Recombinant Ashbya gossypii Diphthamide biosynthesis protein 3 (DPH3)

Basic Research Questions

• What is Diphthamide biosynthesis protein 3 (DPH3) and what is its functional role in fungal species?

Diphthamide biosynthesis protein 3 (DPH3) is a critical component of the diphthamide modification pathway of eukaryotic translation elongation factor 2 (eEF2). Based on studies in Saccharomyces cerevisiae and Schizosaccharomyces pombe, DPH3 functions as an electron donor in the initial step of diphthamide biosynthesis.

The diphthamide biosynthesis pathway follows a multi-step process:

• DPH3 provides electrons to the Dph1–Dph2 complex for the catalysis of 3-amino-carboxypropyl on a specific histidine of eEF2

• Dph5 subsequently tetra-methylates the 3-amino-carboxypropyl-histidine

• Dph7 converts this intermediate to diphthine

• Finally, Dph6 modifies diphthine to diphthamide

In S. pombe, DPH3 has been shown to be required for cellular resistance to cytotoxic drugs like hydroxyurea (HU) and methyl methanesulfonate (MMS), as well as to the fungicide sordarin, which specifically inhibits diphthamide-modified eEF2 . The diphthamide modification is crucial for translation fidelity and cellular stress responses.

• How is the DPH3 gene typically organized in fungal genomes and what can we infer about A. gossypii?

In S. pombe, the dph3 and msh3 genes share a remarkably compact intergenic region of only 268 base pairs between their respective ATG start codons, with divergent and likely overlapping promoters . This tight genomic organization can lead to transcriptional interference when one gene is disrupted, as demonstrated when replacement of the S. pombe msh3 by gene disruption cassettes interfered with dph3 functions .

Given the evolutionary relationship between A. gossypii and other fungi like S. cerevisiae, it's likely that A. gossypii DPH3 might also be closely positioned to neighboring genes. A. gossypii has a compact genome compared to S. cerevisiae, having evolved from a common ancestor but not undergone the whole-genome duplication event experienced by S. cerevisiae .

For researchers working with A. gossypii DPH3, it's crucial to examine its genomic context using the Ashbya Genome Database (AGD), which provides comprehensive annotation information . When designing gene disruption strategies, researchers should carefully consider potential effects on adjacent genes, similar to the observed interference between msh3 and dph3 in S. pombe.

• What expression systems and promoters are most suitable for producing recombinant A. gossypii DPH3?

For optimal expression of recombinant A. gossypii DPH3, several proven expression systems can be employed:

Strong constitutive promoters:

• AgTEF promoter (P_TEF): Derived from the translation elongation factor gene

• AgGPD promoter (P_GPD): Has shown up to 8-fold improvement in recombinant protein expression compared to heterologous promoters

• Newly identified strong promoters: P_CCW12, P_CDA2, and P_SED1

Medium/weak promoters for controlled expression:

• P_TSA1, P_HSP26, P_AGL366C, P_TMA10, P_CWP1, P_AFR038W, and P_PFS1

The most effective expression system uses integrative cassettes rather than episomal vectors due to the multinucleated nature of A. gossypii which can lead to plasmid instability. A recently developed adaptation of the Dual Luciferase Reporter Assay for A. gossypii using integrative cassettes provides a reliable method for assessing promoter strength .

Table 1: Comparison of Promoter Strengths in A. gossypii

When designing expression systems, using native A. gossypii promoters rather than heterologous promoters from S. cerevisiae is recommended, as studies have shown significantly higher expression with native promoters .

• What phenotypic consequences are associated with DPH3 mutations in fungi?

Based on studies in S. pombe, several distinct phenotypes have been associated with DPH3 mutations:

Drug and toxin sensitivity:

• DPH3-deficient strains show increased sensitivity to hydroxyurea (HU), which inhibits ribonucleotide reductase and depletes dNTP pools

• Increased sensitivity to methyl methanesulfonate (MMS), an alkylating agent that causes DNA breaks

• Sensitivity to the fungicide sordarin, which specifically targets diphthamide-modified eEF2

Molecular basis of phenotypes:
When DPH3 function is impaired, the diphthamide modification of eEF2 is blocked, leading to:

• Altered translation fidelity

• Compromised cellular response to DNA damage and replication stress

• Increased susceptibility to toxins targeting eEF2

Importantly, the S. pombe study demonstrated that these phenotypes were specifically associated with dph3 mutations and not with mutations in neighboring genes like msh3. Researchers created strains with mutated ATG start codons (ATGmut) to verify that drug sensitivity was due to impaired dph3 function rather than positional effects of marker insertions .

For A. gossypii, similar phenotypes would be expected in DPH3 mutants, though the specific severity and manifestation might differ based on its unique physiology as a filamentous fungus.

• What are the advantages of A. gossypii as an expression system for recombinant proteins?

