Recombinant Ashbya gossypii Ubiquitin-conjugating enzyme E2 6 (UBC6), partial

What is Ashbya gossypii and why is it relevant for UBC6 research?

Ashbya gossypii is a filamentous hemiascomycete fungus primarily known for industrial riboflavin production. It has emerged as an important biotechnological chassis due to its favorable characteristics including: (i) availability of extensive molecular toolboxes for systems metabolic engineering, such as gene-targeting methods and CRISPR/Cas9/Cas12 adapted systems; (ii) ability to utilize various carbon sources including industrial residues; and (iii) ease of mycelial harvesting through simple filtration . These features make A. gossypii an excellent model organism for studying eukaryotic ubiquitin system components, including UBC6, with potential biotechnological applications beyond its traditional use in riboflavin production.

How does A. gossypii UBC6 compare structurally and functionally to other yeast UBC6 homologs?

A. gossypii UBC6 shares significant structural and functional similarities with other yeast UBC6 homologs, particularly that of Saccharomyces cerevisiae. This similarity is explained by the evolutionary relationship between A. gossypii and S. cerevisiae, which show remarkable similarities at the synteny level, though A. gossypii lacks the sequence duplications present in S. cerevisiae . As part of the E2 ubiquitin-conjugating enzyme family, UBC6 likely directs polyubiquitination to specific lysine residues on target proteins. Research with other E2 enzymes has shown that they can direct ubiquitination to distinct subsets of ubiquitin lysines even in the absence of E3 enzymes . In yeast, Ubc7 (related to the human E2G2) participates in endoplasmic reticulum-associated degradation (ERAD), and evidence suggests its ortholog in A. gossypii may serve similar functions.

What are the current methods for genomic characterization of UBC6 in A. gossypii?

Current genomic characterization of UBC6 in A. gossypii typically employs PCR amplification of the target gene region followed by sequencing. The genomic DNA can be isolated using standard fungal DNA extraction protocols. For more advanced characterization, researchers can utilize the fully sequenced and annotated A. gossypii genome . Gene-targeting methods specific to A. gossypii have been developed, allowing precise genetic modifications. Recently, CRISPR/Cas9 and Cas12 systems have been adapted for A. gossypii, enabling more efficient genetic engineering approaches . These tools facilitate precise genomic modifications for characterizing UBC6 function through targeted mutations or regulatory element adjustments.

What expression systems are most effective for producing recombinant A. gossypii UBC6?

The most effective expression systems for recombinant A. gossypii UBC6 production include:

For homologous expression in A. gossypii, the TEF1 promoter (AgPTEF1) has proven effective for strong constitutive expression . When expressing in A. gossypii, cultivation in defined media at pH 6.5 with ammonium as the nitrogen source is recommended, as the organism cannot utilize nitrate . For heterologous expression, codon optimization based on the target expression system is advisable for optimal protein production.

What are the optimized conditions for cultivating A. gossypii for UBC6 expression studies?

For optimal cultivation of A. gossypii for UBC6 expression studies, the following conditions are recommended:

• Media composition: Chemically defined media with glucose, glycerol, or starch as carbon sources. Ammonium serves as an effective nitrogen source at pH 6.5, while nitrate cannot be utilized by A. gossypii .

• pH considerations: Maintain pH at 6.5, as substantial growth at pH 4.5 is observed only on complex medium .

• Temperature: 28-30°C is optimal for A. gossypii growth.

• Aeration: Good aeration is essential for optimal growth as A. gossypii is an aerobic organism.

• Strain selection: Consider strain differences, as phenotypic differences between related A. gossypii strains can be significant. ATCC 10895 (the sequenced strain) is commonly used for molecular biology applications .

• Carbon source: For studies requiring alternative carbon sources, A. gossypii can be engineered to utilize xylose, which may be advantageous for certain experimental designs .

What purification strategy yields the highest purity and activity for recombinant A. gossypii UBC6?

A multi-step purification strategy is recommended for obtaining high-purity, active recombinant A. gossypii UBC6:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar resin if the recombinant UBC6 contains a His-tag.

• Intermediate Purification: Ion exchange chromatography (typically anion exchange) to separate the target protein from contaminants with different charge properties.

• Polishing: Size exclusion chromatography to achieve final purity and remove aggregates.

