Recombinant Ashbya gossypii Required for respiratory growth protein 7, mitochondrial (RRG7)

What is Ashbya gossypii and why is it significant for recombinant protein research?

Ashbya gossypii is a filamentous fungus belonging to the Saccharomycete family that has been industrially exploited for riboflavin (vitamin B2) production. In recent years, it has gained significance as a valuable biocatalyst for the production of various metabolites including folic acid, nucleosides, and biolipids through metabolic engineering approaches . A. gossypii is particularly valuable for recombinant protein research because:

• It possesses a genome that shares high synteny with Saccharomyces cerevisiae, making genetic manipulations relatively straightforward

• It can secrete proteins into the culture medium

• It can be grown in large-scale bioreactors

• It has GRAS (Generally Recognized As Safe) status

• It has efficient secretory pathways

Analysis of the A. gossypii secretome has revealed that approximately 1-4% of its proteins are likely secreted, with most secreted proteins having an isoelectric point between 4 and 6 and a molecular mass above 25 kDa . This characteristic makes A. gossypii an attractive host for heterologous protein expression, particularly for proteins requiring post-translational modifications.

How does RRG7 expression relate to riboflavin production in A. gossypii?

The relationship between RRG7 and riboflavin production can be understood by examining the connection between mitochondrial function and flavin metabolism in A. gossypii.

Riboflavin serves as a precursor for flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which function as cofactors for various flavoproteins including those in the mitochondrial respiratory chain. Succinate dehydrogenase (SDH), a component of respiratory complex II, is a flavoprotein that has FAD covalently attached to its Sdh1p subunit . Studies have shown that disruption of mitochondrial flavin metabolism affects SDH activity and riboflavin production in A. gossypii.

When SDH activity is inhibited by malonate (an SDH inhibitor), specific riboflavin production decreases to 78% compared to controls, and heterozygous AgSDH1 gene-disrupted mutants (AgSDH1−/+) show reduced riboflavin production (63% compared to wild type) . This suggests that mitochondrial respiratory proteins, including potentially RRG7, are interconnected with the riboflavin biosynthetic pathway.

What promoter systems are most effective for recombinant RRG7 expression in A. gossypii?

The selection of appropriate promoters is critical for successful expression of recombinant proteins like RRG7 in A. gossypii. Recent research has expanded the molecular toolbox for A. gossypii with several new promoters that offer different expression strengths and regulatory properties.

For constitutive expression of RRG7, the following strong promoters could be considered:

• P_GPD1 (strong constitutive promoter commonly used as a standard)

• P_CCW12 (novel strong promoter)

• P_SED1 (novel strong promoter)

For regulated expression, depending on experimental needs, the following alternatives are available:

• Carbon source-regulatable promoters

• Medium to weak promoters such as P_TSA1, P_HSP26, P_AGL366C, P_TMA10, P_CWP1, P_AFR038W, and P_PFS1

The Dual Luciferase Reporter (DLR) Assay has been adapted for promoter analysis in A. gossypii using integrative cassettes, providing a reliable method to evaluate promoter strength and regulation . This system utilizes genomic integrative cassettes comprising recombinogenic flanks, a loxP-KanMX-loxP marker, the promoter sequence, the reporter luciferase CDS, and the terminator sequence of PGK1 .

Table 1: Relative Strengths of Selected Promoters in A. gossypii

How can I design experiments to evaluate the impact of RRG7 disruption on A. gossypii respiration?

Designing experiments to evaluate the impact of RRG7 disruption on A. gossypii respiration requires careful consideration of methodologies for gene disruption, phenotypic assessment, and respiratory function analysis.

Experimental Design Approach:

• Gene Disruption Strategy:

  • Create heterozygous (RRG7−/+) and, if viable, homozygous (RRG7−/−) disruption mutants using a kanamycin (geneticin) resistance gene expression cassette

  • Confirm disruption by PCR analysis

  • Isolate spores and verify the disruption in resulting colonies

• Growth Assessment under Different Conditions:

  • Compare growth rates on fermentable (e.g., glucose) versus non-fermentable (e.g., glycerol, ethanol) carbon sources

  • Assess growth under aerobic versus microaerobic conditions

  • Monitor colony morphology and sporulation patterns

• Respiratory Function Analysis:

  • Measure oxygen consumption rates using oxygen electrodes

  • Assess mitochondrial membrane potential using fluorescent dyes

  • Determine activities of respiratory chain complexes, particularly Complex II (SDH)

• Metabolic Impact Evaluation:

  • Analyze riboflavin production levels

  • Measure intracellular ROS accumulation

  • Assess levels of ubiquitinated proteins as indicators of protein homeostasis disruption

Based on previous studies with SDH1 disruption, if RRG7 is essential for respiratory growth, homozygous knockout strains may not be viable on glucose, necessitating the use of heterozygous mutants or conditional expression systems for analysis .