A. gossypii offers several distinct advantages as an expression system for recombinant proteins:

Protein secretion capabilities:

• Efficiently secretes native and heterologous enzymes to the extracellular medium

• Recognizes signal peptides from other organisms as secretion signals

Post-translational modification capacity:

• Performs protein post-translational modifications including glycosylation

• Produces N-glycans similar to those from non-conventional yeasts like Pichia pastoris

Genetic tractability:

• High efficiency of homologous recombination facilitates genetic engineering

• Well-established molecular toolkit including selectable markers and promoters

• Golden Gate modular cloning systems adapted for A. gossypii

Growth characteristics:

• Fast growth on inexpensive media

• Ability to utilize various carbon sources, including industrial byproducts like crude glycerol

Industrial relevance:

• Already used in industrial processes (riboflavin production)

• Has GRAS (Generally Recognized As Safe) status

Genomic resources:

• Fully sequenced and annotated genome

• Comprehensive Ashbya Genome Database (AGD)

• Close relationship to S. cerevisiae allows leveraging knowledge from this well-studied organism

A study demonstrated that using glycerol instead of glucose as carbon source resulted in 1.5-fold higher recombinant production of β-galactosidase by A. gossypii , indicating that optimization of culture conditions can further enhance recombinant protein yields.

Advanced Research Questions

• How can transcriptional interference between DPH3 and neighboring genes be assessed and mitigated?

Based on findings in S. pombe, where disruption of the msh3 gene interfered with dph3 function due to their close genomic proximity, understanding and mitigating transcriptional interference is crucial when working with DPH3.

Methods to assess transcriptional interference:

• RT-PCR and qRT-PCR Analysis:

  • Design primers spanning different regions of the transcript

  • Use intron-spanning primers to specifically detect mature mRNA

  • In S. pombe studies, researchers used primers spanning intron I (dph3q-F) and different reverse primers covering most of the dph3 mRNA

• Expression Analysis Under Different Conditions:

  • Compare expression in wild-type and mutant strains under both normal and stress conditions

  • In S. pombe, researchers examined dph3 expression in wild-type and msh3::kanMX strains, both untreated and treated with HU

Strategies to mitigate transcriptional interference:

• Precise Genetic Modifications:

  • Use ATG start codon mutations (ATGmut) rather than complete gene replacements

  • Design gene deletion cassettes that preserve regulatory elements of neighboring genes

  • Select appropriate marker systems that minimize interference

• Marker Selection:

  • In S. pombe, integration of kanMX or hphMX at the msh3 locus interfered with dph3 function, while loxP-ura4-loxM integration did not cause interference

  • The loxP-marker-loxM system allows for subsequent marker removal, further reducing potential interference

• Validation Approaches:

  • Perform complementation tests with wild-type copies of affected genes

  • Verify phenotypes with multiple independent mutants

  • Use functional assays specific to each gene to confirm independent effects

When working with A. gossypii DPH3, these lessons from S. pombe highlight the importance of carefully designing genetic modifications to avoid unintended effects on neighboring genes.

• What are the methodological approaches for studying DPH3's role in stress response and drug resistance?

To investigate DPH3's function in stress response and drug resistance, several complementary approaches can be employed:

Genetic manipulation strategies:

• Precise gene knockouts:

  • Create clean deletions using homologous recombination

  • Use marker-free systems where possible to avoid interference with neighboring genes

  • Consider ATG start codon mutations to specifically block translation while maintaining genomic context

• Drug sensitivity assays:

  • Perform plate-based growth assays with varying concentrations of:

    • Hydroxyurea (HU), which depletes dNTP pools

    • Methyl methanesulfonate (MMS), which causes DNA breaks

    • Sordarin, which specifically targets diphthamide-modified eEF2

  • Use quantitative measurements of growth inhibition zones or survival rates

• Stress response analysis:

  • Expose cells to different stressors (oxidative, temperature, osmotic)

  • Monitor gene expression changes using RT-PCR or RNA-seq

  • Analyze protein levels and modifications under stress conditions

• Translational fidelity measurements:

  • Implement reporter systems to measure translational accuracy

  • Assess rates of frameshifting or stop codon readthrough

  • Correlate with diphthamide modification status

• Complementation studies:

  • Test whether DPH3 from other species can restore function in A. gossypii dph3 mutants

  • Create chimeric proteins to identify functional domains

  • Express mutated versions of DPH3 to identify critical residues

In S. pombe studies, researchers found that dph3Δ mutants, but not msh3Δ mutants, were sensitive to HU, MMS, and sordarin, demonstrating the specific role of DPH3 in stress response and drug resistance . Similar methodological approaches would be applicable to studying A. gossypii DPH3.

• How can we design experiments to study the electron transfer function of DPH3 in the diphthamide biosynthesis pathway?