• Buffer Optimization: Maintain pH 7.5-8.0 with 150 mM NaCl and 1-5 mM DTT to preserve enzymatic activity. Consider adding glycerol (10%) for long-term storage stability.

Throughout purification, it's critical to monitor UBC6 activity using ubiquitination assays. Based on studies with other E2 enzymes, UBC6 activity may depend on its dimerization status, as functional units of E2 enzymes often operate as dimers . Consider activity assays that detect the formation of ubiquitin-conjugated products using western blotting or fluorescence-based methods.

How can one assess the catalytic activity of A. gossypii UBC6 in vitro?

To assess the catalytic activity of A. gossypii UBC6 in vitro, several complementary approaches can be used:

• Thioester formation assay: Monitor the formation of a thioester bond between UBC6 and ubiquitin, which requires ATP, E1 (ubiquitin-activating enzyme), and ubiquitin. This can be detected by non-reducing SDS-PAGE followed by western blotting.

• Autoubiquitination assay: Assess UBC6's ability to catalyze its own ubiquitination. Similar to the systematic study of E2 enzymes described in the literature, this can be performed in the absence of E3 to evaluate the intrinsic activity of UBC6 .

• Mass spectrometry analysis: Determine the lysine specificity in ubiquitin conjugates generated by UBC6. This approach can reveal which lysine residues in ubiquitin are preferentially used for chain formation, providing insights into the type of polyubiquitin chains UBC6 can build .

• Ubiquitin chain assembly assay: Using a gallery of single lysine or arginine ubiquitin derivatives, evaluate which lysine residues are utilized by UBC6 for polyubiquitin chain assembly .

• Substrate ubiquitination assay: If known substrates exist, assess UBC6's ability to ubiquitinate these substrates in conjunction with appropriate E3 ligases.

The activity assays should include proper controls, such as catalytically inactive UBC6 mutants (e.g., active site cysteine to alanine mutations) and reactions lacking ATP or E1.

What is known about the substrate specificity of A. gossypii UBC6?

• UBC6 homologs typically participate in endoplasmic reticulum-associated degradation (ERAD) pathways, targeting misfolded or unassembled proteins for degradation.

• The E2 enzyme family plays a crucial role in determining the topology of polyubiquitin chains by directing ubiquitination to distinct subsets of ubiquitin lysines . This lysine preference can occur even in the absence of E3 enzymes, though E3s may further modulate this specificity.

• Substrate recognition often involves cooperation with specific E3 ubiquitin ligases, which provide much of the substrate specificity. Identifying the E3 partners of A. gossypii UBC6 would provide valuable insights into its substrate range.

• In S. cerevisiae, Ubc6 (along with Ubc7) is involved in the degradation of specific ERAD substrates. Given the evolutionary relationship between S. cerevisiae and A. gossypii , similar substrate specificity might be expected.

To definitively characterize UBC6 substrate specificity, proteomics approaches such as ubiquitin remnant profiling in UBC6 knockout versus wild-type strains could be employed to identify substrates in vivo.

How does the dimeric state of UBC6 affect its function in the ubiquitination pathway?

The dimeric state of UBC6 likely plays a significant role in its function within the ubiquitination pathway. Research on E2 enzymes suggests that the functional unit of E2 is often a dimer . The effects of dimerization on UBC6 function may include:

• Enhanced catalytic efficiency: Dimerization may position the active sites of both UBC6 molecules optimally for sequential transfer of ubiquitin molecules during polyubiquitin chain formation.

• Chain assembly mechanism: One model for polyubiquitin chain assembly involves the construction of the chain on the active site cysteine of one E2 molecule, presumably by the action of additional E2 molecules, before transfer to the substrate . This process could be facilitated by E2 dimerization.

• Regulation of activity: Dimerization may serve as a regulatory mechanism, controlling UBC6 activity in response to cellular conditions.

• E3 interaction: The dimeric state may affect how UBC6 interacts with E3 ubiquitin ligases, potentially influencing substrate specificity and ubiquitination efficiency.

To experimentally assess the importance of dimerization, size exclusion chromatography, analytical ultracentrifugation, or native PAGE could be used to determine the oligomeric state of UBC6 under various conditions. Mutations at the dimer interface could be introduced to generate obligate monomers, allowing comparison of monomeric versus dimeric UBC6 activity.

