Recombinant Pseudomonas putida Fumarate hydratase class II (fumC-2)

What is Fumarate Hydratase Class II (fumC-2) and how does it differ from other fumarase classes?

Fumarate hydratase class II (fumC-2) is an iron-independent enzyme that catalyzes the stereospecific interconversion of fumarate to L-malate in the tricarboxylic acid (TCA) cycle. Unlike class I fumarases (fumA and fumB), fumC-2 is iron-independent and remains active under oxidative stress conditions, serving as a backup enzyme under such circumstances. In P. putida, fumC-2 (encoded by PP_0897) is crucial for connecting central metabolism to many cellular functions that extend beyond those captured by genome-scale metabolic models .

The primary differences between fumC and other fumarase classes include:

• Iron dependence: FumC is iron-independent, while FumA and FumB require iron for activity

• Oxidative stress resistance: FumC remains functional during oxidative stress

• Subcellular localization: In many bacteria, FumC is predominantly cytosolic

• Sequence conservation: FumC shows high sequence conservation across different bacterial species

How is fumC expression regulated in P. putida under different environmental conditions?

In P. putida, fumC expression is dynamically regulated in response to various environmental stimuli. Under iron limitation and oxidative stress conditions, fumC expression is upregulated to compensate for decreased activity of iron-dependent fumarases . Proteomics data has revealed that altering fumC levels affects the expression of numerous other proteins, indicating its integration into complex regulatory networks .

Studies have shown that fumC expression changes in response to:

• Oxidative stress: Significantly upregulated

• Iron limitation: Expression increases as a compensatory mechanism

• Carbon source variation: Different expression patterns observed depending on carbon substrate

• Growth phase: Expression levels vary between exponential and stationary phases

Experimental evidence suggests that maintaining precise fumC activity is critical, as both complete deletion and overexpression can negatively impact cellular metabolism .

What are the kinetic properties of P. putida FumC compared to other bacterial fumarases?

P. putida FumC exhibits distinct kinetic properties compared to fumarases from other bacterial species. While specific kinetic data for P. putida FumC is limited in the provided search results, comparative analysis with other bacterial fumarases provides valuable insights:

*Values estimated based on similar bacterial class II fumarases

Unlike iron-dependent fumarases, P. putida FumC retains activity under oxidative stress conditions, making it particularly important in environments where reactive oxygen species are present .

What are the most effective methods for heterologous expression of fumC in P. putida?

Several effective methods have been developed for heterologous expression of genes like fumC in P. putida, each with specific advantages depending on research objectives:

• Plasmid-based expression systems: Various plasmid vectors have been developed for strong expression of genes in P. putida. The pSEVA platform offers modular vectors with different origins of replication, antibiotic resistance markers, and promoters . For fumC expression, promoters like Ptrc or PEM7 can be used for constitutive expression, while inducible systems like the 3-methylbenzoate-inducible system offer controlled expression .

• Chromosomal integration: For stable expression without antibiotic selection, chromosomal integration is preferred. The I-SceI-based system allows for marker-free integration into the P. putida genome . This approach can be enhanced with CRISPR-Cas9 counterselection to improve efficiency, as demonstrated in recent studies .

• Tunable expression systems: When precise control of fumC expression is required, CRISPRi-based systems offer tunable, tightly controlled gene repression . This approach is particularly valuable when studying the effect of different fumC expression levels on metabolism.

Implementation steps for chromosomal integration using the I-SceI system with CRISPR-Cas9 counterselection:

• Design homologous flanking regions (500-700 bp) upstream and downstream of the integration site

• Clone these regions into a non-replicative vector (e.g., pEMG)

• Introduce the vector into P. putida via triparental mating or electroporation

• Select co-integrants using appropriate antibiotics

• Introduce pSW-2 plasmid carrying the I-SceI gene

• Induce I-SceI expression to force homologous recombination

• Screen for successful recombinants using colony PCR

• Cure cells of the pSW-2 plasmid

What strategies can be used to modify fumC activity without complete gene deletion?

