent degradation from freeze-thaw cycles.

Quality Control Metrics:

• Verify purity using SDS-PAGE (should exceed 90%)

• Confirm identity by mass spectrometry

• Assess proper folding via circular dichroism

• Verify activity through functional assays such as mitochondrial binding studies

This protocol typically yields 2-5 mg of highly pure protein per liter of bacterial culture, suitable for structural and biochemical analyses.

How can researchers verify the structural integrity and activity of purified AIM39?

Comprehensive validation of purified AIM39 requires multiple complementary approaches:

Structural Integrity Assessment:

• Circular Dichroism (CD) Spectroscopy:

  • Measure spectra between 190-260 nm

  • Compare secondary structure content with prediction algorithms

  • Monitor thermal stability by tracking CD signal at 222 nm during temperature ramping

• Thermal Shift Assays:

  • Use SYPRO Orange dye to monitor protein unfolding

  • Calculate melting temperature (Tm) to assess stability

  • Compare stability under different buffer conditions

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS):

  • Determine oligomeric state in solution

  • Verify monodispersity and absence of aggregation

Functional Validation:

• Mitochondrial Binding Assays:

  • Isolate mitochondria from Z. rouxii cells

  • Incubate with fluorescently labeled AIM39

  • Analyze binding using fluorescence microscopy or flow cytometry

• Complementation Studies:

  • Generate AIM39-knockout Z. rouxii strains

  • Transform with wild-type or mutant AIM39 constructs

  • Assess rescue of mitochondrial inheritance phenotypes

• Stress Response Analysis:

  • Compare mitochondrial morphology between wild-type and AIM39-deficient strains under osmotic stress

  • Evaluate mitochondrial membrane potential using potential-sensitive dyes

  • Measure ROS production with fluorescent probes like MitoSOX

A protein with proper structural integrity should demonstrate the predicted secondary structure content, thermal stability consistent with a well-folded protein, and functional activity in at least one of the assays described above.

How does AIM39 contribute to Z. rouxii's exceptional osmotolerance and stress adaptation?

Z. rouxii's remarkable ability to grow in environments containing up to 60% glucose suggests specialized mitochondrial adaptations, potentially involving AIM39. Evidence indicates several mechanisms through which AIM39 may contribute to stress tolerance:

Mitochondrial Dynamics Regulation:
Osmotic stress significantly alters mitochondrial morphology and distribution. Research suggests AIM39 likely participates in maintaining proper mitochondrial network organization under stress conditions. Fluorescence microscopy studies of GFP-tagged AIM39 show redistribution during exposure to high sugar environments .

Metabolic Adaptation:
Z. rouxii demonstrates unique metabolic responses to osmotic stress. For example, trehalose supplementation enhances high-temperature resistance through mechanisms involving gene activation rather than direct metabolic utilization . AIM39 may participate in this adaptation by:

• Modulating mitochondrial respiratory capacity

• Facilitating metabolic shifts between fermentation and respiration

• Maintaining ATP production under stress conditions

Integration with Stress Response Pathways:
Transcriptomic analyses reveal that under salt stress, Z. rouxii undergoes significant changes in gene expression patterns related to mitochondrial function . AIM39 likely interacts with these stress response pathways, potentially serving as a sensor or effector in mitochondrial stress signaling networks.

To definitively establish AIM39's role in osmotolerance, researchers should employ:

• Comparative phenotypic analysis of wild-type versus AIM39-knockout strains under various stress conditions

• Metabolomic profiling to identify AIM39-dependent metabolic adaptations

• Transcriptomic analysis to identify genes co-regulated with AIM39 during stress

What experimental approaches can elucidate AIM39's role in mitochondrial inheritance?

To investigate AIM39's specific functions in mitochondrial inheritance, researchers should implement a multi-faceted experimental strategy:

Genetic Manipulation Approaches:

• CRISPR-Cas9 Gene Editing:

  • Generate precise AIM39 knockout strains

  • Create domain-specific mutants to identify functional regions

  • Develop conditional expression systems to study temporal requirements

• Fluorescent Protein Tagging:

  • Create C-terminal or N-terminal GFP fusions (avoiding disruption of targeting sequences)

  • Implement dual-color imaging with mitochondrial markers

  • Apply time-lapse microscopy to track mitochondrial movement during cell division

Advanced Imaging Techniques:

• Super-Resolution Microscopy:

  • Employ techniques like STORM or PALM for nanoscale localization

  • Analyze AIM39 distribution within mitochondrial subcompartments

  • Track movement during cell division with 3D reconstruction

• Live-Cell Dynamics Analysis:

  • Use photoactivatable fluorescent proteins to track subpopulations of mitochondria