How can UBC6 be manipulated to enhance protein production in A. gossypii?

UBC6 manipulation can potentially enhance protein production in A. gossypii through several strategic approaches:

• Modulating ERAD efficiency: As UBC6 likely participates in ERAD, modifying its expression or activity could reduce degradation of recombinant proteins that might otherwise be targeted for destruction due to misfolding or slow assembly. This could be achieved through:

  • Conditional knockdown of UBC6 during protein expression phases

  • Engineering UBC6 variants with altered substrate specificity

  • Co-expression of UBC6 inhibitors during production phases

• Improving folding capacity: Instead of directly modifying UBC6, enhancing the cell's protein folding capacity could reduce ERAD activation. This might involve:

  • Overexpression of chaperones specific to the secretory pathway

  • Optimizing cultivation conditions to reduce ER stress

• Leveraging strain differences: Given the documented strain heterogeneity in A. gossypii , screening different strains for UBC6 variants with naturally altered activity could identify strains with improved protein production capabilities.

• Integration with existing metabolic engineering tools: Combine UBC6 manipulation with established A. gossypii engineering approaches, including the molecular toolbox for systems metabolic engineering, gene-targeting methods, and CRISPR/Cas9/Cas12 adapted systems .

When implementing these strategies, it's important to monitor not just protein yields but also cell viability and growth rates, as significant disruption of the ubiquitin-proteasome system can impair essential cellular functions.

What role might UBC6 play in riboflavin production pathways in A. gossypii?

UBC6 might influence riboflavin production in A. gossypii through several mechanisms, although direct evidence is not provided in the search results:

• Regulation of enzyme stability: UBC6-mediated ubiquitination could regulate the turnover of key enzymes in the riboflavin biosynthetic pathway, affecting flux through the pathway.

• Response to metabolic stress: As A. gossypii is a natural producer of riboflavin , the production of this vitamin at high levels might impose metabolic stress. UBC6 could be involved in the cellular response to this stress by targeting misfolded or damaged proteins for degradation.

• Indirect effects on transcriptional regulation: The ubiquitin-proteasome system often regulates transcription factors. UBC6 might participate in controlling the stability or activity of transcription factors that regulate riboflavin biosynthetic genes.

• Quality control of riboflavin biosynthetic machinery: UBC6, as part of the ERAD system, likely ensures the quality of ER-associated proteins, potentially including transporters or other proteins indirectly necessary for efficient riboflavin production.

To investigate these potential roles, comparative studies examining riboflavin production in UBC6 knockout or overexpression strains could be conducted. Proteomic analysis could identify changes in the stability of proteins involved in riboflavin biosynthesis when UBC6 levels are altered.

How can CRISPR/Cas9 technology be optimized for UBC6 modification in A. gossypii?

CRISPR/Cas9 technology can be optimized for UBC6 modification in A. gossypii through the following approaches:

• Guide RNA design: Design highly specific guide RNAs targeting UBC6 using A. gossypii genome sequence data. Consider:

  • GC content optimization (40-60%)

  • Minimizing off-target effects by selecting unique target sequences

  • Proximity to desired modification site

• Delivery method optimization:

  • Transform A. gossypii with plasmids containing both Cas9 and gRNA expression cassettes

  • Consider using the TEF1 promoter (AgPTEF1) for strong expression of Cas9

  • Optimize transformation protocols specific to A. gossypii, accounting for its filamentous nature

• Repair template design:

  • For precise modifications, design repair templates with homology arms of 40-60 bp flanking the cut site

  • For specific mutations (e.g., active site mutations), incorporate silent mutations in the PAM site to prevent re-cutting of modified DNA

• Screening strategy:

  • Develop PCR-based screening methods to identify successful editing events

  • Consider phenotypic screens if UBC6 modification results in visible phenotypes

  • For subtle modifications, sequence verification is essential

• Validation of modifications:

  • Confirm the intended genetic modifications at the DNA sequence level

  • Verify changes in mRNA expression by qRT-PCR (similar to methods used for other genes in A. gossypii )

  • Assess protein expression and function to ensure the modification achieved the desired effect

This approach can be used to generate various UBC6 variants, including knockout strains, strains expressing tagged versions for localization studies, or strains with specific mutations in catalytic residues or interaction domains.

What are common challenges in expressing functional A. gossypii UBC6 and how can they be overcome?