Complete deletion of fumC in P. putida can severely impact growth and metabolism, making it challenging to study its function directly . Instead, researchers can employ several strategies to modulate fumC activity without complete deletion:

• CRISPRi-based repression: CRISPR interference allows for tunable downregulation of fumC expression. By designing guide RNAs targeting different regions of the fumC gene or its promoter and using a catalytically inactive Cas9 (dCas9), researchers can achieve varying levels of repression . This system enables:

  • Temporal control of repression through inducible promoters

  • Different repression levels by targeting various regions of the gene

  • Simultaneous repression of multiple genes if needed

• Promoter engineering: Replacing the native fumC promoter with synthetic promoters of varying strengths allows for controlled expression levels. Libraries of characterized promoters for P. putida are available, enabling precise tuning of fumC expression .

• Base editing: Novel CRISPR base-editing techniques permit specific nucleotide substitutions without double-strand breaks. This approach can be used to introduce point mutations in fumC that alter enzyme activity or regulation without removing the gene entirely . Recent advancements include:

  • PAM-relaxed nCas9 variants that increase genome targetability

  • Self-curing vectors that facilitate multiple rounds of editing

  • Cytidine and adenine base editors for C→T and A→G conversions, respectively

• Conditional expression systems: Placing fumC under the control of inducible promoters allows for temporal control of expression. Systems responsive to chemical inducers (e.g., IPTG, 3-methylbenzoate) or environmental signals can be employed .

How can researchers verify successful genetic modification of fumC in P. putida?

Verification of genetic modifications to fumC in P. putida requires a multi-faceted approach:

• Molecular verification:

  • PCR amplification using primers flanking the modified region

  • Sanger sequencing to confirm the precise modification

  • Whole-genome sequencing to verify the modification and detect any off-target effects

  • Restriction enzyme digestion when appropriate restriction sites are present

• Transcriptional verification:

  • RT-qPCR to quantify fumC mRNA levels

  • RNA sequencing to assess global transcriptional changes resulting from fumC modification

  • Northern blotting for qualitative assessment of transcript size and abundance

• Protein verification:

  • Western blotting using anti-FumC antibodies

  • Proteomics analysis to quantify FumC protein levels and identify changes in related proteins

  • Enzyme activity assays measuring the conversion of fumarate to malate

• Metabolic verification:

  • Measurement of TCA cycle intermediates, particularly fumarate and malate ratios

  • Growth characterization under different carbon sources

  • Metabolic flux analysis to determine changes in central carbon metabolism

• Phenotypic verification:

  • Assessment of growth rates and biomass yields

  • Stress tolerance evaluation, particularly under oxidative stress

  • Product formation capabilities when relevant

How does modulation of fumC expression affect central carbon metabolism in P. putida?

Modulation of fumC expression has profound effects on P. putida's central carbon metabolism, with impacts extending beyond the TCA cycle. Experimental data has revealed that:

• TCA cycle flux: Altering fumC levels directly impacts the flux through the TCA cycle. Proteomics data and context-specific metabolic models confirm that a narrow flux range through the FUM reaction is required for optimal cell function . Both excessive and insufficient fumC activity can disrupt metabolic balance.

• Amino acid metabolism: Studies have shown that deletion or downregulation of fumC affects cellular pools of glutamate and glutamine . This connection arises because:

  • The TCA cycle intermediate α-ketoglutarate is a precursor for glutamate synthesis

  • Fumarate feeds into aspartate metabolism via the conversion of oxaloacetate

  • Serine biosynthesis pathways are connected to TCA cycle intermediates

• Cofactor balancing: FumC activity influences the redox balance within the cell by affecting the generation and consumption of reducing equivalents (NADH, FADH2) through the TCA cycle. This, in turn, impacts numerous cellular processes dependent on these cofactors .

• Carbon flux distribution: When fumC activity is limited, carbon flux is redistributed through alternative pathways, potentially leading to:

  • Increased flux through anaplerotic reactions

  • Altered byproduct formation

  • Changes in carbon storage compound synthesis (e.g., polyhydroxyalkanoates)

• Energy metabolism: As a key enzyme in the TCA cycle, fumC indirectly affects ATP generation through oxidative phosphorylation. Perturbations in fumC activity can significantly impact cellular energetics .

What metabolic engineering strategies can compensate for altered fumC activity in P. putida?

