Recombinant Ashbya gossypii Phosphoenolpyruvate carboxykinase [ATP] (PCK1), partial

What is the biochemical function of PCK1 and why is Ashbya gossypii used as an expression host?

Phosphoenolpyruvate carboxykinase (PCK1) catalyzes the reversible conversion of oxaloacetate (OAA) into phosphoenolpyruvate (PEP), serving as a major regulator in multiple metabolic pathways including gluconeogenesis, glyceroneogenesis, and cataplerosis in the tricarboxylic acid (TCA) cycle. While primarily recognized for its gluconeogenic function, PCK1 can also perform an anaplerotic role by synthesizing OAA from PEP, thereby replenishing the TCA cycle under specific cellular conditions .

Ashbya gossypii serves as an advantageous expression host due to its established industrial applications in producing various high-value compounds such as riboflavin (vitamin B2), folic acid, nucleosides, and biolipids . This filamentous fungus possesses a sophisticated molecular toolbox for genetic manipulation, including targeted gene insertion methods, heterologous expression modules, and precise CRISPR-Cas9 and CRISPR-Cpf1 systems that facilitate marker-free engineering strategies . These features make A. gossypii particularly suitable for recombinant protein production, including enzymes like PCK1, especially when complex post-translational modifications are required.

How does PCK1 enzymatic activity differ between its gluconeogenic and anaplerotic functions?

The dual functionality of PCK1 is governed by distinct kinetic parameters that vary depending on reaction direction. In its gluconeogenic mode (OAA→PEP), unmodified PCK1 demonstrates higher catalytic efficiency. Research indicates that post-translational modifications, particularly acetylation, significantly alter these kinetic parameters .

Quantitative kinetic studies reveal that acetylation of PCK1 decreases the Km value (2-fold reduction) for the gluconeogenic reaction while slightly improving efficiency for the anaplerotic (PEP→OAA) direction . This bidirectional capability allows PCK1 to function as a metabolic switch, adapting to cellular energy requirements. The reversal between gluconeogenic and anaplerotic functions is primarily controlled by acetylation status, with specific lysine residues (particularly Lys91) playing crucial roles in determining reaction preference through structural changes in the active site .

What are the optimal promoters and expression strategies for recombinant PCK1 production in A. gossypii?

Research demonstrates that promoter selection significantly impacts recombinant protein expression in A. gossypii. Initial attempts using Saccharomyces cerevisiae promoters (ScPGK1) proved inefficient for heterologous protein expression . For optimal PCK1 expression, native A. gossypii promoters, particularly AgTEF and AgGPD, have shown superior performance, achieving up to 8-fold improvement in recombinant protein secretion compared to heterologous promoters .

The expression strategy should incorporate:

• Native A. gossypii promoters (AgTEF or AgGPD) rather than heterologous ones

• Integration of stable expression cassettes rather than episomal vectors

• Careful selection of terminator sequences (avoiding ScADH1 terminator which exhibits autonomous replicating sequence activity in A. gossypii)

• Optimization of carbon source (glycerol rather than glucose has demonstrated 1.5-fold higher recombinant protein yields)

For stable genome integration, the CRISPR-Cas9 one-vector system developed specifically for A. gossypii provides an efficient method, containing all required modules (Cas9 expression, sgRNA expression, and donor DNA) in a single construct with reported editing efficiencies averaging 60% .

How can researchers design an effective purification protocol for recombinant PCK1 expressed in A. gossypii?

An effective purification strategy for recombinant PCK1 from A. gossypii should accommodate both the properties of the protein and the characteristics of the fungal expression system. Based on structural and biochemical data on PCK1, a multi-step purification approach is recommended:

• Initial clarification: Harvest and disrupt A. gossypii cells under conditions that prevent proteolytic degradation and maintain enzyme stability (typically 4°C with protease inhibitors)

• Ammonium sulfate fractionation: PCK1 typically precipitates between 35-55% saturation

• Ion exchange chromatography: PCK1 (pI approximately 6.5) binds efficiently to anion exchangers (e.g., Q-Sepharose) at pH 7.5-8.0

• Affinity chromatography: If tagged PCK1 variants are expressed, appropriate affinity resins can be employed

• Size exclusion chromatography: A final polishing step to ensure homogeneity

Throughout purification, enzyme activity should be monitored using both forward (gluconeogenic) and reverse (anaplerotic) reaction assays to ensure that the purified enzyme retains dual functionality. Purified PCK1 can be analyzed by SDS-PAGE, with expected molecular weight approximately 69-70 kDa, and authenticity confirmed by western blotting or mass spectrometry.

