Recombinant Ashbya gossypii Endopolyphosphatase (PPN1), partial

What is Endopolyphosphatase (PPN1) and what is its functional role in fungi?

Endopolyphosphatase (PPN1) is an enzyme that hydrolyzes inorganic polyphosphate (poly P) chains of many hundreds of phosphate residues into shorter lengths. In Saccharomyces cerevisiae, the enzyme has been extensively characterized as a homotetramer of 35-kDa subunits derived from a 75-kDa (674-aa) precursor polypeptide through protease activation . The primary function of PPN1 is to regulate poly P metabolism by internally cleaving long poly P chains, which distinguishes it from exopolyphosphatases that degrade poly P from the termini .

Research in S. cerevisiae has demonstrated that PPN1's activity produces specific products - primarily inorganic phosphate (Pi) and triphosphate (P3) - rather than the previously reported P60 chains . This enzymatic activity appears essential for normal growth, as null mutants show defective growth in minimal media and accumulate long-chain poly P .

How does the structure and expression of PPN1 in Ashbya gossypii compare to its homologs in Saccharomyces cerevisiae?

While the search results primarily focus on S. cerevisiae PPN1, comparative genomic analysis would suggest structural and functional similarities between A. gossypii and S. cerevisiae homologs, given the evolutionary relationship between these fungi.

A. gossypii possesses many genes with syntenic homology to S. cerevisiae, as demonstrated with other genes such as BAS1 . Like S. cerevisiae, A. gossypii's growth cycle consists of distinct trophic and production phases, where differential gene expression occurs . This suggests that PPN1's expression pattern might similarly vary throughout growth phases in A. gossypii.

When studying recombinant A. gossypii PPN1, researchers should consider potential differences in post-translational processing mechanisms, as the activation of PPN1 in S. cerevisiae requires specific vacuolar proteases . These proteolytic processing steps may differ between the species, potentially affecting recombinant protein activity.

What experimental approaches are recommended for initial characterization of PPN1 activity?

For initial characterization of recombinant A. gossypii PPN1 activity, researchers should:

• Substrate preparation: Use purified long-chain poly P (several hundred residues) as the substrate.

• Activity assay: Measure enzyme activity by:

  • Monitoring release of inorganic phosphate using malachite green or molybdate assays

  • Analyzing poly P chain length distribution before and after enzyme treatment via gel electrophoresis

  • Using improved separation methods such as ion-exchange chromatography to identify specific products (Pi and P3)

• Optimizing assay conditions:

  • Test pH range (typically 6.5-7.5)

  • Evaluate divalent cation requirements (Mg2+, Mn2+)

  • Determine temperature optima (likely 30-37°C)

• Control experiments:

  • Include heat-inactivated enzyme controls

  • Compare activity to known S. cerevisiae PPN1

  • Test enzyme on masked poly P chains (those resistant to exopolyphosphatase action)

What are the most effective methods for expressing and purifying recombinant Ashbya gossypii PPN1?

Based on protocols used for S. cerevisiae PPN1, the following approach is recommended:

Expression System Selection:

• Yeast-based expression: Consider using S. cerevisiae expression systems such as those employing the GAL promoter, which has proven effective for PPN1 overexpression (yielding 25-fold increase in activity)

• E. coli expression: May require optimization due to potential issues with proper folding and post-translational modifications

Cloning Strategy:

• Amplify the PPN1 coding sequence from A. gossypii genomic DNA using high-fidelity polymerase

• Consider multiple construct designs:

  • Full-length native PPN1

  • N-terminal His-tagged PPN1

  • C-terminal His-tagged PPN1

  • Truncated versions focusing on the catalytic domain

Expression and Purification Protocol:

• Transform host cells with verified expression constructs

• Induce expression under optimized conditions

• For yeast expression, cell disruption using glass beads yields higher specific activity (20-fold) compared to sonication

• Employ a purification scheme similar to that used for S. cerevisiae PPN1:

  • DEAE-Sepharose chromatography

  • Hydroxyapatite chromatography

  • Size exclusion chromatography

  • Affinity chromatography (if tagged constructs are used)

Activity Verification:

• Verify enzyme functionality using poly P hydrolysis assays

• Confirm identity via mass spectrometry of purified protein

• Analyze protease processing if using full-length constructs

How does PPN1 deletion affect growth, metabolism, and polyphosphate accumulation in fungi?

Studies in S. cerevisiae have revealed significant phenotypic consequences of PPN1 deletion. Similar effects might be expected in A. gossypii, though with potential variations due to its unique metabolism.