Common challenges in expressing functional A. gossypii UBC6 include:

Additionally:

• For improved expression in A. gossypii, consider the strain heterogeneity factor, as different A. gossypii strains may show varying expression efficiencies .

• When using heterologous expression systems, remember that A. gossypii cannot utilize nitrate as a nitrogen source, but grows well on ammonium or yeast extract at pH 6.5 .

• If expressing UBC6 for functional studies, maintain reducing conditions throughout purification to protect the active site cysteine.

How can researchers distinguish between the roles of different E2 enzymes in A. gossypii?

Researchers can distinguish between the roles of different E2 enzymes in A. gossypii through a multi-faceted approach:

• Systematic gene knockout studies:

  • Generate single and combinatorial knockouts of E2 enzyme genes

  • Perform phenotypic characterization to identify specific defects

  • Use growth assays under various stress conditions to identify condition-specific requirements

• In vitro biochemical specificity assays:

  • Purify multiple E2 enzymes from A. gossypii

  • Compare their lysine specificities in ubiquitin chain formation using mass spectrometry

  • Test their activities with various E3 ligases to identify specific E2-E3 pairs

• Substrate identification approaches:

  • Use proteomics to identify proteins with altered ubiquitination or stability in specific E2 knockout strains

  • Perform ubiquitin remnant profiling to map ubiquitination sites that depend on specific E2 enzymes

  • Develop in vitro ubiquitination assays with candidate substrates

• Localization studies:

  • Generate fluorescently tagged versions of each E2

  • Determine their subcellular localization patterns

  • Identify colocalization with potential substrates or E3 partners

• Evolutionary analysis:

  • Compare E2 sequences across fungal species to identify conserved and divergent features

  • Use the evolutionary relationship between A. gossypii and S. cerevisiae to infer potential functions based on better-characterized S. cerevisiae E2 enzymes

This systematic approach would help delineate the specific roles of UBC6 versus other E2 enzymes in A. gossypii protein quality control and cellular regulation.

What are the implications of UBC6 research for understanding protein quality control in filamentous fungi?

Research on A. gossypii UBC6 has several important implications for understanding protein quality control in filamentous fungi:

• Evolutionary insights: A. gossypii represents an evolutionary link between unicellular yeasts and filamentous fungi . Understanding its UBC6 function could reveal how protein quality control mechanisms evolved to accommodate the unique challenges of filamentous growth.

• Biotechnological applications: As filamentous fungi are important production hosts for various compounds (including riboflavin by A. gossypii ), understanding UBC6's role in protein quality control could lead to engineered strains with enhanced production capabilities through reduced protein degradation or improved folding capacity.

• Stress response mechanisms: Filamentous fungi encounter various environmental stresses that can cause protein misfolding. UBC6, as part of the ERAD system, likely plays a key role in managing these stresses, with implications for fungal survival and adaptation.

• Morphogenesis regulation: The transition between yeast-like and filamentous growth in many fungi involves significant proteome remodeling. UBC6 might contribute to this process by selectively degrading proteins that inhibit filamentous growth or by stabilizing filament-promoting factors.

• Comparative biology: Comparing UBC6 function across unicellular yeasts (S. cerevisiae), hemiascomycetes with filamentous growth (A. gossypii), and true filamentous fungi could reveal how protein quality control is adapted to different growth morphologies.

Future research should focus on identifying UBC6-dependent substrates in A. gossypii and determining how these differ from those in non-filamentous relatives, potentially revealing unique aspects of protein quality control in filamentous fungi.

How does UBC6 function integrate with metabolic engineering approaches in A. gossypii?

UBC6 function can be integrated with metabolic engineering approaches in A. gossypii in several ways:

• Enhancing heterologous protein expression:

  • UBC6 modulation could reduce degradation of recombinant proteins

  • This approach could complement existing A. gossypii expression systems that use tools like the TEF1 promoter

• Improving stress tolerance during fermentation:

  • UBC6's role in protein quality control likely affects how A. gossypii handles various stresses during fermentation

  • Engineering UBC6 activity could enhance tolerance to stresses encountered during industrial production of riboflavin or other compounds

• Coordinating with carbon source utilization pathways:

  • As A. gossypii can be engineered to utilize alternative carbon sources like xylose , UBC6-mediated protein quality control might affect the stability and function of the enzymes involved in these pathways

