Recombinant Pseudomonas putida Transaldolase (tal)

What is transaldolase (tal) and what is its function in Pseudomonas putida metabolism?

Transaldolase (tal) is a key enzyme in the non-oxidative branch of the pentose phosphate pathway (PPP) that catalyzes the reversible transfer of a three-carbon dihydroxyacetone moiety from sedoheptulose 7-phosphate to glyceraldehyde 3-phosphate, yielding erythrose 4-phosphate and fructose 6-phosphate. In engineered P. putida strains, tal plays a critical role in the assimilation of non-native pentose sugars, particularly D-xylose. The enzyme works in concert with transketolase (tktA) to balance carbon flow through the PPP, enabling the conversion of pentose sugars into metabolites that can enter central carbon metabolism. Research demonstrates that enhanced expression of transaldolase is one of the key events during adaptive evolution of P. putida strains to efficiently utilize D-xylose .

What are the main genetic modifications required to enable P. putida to utilize D-xylose?

The basic genetic modifications required for P. putida to utilize D-xylose typically include:

• Introduction of a functional D-xylose transport system (such as XylE from E. coli)

• Expression of the xylose isomerase pathway genes (xylAB) for the initial conversion of D-xylose to D-xylulose

• Deletion of the hexR transcriptional regulator to derepress glycolysis

• Enhancement of the pentose phosphate pathway by overexpression of transaldolase (tal) and transketolase (tktA)

These modifications establish the core machinery for D-xylose uptake and conversion, though the resulting strains often exhibit slow growth and long lag phases, requiring further optimization through adaptive laboratory evolution (ALE) .

How does overexpression of transaldolase affect the metabolic flux in engineered P. putida?

Overexpression of transaldolase significantly influences metabolic flux distribution in engineered P. putida strains growing on D-xylose. When tal is overexpressed together with transketolase (tktA), it enhances the capacity of the non-oxidative branch of the pentose phosphate pathway, which is critical for effectively channeling D-xylose-derived metabolites into central carbon metabolism.

In P. putida S12ΔhexR_xylAB, overexpression of tktA and tal reduced the time required to consume 12 mM D-xylose from 13 days to only 4 days, while maintaining or slightly improving biomass yield (56.0 ± 2.6 cmol% compared to 52.9 ± 0.9 cmol%) . This significant improvement suggests that tal overexpression relieves a critical bottleneck in the non-oxidative PPP.

The enhanced non-oxidative PPP creates a metabolic "pull" that stimulates the oxidative branch without the negative effects on biomass yield associated with 6-phosphogluconate dehydrogenase (gnd) overexpression. Specifically, overexpression of tal and tktA increases the drain on ribose-5-phosphate, which promotes its replenishment via the oxidative branch .

What is the significance of hexR deletion in P. putida xylose metabolism?

The deletion of hexR, a local transcriptional regulator, plays a crucial role in optimizing D-xylose metabolism in engineered P. putida strains for several reasons:

• HexR acts as a repressor of key enzymes in the Entner-Doudoroff (ED) pathway and parts of the EDEMP cycle (a metabolic architecture combining the ED pathway, Embden-Meyerhof-Parnas pathway, and pentose phosphate pathway).

• Deletion of hexR derepresses glycolysis and several catabolic enzymes, allowing for more efficient carbon flow from D-xylose into central metabolism.

• In P. putida S12, hexR deletion (S12ΔhexR_xylAB) improved biomass yield on D-xylose from 46.5 ± 1.2 cmol% to 52.9 ± 0.9 cmol% compared to the Δgcd_xylAB strain .

• The hexR deletion significantly reduces the lag phase on D-xylose in engineered strains, as demonstrated in both P. putida EM42 and P. putida S12 strains .

The impact of hexR deletion underscores the importance of not only introducing the necessary genes for D-xylose metabolism but also modifying the regulatory network to accommodate the new metabolic capabilities .

How do different pentose phosphate pathway modifications affect D-xylose utilization in P. putida?

Different modifications to the pentose phosphate pathway have distinct effects on D-xylose utilization in engineered P. putida strains, as summarized in the following table based on experimental data:

Key observations from these data:

• Overexpression of tktA and tal (transketolase and transaldolase) in the hexR deletion background produces the best combination of yield and growth rate.

• Overexpression of gnd (6-phosphogluconate dehydrogenase) increases the supply of ribose-5-phosphate but significantly decreases biomass yield, likely due to deregulation of carbon flux distribution.

• Combined overexpression of gnd with tktA-tal compromises growth performance, requiring 8 days to consume the same amount of D-xylose that the tktA-tal strain consumes in 3 days .

These results demonstrate that balanced enhancement of both oxidative and non-oxidative branches of the PPP is critical for optimal D-xylose utilization, with the non-oxidative branch (particularly tal and tktA) playing the more crucial role .

What approaches are most effective for characterizing transaldolase activity in recombinant P. putida strains?