Growth Phenotypes:

• PPN1-deficient S. cerevisiae strains show poor growth in rich media, reaching only half the growth of wild-type after 24 hours and even less after 72 hours

• In synthetic media, PPN1 mutants exhibit extended lag phases, with double mutants (PPN1 and PPX1) failing to grow even after 72 hours

• Colony size is reduced when mutants are plated on solid media

Viability Effects:

• Double mutants of PPN1 and PPX1 show rapid viability loss in stationary phase, with sharp decline observed around 40 hours after inoculation

• Single PPN1 mutants show less severe but still noticeable viability reduction

Polyphosphate Metabolism:

• PPN1 mutants accumulate long-chain poly P at levels at least double those in wild-type strains

• This accumulation may contribute to the observed growth defects, though whether through toxicity of long chains or lack of shorter chains remains unclear

What is the relationship between polyphosphate metabolism and riboflavin production in Ashbya gossypii?

• Purine pathway connection: Riboflavin production in A. gossypii is closely linked to purine metabolism . The BAS1 transcription factor regulates both purine biosynthesis and riboflavin production . Since polyphosphates serve as phosphate reserves that could influence purine biosynthesis, PPN1 activity might indirectly affect riboflavin production.

• Growth phase regulation: Riboflavin overproduction in A. gossypii occurs during the productive phase after active growth (trophic phase) has ceased . PPN1 deletion in S. cerevisiae causes growth defects and extends the trophic phase . A similar effect in A. gossypii could potentially alter the timing and extent of riboflavin production.

• Stress response: Polyphosphate metabolism is often linked to stress responses in fungi. Riboflavin overproduction in A. gossypii has been suggested to function as a detoxifying and protective mechanism . PPN1's role in polyphosphate homeostasis might therefore influence stress-related riboflavin production.

Experimental approaches to investigate this relationship could include:

• Creating PPN1 deletion strains in A. gossypii and measuring riboflavin production

• Analyzing gene expression correlations between polyphosphate metabolism genes and riboflavin biosynthesis genes across growth phases

• Studying the effect of exogenous polyphosphates of different chain lengths on riboflavin production

How do post-translational modifications affect recombinant PPN1 activity and what approaches can ensure proper enzyme processing?

Post-translational modifications play a crucial role in PPN1 activity, particularly proteolytic processing and glycosylation.

Proteolytic Processing:

• In S. cerevisiae, PPN1 is synthesized as a 78-kDa (674-aa) precursor that requires protease activation to yield the mature 35-kDa active enzyme

• Vacuolar proteases are essential for this activation, as strains lacking vacuolar proteases show no PPN1 activity

• Both N and C termini of the precursor are critical for proper protease-mediated maturation

Glycosylation:

• N-glycosylation is essential for the proper maturation of PPN1

• Potential N-glycosylation sites have been identified at Asn-11 and Asn-505 in the S. cerevisiae protein

Experimental Approaches:

• For proper processing of recombinant PPN1:

  • Express full-length protein in protease-proficient yeast strains

  • Consider co-expression with relevant proteases if using heterologous systems

  • Maintain proper targeting to vacuolar compartments if expressing in yeast

• For analytical studies:

  • Use site-directed mutagenesis to modify glycosylation sites and assess effects

  • Create truncated variants to identify minimal catalytic domains

  • Employ proteomics to map exact cleavage sites in the mature enzyme

• For maximizing active enzyme production:

  • Design expression constructs that mimic the mature processed form, bypassing the need for proteolytic activation

  • Include appropriate secretion signals if aiming for extracellular production

  • Consider N-terminal sequencing of purified active enzyme to confirm proper processing

What are the methodological considerations for studying PPN1 in the context of genome-scale metabolic modeling of Ashbya gossypii?

With the recent availability of genome-scale metabolic models for A. gossypii , integrating PPN1 into such models presents both opportunities and challenges:

Integration Considerations:

• Stoichiometric representation: Polyphosphate metabolism must be represented with appropriate reactions including:

  • Polyphosphate synthesis (via polyphosphate kinase)

  • Exopolyphosphatase-mediated degradation

  • Endopolyphosphatase (PPN1) internal cleavage reactions

  • Transport reactions between cellular compartments

• Chain length representation: Unlike most metabolic reactions, polyphosphate metabolism involves polymers of varying lengths. Models must either:

  • Use lumped reactions representing average behavior

  • Explicitly model different chain-length classes with separate metabolites

  • Employ hybrid approaches with key chain-length thresholds

• Compartmentalization: PPN1 in S. cerevisiae is localized to vacuoles . Models should account for:

  • Vacuolar localization of enzyme activity

  • Compartment-specific pools of polyphosphates

  • Transport processes between compartments

Experimental Data Requirements:

• Kinetic parameters:

  • Measure PPN1 activity against polyphosphates of defined length

  • Determine Km and Vmax values under various conditions

  • Characterize product distribution patterns

• Flux measurements:

  • Use isotope-labeled phosphate to trace polyphosphate metabolism

  • Quantify polyphosphate pools in different compartments

  • Measure turnover rates in wild-type and mutant strains

• Phenotypic data:

  • Growth rates under various media conditions

  • Metabolite profiling with emphasis on phosphate-related compounds

  • Transcriptional responses to phosphate limitation

Model Validation Approaches:

• Compare model predictions with experimental growth phenotypes of PPN1 deletion mutants

• Test model predictions regarding the effect of polyphosphate metabolism on riboflavin production

• Use the model to design experiments that could reveal non-obvious connections between polyphosphate metabolism and other cellular processes

How can CRISPR-Cas9 gene editing be optimized for studying PPN1 function in Ashbya gossypii?