When fumC activity is altered in P. putida, several metabolic engineering strategies can be employed to restore metabolic balance and maintain desired phenotypes:

• Nutrient supplementation: Studies have shown that supplementing growth media with specific amino acids (aspartic acid, asparagine, or serine) can partially compensate for reduced fumC activity, similar to observations in yeast FUM1 deletion strains . This approach addresses specific metabolic deficiencies resulting from altered TCA cycle flux.

• Alternative pathway activation: Redirecting carbon flux through alternative pathways can bypass metabolic bottlenecks created by altered fumC activity. For example:

  • Enhancing glyoxylate shunt activity to bypass part of the TCA cycle

  • Upregulating anaplerotic reactions to replenish TCA cycle intermediates

  • Activating pathways that generate fumarate or malate from other sources

• Cofactor engineering: Manipulating the expression of transhydrogenases or introducing heterologous cofactor-regenerating enzymes can help maintain redox balance when fumC modulation disrupts normal cofactor ratios .

• Fine-tuning of related pathways: Adjusting the expression of enzymes catalyzing reactions connected to fumC can help maintain metabolic homeostasis:

  • Modulating other TCA cycle enzymes

  • Adjusting expression of enzymes involved in amino acid metabolism

  • Engineering regulatory proteins that control related metabolic pathways

• Adaptive laboratory evolution: Allowing P. putida strains with altered fumC activity to evolve under specific selective pressures can result in compensatory mutations that restore growth and desired phenotypes . This approach has proven particularly effective for recovering function in strains with significant metabolic perturbations.

How does fumC activity in P. putida influence stress response mechanisms?

FumC activity in P. putida is intricately linked to various stress response mechanisms, making this enzyme not only a metabolic component but also a factor in cellular resilience:

• Oxidative stress response: FumC serves as a backup enzyme under oxidative stress conditions when iron-dependent fumarases become inactivated . Studies in related organisms suggest that fumarate itself may function as a signaling molecule that modulates the bacterial response to oxidative stress .

• DNA damage response: Research on fumC homologs in other species, including E. coli and yeast, has revealed a connection between fumarate hydratase activity and the DNA damage response, particularly to double-strand breaks . This suggests that functional fumC in P. putida may similarly enhance DNA repair mechanisms.

• Iron limitation adaptation: As an iron-independent enzyme, fumC becomes particularly important under iron-limited conditions. Maintaining TCA cycle functionality during iron starvation is critical for P. putida's survival in environments where this essential metal is scarce .

• Antibiotic resistance: Interestingly, studies have shown connections between metabolic adaptations and antibiotic resistance in P. putida. Strains with altered metabolic profiles often exhibit changed susceptibility to antibiotics. While not directly linked to fumC in the search results, modifications to central metabolism through fumC modulation could potentially influence antibiotic tolerance mechanisms .

• Predator avoidance: Research has demonstrated that metabolic adaptations in P. putida can contribute to predator avoidance strategies. Changes in central metabolism affect production of protective compounds like pyoverdine and conversion to mucoid phenotypes, which could be indirectly influenced by fumC activity .

How can recombinant fumC expression be utilized for enhanced production of TCA cycle-derived compounds?

Recombinant expression of fumC in P. putida offers several strategies for enhancing the production of TCA cycle-derived compounds:

• Optimizing precursor supply: By fine-tuning fumC expression, researchers can control the flux through the TCA cycle and optimize the supply of precursors for target compounds. For example:

  • Increased fumC activity can enhance malate availability for malate-derived products

  • Controlled fumC expression can balance the flux between biomass formation and product synthesis

  • Coordinated expression with other TCA cycle enzymes can direct carbon flux toward specific branch points

• Production of organic acids: P. putida has been engineered for the production of various organic acids, including muconic acid, which can be derived from TCA cycle intermediates . By modulating fumC expression alongside other metabolic modifications, the yield and productivity of these compounds can be enhanced.

• Integration with heterologous pathways: When expressing heterologous biosynthetic pathways that utilize TCA cycle intermediates as precursors, coordinated expression with fumC can ensure adequate supply without disrupting central metabolism. Examples include:

  • Rhamnolipid production pathways that draw on TCA cycle-derived precursors

  • Polyketide biosynthesis routes that utilize malonyl-CoA, which is indirectly connected to TCA cycle metabolism

• Growth-coupled production strategies: Design strategies that couple product formation to biomass synthesis require precise control of central metabolism. Experimental evidence shows that maintaining specific fumC activity levels is crucial for successful implementation of such designs . This approach ensures stable and continuous production throughout the cultivation process.