How do specific acetylation sites affect PCK1 catalytic properties and reaction directionality?

PCK1 function is significantly influenced by acetylation at specific lysine residues, with distinct effects on reaction kinetics and directionality. Comprehensive kinetic analysis has identified several critical acetylation sites with the following functional impacts:

Among these sites, Lys91 acetylation exerts the most profound effect on redirecting PCK1 toward anaplerotic function by catalyzing more efficiently both PEP and GDP utilization . This acetylation notably destabilizes the active site structure, as evidenced by a 5-fold increase in pyruvate kinase activity, which serves as a probe for active site alterations . These structural changes facilitate the reverse reaction while making the gluconeogenic reaction less favorable.

What methodological approaches can accurately measure PCK1 activities in both reaction directions?

Accurate measurement of bidirectional PCK1 activity requires distinct assay systems tailored to each reaction direction:

For gluconeogenic activity (OAA→PEP):

• Direct assay: Monitor the formation of PEP spectrophotometrically at 240 nm (ε = 1.7 mM⁻¹cm⁻¹)

• Coupled assay: Link PEP formation to NADH oxidation via pyruvate kinase and lactate dehydrogenase

  • Reaction mixture: OAA, GTP, MgCl₂, KHCO₃, NADH, pyruvate kinase, lactate dehydrogenase

  • Monitor NADH decrease at 340 nm (ε = 6.22 mM⁻¹cm⁻¹)

For anaplerotic activity (PEP→OAA):

• Direct assay: Monitor OAA formation spectrophotometrically at 290 nm (ε = 0.7 mM⁻¹cm⁻¹)

• Coupled assay: Link OAA formation to NADH oxidation via malate dehydrogenase

  • Reaction mixture: PEP, GDP, MgCl₂, KHCO₃, NADH, malate dehydrogenase

  • Monitor NADH decrease at 340 nm

For pyruvate kinase activity (probe for active site integrity):

• Measure pyruvate formation from PEP in the absence of a phosphoryl donor (GDP instead of GTP)

• Link to NADH oxidation via lactate dehydrogenase and monitor at 340 nm

When comparing acetylated and non-acetylated PCK1 variants, it is essential to normalize enzyme concentrations and perform comprehensive kinetic analyses (Km, kcat, and kcat/Km) for all substrates in both reaction directions to accurately characterize changes in enzyme preference .

How do posttranslational modifications interconvert PCK1 between gluconeogenic and anaplerotic functions?

PCK1 functions are regulated through a sophisticated network of post-translational modifications, creating a metabolic switch mechanism that responds to cellular energy status:

• Acetylation-mediated regulation:

  • Under high glucose conditions, p300-dependent hyperacetylation occurs, particularly at Lys91, which structurally reconfigures the active site to favor the anaplerotic reaction (PEP→OAA)

  • This acetylation does not trigger protein degradation but instead represents a functional shift in enzymatic activity

  • Kinetic analysis shows acetylated PCK1 exhibits decreased Km values for PEP and increased catalytic efficiency in the anaplerotic direction

• Deacetylation-mediated regulation:

  • Under low energy conditions, SIRT1 (but not SIRT2) specifically deacetylates PCK1, fully restoring its gluconeogenic capability

  • This sirtuin-mediated deacetylation represents a critical energy-sensing mechanism linking cellular NAD+ levels to PCK1 function

• Phosphorylation:

  • GSK3β-mediated phosphorylation of PCK1 has a dual regulatory effect, decreasing acetylation while increasing ubiquitination

  • This creates an additional layer of regulation that can influence both activity and protein stability

This regulatory network allows PCK1 to function as a metabolic sensor, shifting between gluconeogenic and anaplerotic roles based on cellular energy requirements, with specific post-translational modifications serving as the molecular switches that determine reaction directionality.

What experimental approaches can be used to study PCK1 acetylation in A. gossypii?