  • Integrating UBC6 modifications with carbon utilization engineering could lead to more robust production strains

• Supporting terpene biosynthesis:

  • For applications such as limonene production , UBC6 could affect the stability of heterologous enzymes like limonene synthase

  • Modulating UBC6 activity might enhance the expression and stability of rate-limiting enzymes in engineered pathways

• Leveraging strain differences:

  • Given the documented strain heterogeneity in A. gossypii , different strains might have naturally varying UBC6 activities

  • Screening for strains with optimal UBC6 activity profiles could complement metabolic engineering efforts

What experimental contradictions exist in current UBC6 research and how might they be resolved?

Current research on UBC6 in A. gossypii and related E2 enzymes contains several experimental contradictions that require resolution:

• Dimeric state versus monomeric function:

  • Contradiction: While evidence suggests E2 enzymes function as dimers , structural studies often show monomeric E2s.

  • Resolution approach: Conduct in vitro studies with purified A. gossypii UBC6 under various conditions to determine its oligomeric state during catalysis. Use techniques like analytical ultracentrifugation or native mass spectrometry to resolve this contradiction.

• Lysine specificity determinants:

  • Contradiction: Some studies suggest E2s determine lysine specificity in ubiquitin chains , while others attribute this role primarily to E3 ligases.

  • Resolution approach: Perform systematic in vitro ubiquitination reactions with A. gossypii UBC6 alone and in combination with various E3 ligases, followed by mass spectrometry analysis to determine the relative contributions of each to lysine specificity.

• Roles in ERAD versus other cellular pathways:

  • Contradiction: While UBC6 homologs are typically associated with ERAD, evidence in some systems suggests broader functions.

  • Resolution approach: Conduct comprehensive proteomics of UBC6 knockout A. gossypii strains to identify the full range of substrates and pathways affected.

• Growth conditions effect on UBC6 function:

  • Contradiction: A. gossypii shows significant phenotypic differences depending on media composition and pH , but how these affect UBC6 function is unclear.

  • Resolution approach: Systematically characterize UBC6 expression, localization, and activity under various growth conditions to determine environmental influences on its function.

• Strain variability impact:

  • Contradiction: A. gossypii shows substantial strain heterogeneity , but its impact on UBC6 function is unknown.

  • Resolution approach: Compare UBC6 sequences, expression levels, and activities across multiple A. gossypii strains (ATCC10895, MUCL29450, IMI31268, and CBS109.26) to determine strain-specific variations.

Resolving these contradictions will advance our understanding of UBC6 function and improve its application in biotechnological contexts.

How can advanced imaging techniques contribute to understanding UBC6 dynamics in A. gossypii?

Advanced imaging techniques can significantly enhance our understanding of UBC6 dynamics in A. gossypii through multiple approaches:

• Super-resolution microscopy for localization studies:

  • Techniques like STORM or PALM can resolve UBC6 distribution at nanoscale resolution

  • This would reveal precise localization patterns within the ER membrane beyond the diffraction limit

  • Co-localization studies with other ERAD components could map the spatial organization of quality control machinery

• Live-cell imaging for dynamic analysis:

  • Tagging UBC6 with photoconvertible fluorescent proteins would enable monitoring of protein movement and turnover

  • This approach could reveal how UBC6 responds to ER stress or changes in nutrient availability

  • Particularly valuable given A. gossypii's filamentous nature, allowing analysis of protein dynamics along hyphae

• FRET/FLIM for protein-protein interaction studies:

  • Förster Resonance Energy Transfer (FRET) could detect interactions between UBC6 and putative E3 partners or substrates

  • Fluorescence Lifetime Imaging (FLIM) would provide quantitative measures of these interactions in living cells

  • These techniques could resolve the question of UBC6 dimerization in its native context

• Correlative light and electron microscopy (CLEM):

  • Combining fluorescence microscopy of tagged UBC6 with electron microscopy

  • Would reveal the ultrastructural context of UBC6 localization

  • Particularly valuable for understanding UBC6's relationship to ER morphology in filamentous fungi

• Single-molecule tracking:

  • Following individual UBC6 molecules in living cells

  • Could reveal different mobility populations corresponding to active versus inactive states

  • Would provide insights into the diffusion properties of membrane-bound E2 enzymes