Recombinant Ashbya gossypii Adenylyltransferase and sulfurtransferase UBA4 (UBA4)

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

Ashbya gossypii is a filamentous fungus that has gained prominence as a model organism in biotechnology, primarily due to its ability to produce riboflavin (vitamin B2) at industrial scales. It has long been considered a paradigm of White Biotechnology for riboflavin production . Its industrial relevance has led to the development of significant molecular toolkits and in silico modeling capabilities that facilitate genetic manipulation and metabolic engineering . While initially valued for riboflavin production, A. gossypii has emerged as a versatile host for producing recombinant proteins, including enzymes like UBA4, due to several advantageous characteristics:

• A fully sequenced and well-annotated genome

• Genetic similarity to Saccharomyces cerevisiae, allowing application of yeast genetic tools

• Efficient secretion machinery for protein expression

• Established protocols for transformation and gene integration

• Ability to grow on simple, cost-effective media

The genomic knowledge and molecular tools available for A. gossypii make it particularly suitable for expression of proteins like UBA4 with experimental manipulations possible through techniques such as homologous recombination in S. cerevisiae .

What are the primary functions of Adenylyltransferase and Sulfurtransferase UBA4 in A. gossypii?

UBA4 in A. gossypii (UBA4_ASHGO) is an enzyme with dual catalytic functions of adenylyltransferase and sulfurtransferase activities . Based on ortholog analysis, UBA4 appears to be involved in essential cellular functions that are conserved across diverse species from fungi to humans . Specifically:

• Adenylyltransferase activity: Catalyzes the transfer of an adenylyl group from ATP to target substrates, an essential step in various metabolic pathways.

• Sulfurtransferase activity: Mediates the transfer of sulfur to various acceptor molecules, playing a crucial role in pathways such as tRNA thiolation and molybdenum cofactor biosynthesis.

The dual functionality of UBA4 positions it as a key player in multiple biochemical pathways that contribute to cellular homeostasis and survival in A. gossypii. Based on comparative genomics data, the protein appears to be highly conserved, suggesting its fundamental importance in fungal metabolism .

How does UBA4 conservation compare across different species relative to A. gossypii?

Ortholog analysis reveals that A. gossypii UBA4 is part of at least 522 full-length protein ortholog groups, indicating significant conservation across diverse species . This extensive conservation suggests that UBA4 serves essential biological functions. Notable orthologous relationships include:

The high bitscore (593) between A. gossypii UBA4 and the Kazachstania africana ortholog indicates particularly strong sequence similarity between these fungal species . The conservation extends beyond fungi to diverse organisms including nematodes (Loa loa), bacteria (Micromonospora echinospora), and cnidarians (Actinia tenebrosa), highlighting the evolutionary importance of this enzyme .

What expression systems are optimal for recombinant production of A. gossypii UBA4?

For recombinant production of A. gossypii UBA4, researchers should consider several expression systems, each with distinct advantages depending on research objectives:

Homologous Expression in A. gossypii:

• Benefits: Native post-translational modifications, proper folding, and physiological relevance

• Methodology: Integration of expression cassettes can be achieved through homologous recombination techniques similar to those used for other A. gossypii genes, as demonstrated in septin studies

• Considerations: Growth conditions should be optimized based on established protocols for A. gossypii cultivation, potentially using media compositions similar to those used for riboflavin production experiments (RPM media)

Heterologous Expression in S. cerevisiae:

• Benefits: Genetic similarity to A. gossypii, well-established tools, suitable for protein interaction studies

• Methodology: Yeast homologous recombination can be employed to generate expression constructs, as shown for other A. gossypii proteins

• Considerations: May require codon optimization based on S. cerevisiae preferences

E. coli Expression System:

• Benefits: High yields, simple cultivation, rapid results

• Methodology: Standard protocols for recombinant protein expression in E. coli with appropriate affinity tags

• Considerations: May lack post-translational modifications; refolding might be necessary if inclusion bodies form

Comparative Expression Performance:

The selection of an appropriate expression system should be guided by specific research requirements, particularly concerning protein authenticity, yield, and downstream applications.