To investigate PCK1 acetylation in A. gossypii, researchers should implement a multi-faceted experimental strategy:

• Generation of site-specific acetylated PCK1 variants:

  • Genetic code expansion technology using orthogonal aminoacyl-tRNA synthetase/tRNA pairs to incorporate acetyllysine at specific positions (Lys91, Lys473, Lys521)

  • Expression in BL21 ΔCobB E. coli strain to prevent deacetylation

  • Alternative approach: Site-directed mutagenesis to create acetylation-mimicking mutations (K→Q) or acetylation-preventing mutations (K→R)

• Detection and quantification of acetylation:

  • Immunoblotting with anti-acetyllysine antibodies

  • Mass spectrometry analysis (LC-MS/MS) following tryptic digestion to identify and quantify site-specific acetylation

  • Targeted selective reaction monitoring (SRM) mass spectrometry for quantitative analysis of acetylation at specific lysine residues

• Functional characterization:

  • Enzymatic assays measuring both gluconeogenic and anaplerotic activities

  • Determination of kinetic parameters (Km, kcat, kcat/Km) for all substrates

  • Pyruvate kinase activity assays as a probe for active site integrity

• Structural analysis:

  • Gel filtration chromatography to verify oligomeric state

  • X-ray crystallography or cryo-EM to determine structural changes induced by acetylation

  • Molecular dynamics simulations to understand the dynamic effects of acetylation

• In vivo studies in A. gossypii:

  • CRISPR-Cas9 genome editing to create strains with PCK1 mutations at key acetylation sites

  • Metabolomic analysis to assess the impact on cellular metabolism

  • Measurement of OAA/PEP ratios under different growth conditions

This comprehensive approach enables detailed mechanistic understanding of how acetylation regulates PCK1 function in the context of A. gossypii metabolism.

How can CRISPR-Cas9/Cpf1 systems be optimized for PCK1 modifications in A. gossypii?

For efficient PCK1 gene modifications in A. gossypii, CRISPR-Cas systems should be tailored specifically for this organism and gene:

• Selection of appropriate CRISPR system:

  • CRISPR-Cas9 requires 5'-NGG-3' PAM sequences and is suitable when these are available near the target site

  • CRISPR-Cpf1 from Lachnospiraceae bacterium recognizes T-rich PAM sequences (5'-TTTN-3') and offers an alternative when NGG PAMs are unavailable

  • Cpf1 also facilitates multiplexing, allowing simultaneous editing of PCK1 and related metabolic genes

• Vector design optimization:

  • One-vector strategy containing all required components (Cas9/Cpf1, sgRNA, donor DNA) is critical for efficient editing in the multinucleated A. gossypii

  • For PCK1 modifications, the system should include:

    • Human codon-optimized Cas9/Cpf1 under control of the TEF1 promoter

    • sgRNA expression module under control of A. gossypii SNR52 promoter

    • Donor DNA template with homology arms for precise PCK1 modifications

    • loxP-KanMX-loxP marker for selection

• Target site selection:

  • Target sequence selection significantly affects editing efficiency

  • For PCK1 acetylation studies, design sgRNAs targeting regions encoding Lys91, Lys473, and Lys521

  • For each target, design and test multiple sgRNAs to identify optimal targeting efficiency

• Donor DNA design for precise modifications:

  • 40-60 bp homology arms flanking the modification site

  • For acetylation studies, introduce specific mutations to replace lysine codons with glutamine (acetylation-mimicking) or arginine (acetylation-preventing)

  • Include silent mutations in the PAM or seed sequence to prevent re-cutting after editing

The average editing efficiency with optimized one-vector CRISPR-Cas9 systems in A. gossypii reaches approximately 60% , making this an effective approach for PCK1 gene modifications.

What strategies can enhance recombinant PCK1 expression levels in A. gossypii?