Investigating the electron transfer function of DPH3 requires specialized experimental approaches:

In vitro biochemical approaches:

• Reconstitution system:

  • Express and purify recombinant A. gossypii Dph1, Dph2, and DPH3

  • Set up in vitro reactions with purified components

  • Monitor the formation of modified eEF2 using mass spectrometry

  • Use spectroscopic methods to track electron transfer from DPH3 to Dph1-Dph2

• Spectroscopic characterization:

  • Employ UV-visible spectroscopy to characterize the redox state of DPH3

  • Use electron paramagnetic resonance (EPR) to detect changes in redox-active centers

  • Apply circular dichroism (CD) spectroscopy to assess structural changes associated with electron transfer

• Electrochemical analysis:

  • Determine the redox potential of DPH3 using protein film voltammetry

  • Compare with redox potentials of proposed partners

Cellular and genetic approaches:

• Structure-function analysis:

  • Generate DPH3 variants with mutations in predicted electron transfer residues

  • Test their ability to complement dph3Δ phenotypes

  • Correlate functional deficits with biochemical properties

• Interaction studies:

  • Perform pull-down assays or co-immunoprecipitation with Dph1-Dph2

  • Use yeast two-hybrid or split-protein complementation assays to detect interactions

  • Identify residues critical for protein-protein interactions

• Sordarin sensitivity as readout:

  • Use the fungicide sordarin, which targets diphthamide-modified eEF2

  • Measure growth inhibition as a proxy for functional diphthamide synthesis

  • Compare wild-type with various DPH3 mutants

Since the electron transfer function of DPH3 is critical for the first step of diphthamide biosynthesis, these approaches would allow researchers to characterize the molecular mechanisms of this important process in A. gossypii.

• What strategies can be employed for site-directed mutagenesis of A. gossypii DPH3 to study structure-function relationships?

To investigate structure-function relationships of A. gossypii DPH3 through site-directed mutagenesis, several sophisticated approaches can be utilized:

Mutagenesis strategies:

• PCR-based site-directed mutagenesis:

  • Design primers containing the desired mutations

  • Amplify the entire plasmid containing the DPH3 gene

  • Digest template DNA with DpnI and transform into E. coli

  • Verify mutations by sequencing before transforming into A. gossypii

• Golden Gate assembly:

  • Utilize the Golden Gate modular cloning system adapted for A. gossypii

  • Enables seamless assembly of multiple fragments with mutations

  • Facilitates the creation of complex variants

• Genomic editing considerations:

  • Account for A. gossypii's multinucleated nature when planning transformations

  • Multiple rounds of selection may be required to obtain homokaryotic mutants

  • Consider genomic context, particularly if DPH3 shares regulatory elements with neighboring genes

Selection of mutation targets:

• Conserved residues:

  • Identify residues conserved across fungal species through multiple sequence alignment

  • Focus on residues in the S. pombe DPH3 known to affect drug sensitivity

• Functional domains:

  • Target residues potentially involved in electron transfer

  • Identify amino acids likely involved in protein-protein interactions with Dph1-Dph2

• Systematic approaches:

  • Consider alanine-scanning mutagenesis of specific regions

  • Create chimeric proteins with DPH3 from other species

  • Use domain swapping to identify functional modules

Functional assessment:

• Drug sensitivity assays:

  • Test mutant strains for sensitivity to HU, MMS, and sordarin

  • Quantify growth rates under different conditions

• Biochemical characterization:

  • Express and purify mutant proteins

  • Assess electron transfer activity

  • Evaluate protein-protein interactions

As demonstrated in S. pombe, even single amino acid changes can have significant effects on DPH3 function . A systematic mutagenesis approach would provide valuable insights into the structure-function relationships of A. gossypii DPH3.

• What are the optimal culture conditions for producing recombinant DPH3 in A. gossypii?

Based on the literature on recombinant protein production in A. gossypii, the following culture conditions can be optimized for DPH3 expression:

Media and growth parameters:

• Media composition:

  • Rich media for high biomass production

  • Consider using glycerol instead of glucose as carbon source, which has been shown to yield 1.5-fold higher recombinant protein production in A. gossypii

  • If DPH3 contains redox-active centers, consider adding reducing agents to maintain protein integrity

• Growth parameters:

  • Standard growth temperature: 28-30°C

  • Consider lowering to 24-26°C during expression phase to improve protein folding

  • Maintain pH between 6.0-6.5

  • Ensure adequate aeration without excessive shear stress

• Inoculation strategy:

  • Use standardized spore suspensions (approximately 10^7 spores/L)

  • Consider pre-cultures to standardize the physiological state

Expression strategies:

• Promoter selection:

  • For high-level expression: Use strong native promoters like P_TEF, P_GPD, P_CCW12, P_CDA2, or P_SED1

  • For moderate expression: Consider P_TSA1, P_HSP26, or P_AGL366C

  • For fine-tuned expression: Use weaker promoters like P_TMA10, P_CWP1, P_AFR038W, or P_PFS1

• Harvest timing:

  • Monitor protein expression over time to determine optimal harvest point

  • Balance protein yield with potential degradation

  • For secreted variants, monitor both cellular and extracellular fractions

• Purification considerations:

  • If DPH3 contains redox-active centers, maintain reducing conditions throughout purification

  • Include appropriate affinity tags for simplified purification

  • Consider the predicted properties of DPH3 (size, charge, hydrophobicity) in designing purification steps

An experimental design approach using multi-factorial optimization is recommended to systematically identify the optimal combination of culture conditions for maximum yield of functional DPH3.