What methods can be used to assess respiratory capacity in RRG7-modified A. gossypii strains?

Several methodological approaches can be employed to assess respiratory capacity in RRG7-modified A. gossypii strains:

• Enzymatic Activity Assays:

  • NADH dehydrogenase activity measurement: Spectrophotometric assay monitoring NADH oxidation

  • Succinate dehydrogenase activity measurement: Spectrophotometric assay using 2,6-dichlorophenolindophenol (DCPIP) as an electron acceptor

  • Cytochrome c oxidase activity: Polarographic or spectrophotometric methods

• Respiration Measurements:

  • Oxygen consumption rate determination using oxygen electrodes or optical oxygen sensors

  • Measurement of respiratory quotient (CO₂ produced/O₂ consumed)

• Mitochondrial Function Assessment:

  • Mitochondrial membrane potential using fluorescent dyes like JC-1 or TMRM

  • Mitochondrial morphology evaluation using fluorescence microscopy

  • Assessment of reactive oxygen species (ROS) accumulation using specific dyes such as DCFH-DA

• Molecular Analysis:

  • Western blot analysis to quantify the levels of respiratory complex components

  • Blue Native PAGE to assess respiratory supercomplex assembly

  • RNA-seq or RT-qPCR to analyze expression of respiratory genes

• Metabolic Analysis:

  • Measurement of riboflavin production as an indicator of respiratory/metabolic status

  • Analysis of intracellular ATP levels

  • Determination of NAD+/NADH and FAD/FADH₂ ratios

When applying these methods, it's important to use appropriate controls, including wild-type strains and strains with known respiratory deficiencies, to properly interpret the results in the context of RRG7 function.

What role does RRG7 play in the FAD utilization pathway and how does this impact recombinant protein production?

While specific information about RRG7's role in the FAD utilization pathway is not directly addressed in the provided literature, we can propose a conceptual framework based on the known relationships between mitochondrial function, flavin metabolism, and recombinant protein production in A. gossypii.

Riboflavin is a precursor of FAD, which serves as a critical cofactor for various flavoproteins, including components of the mitochondrial respiratory chain. In yeast, the mitochondrial FAD transporter Flx1p maintains the function of the SDH flavoprotein subunit Sdh1p . Given that RRG7 is required for respiratory growth, it might interact with or regulate components of the FAD utilization pathway.

Potential roles for RRG7 in the FAD utilization pathway could include:

• Regulation of mitochondrial FAD transport

• Assembly or stability of flavoprotein complexes

• Coordination of flavin metabolism with respiratory chain function

• Protection of flavoenzymes from oxidative damage

The impact on recombinant protein production could be manifested through:

These hypotheses could be tested through:

• Metabolomic analysis of flavin intermediates in RRG7 mutants

• Interaction studies between RRG7 and known components of flavin metabolism

• Assessment of recombinant protein yields in strains with modulated RRG7 expression

How does oxidative stress affect RRG7 function and what are the implications for experimental design?

Oxidative stress likely influences RRG7 function given the protein's mitochondrial localization and role in respiratory growth. Mitochondria are both generators and targets of reactive oxygen species (ROS), creating a complex relationship between respiratory function and oxidative stress.

Studies with proteasome inhibitors in A. gossypii provide indirect insights into this relationship. When A. gossypii was treated with MG-132 (a proteasome inhibitor), researchers observed:

• Accumulation of ROS

• Accumulation of ubiquitinated proteins

• Decreased riboflavin production

• Reduced SDH activity

These findings suggest that protein homeostasis, mitochondrial function, and oxidative stress are interconnected in A. gossypii.