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to measure AIM39 mobility

  • Apply correlation analysis to identify coordinated movements with other cellular structures

Biochemical Interaction Studies:

• Proximity Labeling:

  • Use BioID or APEX2 fusions to identify proximally located proteins

  • Compare interaction networks under normal and stress conditions

  • Identify stress-specific interaction partners

• Crosslinking Mass Spectrometry:

  • Apply chemical crosslinkers to capture transient interactions

  • Identify AIM39 binding partners through MS/MS analysis

  • Map interaction sites through crosslink identification

A systematic integration of these approaches can establish AIM39's precise role in ensuring proper mitochondrial inheritance during cell division and under various stress conditions relevant to Z. rouxii's natural environment.

How can researchers investigate the potential relationship between AIM39 and Z. rouxii's unique metabolic capabilities?

Z. rouxii exhibits distinctive metabolic properties, including the production of flavor compounds like HDMF (4-hydroxy-2,5-dimethyl-3(2H)-furanone) and adaptation to high sugar environments . To explore AIM39's potential involvement in these processes:

Metabolic Flux Analysis:

• 13C-Isotope Labeling:

  • Culture cells with 13C-labeled glucose or fructose

  • Compare metabolite labeling patterns between wild-type and AIM39-deficient strains

  • Identify shifts in carbon flux through central metabolic pathways

• Real-time Metabolic Monitoring:

  • Measure oxygen consumption rates using Seahorse extracellular flux analysis

  • Compare ATP production under various carbon sources

  • Assess metabolic flexibility in response to nutrient shifts

Integration with Known Metabolic Pathways:

The enhanced production of HDMF in Z. rouxii involves key glycolytic enzymes, including fructose-1,6-bisphosphate aldolase (FBA) and triose phosphate isomerase (TPI) . To investigate potential connections with AIM39:

• Co-expression Analysis:

  • Compare expression patterns of AIM39 with FBA and TPI under various conditions

  • Identify potential co-regulation mechanisms

  • Analyze promoter regions for common regulatory elements

• Protein-Protein Interaction Studies:

  • Perform co-immunoprecipitation experiments with AIM39 and metabolic enzymes

  • Use yeast two-hybrid screening to identify direct interactions

  • Apply proximity labeling to identify functional associations

Experimental Setup for Metabolic Studies:

These approaches can reveal whether AIM39 plays a direct or indirect role in Z. rouxii's specialized metabolic capabilities, potentially identifying new targets for metabolic engineering of this industrially relevant yeast.

What are common challenges in recombinant AIM39 expression and how can they be addressed?

Researchers often encounter specific challenges when working with AIM39. Here are evidence-based solutions to common problems:

Challenge 1: Protein Insolubility and Aggregation

Mitochondrial proteins like AIM39 frequently form inclusion bodies due to hydrophobic regions. To overcome this:

• Optimize Expression Conditions:

  • Lower induction temperature to 16-18°C

  • Reduce IPTG concentration to 0.1-0.3 mM

  • Implement autoinduction media for gradual protein expression

• Modify Buffer Composition:

  • Include 0.5-1% Triton X-100 or 0.1% Tween-20 in lysis buffer

  • Add 6% trehalose as indicated in storage conditions

  • Include 1-5 mM β-mercaptoethanol to prevent disulfide-mediated aggregation

• Use Solubility-Enhancing Tags:

  • Test MBP, SUMO, or TRX fusion constructs

  • Consider dual-tagging strategies (His-MBP-AIM39)

  • Optimize tag position (N vs. C-terminal)

Challenge 2: Low Expression Yields

When AIM39 expression levels are insufficient:

• Address Codon Bias:

  • Use codon-optimized sequences for the expression host

  • Select Rosetta or CodonPlus E. coli strains

  • Supplement rare tRNAs in the expression system

• Optimize Promoter Selection:

  • Compare T7 vs. tac promoters in bacterial systems

  • Test GAL1 vs. ADH1 promoters in yeast systems

  • Evaluate constitutive vs. inducible expression strategies

• Adjust Media Composition:

  • Implement enriched media formulations (2XYT, TB)

  • Add glucose during initial growth phase to suppress leaky expression

  • Use fed-batch cultivation to achieve higher cell densities

Challenge 3: Protein Instability

To enhance AIM39 stability during purification and storage:

• Buffer Optimization:

  • Include the reported 6% trehalose in all buffers

  • Maintain pH between 7.5-8.0

  • Add 5-10% glycerol to prevent aggregation

• Storage Protocol:

  • Aliquot in small volumes to avoid freeze-thaw cycles

  • Flash-freeze in liquid nitrogen

  • Store at -80°C rather than -20°C for long-term stability

Implementing these specific, evidence-based solutions can significantly improve recombinant AIM39 production and quality.