To effectively characterize transaldolase activity in recombinant P. putida strains, researchers should employ a multi-faceted approach:

These complementary approaches provide a comprehensive understanding of transaldolase activity and its role in P. putida metabolism, enabling more effective metabolic engineering strategies.

How can adaptive laboratory evolution (ALE) be optimized for improving transaldolase function in P. putida?

Optimizing adaptive laboratory evolution (ALE) for improving transaldolase function in P. putida requires a strategic approach:

This integrated approach harnesses evolutionary processes while maintaining focus on transaldolase function improvement, leading to more successful outcomes in metabolic engineering efforts.

What are the best methods for integrating transaldolase genes into the P. putida genome?

Several methods can be employed for integrating transaldolase genes into the P. putida genome, each with specific advantages depending on the research goals:

• Plasmid-based expression systems: For initial testing and proof-of-concept studies, transaldolase genes can be introduced using broad-host-range plasmids. For example, plasmid pJTxylAB_tkt-tal was constructed by cloning the tktA-tal genes from P. putida S12 into plasmid pJTxylAB, providing a convenient way to express these genes in combination with xylose utilization genes . While this approach is straightforward, plasmid instability or variability in copy number may affect consistent expression.

• Transposon-based integration: Transposon systems like Tn5 or Tn7 can be used for stable chromosomal integration. The characterized P. putida insertion element ISPpu10 can potentially be engineered for this purpose, though its high target specificity (it inserts within specific repetitive extragenic palindromic sequences) should be considered .

• CRISPR-Cas9 genome editing: This approach allows precise integration of transaldolase genes at specific genomic loci without leaving selection markers. This method is particularly useful when multiple modifications are needed or when targeting specific genomic locations is important for expression levels or metabolic function.

• Homologous recombination-based methods: Traditional allelic exchange using suicide vectors with homology regions flanking the insertion site. This approach was likely used for constructing some of the strains mentioned in the research, such as P. putida S12ΔhexR_xylAB_tkt-tal .

• Landing pad systems: Pre-integrating recombination sites into the genome to facilitate subsequent integration of genes of interest, which can be particularly useful for iterative strain engineering.

When designing integration strategies, consider:

• Expression level requirements (constitutive vs. inducible promoters)

• Genomic context effects on expression

• Potential disruption of native genes or regulatory elements

• Stability of the integrated constructs during long-term cultivation or ALE

The choice of integration method should align with specific experimental goals, whether testing transaldolase variants, optimizing expression levels, or creating stable production strains.

How does the interplay between transaldolase and other enzymes in the pentose phosphate pathway affect carbon flux in P. putida?

The interplay between transaldolase and other enzymes in the pentose phosphate pathway creates a complex metabolic network that significantly influences carbon flux in P. putida engineered for D-xylose utilization:

This complex interplay underscores the importance of considering the entire metabolic network when engineering transaldolase expression or activity in P. putida.

What genomic adaptations occur during adaptive laboratory evolution of D-xylose-utilizing P. putida strains with modified transaldolase expression?

Adaptive laboratory evolution (ALE) of D-xylose-utilizing P. putida strains leads to various genomic adaptations that optimize metabolism and growth. Key genomic changes include:

• Large genomic rearrangements: ALE can trigger substantial structural changes in the genome. Search result mentions "the importance of a large genomic re-arrangement" among the changes observed in evolved mutants. These rearrangements may affect gene expression patterns across multiple pathways or restore genomic stability in the engineered strains.

• Transaldolase expression enhancement: Evolved strains consistently show improved expression of transaldolase, highlighting its critical role in D-xylose metabolism. This enhancement may occur through mutations in promoter regions, regulatory elements, or changes in gene copy number .

• Transporter modifications: Mutations in sugar transporters are common adaptive responses. In P. putida S12xylAB2, two mutations in the glucose ABC transporter gene gtsA (resulting in amino acid substitutions T74A and P85L) were identified, potentially improving affinity for D-xylose . Transport efficiency is critical as it can be a rate-limiting step in sugar utilization.

• Regulatory network adjustments: Beyond engineered changes like hexR deletion, additional mutations in transcriptional regulators may occur during ALE to fine-tune expression of key metabolic genes, optimizing the balance between different pathways .

• Expression balancing of synthetic pathways: ALE helps optimize the expression levels of introduced heterologous genes. Search result specifically mentions "balancing gene expression in the synthetic" pathway as an important outcome of evolution, suggesting that appropriate stoichiometry between pathway components is critical for optimal function.

• Metabolic flux redistribution: While not strictly genomic adaptations, changes in enzyme activities and metabolic flux often result from genomic mutations. The redistribution of carbon flux through different pathways, particularly between the ED pathway and PPP, is a consistent feature of adapted strains .

These genomic adaptations demonstrate P. putida's remarkable genomic and metabolic plasticity, enabling it to find diverse solutions to the challenge of utilizing non-native substrates like D-xylose .

How do cofactor balancing and redox state affect transaldolase function in engineered P. putida strains?