For RRG7, this has several implications:

• RRG7 function may be sensitive to oxidative damage

• RRG7 may participate in adaptive responses to oxidative stress

• RRG7 regulation could be linked to protein quality control mechanisms

Implications for Experimental Design:

When designing experiments to study RRG7 under conditions of oxidative stress, researchers should consider:

• Stress Induction Methods:

  • Chemical inducers (H₂O₂, paraquat, menadione)

  • Genetic approaches (SOD or catalase deletions)

  • Growth conditions promoting oxidative stress

• Stress Monitoring:

  • ROS detection methods (fluorescent probes like DCFH-DA)

  • Oxidative damage markers (protein carbonylation, lipid peroxidation)

  • Antioxidant enzyme activities

• RRG7-Specific Considerations:

  • Protein stability under oxidative conditions

  • Post-translational modifications in response to stress

  • Interaction partners under normal versus oxidative conditions

• Controls and Variables:

  • Time-course experiments to distinguish acute versus chronic effects

  • Dose-response relationships for stress inducers

  • Comparison with other mitochondrial proteins

Table 2: Experimental Approaches for Studying RRG7 Under Oxidative Stress

What expression systems are most suitable for producing recombinant RRG7 in A. gossypii?

For the production of recombinant RRG7 in A. gossypii, several expression systems can be considered, each with specific advantages depending on the research objectives:

• Integrative Expression Systems:
  A. gossypii lacks stable episomic vectors, making genomic integration the preferred approach for stable expression . Key components of an effective integrative expression system include:

  • Targeting sequences: Recombinogenic flanks for specific genomic loci (e.g., ADR304W or AGL034C loci)

  • Selection markers: loxP-KanMX-loxP cassette conferring resistance to G418, which can be later eliminated using Cre recombinase

  • Promoter: Selection based on desired expression level (see Table 1 for options)

  • Terminator: PGK1 terminator has been shown to be effective

• Dual Reporter Systems:
  For studies requiring precise quantification of expression levels, the adapted Dual Luciferase Reporter (DLR) Assay provides a reliable platform . This system involves:

  • An integrative module for Renilla luciferase expression as an internal control

  • An integrative cassette for firefly luciferase as the experimental reporter

  • Normalization of expression data to account for variations in transformation efficiency

• Secretion-Based Systems:
  If the goal is to study secreted forms of RRG7 or its interaction with the secretory pathway:

  • Analysis of the A. gossypii secretome indicates that 1-4% of proteins are likely secreted

  • Most secreted proteins have an isoelectric point between 4 and 6, and a molecular mass above 25 kDa

  • Less than 33% of secreted proteins are putative hydrolases

For optimal expression system design, consider:

• Using strong constitutive promoters (P_GPD1, P_CCW12, P_SED1) for high-level expression

• Employing regulatable promoters for studies requiring conditional expression

• Incorporating appropriate signal sequences if secretion is desired

• Implementing codon optimization based on A. gossypii codon usage preferences

How can I optimize respiratory growth conditions for studying RRG7 function in A. gossypii?

Optimizing respiratory growth conditions is crucial for studying RRG7 function in A. gossypii. Based on the available literature, the following approaches are recommended:

• Carbon Source Selection:

  • Glucose supports both fermentative and respiratory metabolism in A. gossypii

  • Non-fermentable carbon sources (glycerol, ethanol, acetate) force respiratory growth

  • A transitional approach using glucose followed by shift to non-fermentable carbon sources can help study the adaptation process

• Oxygen Availability:

  • Ensure consistent aeration through appropriate flask design (baffled flasks) or controlled bioreactors

  • Monitor dissolved oxygen levels to verify respiratory conditions

  • Compare growth under fully aerobic versus oxygen-limited conditions

• Media Composition:

  • Complex media versus minimal media can influence respiratory metabolism

  • Supplement with appropriate concentrations of trace elements important for respiratory chain assembly

  • Consider the impact of nitrogen source on respiratory metabolism

• Growth Monitoring:

  • Track growth rates using optical density measurements

  • Monitor filamentous growth patterns and morphological characteristics

  • Assess sporulation as an indicator of metabolic state

• Metabolic Indicators:

  • Measure riboflavin production as an indicator of respiratory/metabolic status

  • Monitor pH changes that might reflect metabolic shifts

  • Analyze consumption patterns of carbon sources

It's important to note that riboflavin production in A. gossypii requires aerobic conditions when glucose is the carbon source . This suggests that optimizing for respiratory growth may also enhance the production of recombinant proteins that depend on similar metabolic pathways.