To maximize recombinant PCK1 expression in A. gossypii, researchers should implement a comprehensive optimization strategy:

• Promoter and terminator optimization:

  • Replace heterologous promoters with native A. gossypii promoters; AgTEF and AgGPD promoters have demonstrated up to 8-fold improvement in recombinant protein expression

  • Avoid the ScADH1 terminator, which exhibits autonomous replicating sequence activity in A. gossypii

• Codon optimization:

  • Adapt the PCK1 coding sequence to A. gossypii codon usage preferences

  • Eliminate rare codons and optimize GC content for improved translation efficiency

• Carbon source modification:

  • Use glycerol instead of glucose as carbon source, which has shown 1.5-fold higher recombinant protein production

  • Consider testing other carbon sources like maltose or mixed carbon strategies

• Secretion enhancement:

  • Test various signal sequences for optimal secretion efficiency

  • Consider engineering strains with enhanced secretory capacity through modifications of the secretory pathway

• Strain improvement:

  • Apply random mutagenesis followed by screening for enhanced expression (2-fold improvements have been reported)

  • Use CRISPR-Cas9/Cpf1 multiplexing to simultaneously modify multiple genes involved in protein production and secretion

• Cultivation conditions optimization:

  • Develop fed-batch strategies to maintain optimal nutrient levels

  • Optimize temperature, pH, and dissolved oxygen levels for maximum protein production

  • Consider supplementation with specific amino acids or vitamins to enhance protein synthesis

The combination of these strategies, particularly utilizing native promoters, alternative carbon sources, and targeted strain engineering, has demonstrated significant potential for improving recombinant protein production in A. gossypii .

How can researchers address issues with PCK1 activity loss during purification from A. gossypii?

Activity loss during purification of recombinant PCK1 from A. gossypii can result from several factors, each requiring specific troubleshooting approaches:

• Post-translational modification changes:

  • Problem: Acetylation status changes during purification, altering activity profile

  • Solution: Add HDAC inhibitors (e.g., nicotinamide) to preserve acetylation or sirtuin inhibitors to maintain deacetylation state

  • Verification: Monitor acetylation status using anti-acetyllysine antibodies at each purification step

• Active site integrity issues:

  • Problem: Destabilization of the active site structure during purification

  • Solution: Include stabilizing agents (glycerol 10-20%, reducing agents like DTT or β-mercaptoethanol)

  • Verification: Measure pyruvate kinase activity as a sensitive probe for active site alterations

• Cofactor loss:

  • Problem: Loss of essential metal ions required for PCK1 activity

  • Solution: Supplement buffers with low concentrations of Mg²⁺ and Mn²⁺ (1-2 mM)

  • Verification: Test activity restoration by adding metal ions to purified enzyme

• Proteolytic degradation:

  • Problem: A. gossypii proteases cleaving PCK1 during extraction

  • Solution: Use comprehensive protease inhibitor cocktails; maintain low temperatures (4°C); minimize purification duration

  • Verification: Analyze samples by SDS-PAGE to detect degradation products

• Incorrect oligomeric state:

  • Problem: PCK1 aggregation or monomerization affecting activity

  • Solution: Include mild detergents or optimize ionic strength to maintain proper oligomeric state

  • Verification: Use gel filtration chromatography to verify oligomeric state before and after each purification step

• Activity assay incompatibilities:

  • Problem: Components in the purification buffers interfering with activity assays

  • Solution: Dialyze or buffer-exchange samples before activity measurements

  • Verification: Include buffer-only controls in activity assays to detect interference

For optimal results, researchers should monitor both gluconeogenic and anaplerotic activities throughout purification, as these may be affected differently by various factors.

What experimental controls are essential when studying acetylation effects on PCK1 activity?

Rigorous experimental controls are critical for accurately interpreting acetylation effects on PCK1 activity:

• Protein concentration and purity controls:

  • Precisely quantify protein concentration using multiple methods (Bradford, BCA, and A280)

  • Verify purity by SDS-PAGE with Coomassie and silver staining

  • Confirm identity by western blotting or mass spectrometry

• Acetylation status controls:

  • Include non-acetylated PCK1 as negative control

  • Include chemically acetylated PCK1 (using acetic anhydride) as maximum acetylation control

  • Verify site-specific acetylation using mass spectrometry or site-specific antibodies

  • Include acetylation-mimicking (K→Q) and acetylation-preventing (K→R) mutants as complementary approaches

• Enzyme activity controls:

  • Perform parallel assays for both directions (gluconeogenic and anaplerotic)

  • Include time-zero controls to account for background activity

  • Run substrate-minus controls to detect non-specific activity

  • Include commercial PCK1 as reference standard when available

• Deacetylation controls:

  • Include SIRT1 treatment to verify reversibility of acetylation effects

  • Include SIRT1 inhibitors (e.g., EX-527) as negative controls

  • Test SIRT2 as specificity control (should not affect PCK1 acetylation)

• Structural integrity controls:

  • Measure pyruvate kinase activity as indicator of active site alterations

  • Perform thermal shift assays to detect stability differences

  • Verify oligomeric state using size exclusion chromatography

• Data analysis controls:

  • Calculate and compare complete kinetic parameters (Km, kcat, kcat/Km) for all substrates

  • Perform statistical analysis with appropriate tests (t-test, ANOVA)

  • Generate Lineweaver-Burk and Eadie-Hofstee plots to detect irregular kinetic behavior

These comprehensive controls ensure that observed changes in PCK1 activity can be confidently attributed to specific acetylation events rather than experimental artifacts.

What are the potential applications of engineered A. gossypii strains expressing modified PCK1 variants?

Engineered A. gossypii strains expressing modified PCK1 variants offer several promising research and biotechnological applications:

• Metabolic flux engineering:

  • Strains with acetylation-mimicking PCK1 variants (K91Q) could enhance anaplerotic flux into the TCA cycle, potentially improving production of TCA-derived compounds

  • Conversely, deacetylation-mimicking variants (K91R) could enhance gluconeogenic flux, potentially improving production of glucose-derived compounds

• Bioproduction of specialized metabolites:

  • PCK1-modified strains could be engineered for enhanced production of high-value nucleosides, as demonstrated with strains achieving up to 150-fold increased inosine excretion through disruption of only two genes (AgADE12 and AgPNP1)

  • The metabolic redirection through altered PCK1 activity could be harnessed for production of other metabolites requiring precise carbon flux control

• Fundamental metabolism research:

  • Creation of strains with specific PCK1 acetylation states would provide valuable in vivo models to study the physiological significance of PCK1 post-translational modifications

  • Multi-omics analysis of these strains could reveal unexpected metabolic adaptations and regulatory networks

• Protein production enhancement:

  • Optimizing PCK1-mediated carbon flux could potentially improve the cellular energetics and precursor availability for recombinant protein production

  • Integration with established A. gossypii expression systems using native promoters like AgTEF and AgGPD could create superior production strains

• Synthetic biology applications:

  • PCK1 variants with altered kinetic properties could serve as metabolic switches in synthetic circuits, responding to specific cellular conditions

  • CRISPR-Cpf1 multiplexing technology enables simultaneous editing of multiple metabolic genes along with PCK1, facilitating complex pathway engineering

The development of these applications would benefit substantially from the established molecular toolbox for A. gossypii, including CRISPR-Cas9/Cpf1 systems with demonstrated efficiencies of approximately 60% .

What methodological advances are needed to better understand PCK1 regulatory networks in A. gossypii?

Advancing our understanding of PCK1 regulatory networks in A. gossypii requires several methodological improvements:

• Advanced genomic engineering tools:

  • Development of inducible CRISPR-Cas9/Cpf1 systems for temporal control of PCK1 modifications

  • Establishment of base editing and prime editing capabilities for precise nucleotide substitutions without double-strand breaks

  • Creation of genome-wide knockout/knockdown libraries in A. gossypii to identify novel PCK1 regulators

• Improved protein modification analysis:

  • Development of A. gossypii-specific antibodies against acetylated PCK1 at specific lysine residues

  • Implementation of targeted proteomics approaches to quantify site-specific PCK1 modifications in complex samples

  • Adaptation of proximity labeling techniques (BioID, APEX) to identify PCK1 interaction partners under different metabolic conditions

• Enhanced metabolic analysis:

  • Development of 13C-metabolic flux analysis protocols specifically for A. gossypii

  • Implementation of real-time metabolite sensing technologies to monitor PCK1 substrates and products in vivo

  • Integration of metabolomics with transcriptomics and proteomics for multi-omics analysis of PCK1 regulatory networks

• Improved in vivo imaging:

  • Development of fluorescent biosensors for PCK1 activity in A. gossypii

  • Adaptation of techniques to visualize protein acetylation dynamics in living cells

  • Implementation of super-resolution microscopy to study PCK1 localization and interactions

• Systems biology approaches:

  • Construction of A. gossypii-specific metabolic models incorporating PCK1 regulation

  • Development of machine learning algorithms to predict PCK1 behavior under various conditions

  • Integration of experimental data with computational models to identify emergent properties of PCK1 regulatory networks

These methodological advances would enable researchers to move beyond studying isolated PCK1 molecules to understanding their function within the complex metabolic and regulatory networks of A. gossypii, ultimately facilitating more effective metabolic engineering strategies.

