Recombinant Ashbya gossypii Succinate dehydrogenase assembly factor 2, mitochondrial (EMI5), partial

What is Ashbya gossypii and why is it significant in biotechnology?

Ashbya gossypii is a filamentous fungus belonging to the Saccharomycetaceae family that has long been considered a paradigm of White Biotechnology, particularly for riboflavin (vitamin B2) production . Its industrial relevance has led to the development of significant molecular and in silico modeling tools for its manipulation . Beyond riboflavin production, A. gossypii has emerged as a versatile microbial chassis for producing various valuable metabolites including recombinant proteins, single cell oils (SCOs), and flavor compounds .

What is the function of EMI5 (Succinate dehydrogenase assembly factor 2) in A. gossypii?

EMI5, also known as Succinate dehydrogenase assembly factor 2 (SDHAF2), plays a crucial role in the assembly and function of the succinate dehydrogenase complex in mitochondria . This complex (Complex II) is essential for both the tricarboxylic acid (TCA) cycle and the electron transport chain, making EMI5 vital for proper mitochondrial function and cellular energy metabolism. In A. gossypii, EMI5 (gene name AGOS_AER200C or SDH5) ensures the correct assembly of the succinate dehydrogenase complex, which is critical for the organism's robust metabolic capabilities that support its biotechnological applications .

How does A. gossypii EMI5 compare to homologous proteins in other organisms?

EMI5 from A. gossypii shares significant structural and functional similarities with homologous proteins found in other fungi, particularly those in the Saccharomycetaceae family. The protein contains conserved domains typical of succinate dehydrogenase assembly factors, including regions that facilitate protein-protein interactions necessary for complex assembly . Comparative analysis with SDH5 from Saccharomyces cerevisiae shows high sequence conservation, reflecting their shared evolutionary history and functional importance . This conservation extends to key functional domains but may exhibit species-specific variations that could influence protein-protein interactions and assembly efficiency.

What expression systems are most effective for producing recombinant A. gossypii EMI5?

For recombinant A. gossypii EMI5 production, several expression systems have been employed with varying degrees of success:

The most efficient system appears to be yeast-based expression, which balances yield with proper protein folding . For studies requiring post-translational modifications, insect cell systems offer a viable compromise between authenticity and yield. The choice of host system should be guided by the specific research requirements, particularly whether native conformation and post-translational modifications are critical for the planned experiments .

What purification strategies optimize recovery of functional recombinant EMI5?

Purification of recombinant EMI5 requires careful consideration of its mitochondrial nature and potential hydrophobic domains. An effective purification protocol includes:

• Cell lysis using gentle detergents (e.g., 0.5-1% Triton X-100) to preserve protein structure

• Initial clarification by centrifugation (10,000-15,000 × g for 20-30 minutes)

• Affinity chromatography using either:

  • His-tag affinity if a histidine tag was incorporated

  • Immobilized metal affinity chromatography (IMAC) for native protein

• Size exclusion chromatography to remove aggregates and improve purity

• Concentration using centrifugal filters with appropriate molecular weight cutoffs

This approach typically yields protein with ≥85% purity as determined by SDS-PAGE . For highest activity, purification should be performed at 4°C with protease inhibitors to minimize degradation. Buffer optimization is critical, with most protocols using phosphate or Tris-based buffers (pH 7.0-8.0) containing 100-300 mM NaCl and 5-10% glycerol to stabilize the protein .

How can mitochondrial membrane potential be measured when studying EMI5 function?

Mitochondrial membrane potential (ΔΨm) measurement is crucial when evaluating EMI5 function since properly assembled succinate dehydrogenase contributes to maintaining this potential. Several methodologies can be employed:

• Fluorescent probe-based assays:

  • TMRM/TMRE (tetramethylrhodamine methyl/ethyl ester) assays provide dynamic monitoring of membrane potential in live cells

  • MitoTracker dyes (particularly MitoTracker Red CMXRos) allow visualization of potential-dependent mitochondrial staining

  • The Incucyte® Mitochondrial Membrane Potential Assay enables real-time detection of changes in MMP in live cells

• Control compounds for validation:

  • FCCP (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone) serves as a negative control by dissipating membrane potential

  • Oligomycin A can be used as a positive control to enhance membrane potential by inhibiting ATP synthase

• Detection platforms:

  • Flow cytometry for quantitative population analysis

  • Fluorescence microscopy for spatial resolution and subcellular localization

  • Microplate readers for high-throughput screening

For the most robust results, researchers should employ multiple complementary techniques and include proper controls to account for variations in mitochondrial mass versus membrane potential .

How can recombinant EMI5 be used to study mitochondrial function in A. gossypii?