What analytical methods are most effective for characterizing RRG7 interactions with the mitochondrial respiratory chain?

Characterizing the interactions between RRG7 and components of the mitochondrial respiratory chain requires a combination of biochemical, genetic, and biophysical approaches. Based on methodologies used in related studies, the following analytical methods are recommended:

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation (Co-IP) with tagged RRG7

  • Yeast two-hybrid screening using RRG7 as bait

  • Proximity labeling approaches (BioID or APEX2) for in vivo interaction mapping

  • Blue Native PAGE to identify respiratory complex associations

• Functional Association Analysis:

  • Genetic interaction screening (synthetic lethality/sickness with respiratory chain components)

  • Suppressor screening to identify genes that can compensate for RRG7 dysfunction

  • Epistasis analysis with respiratory chain component mutations

• Localization and Dynamics:

  • Fluorescent protein tagging and confocal microscopy

  • Immunogold electron microscopy for precise submitochondrial localization

  • Fractionation studies to determine membrane association

• Biochemical Function Assays:

  • Enzyme activity measurements of respiratory complexes in RRG7 mutants

  • Specific activity measurements of succinate dehydrogenase and NADH dehydrogenase

  • Oxygen consumption rates using oxygen electrodes

  • Measurement of mitochondrial membrane potential

• Structural Studies:

  • Cross-linking mass spectrometry to map interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry to identify conformational changes

  • Cryo-EM of respiratory complexes with and without RRG7

Table 3: Analytical Methods for RRG7 Characterization

When applying these methods, it's important to consider:

• The potential impact of tags or modifications on RRG7 function

• The need for appropriate controls, including wild-type and known respiratory mutants

• The possibility of condition-specific interactions that may only occur under certain metabolic states

• The integration of multiple approaches to build a comprehensive understanding of RRG7 function

How can I troubleshoot poor expression of recombinant RRG7 in A. gossypii?

Troubleshooting poor expression of recombinant RRG7 in A. gossypii requires a systematic approach addressing various aspects of the expression system:

• Genetic Construct Design:

  • Verify the integrity of the expression cassette through sequencing

  • Ensure proper codon optimization for A. gossypii

  • Check for unintended regulatory elements or secondary structures in the mRNA

  • Confirm that the promoter-gene-terminator junctions are correct

• Integration Verification:

  • Perform PCR verification of correct integration into the genome

  • Use analytical PCR with primers spanning integration junctions

  • Consider Southern blot analysis for complex integration patterns

  • Check for potential gene copy number effects

• Transcription Analysis:

  • Measure mRNA levels using RT-qPCR

  • Evaluate mRNA stability

  • Consider different promoters if transcription is limiting

  • Assess whether the promoter is functioning as expected under your growth conditions

• Protein Stability and Folding:

  • Check for protein degradation using Western blot analysis

  • Consider fusion tags that might enhance stability

  • Evaluate the impact of growth temperature on protein folding

  • Test the addition of chemical chaperones to the growth medium

• Growth and Induction Conditions:

  • Optimize media composition

  • Adjust aeration and mixing in culture vessels

  • Evaluate different carbon sources

  • Consider the timing of harvest relative to growth phase

A particular consideration for RRG7, given its mitochondrial localization, is to ensure proper targeting to mitochondria. Verify that any fusion constructs or modifications do not interfere with mitochondrial import signals. Additionally, given that A. gossypii does not display a conventional unfolded protein response , the organism may have unique requirements for handling overexpressed proteins.

What are the key considerations for designing gene disruption experiments for RRG7 in A. gossypii?

Designing gene disruption experiments for RRG7 in A. gossypii requires careful consideration of several factors specific to this organism and to mitochondrial proteins:

• Disruption Strategy:

  • Create a disruption cassette with the kanamycin (geneticin) resistance gene flanked by loxP sites

  • Include recombinogenic sequences matching the target locus (typically 45-50 bp)

  • Consider whether to disrupt the entire gene or specific functional domains

  • Prepare for the possibility that complete disruption may be lethal if RRG7 is essential for respiratory growth

• Verification Methods:

  • Design PCR primers to verify correct integration

  • Consider Southern blot analysis for complex situations

  • Sequence across junction points to confirm precise targeting

  • Verify at the protein level using Western blot if antibodies are available

• Strain Selection and Propagation:

  • Start with appropriate wild-type strains

  • Consider the ploidy of your working strain (A. gossypii is typically haploid but multinucleate)

