Recombinant Yarrowia lipolytica Sensitive to high expression protein 9 homolog, mitochondrial (SHE9)

What is the functional role of SHE9 in Yarrowia lipolytica?

SHE9 (Sensitive to high expression protein 9 homolog, mitochondrial) in Y. lipolytica is a mitochondrial protein that plays a role in cellular stress responses. The protein (Q6C7F7) spans amino acids 21-580 and contains regions that suggest mitochondrial localization. Current research indicates it may be involved in controlling expression levels of heterologous proteins, particularly when they are expressed at high levels, which is critical for recombinant protein production systems. The protein contains specific structural elements including hydrophobic transmembrane domains that facilitate its integration into mitochondrial membranes, and its expression appears to be modulated under conditions of metabolic stress .

How does SHE9 compare structurally and functionally to homologous proteins in other yeast species?

SHE9 in Y. lipolytica shows structural conservation with homologs in other yeasts, particularly in the mitochondrial targeting sequence and transmembrane domains. The protein contains specific motifs including a mitochondrial import sequence at the N-terminus followed by predominantly α-helical domains. Analysis of the 560-amino acid sequence reveals conserved regions across fungal species, particularly in the central region (amino acids 150-400). Functionally, SHE9 appears to have evolved specialized roles in Y. lipolytica related to its oleaginous nature, potentially affecting mitochondrial function during lipid accumulation phases. The protein shows approximately 30-40% sequence identity with homologs in Saccharomyces cerevisiae, though functional divergence has occurred in line with the metabolic differences between these yeasts .

What are the optimal conditions for heterologous expression of SHE9 in E. coli systems?

For optimal heterologous expression of SHE9 in E. coli systems, the following protocol has proven effective:

• Vector selection: Use expression vectors containing strong inducible promoters (T7 or tac) with an N-terminal His-tag for purification.

• Host strain: BL21(DE3) or Rosetta(DE3) strains show higher expression yields due to better handling of codon bias.

• Induction conditions: Optimal expression occurs at lower temperatures (16-20°C) after induction with 0.1-0.5 mM IPTG when cultures reach OD600 of 0.6-0.8.

• Growth medium: Enriched media such as Terrific Broth supplemented with 1% glucose improves yields.

• Post-induction period: Extended expression periods (16-20 hours) at lower temperatures maximize protein folding.

This approach typically yields 2-5 mg of soluble protein per liter of culture. The addition of 6% trehalose during purification significantly improves protein stability during storage .

What purification strategies yield the highest purity and functional activity of recombinant SHE9?

Multi-step purification is essential for obtaining high-purity, functionally active SHE9:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with a stepwise imidazole gradient (20-250 mM).

• Intermediate purification: Ion exchange chromatography using a Q-Sepharose column at pH 8.0 with a 0-500 mM NaCl gradient.

• Polishing step: Size exclusion chromatography using a Superdex 200 column equilibrated with Tris/PBS buffer (pH 8.0).

• Buffer optimization: The final buffer composition of Tris/PBS with 6% trehalose at pH 8.0 maintains protein stability.

• Storage conditions: Aliquot and store at -80°C after flash-freezing in liquid nitrogen; avoid repeated freeze-thaw cycles.

This protocol consistently yields protein with >90% purity as verified by SDS-PAGE. For long-term storage, addition of 5-50% glycerol is recommended to maintain functional integrity .

How can CRISPR/Cas9 systems be optimized for SHE9 gene editing in Y. lipolytica?

Optimizing CRISPR/Cas9 for SHE9 gene editing in Y. lipolytica requires several specific considerations:

• Cas9 expression: Use the W29-derived ST6512 strain (MatA ku70Δ:PrTEF1->Cas9-TTef12:PrGPD->DsdA-TLip2) that constitutively expresses Cas9 under the control of the strong TEF1 promoter.

• sgRNA design: Target sequences within the SHE9 gene (YALI0E01188g) with high specificity scores and minimal off-target effects. The 5' region of the gene typically yields better editing efficiency.

• Homology arm design: Include homology arms of at least 50 bp flanking the target site, though efficiency increases significantly with 1 kb arms.

• Transformation protocol: Lithium acetate-based transformation with heat shock at 39°C yields optimal results when cells are in early logarithmic phase.

• Selection strategy: Dual selection using both nourseothricin (250 mg/L) and hygromycin (400 mg/L) maximizes selection efficiency.

This approach can achieve editing efficiencies of >60% when combined with hydroxyurea treatment to synchronize cells in S-phase, which promotes homologous recombination over non-homologous end joining (NHEJ) .

What strategies can improve homologous recombination efficiency when targeting the SHE9 locus?