Recombinant EMI5 can serve as a powerful tool for investigating mitochondrial function in A. gossypii through several advanced approaches:

• Complementation studies:

  • EMI5-deficient strains can be generated through genetic manipulation

  • Recombinant wild-type or mutated EMI5 can be introduced to assess functional recovery

  • This approach allows structure-function relationship mapping of critical domains

• Protein-protein interaction studies:

  • Co-immunoprecipitation using tagged recombinant EMI5 can identify interaction partners

  • Proximity labeling approaches (BioID or APEX) with EMI5 as the bait protein

  • Yeast two-hybrid screening using EMI5 as bait against an A. gossypii cDNA library

• Respiratory function assessment:

  • Oxygen consumption rate (OCR) measurements in the presence of various EMI5 variants

  • Respirometry assays using reconstituted systems (RIFS) to evaluate succinate-driven respiration

  • Blue native gel electrophoresis to assess complex II assembly and stability

These methodologies can provide insights into how EMI5 contributes to mitochondrial respiration and energy metabolism in A. gossypii, which may have implications for optimizing biotechnological applications of this fungus .

What genetic engineering approaches can enhance EMI5 functionality for metabolic engineering applications?

Several genetic engineering strategies can be employed to enhance EMI5 functionality for metabolic engineering applications in A. gossypii:

• Promoter optimization:

  • Substituting native promoters with strong constitutive promoters (e.g., AgTEF, AgGPD) can increase EMI5 expression by up to 8-fold compared to heterologous promoters

  • Inducible promoter systems enable controlled expression timing

• Codon optimization:

  • Adapting the EMI5 coding sequence to A. gossypii codon usage preferences can improve translation efficiency

  • This approach has shown significant improvement in heterologous protein expression in A. gossypii

• Domain engineering:

  • Targeted mutagenesis of key residues based on structural analysis

  • Creation of chimeric proteins combining domains from EMI5 homologs with superior properties

  • Directed evolution approaches to select for variants with enhanced stability or activity

• Integration strategies:

  • Development of stable expression cassettes through genomic integration rather than episomal vectors

  • Selection of optimal integration sites to maximize expression and minimize fitness costs

  • Multiple integration events to increase gene dosage when beneficial

• Co-expression strategies:

  • Co-overexpression of EMI5 with other mitochondrial proteins that may be limiting for respiratory function

  • Integration of EMI5 modifications into broader metabolic engineering strategies, such as those used for monoterpene production in A. gossypii

These approaches can be particularly valuable when EMI5 optimization is part of a larger metabolic engineering strategy aimed at enhancing A. gossypii's capabilities as a bioproduction platform .

How does EMI5 function relate to A. gossypii's ability to produce secondary metabolites?

EMI5's role in mitochondrial function has significant implications for A. gossypii's secondary metabolite production capabilities:

• Energy metabolism connection:

  • Proper succinate dehydrogenase assembly, facilitated by EMI5, ensures efficient TCA cycle operation

  • This provides essential energy and reducing equivalents (NADH, FADH₂) needed for biosynthetic pathways

  • Secondary metabolite production, including riboflavin and monoterpenes, is energetically demanding and requires robust mitochondrial function

• Precursor availability:

  • TCA cycle intermediates serve as precursors for various secondary metabolites

  • Succinate dehydrogenase activity influences the flux through the TCA cycle and the availability of these precursors

  • EMI5 function may therefore indirectly impact precursor pools for biosynthetic pathways

• Redox balance:

  • Mitochondrial respiratory activity maintained by proper complex assembly affects cellular redox balance

  • Secondary metabolite pathways are often redox-sensitive and respond to changes in NAD⁺/NADH ratios

  • EMI5 dysfunction could alter these ratios and consequently affect biosynthetic outcomes

• Specific pathway effects:

  • For riboflavin production, purine biosynthesis (a precursor pathway) requires intact mitochondrial function

  • Monoterpene production via the mevalonate pathway is linked to acetyl-CoA availability, which is influenced by TCA cycle function

  • Recombinant protein production capability may be enhanced by the robust energy metabolism supported by properly assembled respiratory complexes

Research indicates that engineering mitochondrial function through proteins like EMI5 could be a valuable approach for enhancing A. gossypii's capabilities as a bioproduction platform, particularly for metabolites with high energy requirements .

What are the major challenges in working with recombinant A. gossypii EMI5?