What key factors should researchers consider when designing experiments with recombinant A. gossypii PCK1?

When designing experiments with recombinant A. gossypii PCK1, researchers should carefully consider several critical factors:

• Expression system design:

  • Select native A. gossypii promoters (AgTEF, AgGPD) rather than heterologous promoters for optimal expression

  • Consider glycerol as a carbon source instead of glucose to potentially increase protein yields by up to 1.5-fold

  • Utilize the one-vector CRISPR-Cas9 system for efficient genomic integration with reported 60% editing efficiency

• Post-translational modification control:

  • Design experiments to control or monitor acetylation status, as this critically affects the reaction directionality of PCK1

  • Consider co-expression with or inhibition of relevant modifying enzymes (p300 for acetylation, SIRT1 for deacetylation)

  • When studying specific acetylation sites, validate findings using multiple approaches (site-directed mutagenesis, genetic code expansion for acetyllysine incorporation)

• Activity measurement considerations:

  • Always measure activity in both reaction directions (gluconeogenic and anaplerotic)

  • Select appropriate assay conditions that account for the altered kinetic parameters of modified PCK1 variants

  • Include pyruvate kinase activity measurements as a sensitive probe for active site integrity

• Experimental controls:

  • Include wild-type PCK1 as baseline control

  • For acetylation studies, include both acetylation-mimicking (K→Q) and acetylation-preventing (K→R) mutants

  • Verify protein concentration, purity, and oligomeric state before activity comparisons

• Genetic modification strategy:

  • Choose between CRISPR-Cas9 (requiring NGG PAM) and CRISPR-Cpf1 (requiring TTTN PAM) based on target sequence

  • For multiplex modifications affecting PCK1 and related metabolic enzymes, consider CRISPR-Cpf1 which facilitates the use of crRNA and dDNA arrays

  • Design donor DNA with appropriate homology arms (40-60 bp) for precise modifications

Careful consideration of these factors will significantly improve experimental outcomes and data quality when working with recombinant A. gossypii PCK1, facilitating meaningful advances in our understanding of this metabolically important enzyme.

How can contradictory findings about PCK1 function be reconciled through experimental design?

Contradictory findings regarding PCK1 function can be reconciled through carefully designed experiments that address common sources of discrepancy:

• Acetylation status variation:

  • Challenge: Inconsistent or unreported acetylation states leading to contradictory activity measurements

  • Resolution strategy: Systematically quantify acetylation at specific lysine residues (particularly Lys91, Lys473, and Lys521) using mass spectrometry

  • Experimental approach: Perform parallel activity assays with characterized acetylated, deacetylated, and site-specific mutant PCK1 variants

• Reaction condition differences:

  • Challenge: Variations in assay conditions affecting the equilibrium of the reversible PCK1 reaction

  • Resolution strategy: Standardize reaction conditions and perform comprehensive kinetic analyses

  • Experimental approach: Determine complete kinetic parameters (Km, kcat, kcat/Km) for all substrates under identical conditions for fair comparisons

• Species-specific variations:

  • Challenge: Extrapolating findings between mammalian PCK1 and A. gossypii PCK1

  • Resolution strategy: Conduct comparative studies with PCK1 from multiple species under identical conditions

  • Experimental approach: Express and purify PCK1 from different organisms in the same expression system with identical tags and purification methods

• In vitro versus in vivo discrepancies:

  • Challenge: Differences between purified enzyme behavior and cellular function

  • Resolution strategy: Complement in vitro studies with carefully designed in vivo experiments

  • Experimental approach: Create A. gossypii strains expressing acetylation site mutants and perform metabolic flux analysis to measure actual metabolic outcomes

• Isoform confusion:

  • Challenge: Mixing findings from cytosolic (PCK1) and mitochondrial (PCK2) isozymes

  • Resolution strategy: Clearly distinguish between isozymes in experimental design and reporting

  • Experimental approach: Include both isozymes in comparative studies with explicit labeling and separate analyses