• Multiple carbon source utilization: When engineering P. putida strains to utilize mixed carbon sources (e.g., glucose and xylose) , adjusting fumC expression can help balance the metabolic fluxes resulting from different uptake pathways, thereby optimizing the conversion of these substrates to desired products.

What role does fumC play in recombinant P. putida strains engineered for C1 compound metabolism?

FumC plays several important roles in P. putida strains engineered for metabolism of C1 compounds (methanol, formaldehyde, formate):

• Metabolic integration of C1 pathways: When introducing synthetic C1 assimilation pathways like the reductive glycine (rGly) pathway into P. putida, proper functioning of central carbon metabolism, including fumC-catalyzed reactions, is essential for successful pathway integration . FumC activity affects:

  • The balance between assimilation and energy generation

  • Cofactor availability for C1 conversion reactions

  • Anaplerotic reactions that connect C1 metabolism to central carbon pathways

• Stress management during C1 metabolism: C1 compounds, particularly formaldehyde, can induce oxidative stress in cells. As an oxidative stress-resistant enzyme, fumC helps maintain TCA cycle functionality under these conditions .

• Energy metabolism in formatotrophic strains: In engineered formatotrophic P. putida strains, the TCA cycle plays a critical role in energy generation. Proper fumC functioning ensures efficient energy production to support the metabolic demands of C1 assimilation .

• Biomass formation from C1-derived metabolites: FumC is essential for biomass formation even when cells are utilizing non-traditional carbon sources. In engineered strains using C1 compounds, fumC activity affects the conversion of assimilated carbon into biomass precursors .

• Flux balancing in hybrid metabolism: When P. putida is engineered to simultaneously utilize C1 compounds and traditional carbon sources, fumC helps maintain balanced flux distribution between these different metabolic modules . This balance is critical for stable growth and production.

How does fumC activity influence recombinant protein production and heterologous pathway expression in P. putida?

FumC activity significantly impacts recombinant protein production and heterologous pathway expression in P. putida through several mechanisms:

• Energy supply for protein synthesis: As a key enzyme in the TCA cycle, fumC influences the cell's energy status by affecting ATP generation through oxidative phosphorylation. Protein synthesis is an energy-intensive process, requiring adequate ATP supply for efficient translation .

• Precursor availability for amino acid synthesis: FumC activity affects the levels of TCA cycle intermediates that serve as precursors for amino acid biosynthesis. Altered fumC expression changes the availability of:

  • Oxaloacetate, a precursor for aspartate-family amino acids

  • α-Ketoglutarate, a precursor for glutamate-family amino acids

  • Pyruvate (indirectly), a precursor for alanine, valine, and leucine

• Redox balance impact on protein folding: By influencing the cell's redox state, fumC activity can affect protein folding, which depends on proper oxidizing conditions in the cell. This is particularly important for recombinant proteins containing disulfide bonds .

• Growth rate effects on expression systems: FumC activity influences growth rate, which in turn affects the performance of different promoters used for heterologous expression. Growth rate-dependent promoters will show variable expression levels depending on the metabolic state influenced by fumC .

• Metabolic burden management: When expressing heterologous pathways, cells face increased metabolic burden. Proper fumC activity helps maintain central metabolism functionality despite these additional demands. Studies show that strains with optimal fumC activity can better sustain heterologous pathway expression without compromising growth .

What are common challenges in expressing and studying recombinant fumC in P. putida?

Researchers face several challenges when expressing and studying recombinant fumC in P. putida:

• Balancing expression levels: As demonstrated in experimental studies, both excessive and insufficient fumC activity can negatively impact cell metabolism . Finding the optimal expression level for specific applications requires careful titration using:

  • Promoters of varying strengths

  • Inducible systems with dose-dependent response

  • Copy number variations of expression vectors

• Metabolic instability: Altering fumC expression can create metabolic imbalances that result in genetic instability, leading to:

  • Accumulation of suppressor mutations

  • Loss of expression over time

  • Unpredictable phenotypic variations

  These challenges necessitate careful strain monitoring and potential adaptive laboratory evolution to stabilize desired phenotypes .