  • Plan for spore isolation steps to ensure genetic homogeneity

  • Be prepared for the possibility of heterokaryons

• Phenotypic Analysis Planning:

  • Design assays to measure respiratory growth on different carbon sources

  • Plan for mitochondrial function assessment

  • Include methods to quantify riboflavin production

  • Consider stress response analyses, particularly oxidative stress

• Controls and Rescue Experiments:

  • Include wild-type controls grown under identical conditions

  • Consider creating heterozygous strains if homozygous knockouts are lethal

  • Design complementation experiments with the wild-type gene

  • Plan for controlled expression using regulatable promoters

Based on experiences with SDH1 disruption in A. gossypii, where homozygous knockouts (AgSDH1−/−) could not be isolated during respiratory growth on glucose, it may be necessary to use alternative approaches if RRG7 is similarly essential . These might include:

• Conditional expression systems

• Partial disruptions targeting specific domains

• Heterozygous disruptions

• Temperature-sensitive alleles

How can I integrate RRG7 studies with broader investigations of mitochondrial function in A. gossypii?

Integrating RRG7 studies with broader investigations of mitochondrial function in A. gossypii creates opportunities for comprehensive understanding of respiratory metabolism and its relationship to biotechnological applications. Here are strategic approaches:

• Comparative Analysis with Known Mitochondrial Proteins:

  • Study RRG7 in parallel with well-characterized proteins like SDH components

  • Create a panel of mutants affecting different aspects of mitochondrial function

  • Perform transcriptomic or proteomic analyses to identify co-regulated genes

  • Look for common phenotypes across different mitochondrial mutants

• Systems Biology Approaches:

  • Develop metabolic models incorporating RRG7 function

  • Use multi-omics approaches (transcriptomics, proteomics, metabolomics)

  • Apply network analysis to position RRG7 within mitochondrial functional networks

  • Identify hubs and bottlenecks in mitochondrial pathways

• Connection to Industrial Applications:

  • Link mitochondrial function studies to riboflavin production

  • Investigate how RRG7 and other mitochondrial proteins affect recombinant protein yields

  • Explore the relationship between respiratory metabolism and product formation

  • Develop strategies to modulate mitochondrial function for improved bioproduction

• Methodological Integration:

  • Standardize growth conditions and analytical methods across studies

  • Develop reporter systems for mitochondrial function assessment

  • Create strain collections with defined mitochondrial mutations

  • Establish databases of mitochondrial protein interactions and functions

• Translational Research Directions:

  • Compare findings in A. gossypii with other industrial organisms

  • Explore whether insights from RRG7 studies can improve recombinant protein production

  • Investigate potential applications in metabolic engineering

  • Connect findings to fundamental questions in mitochondrial biology

Table 4: Framework for Integrated Mitochondrial Function Studies in A. gossypii

This integrated approach enables researchers to position RRG7 studies within the broader context of mitochondrial function while maintaining focus on specific mechanistic questions relevant to both basic science and biotechnological applications.

What are the most promising future research directions for RRG7 in A. gossypii?

Based on current understanding of A. gossypii biology and the role of mitochondrial proteins in respiratory growth, several promising research directions emerge for RRG7 studies:

• Mechanistic Understanding:

  • Detailed characterization of RRG7's specific role in respiratory chain assembly or function

  • Identification of direct interaction partners in the mitochondrial proteome

  • Elucidation of its potential role in mitochondrial genome maintenance

  • Investigation of post-translational modifications regulating RRG7 function

• Biotechnological Applications:

  • Exploration of RRG7 modulation for enhanced riboflavin production

  • Development of RRG7-based reporters for mitochondrial function assessment

  • Evaluation of RRG7 expression levels as predictors of recombinant protein yields

  • Engineering of RRG7 variants with improved function under industrial conditions

• Stress Response Integration:

  • Investigation of RRG7's role in adaptation to oxidative stress

  • Analysis of connections between secretion stress and mitochondrial function

  • Examination of RRG7 involvement in protein quality control mechanisms

  • Study of RRG7 regulation under various environmental stresses

• Comparative Biology:

  • Comparison of RRG7 function across fungal species

  • Evolutionary analysis of respiratory growth requirements

  • Investigation of potential functional homologs in other biotechnologically relevant organisms

  • Translation of findings from model organisms to A. gossypii