Understanding and optimizing these redox interactions is crucial for effective metabolic engineering, especially when introducing or enhancing transaldolase activity in P. putida for D-xylose utilization or other biotechnological applications.

How can researchers troubleshoot poor growth phenotypes in P. putida strains with recombinant transaldolase expression?

When troubleshooting poor growth phenotypes in P. putida strains with recombinant transaldolase expression, researchers should consider a systematic approach:

By systematically addressing these factors, researchers can identify and resolve issues limiting growth in P. putida strains with recombinant transaldolase expression.

What strategies can improve the stability and activity of recombinant transaldolase in P. putida?

To improve the stability and activity of recombinant transaldolase in P. putida, researchers can implement several strategies:

• Codon optimization: Adapt the coding sequence to P. putida's codon usage preferences, which can enhance translation efficiency and protein folding. This is particularly important when expressing heterologous transaldolase genes from organisms with different GC content.

• Promoter selection: Choose appropriate promoters based on desired expression levels. Strong constitutive promoters like P EM7 (used for xylE expression in strain PD310 ) can ensure robust expression, while inducible promoters offer control over expression timing and level.

• Ribosome binding site (RBS) engineering: Optimize the RBS to enhance translation initiation. Strong synthetic RBSs have been successfully used in P. putida, as mentioned for xylE expression .

• Genomic integration location: Select integration sites that minimize disruption of native genes while providing stable expression. The location can affect expression levels due to chromosomal context effects.

• Co-expression of chaperones: Express molecular chaperones to assist in proper protein folding, which may enhance the activity and stability of recombinant transaldolase.

• Fusion proteins or tags: Consider adding stability-enhancing tags or domains, or creating fusion proteins that can improve folding, stability, or catalytic properties.

• Protein engineering: Apply rational design or directed evolution approaches to improve specific properties of transaldolase, such as thermostability, substrate specificity, or catalytic efficiency.

• Balance with partner enzymes: Co-express transaldolase with partner enzymes at appropriate ratios. The successful co-expression of tktA and tal genes in P. putida demonstrated significant improvements in D-xylose utilization .

• Adaptive laboratory evolution: Subject engineered strains to ALE to select for mutations that enhance transaldolase stability and activity in the specific cellular context of P. putida .

• Metabolic context optimization: Modify the surrounding metabolic network to better accommodate transaldolase function, such as adjusting glycolysis regulation through hexR deletion .

By combining these approaches, researchers can develop P. putida strains with robust, stable, and highly active recombinant transaldolase expression tailored to specific biotechnological applications.

How can transcriptomic and proteomic analyses guide optimization of transaldolase expression in P. putida?

Transcriptomic and proteomic analyses provide powerful insights that can guide optimization of transaldolase expression in P. putida:

• Expression level optimization: Proteomic analysis can determine the actual abundance of transaldolase protein, helping to identify if expression is too low or too high. In evolved D-xylose-utilizing strains, enhanced expression of transaldolase was identified as a key event during adaptation , providing a target expression level for optimization.

• Regulatory network mapping: Transcriptomic analysis reveals how genetic modifications affect global gene expression patterns. For example, analysis of P. putida S12xylAB2 showed that not only the introduced genes but also native genes like gtsABCD and oprB-1 were up-regulated during growth on D-xylose . This information can identify unexpected regulatory interactions affecting transaldolase expression.

• Balancing metabolic pathways: By examining expression levels of enzymes throughout central carbon metabolism, researchers can identify imbalances between connected pathways. This allows rational adjustments to expression levels of transaldolase relative to other enzymes in the PPP and connected pathways.

• Identifying bottlenecks: Transcriptomic and proteomic data can reveal bottlenecks in metabolic networks. For instance, if transaldolase is expressed at high levels but connected pathways show low expression, this suggests potential targets for additional engineering.

• Stress response identification: Heterologous protein expression can trigger stress responses that affect cellular physiology. Omics analyses can identify these responses, enabling strategies to mitigate adverse effects on growth and metabolism.

• Translation efficiency assessment: Comparing transcriptomic and proteomic data can reveal discrepancies between mRNA and protein levels, indicating potential issues with translation efficiency that could be addressed through codon optimization or RBS engineering.

• Post-translational modifications: Proteomic analysis can identify post-translational modifications that might affect transaldolase activity, providing insights for protein engineering strategies.

• Temporal dynamics: Time-course analyses during growth on D-xylose can reveal how expression patterns change over time, informing decisions about when and how strongly transaldolase should be expressed.

• Strain comparison: Comparing omics data between strains with different growth characteristics (e.g., evolved vs. non-evolved, hexR deletion vs. wild-type) helps identify expression patterns associated with improved performance .

• Integration with metabolic modeling: Omics data can be integrated with genome-scale metabolic models to predict the systemic effects of altered transaldolase expression and guide further engineering efforts.

By leveraging these multi-omics approaches, researchers can develop a holistic understanding of how transaldolase expression affects and is affected by the broader cellular context, enabling more precise and effective metabolic engineering strategies.