CRISPR-Cas9 provides powerful tools for precise genetic manipulation of A. gossypii PPN1. Implementation requires consideration of several factors specific to this filamentous fungus:

sgRNA Design Considerations:

• Target unique regions of the PPN1 gene to avoid off-target effects

• Consider targeting:

  • Early coding sequence for complete disruption

  • Specific domains for functional analysis

  • Regulatory regions for expression studies

Delivery Methods:

• Optimize protoplast transformation protocols specifically for A. gossypii

• Consider using Agrobacterium-mediated transformation as an alternative

• Develop electroporation parameters suited to A. gossypii's filamentous nature

Editing Strategies:

• Gene disruption:

  • Design repair templates with selectable markers

  • Create scarless deletions by incorporating microhomology-mediated end joining

  • Generate conditional knockouts using inducible promoters

• Domain editing:

  • Introduce point mutations in key catalytic residues

  • Create chimeric proteins with domains from other species

  • Develop truncated variants to isolate functional domains

• Regulatory modifications:

  • Edit native promoter elements to alter expression patterns

  • Create reporter fusions to monitor expression dynamics

  • Engineer inducible expression systems

Phenotypic Analysis:

• Evaluate effects on growth, morphology, and polyphosphate accumulation

• Assess impact on riboflavin production throughout growth phases

• Analyze transcriptional responses using RNA-seq

What computational approaches can predict the interaction network of PPN1 in Ashbya gossypii metabolism?

Understanding PPN1's role in the broader metabolic network requires sophisticated computational approaches:

Network Inference Approaches:

• Homology-based predictions:

  • Leverage known interactions of S. cerevisiae PPN1

  • Account for genomic context conservation (gene neighborhoods)

  • Consider protein domain architecture conservation

• Co-expression analysis:

  • Generate RNA-seq data across multiple conditions

  • Identify genes with expression patterns correlated with PPN1

  • Construct condition-specific co-expression networks

• Metabolic modeling:

  • Incorporate PPN1 reactions into genome-scale metabolic models

  • Perform flux balance analysis with varying constraints

  • Predict metabolic changes upon PPN1 perturbation

Experimental Validation Strategies:

• Protein-protein interaction studies:

  • TAP-tagging of PPN1 to identify physical interaction partners

  • Yeast two-hybrid screening with PPN1 as bait

  • Co-immunoprecipitation followed by mass spectrometry

• Metabolic profiling:

  • Compare metabolite levels between wild-type and PPN1 mutants

  • Focus on phosphate-containing metabolites and energy carriers

  • Track metabolic fluxes using 13C-labeled substrates

• Genetic interaction mapping:

  • Perform synthetic genetic array analysis with PPN1 deletion

  • Identify synthetic lethal and synthetic rescue interactions

  • Construct genetic interaction networks

How can transcriptomic and proteomic analyses be integrated to understand PPN1 regulation during different growth phases in Ashbya gossypii?

Multi-omics approaches offer powerful insights into PPN1 regulation across A. gossypii's distinct growth phases:

Experimental Design:

• Sampling strategy:

  • Collect samples at defined points during:

    • Germination

    • Trophic phase

    • Transition phase

    • Productive/riboflavin overproduction phase

  • Maintain precise control of culture conditions

• Transcriptomic analysis:

  • Perform RNA-seq with sufficient replication

  • Consider strand-specific sequencing to detect antisense transcription

  • Include small RNA sequencing to identify potential regulatory RNAs

• Proteomic analysis:

  • Use both shotgun proteomics and targeted approaches

  • Apply phosphoproteomics to detect regulatory modifications

  • Consider subcellular fractionation to track protein localization

Integration Approaches:

• Correlation analysis:

  • Compare mRNA and protein expression profiles

  • Identify time lags between transcription and translation

  • Detect post-transcriptional regulation events

• Pathway enrichment:

  • Map expression changes to metabolic and regulatory pathways

  • Focus on phosphate metabolism and related processes

  • Examine connections to riboflavin biosynthesis

• Network modeling:

  • Construct gene regulatory networks

  • Integrate transcription factor binding data where available

  • Develop predictive models of phase transitions

Expected Insights:

• Identification of transcription factors regulating PPN1 expression

• Discovery of post-translational regulation mechanisms

• Understanding of how PPN1 activity is coordinated with growth phase transitions

• Potential mechanistic links between polyphosphate metabolism and riboflavin production