What are the methodological approaches for studying UBA4's role in the A. gossypii sulfur metabolism network?

To investigate UBA4's role in A. gossypii sulfur metabolism, researchers should employ a multifaceted approach:

1. Gene Deletion/Knockout Studies:

• Generate UBA4 deletion strains using homologous recombination-based gene targeting, similar to methods used for CDC11 studies in A. gossypii

• Assess phenotypic consequences on growth, morphology, and metabolite profiles

• Compare with wild-type strains to identify metabolic pathways affected by UBA4 absence

2. Protein-Protein Interaction Analysis:

• Employ tandem affinity purification (TAP) tagging of UBA4

• Use mass spectrometry to identify interaction partners

• Validate key interactions through co-immunoprecipitation or yeast two-hybrid assays

3. Metabolomic Profiling:

• Compare metabolite profiles between wild-type and UBA4-modified strains

• Focus particularly on sulfur-containing metabolites and molybdenum cofactor synthesis intermediates

• Use liquid chromatography-mass spectrometry (LC-MS) to detect changes in metabolic pathways

4. Enzymatic Activity Assays:

• Develop in vitro assays to measure both adenylyltransferase and sulfurtransferase activities

• Determine kinetic parameters (Km, Vmax) for different substrates

• Assess how mutations in key residues affect catalytic efficiency

5. Transcriptome Analysis:

• Perform RNA sequencing to identify genes differentially expressed in UBA4 mutants

• Look for compensatory changes in expression of other sulfur metabolism genes

• Compare transcript patterns across different growth phases, similar to studies of CDC11a/b expression patterns in A. gossypii

Each of these approaches provides complementary insights into UBA4 function, collectively building a comprehensive understanding of its role in A. gossypii metabolism.

How can I design an efficient gene targeting strategy for UBA4 modification in A. gossypii?

To design an efficient gene targeting strategy for UBA4 modification in A. gossypii, follow this methodological approach:

Step 1: Plasmid Construction using Yeast Homologous Recombination

• Construct targeting vectors in S. cerevisiae using homologous recombination, similar to techniques used for CDC11 modification

• Design primers with 40-50 bp homology arms complementary to sequences flanking your target integration site in the UBA4 locus

• Amplify selection markers (such as G418 or NAT resistance cassettes) with primers containing these homology arms

• Transform S. cerevisiae with the PCR products and a linearized vector backbone

• Extract and verify the resulting plasmids by restriction analysis and sequencing

Step 2: A. gossypii Transformation

• Prepare A. gossypii spores or mycelial fragments for transformation

• Use electroporation or PEG/LiAc-mediated transformation protocols

• Select transformants on appropriate media containing antibiotics (G418 at 200 μg/ml or clonNAT at 50 μg/ml)

• Verify integration by PCR and/or Southern blotting

Step 3: Strain Verification and Characterization

• Confirm proper integration by PCR with primers binding outside the integration site

• Verify protein expression/modification by Western blotting or fluorescence microscopy (for tagged versions)

• Assess strain stability by repeated cultivation and verification of the genetic modification

Specific Modifications for Different Research Goals:

This strategy builds on established A. gossypii genetic manipulation techniques, as demonstrated in the successful modification of CDC11 and other genes .

What are recommended protocols for purifying recombinant A. gossypii UBA4 while preserving enzymatic activity?

Purification of recombinant A. gossypii UBA4 requires careful consideration of protein stability and activity. The following protocol is recommended:

Step 1: Expression System Selection and Optimization

• Express UBA4 with an N-terminal or C-terminal affinity tag (His6, GST, or MBP)

• For E. coli expression, use BL21(DE3) or Rosetta strains to address potential codon bias

• Consider co-expression with chaperones if folding issues are encountered

• Optimize expression conditions: temperature (16-30°C), IPTG concentration (0.1-1.0 mM), and induction time (4-24 hours)

Step 2: Cell Lysis and Initial Clarification

• Resuspend cells in buffer containing:

  • 50 mM Tris-HCl or HEPES, pH 7.5-8.0

  • 150-300 mM NaCl

  • 10% glycerol (stabilizer)

  • 1 mM DTT or 2 mM β-mercaptoethanol (to maintain reduced cysteines)

  • 1 mM PMSF and protease inhibitor cocktail

• Lyse cells by sonication or high-pressure homogenization

• Clarify lysate by centrifugation at 20,000 × g for 30 minutes at 4°C

Step 3: Affinity Chromatography

• Load clarified lysate onto appropriate affinity resin

• For His-tagged UBA4: Ni-NTA or TALON resin

• Wash extensively to remove non-specifically bound proteins

• Elute with imidazole gradient (for His-tag) or reduced glutathione (for GST-tag)

Step 4: Secondary Purification

• Perform ion exchange chromatography based on predicted pI of UBA4

• Follow with size exclusion chromatography for highest purity

• Monitor protein purity by SDS-PAGE after each step

Step 5: Activity Preservation Measures

• Add stabilizing agents to final buffer:

  • 10-20% glycerol

  • 1-5 mM DTT

  • 0.1-0.5 mM EDTA (if metal ions are not required for activity)

• Determine optimal storage conditions (temperature, buffer composition)

• Test enzyme activity after each purification step to identify potential activity loss

Enzymatic Activity Monitoring:

• Develop adenylyltransferase activity assay measuring ATP consumption or AMP formation

• Establish sulfurtransferase activity assay using appropriate sulfur acceptor substrates

• Document specific activity throughout purification process to calculate recovery and purification fold

This protocol integrates approaches used for other recombinant enzymes expressed in A. gossypii and related organisms, with specific adaptations for preserving the dual catalytic activities of UBA4.

How can I analyze orthologous relationships between A. gossypii UBA4 and UBA4 proteins from other species?

Analyzing orthologous relationships for A. gossypii UBA4 requires a systematic bioinformatic approach:

Step 1: Sequence Retrieval and Initial Analysis

• Obtain the amino acid sequence of A. gossypii UBA4 from databases like UniProt (Q756K6)

• Identify conserved domains using tools such as NCBI CDD, Pfam, or InterPro

• Generate a domain architecture diagram highlighting functional motifs

Step 2: Ortholog Identification

• Use ortholog databases like InParanoid, OrthoMCL, or OrthoDB

• As shown in the search results, A. gossypii UBA4 is part of 522 full-length protein ortholog groups

• Perform reciprocal BLAST searches to confirm orthologous relationships

• Focus on key model organisms and closely related fungi for detailed comparison

Step 3: Multiple Sequence Alignment

• Align UBA4 sequences using MUSCLE, MAFFT, or T-Coffee

• Refine alignments manually if necessary, focusing on catalytic domains

• Identify conserved residues across different taxonomic groups

• Pay special attention to adenylyltransferase and sulfurtransferase active sites

Step 4: Phylogenetic Analysis

• Construct phylogenetic trees using Maximum Likelihood or Bayesian methods

• Evaluate branch support using bootstrap or posterior probability values

• Analyze evolutionary rates across different lineages

• Identify potential gene duplication or loss events

Researchers working with recombinant A. gossypii UBA4 often encounter several challenges. Here are common issues and methodological solutions:

Low Expression Levels

Problem: Insufficient protein yields for downstream applications.

Solutions:

• Optimize codon usage for the expression host (particularly important for E. coli)

• Test different promoters with varying strength (constitutive vs. inducible)

• Explore alternative expression hosts (A. gossypii itself, P. pastoris, or mammalian cells)

• Adjust induction conditions (temperature, inducer concentration, timing)

• For A. gossypii expression, consider the temporal regulation pattern observed in other genes like CDC11a/b

Protein Misfolding and Inclusion Body Formation

Problem: Recombinant UBA4 forms insoluble aggregates, particularly in bacterial systems.