• Activity verification: Confirming that recombinant fumC is functionally active can be challenging. Researchers must distinguish between:

  • Protein expression (detectable by Western blot or proteomics)

  • Enzyme activity (requiring specific activity assays)

  • Metabolic impact (necessitating metabolite analysis)

• Background activity interference: Native fumarase activity in P. putida can interfere with studies of recombinant fumC variants. Creating clean genetic backgrounds may require:

  • Deletion of native fumC genes

  • Careful design of fumC variants that can be distinguished from native enzymes

  • Development of specific activity assays for particular fumC variants

• Context-dependent behavior: The impact of recombinant fumC expression varies depending on growth conditions, genetic background, and metabolic state. This context-dependency complicates experimental design and interpretation, requiring comprehensive characterization under various conditions .

How can researchers troubleshoot growth defects in P. putida strains with altered fumC expression?

When P. putida strains with altered fumC expression exhibit growth defects, researchers can employ several troubleshooting strategies:

• Media supplementation: As observed in yeast studies and supported by P. putida research, supplementing growth media with specific amino acids can rescue growth defects associated with fumC modulation :

  • Aspartic acid: Supplements oxaloacetate-derived amino acids

  • Asparagine: Can bypass certain aspartate synthesis limitations

  • Serine: Addresses potential glycine-serine metabolism disruptions

  • Glutamate: Compensates for altered α-ketoglutarate metabolism

• Expression level adjustment: Fine-tuning fumC expression using:

  • Alternative promoters with different strengths

  • Inducible systems with varying inducer concentrations

  • RBS modifications to adjust translation efficiency

  • CRISPRi for precise downregulation rather than complete deletion

• Adaptive laboratory evolution: Allowing strains to evolve under selective pressure can yield compensatory mutations that restore growth while maintaining desired phenotypes . This approach involves:

  • Serial passaging under growth-limiting conditions

  • Gradually increasing selective pressure

  • Whole-genome sequencing to identify beneficial mutations

  • Reconstruction of identified mutations in the original strain

• Metabolic bypasses: Engineering alternative metabolic routes to bypass fumC-dependent reactions:

  • Enhancing glyoxylate shunt activity

  • Expressing heterologous enzymes with similar functions

  • Introducing pathways that generate malate from other metabolites

• Growth condition optimization: Adjusting cultivation parameters to reduce metabolic stress:

  • Testing different carbon sources and concentrations

  • Optimizing oxygen levels

  • Adjusting pH and temperature

  • Implementing fed-batch strategies to control growth rate

What advanced research approaches can uncover new insights about fumC function in P. putida?

Several advanced research approaches can provide deeper insights into fumC function in P. putida:

• Systems biology approaches:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics) to create comprehensive models of fumC's role in cellular networks

  • Constraint-based metabolic modeling to predict the effects of fumC modulation on metabolic fluxes

  • Regulatory network analysis to identify transcriptional and post-transcriptional controls affecting fumC expression

• High-resolution enzyme characterization:

  • Protein crystallography to determine the three-dimensional structure of P. putida fumC

  • Directed evolution to generate fumC variants with enhanced activity or stability

  • Enzyme kinetics under various conditions to fully characterize catalytic properties

  • Protein-protein interaction studies to identify functional partners of fumC

• Advanced genetic approaches:

  • Base editing for precise introduction of point mutations to study structure-function relationships

  • CRISPRi libraries targeting different regions of fumC to create activity gradients

  • CRISPR activation (CRISPRa) to enhance fumC expression from its native locus

  • Synthetic genetic arrays to identify genetic interactions with fumC

• Single-cell analyses:

  • Single-cell RNA sequencing to capture cell-to-cell variability in fumC expression

  • Microfluidics combined with fluorescent reporters to monitor real-time responses to fumC modulation

  • Flow cytometry sorting of phenotypic variants for subsequent characterization

• In vivo metabolic flux analysis:

  • 13C metabolic flux analysis to quantify changes in metabolic fluxes resulting from fumC modulation

  • Isotope tracing experiments to track the fate of carbon through pathways connected to fumC activity

  • Kinetic modeling of TCA cycle dynamics with varying fumC activity levels

These advanced approaches, when combined, can provide unprecedented insights into the complex role of fumC in P. putida metabolism, stress response, and potential biotechnological applications.