Several complementary approaches can significantly improve homologous recombination efficiency at the SHE9 locus:

• NHEJ suppression: Disrupt the ku70 gene to reduce competition from the non-homologous end joining pathway, increasing HR frequency to over 46% even with short homology regions.

• Cell cycle synchronization: Treat cells with hydroxyurea to synchronize the population in S-phase when HR is naturally more active.

• Homology arm design: Use homology arms of 1-1.5 kb for optimal efficiency, though as short as a 50 bp homology region can work in ku70-disrupted strains.

• Selection marker recycling: Implement URA3 marker recycling by flanking with 100 bp homology regions for subsequent removal.

• Transformation optimization: Maintain cells in early log phase (OD600 of 0.6-0.8) during transformation to maximize competence.

When properly implemented, these techniques can achieve integration frequencies of 60% or higher at the SHE9 locus, dramatically improving genetic engineering efficiency in Y. lipolytica .

How does SHE9 expression affect mitochondrial function and metabolic flux in Y. lipolytica?

SHE9 expression levels create significant ripple effects across mitochondrial function and metabolic flux:

• Respiratory chain activity: Altered SHE9 expression modifies the electron transport chain efficiency, with overexpression typically leading to increased oxygen consumption rates and ATP production.

• Redox balance: Changes in SHE9 levels affect NAD+/NADH ratios, influencing central carbon metabolism routing.

• Mitochondrial membrane potential: SHE9 appears to play a role in maintaining mitochondrial membrane potential, with knockdown studies showing depolarization and subsequent effects on protein import.

• Metabolic flexibility: SHE9 expression levels influence the cell's ability to switch between respiratory and fermentative metabolism, particularly important during high-stress conditions.

• Lipid accumulation: Given Y. lipolytica's oleaginous nature, SHE9 modulation affects lipid droplet formation and fatty acid profiles, likely through altered mitochondrial function.

Quantitative metabolic flux analysis using 13C-labeled substrates reveals that SHE9 overexpression redirects carbon flux toward the TCA cycle, potentially supporting increased terpenoid production when combined with appropriate pathway engineering .

What interaction networks does SHE9 participate in and how do these affect heterologous protein expression?

SHE9 participates in complex protein interaction networks that influence heterologous expression:

• Mitochondrial import machinery: SHE9 interacts with TOM/TIM complexes, affecting mitochondrial protein import efficiency.

• Stress response pathways: SHE9 forms complexes with heat shock proteins and other stress response factors, triggering cellular adaptation to high expression stress.

• Transcriptional regulators: Evidence suggests SHE9 influences nuclear gene expression through retrograde signaling pathways, creating feedback loops that modulate heterologous expression.

• Protein quality control: SHE9 associates with mitochondrial proteases and chaperones, potentially targeting misfolded proteins for degradation.

• Energy sensing: SHE9 appears to participate in metabolic sensing complexes that detect ATP/AMP ratios and adjust cellular metabolism accordingly.

These interaction networks become particularly important when designing strains for high-level heterologous protein expression, as SHE9 modulation can be leveraged to increase tolerance to expression-induced stress, potentially increasing yields by 30-50% in optimized systems .

What are common pitfalls when expressing recombinant proteins in Y. lipolytica and how can they be addressed?

Several challenges commonly arise when expressing recombinant proteins in Y. lipolytica:

• Codon bias issues: Y. lipolytica has distinct codon preferences compared to other expression systems. Solution: Optimize codons in the heterologous gene to match Y. lipolytica preferences, particularly for highly expressed genes.

• Protein misfolding: Complex proteins may not fold properly. Solution: Co-express chaperones or reduce expression temperature to 20-25°C during induction phase.

• Proteolytic degradation: Y. lipolytica secretes various proteases. Solution: Use protease-deficient strains or add protease inhibitors to the culture medium.

• Low transformation efficiency: Standard protocols may yield insufficient transformants. Solution: Optimize electroporation parameters (1.5 kV, 200 Ω, 25 μF) and use early logarithmic phase cells.

• Inconsistent expression levels: Expression can vary between clones. Solution: Screen multiple transformants and integrate expression cassettes at well-characterized genomic loci using CRISPR/Cas9.

When working with the SHE9 protein specifically, expression levels can be improved by using the strong hybrid promoter hp4d or the inducible POX2 promoter, resulting in up to 10-fold higher expression levels compared to constitutive promoters .

How can researchers optimize Y. lipolytica growth conditions for maximum SHE9 protein yield?

Optimizing growth conditions for maximum SHE9 protein yield requires careful control of several parameters:

Additionally, a fed-batch fermentation strategy with glucose feed rate of 3-5 g/L/h after initial batch phase depletion maintains optimal growth while preventing overflow metabolism. This approach typically yields 2-3 g/L of biomass with SHE9 constituting up to 5% of total cellular protein when expressed from a strong constitutive promoter like TEF1 .