How should researchers approach experimental design when studying AIM39 function in Z. rouxii?

Robust experimental design for AIM39 studies should address the unique characteristics of Z. rouxii while maintaining scientific rigor:

1. Strain Selection and Validation:

2. Appropriate Growth Conditions:

Z. rouxii's natural environments include high osmolarity settings. Design experiments that:

• Include both standard and stress conditions (high sugar, salt, temperature)

• Account for adaptation periods (24-48h) as noted in trehalose adaptation studies

• Monitor growth over extended periods (up to 98h) to capture full adaptation responses

• Control for osmotic effects using different osmolytes as controls

3. Comprehensive Controls:

For meaningful interpretation of AIM39 function:

• Include isogenic control strains lacking only the AIM39 modification

• Implement empty vector controls for all transformations

• Use appropriate subcellular markers (especially mitochondrial markers)

• Include positive controls for stress responses (known stress-responsive genes)

4. Temporal Considerations:

Z. rouxii demonstrates distinct adaptation phases to stress conditions:

• Short-term responses (0-24h): Initial stress adaptation period

• Medium-term responses (24-60h): Log-phase growth under adapted conditions

• Long-term responses (>60h): Stationary phase and advanced adaptation

Design time-course experiments that capture these phases to differentiate immediate versus adaptive roles of AIM39.

5. Multi-omics Integration:

For holistic understanding:

• Complement transcriptomic data with proteomics

• Integrate metabolomic profiling to capture metabolic shifts

• Correlate phenotypic observations with molecular changes

• Employ statistical methods that account for temporal dynamics

This systematic approach addresses the specific challenges of studying AIM39 in Z. rouxii while ensuring experimental rigor and reproducibility.

What computational approaches can enhance the analysis of AIM39 function and structure?

Computational methods offer powerful tools for investigating AIM39's structure and function:

Structural Analysis:

• Protein Structure Prediction:

  • Apply AlphaFold2 or RoseTTAFold to predict AIM39's tertiary structure

  • Identify potential functional domains and binding sites

  • Compare predicted structures across AIM39 homologs from different yeast species

• Molecular Dynamics Simulations:

  • Simulate AIM39 behavior in membrane environments

  • Assess structural stability under varying conditions (pH, temperature, ionic strength)

  • Identify conformational changes that might occur during function

• Binding Site Prediction:

  • Use CASTp or Fpocket to identify potential binding pockets

  • Perform in silico docking with potential ligands or protein partners

  • Validate predictions through mutagenesis of key residues

Sequence-Based Analysis:

• Multiple Sequence Alignment (MSA) and Conservation Analysis:

  • Align AIM39 sequences across fungal species

  • Identify highly conserved residues as potentially functionally critical

  • Generate conservation scores to guide mutagenesis experiments

• Motif Identification:

  • Search for known functional motifs using databases like PROSITE

  • Identify potential post-translational modification sites

  • Predict subcellular localization signals

Network Analysis:

• Protein-Protein Interaction Networks:

  • Construct interaction networks based on experimental data

  • Apply graph theory algorithms to identify central nodes

  • Compare networks under normal versus stress conditions

• Gene Co-expression Analysis:

  • Analyze transcriptomic data to identify genes co-regulated with AIM39

  • Compare expression patterns across multiple conditions

  • Use clustering approaches to identify functional modules

Integration of Multiple Data Types:

• Machine Learning Approaches:

  • Train models to predict AIM39 interactions based on diverse data types

  • Use feature importance analysis to identify key determinants of function

  • Apply classification algorithms to predict functional impacts of mutations

• Pathway Analysis:

  • Map AIM39 and interacting partners to known biological pathways

  • Identify enriched pathways under different conditions

  • Model the impact of AIM39 perturbation on pathway activity

These computational approaches can guide experimental design, help interpret complex data, and generate testable hypotheses about AIM39 function in Z. rouxii's unique physiological context.

What are promising avenues for exploring the evolutionary significance of AIM39 in osmotolerant yeasts?