Solutions:

• Lower expression temperature (16-20°C) to slow folding

• Co-express with molecular chaperones (GroEL/ES, DnaK/J)

• Use solubility-enhancing fusion partners (MBP, SUMO, or TrxA)

• Develop refolding protocols if inclusion bodies persist:

  • Solubilize in 6-8 M urea or 6 M guanidine-HCl

  • Remove denaturant by dialysis or rapid dilution

  • Include redox pairs (GSH/GSSG) to assist disulfide formation if needed

Step 1: In Silico Analysis and Residue Prediction

• Perform multiple sequence alignments of UBA4 orthologs across different species

• Identify strictly conserved residues, particularly within predicted catalytic domains

• Use homology modeling based on crystallized orthologous proteins to predict structure

• Identify potential catalytic residues based on:

  • Conservation across species

  • Proximity to predicted active sites

  • Known catalytic motifs for adenylyltransferases and sulfurtransferases

Step 2: Site-Directed Mutagenesis Strategy

• Design primers for site-directed mutagenesis of predicted catalytic residues

• Create a panel of mutations including:

  • Conservative substitutions (maintaining chemical properties)

  • Non-conservative substitutions (altering chemical properties)

  • Alanine scanning of potentially important regions

• Generate mutant constructs using PCR-based methods similar to those used for other A. gossypii genes

Step 3: Functional Characterization of Mutants

• Express and purify each mutant protein using identical conditions

• Develop quantitative assays for both enzymatic activities:

  • Adenylyltransferase activity: measure ATP consumption or AMP formation

  • Sulfurtransferase activity: quantify sulfur transfer to acceptor substrates

• Compare kinetic parameters (kcat, Km) between wild-type and mutant proteins

• Document effects on protein stability and folding through thermal shift assays

Step 4: Data Analysis and Structure-Function Correlation

• Create a comprehensive data table correlating mutations with activity changes:

• Map activity-altering mutations onto the structural model

• Distinguish between residues affecting substrate binding versus catalysis

• Identify residues specifically involved in each catalytic function

This systematic approach allows for precise identification of functionally critical residues and provides insights into the mechanistic basis of UBA4's dual catalytic activities.

What are the methodological approaches for investigating UBA4's role in A. gossypii stress response pathways?

To investigate UBA4's role in A. gossypii stress response pathways, implement this comprehensive experimental strategy:

Step 1: Generate UBA4-Modified Strains

• Create UBA4 deletion mutants using homologous recombination techniques

• Develop conditional expression strains (e.g., using regulatable promoters)

• Generate point mutants that specifically affect adenylyltransferase or sulfurtransferase activities

• Include appropriate control strains with similar genetic backgrounds

Step 2: Stress Response Phenotyping

• Expose wild-type and UBA4-modified strains to various stressors:

  • Oxidative stress (H₂O₂, paraquat)

  • Heavy metal stress (cadmium, arsenic)

  • Nutritional stress (sulfur limitation, nitrogen starvation)

  • Temperature stress (heat shock, cold shock)

• Quantify growth parameters (lag phase, doubling time, final biomass)

• Document morphological changes through microscopy

• Measure survival rates under acute stress conditions

Step 3: Transcriptomic Analysis

• Perform RNA-seq on wild-type and UBA4-modified strains under normal and stress conditions

• Identify differentially expressed genes and enriched pathways

• Validate key findings with RT-qPCR

• Compare expression patterns to those observed in studies of other A. gossypii genes like CDC11a/b that show developmental regulation

Step 4: Proteomic and Post-Translational Modification Analysis

• Use mass spectrometry to identify proteins with altered abundance or modification

• Focus particularly on changes in thiolation status of target proteins

• Analyze profiles of oxidatively damaged proteins

• Quantify levels of stress-responsive metabolites

Step 5: Integration and Pathway Mapping

• Construct a network model integrating transcriptomic and proteomic data

• Identify key regulatory nodes linking UBA4 to stress response pathways

• Validate predicted connections through targeted experiments

• Compare findings with known stress response pathways in related fungi

This methodological framework provides a comprehensive approach to understanding UBA4's contribution to stress response mechanisms in A. gossypii, potentially revealing novel regulatory connections and pathway interactions.