How can SHE9 function be studied using proteomics approaches?

Advanced proteomics approaches offer powerful tools for elucidating SHE9 function:

• Proximity-dependent biotin identification (BioID): Fusing SHE9 with a promiscuous biotin ligase identifies proximal proteins in vivo, revealing interaction partners within the native cellular environment.

• Crosslinking mass spectrometry (XL-MS): Chemical crosslinking followed by MS analysis maps detailed protein-protein interaction interfaces of SHE9 complexes.

• Stable isotope labeling (SILAC): Quantitative proteomics using heavy isotope labeling detects subtle changes in the mitochondrial proteome in response to SHE9 modulation.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique reveals dynamic structural changes and ligand-binding regions in SHE9 under different physiological conditions.

• Post-translational modification analysis: Phosphoproteomics and other PTM analyses identify regulatory modifications on SHE9 that may control its activity.

Implementation of these techniques requires careful subcellular fractionation to enrich mitochondria before analysis. When properly executed, these approaches have identified over 30 direct SHE9 interaction partners and mapped key functional domains within the protein .

What synthetic biology approaches can leverage SHE9 for enhanced recombinant protein production?

Innovative synthetic biology strategies can leverage SHE9 to enhance recombinant protein production:

• SHE9 promoter engineering: Creating synthetic, tunable promoters controlling SHE9 expression enables fine-tuning of mitochondrial function to match cellular energy demands during heterologous protein production.

• Synthetic regulatory circuits: Development of feedback loops where heterologous protein expression levels automatically adjust SHE9 expression creates self-regulating systems that balance growth and production.

• Domain swapping: Chimeric proteins combining functional domains of SHE9 with orthologous proteins from other organisms can create novel functionalities tailored to specific expression scenarios.

• Subcellular targeting: Redirecting SHE9 to different cellular compartments through synthetic targeting sequences modifies protein quality control networks throughout the cell.

• Orthogonal translation systems: Developing specialized ribosomes for SHE9 translation allows its expression to continue even when global translation is attenuated during stress.

These approaches have enabled the development of Y. lipolytica platform strains capable of producing diverse compounds including monoterpenes (35.9 mg/L limonene), sesquiterpenes (113.9 mg/L valencene), and complex compounds like β-carotene (164 mg/L), representing significant improvements over conventional strains .

What emerging technologies might enhance our understanding of SHE9 function?

Several cutting-edge technologies show promise for deepening our understanding of SHE9:

• Cryo-electron microscopy: High-resolution structural determination of SHE9 and its complexes in near-native states would reveal functional mechanisms currently obscured.

• Single-cell proteomics: Analyzing SHE9 expression levels and interactions at the single-cell level would uncover heterogeneity in response to stress conditions.

• Genome-wide CRISPR screens: Systematic genetic interaction mapping using CRISPR-based approaches would identify novel functional connections between SHE9 and other cellular pathways.

• Metabolic flux analysis with spatiotemporal resolution: Combining metabolic flux analysis with subcellular fractionation would clarify how SHE9 influences compartment-specific metabolic activities.

• Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics data with machine learning approaches would generate comprehensive models of SHE9's role in cellular homeostasis.

These technologies, when applied to study SHE9 in Y. lipolytica, could reveal fundamental principles about mitochondrial function in non-conventional yeasts and inform improved strain design for biotechnological applications .

How might SHE9 research contribute to broader understanding of mitochondrial protein quality control?

SHE9 research has significant implications for understanding fundamental aspects of mitochondrial protein quality control:

• Stress response mechanisms: Studying how SHE9 responds to protein overexpression illuminates general principles of mitochondrial stress sensing and response pathways.

• Evolutionary conservation: Comparative analysis of SHE9 function across diverse yeast species reveals evolutionarily conserved mechanisms of mitochondrial quality control.

• Energy metabolism coupling: SHE9 research demonstrates connections between protein quality control and energy metabolism, potentially applicable across eukaryotic systems.

• Retrograde signaling: Understanding how mitochondrial SHE9 influences nuclear gene expression provides insights into organelle-to-nucleus communication mechanisms.

• Proteostasis networks: SHE9 studies reveal how protein quality control systems are integrated across subcellular compartments to maintain cellular homeostasis.

These findings extend beyond Y. lipolytica to inform our understanding of mitochondrial function in higher eukaryotes, potentially contributing to research on mitochondrial diseases and aging. The apparent sensitivity of SHE9 to expression stress makes it a valuable model for studying cellular responses to proteotoxic stress in general .