The unusual environmental adaptations of Z. rouxii provide a compelling context for investigating AIM39's evolution:

Comparative Genomics Approaches:

• Phylogenetic Analysis:

  • Construct phylogenetic trees of AIM39 across fungal species

  • Compare evolutionary rates between osmotolerant and non-osmotolerant yeasts

  • Identify signatures of positive selection in Z. rouxii AIM39

• Synteny Analysis:

  • Examine conservation of gene order surrounding AIM39

  • Identify potential co-evolved gene clusters

  • Investigate horizontal gene transfer events that might have contributed to Z. rouxii's adaptations

Functional Evolution Studies:

• Ancestral Sequence Reconstruction:

  • Infer ancestral AIM39 sequences

  • Express and characterize reconstructed proteins

  • Compare functional properties with modern AIM39 variants

• Cross-Species Complementation:

  • Express Z. rouxii AIM39 in AIM39-deficient S. cerevisiae

  • Test whether Z. rouxii AIM39 confers enhanced stress tolerance

  • Identify specific domains responsible for functional differences

Ecological Adaptations:

Z. rouxii's natural habitats include high-sugar environments like honey and fruit juices. Investigating AIM39's role in these ecological contexts could reveal:

• How mitochondrial functions adapt to natural osmotic fluctuations

• Whether AIM39 variants correlate with specific ecological niches

• If similar adaptations evolved independently in other osmotolerant organisms

These evolutionary studies could provide fundamental insights into how essential cellular processes like mitochondrial inheritance adapt to extreme environments, with potential applications in both fundamental biology and biotechnology.

How might engineered AIM39 variants enhance Z. rouxii's biotechnological applications?

Z. rouxii has significant biotechnological value, particularly in flavor compound production . Strategic engineering of AIM39 could enhance these applications:

Enhancing Metabolic Output:

• HDMF Production Improvement:
  Studies show that overexpression of glycolytic enzymes FBA and TPI increases HDMF production in Z. rouxii . Engineering AIM39 could complement these approaches by:

  • Optimizing mitochondrial function to support precursor metabolism

  • Enhancing cellular energy production to drive biosynthetic pathways

  • Improving stress tolerance during high-density fermentation

• Stress Tolerance Engineering:
  AIM39 modifications could potentially enhance Z. rouxii's already impressive stress tolerance:

  • Create variants with improved function under industrial fermentation conditions

  • Develop strains with enhanced temperature tolerance for more efficient processing

  • Engineer osmotolerance for even higher sugar concentration fermentations

Experimental Approaches:

• Directed Evolution:

  • Subject AIM39 to random mutagenesis

  • Screen for variants conferring enhanced stress tolerance or metabolic output

  • Combine beneficial mutations through DNA shuffling

• Rational Design:

  • Modify specific domains based on structural predictions

  • Engineer protein-protein interactions to enhance mitochondrial performance

  • Create synthetic regulatory circuits controlling AIM39 expression

Potential Applications:

The unique properties of Z. rouxii AIM39, particularly its adaptation to extreme conditions, make it a promising target for biotechnological applications requiring robust cellular performance under industrial stress conditions.

What methodological advances would facilitate deeper understanding of AIM39's molecular mechanisms?

Advancing our understanding of AIM39 function will require innovative methodological approaches:

Advanced Imaging Technologies:

• Cryo-Electron Tomography:

  • Visualize AIM39 in its native mitochondrial context

  • Map spatial organization within the mitochondrial network

  • Resolve structural changes under different environmental conditions

• Single-Molecule Tracking:

  • Monitor individual AIM39 molecules in living cells

  • Characterize dynamic behaviors during mitochondrial inheritance

  • Quantify interaction kinetics with partner proteins

Multi-omics Integration:

• Spatially-Resolved Transcriptomics:

  • Map transcriptional responses in relation to AIM39 localization

  • Identify localized effects of AIM39 activity

  • Correlate spatial patterns with functional outcomes

• Proteome-wide Interaction Mapping:

  • Implement BioID or APEX proximity labeling in Z. rouxii

  • Create comprehensive interaction maps under various conditions

  • Identify condition-specific interaction networks

Functional Genomics Approaches:

• CRISPR Interference/Activation Screens:

  • Develop CRISPRi/CRISPRa systems for Z. rouxii

  • Perform genome-wide screens for AIM39 genetic interactions

  • Identify synthetic lethal or synthetic rescue interactions

• Domain-Specific Mutagenesis:

  • Implement high-throughput mutagenesis of AIM39 domains

  • Develop functional assays compatible with variant libraries

  • Map structure-function relationships at high resolution

Real-time Metabolic Monitoring:

• Genetically-Encoded Metabolic Sensors:

  • Develop FRET-based sensors for key metabolites

  • Monitor metabolic changes in response to AIM39 perturbation

  • Track spatiotemporal dynamics of metabolic responses

• In situ Metabolomics:

  • Implement mass spectrometry imaging of metabolites

  • Correlate metabolite distributions with AIM39 activity

  • Identify localized metabolic effects

These methodological advances would enable researchers to move beyond correlative observations to mechanistic understanding of AIM39's functions in Z. rouxii's exceptional stress adaptation capabilities, potentially revealing new paradigms in mitochondrial biology.