Researchers face several significant challenges when working with recombinant A. gossypii EMI5:

• Protein stability issues:

  • As a mitochondrial protein, EMI5 may have hydrophobic domains that complicate handling in aqueous solutions

  • The protein may require specific buffer conditions and stabilizing agents to maintain structure and function

  • Long-term storage often results in activity loss, necessitating fresh preparation for critical experiments

• Functional assessment complexity:

  • Since EMI5 functions in complex assembly rather than having direct enzymatic activity, functional assays are indirect

  • Requires assessment of succinate dehydrogenase complex assembly and activity rather than simple activity measurements

  • May necessitate reconstitution systems to properly evaluate functionality

• Expression and purification hurdles:

  • Heterologous expression systems may not provide proper folding or post-translational modifications

  • Maintaining native conformation during purification requires careful optimization of conditions

  • Yield and purity trade-offs often complicate obtaining sufficient quantities for advanced structural studies

• Integration with cellular systems:

  • When studying EMI5 in cellular contexts, distinguishing its specific effects from broader mitochondrial changes is challenging

  • Requires careful experimental design with appropriate controls to isolate EMI5-specific phenomena

  • May necessitate development of EMI5-specific antibodies or tagged versions that don't compromise function

Addressing these challenges requires interdisciplinary approaches combining protein biochemistry, mitochondrial biology, and A. gossypii-specific molecular biology techniques .

How can EMI5 research contribute to understanding A. gossypii's potential for novel biotechnological applications?

EMI5 research provides unique insights into A. gossypii's biotechnological potential through several avenues:

• Metabolic optimization:

  • Understanding how EMI5 influences respiratory efficiency can inform strategies to enhance energetic capacity

  • This knowledge can be applied to optimize strains for energy-intensive production processes

  • Research suggests that enhanced mitochondrial function correlates with higher productivity in biotechnological applications

• Stress response elucidation:

  • EMI5 function may influence cellular responses to industrial process stresses

  • Investigating how EMI5 variants affect respiratory resilience under stress conditions

  • This knowledge can guide development of more robust production strains

• Growth on alternative substrates:

  • A. gossypii has shown potential to utilize various waste streams and alternative carbon sources

  • EMI5's role in central metabolism may influence the efficiency of alternative substrate utilization

  • Optimizing EMI5 function could enhance growth on economically advantageous substrates

• Integration with emerging applications:

  • Recent research has demonstrated A. gossypii's potential for producing:

    • Monoterpenes like sabinene (684.5 mg/L) and limonene (383 mg/L) from agro-industrial wastes

    • Recombinant proteins with yields comparable to established production hosts

    • Single cell oils (SCOs) and flavor compounds

  • EMI5 optimization could enhance metabolic capacities underpinning these applications

• Genomic and systems biology insights:

  • EMI5 studies contribute to broader understanding of A. gossypii metabolism

  • Integration with genome-scale metabolic models enables predictive strain engineering

  • This systems-level understanding accelerates development of novel applications

The intersection of EMI5 research with these emerging applications represents a promising frontier in industrial biotechnology .

What advanced techniques are emerging for studying EMI5 in the context of mitochondrial respiratory complexes?

Several cutting-edge techniques are emerging for studying EMI5 in the context of mitochondrial respiratory complexes:

• Cryo-electron microscopy (Cryo-EM):

  • Enables visualization of EMI5's interaction with complex II components at near-atomic resolution

  • Can capture different assembly states to elucidate the mechanism of EMI5-assisted complex assembly

  • Requires minimal sample amount compared to crystallography, though protein purity remains critical

• Respiration assays for frozen samples (RIFS):

  • Novel approaches for measuring mitochondrial respiration in frozen samples overcome traditional limitations

  • Enables storage and batch analysis of samples, reducing experimental variability

  • Allows assessment of EMI5 variants' effects on respiratory function in stored samples

  • Protocol involves substrate combinations such as pyruvate + malate (5 mM each), NADH (1 mM), or succinate + rotenone (5 mM + 2 μM)

• Proximity-dependent labeling:

  • BioID or APEX2 fusions with EMI5 enable mapping of its spatial interactome in intact mitochondria

  • Identifies transient interactions that may be missed by traditional co-immunoprecipitation

  • Provides temporal resolution of complex assembly steps when combined with inducible expression systems

• Live-cell imaging techniques:

  • Fluorescently tagged EMI5 variants combined with super-resolution microscopy

  • Enables real-time visualization of EMI5 dynamics during complex assembly

  • Can be multiplexed with membrane potential indicators to correlate assembly with functional outcomes

• Respirometry combined with metabolomics:

  • Simultaneous measurement of oxygen consumption and metabolite changes

  • Connects EMI5 function to broader metabolic consequences

  • Provides insights into how EMI5 variants affect not just respiration but also metabolic flux distributions

These advanced techniques promise to reveal new dimensions of EMI5 function that conventional approaches may miss, potentially uncovering novel strategies for optimizing A. gossypii's biotechnological applications .