Recombinant Synechococcus sp. Phosphoenolpyruvate carboxylase (ppc), partial

What are the structural characteristics of the PEPC gene in Synechococcus PCC 7002?

The pepc gene of Synechococcus PCC 7002 consists of an open reading frame (ORF) of 2988 base pairs encoding a protein of 995 amino acids . The gene has a GC content of 52%, which is consistent with the GC content typically observed in cyanobacterial genomes . The deduced protein has a calculated molecular mass of 114,049 Da . Analysis of the gene sequence reveals no obvious -10 and -35 promoter sequences in the region upstream of the ATG start site, suggesting potentially complex regulation mechanisms . The protein structure contains several conserved domains that are characteristic of PEPCs, including the catalytic site and regulatory regions that govern enzyme activity and allosteric regulation.

How does the amino acid sequence of Synechococcus PCC 7002 PEPC compare with PEPC from other organisms?

Sequence alignment analysis reveals that Synechococcus PCC 7002 PEPC shares significant homology with PEPC from other cyanobacteria but diverges considerably from plant and bacterial PEPCs:

What are the essential functional domains and conserved amino acids in Synechococcus PEPC?

The Synechococcus PCC 7002 PEPC contains several critical functional domains that are conserved across different species:

• Glycine-rich loop (FHGRGGXXGRGG): This loop helps position substrate molecules at the active site and forms a protective lid that shields reaction intermediates from water molecules .

• Aspartate-binding site homology: Composed of three domains - EM(T/V)(L/F)(S/A)K, LRN(G/I)(T/Y), and MRNTG .

• Critical arginine residues: The GRGG repeats contain invariant arginine residues (e.g., R683 in Synechococcus PCC 7002 PEPC, corresponding to R587 in E. coli PEPC) that are essential for catalytic activity. Replacement of these arginines results in loss of enzyme function .

• Regulatory domains: Includes sites for allosteric regulation, though these may differ between cyanobacterial and other bacterial PEPCs. For example, the aspartate-binding site is present in both E. coli and Synechococcus PCC 7002 PEPCs, but interestingly, some cyanobacterial PEPCs (like Anacystis nidulans) contain this site despite not being inhibited by aspartate .

What are the challenges in expressing functional recombinant Synechococcus PEPC in E. coli?

Expressing functional recombinant Synechococcus PCC 7002 PEPC in E. coli presents several significant challenges:

• Low expression levels: Initial attempts to express the native Synechococcus PCC 7002 pepc gene in E. coli resulted in weak expression, with the protein only detectable by Western blotting using antibodies against the His-tag .

• Codon usage bias: The codon usage in Synechococcus differs significantly from E. coli, potentially explaining the low expression levels observed with the native gene sequence .

• Protein solubility issues: Even after codon optimization and improved expression, the recombinant protein formed insoluble inclusion bodies, making it difficult to obtain the active enzyme .

• Refolding challenges: Attempts to resolubilize the protein from inclusion bodies while maintaining its native structure were unsuccessful, indicating challenges in proper protein folding .

• Functionality concerns: Even when expression is achieved, the recombinant enzyme may not be fully functional due to differences in post-translational modifications or protein folding between the native cyanobacterial environment and the E. coli host.

These challenges necessitate careful optimization of expression conditions and potentially the development of alternative expression systems or refolding protocols for obtaining functional recombinant PEPC.

How can codon optimization improve the expression of recombinant Synechococcus PEPC?

Codon optimization significantly enhances the expression of recombinant Synechococcus PCC 7002 PEPC in E. coli through several mechanisms:

• Matching host tRNA abundance: Replacing rare codons in the Synechococcus gene with synonymous codons that are more frequently used in E. coli can dramatically improve translation efficiency.

• Quantitative improvement: As demonstrated in the research, commercially synthesized codon-optimized pepc sequence resulted in "an intense band at the correct size (114 kDa)" compared to the weak expression observed with the native sequence .

• Eliminating rare codon clusters: Clusters of rare codons can cause ribosomal stalling and premature translation termination. Optimizing these regions can improve full-length protein synthesis.

• Adjusting GC content: Modifying the GC content to better match the host organism can improve mRNA stability and translation efficiency.

• Removing problematic secondary structures: Eliminating RNA secondary structures that impede translation initiation or ribosome progression can enhance expression.

While codon optimization successfully increases protein quantity, it does not necessarily address protein solubility issues, as evidenced by the formation of inclusion bodies despite high expression levels of the optimized construct . This suggests that additional strategies focusing on protein folding are necessary for obtaining soluble, active enzyme.

What is the relationship between PEPC activity and free fatty acid production in engineered cyanobacteria?

The relationship between PEPC activity and free fatty acid (FFA) production in engineered cyanobacteria is complex and involves several metabolic intersections:

Experimental evidence from related metabolic engineering efforts suggests that enhancing carbon fixation capacity through overexpression of carbon fixation enzymes (including PEPC or RuBisCO) can significantly improve biofuel precursor production in cyanobacteria .

How does overexpression of PEPC compare to RuBisCO overexpression for enhancing carbon fixation in cyanobacteria?

The comparative effectiveness of PEPC versus RuBisCO overexpression for enhancing carbon fixation in cyanobacteria reveals important strain-specific and pathway-dependent considerations:

Research with Synechococcus sp. PCC 7002 demonstrated that expressing RuBisCO from S. elongatus PCC 7942 with its own promoter (psbA1) resulted in more than threefold improvement in free fatty acid production, while similar engineering in S. elongatus PCC 7942 showed no improvement . This suggests that carbon fixation is rate-limiting in Synechococcus sp. PCC 7002 but not in S. elongatus PCC 7942.

What are the optimal strategies for isolating the pepc gene from Synechococcus sp.?

Successful isolation of the pepc gene from Synechococcus requires a strategic approach combining multiple techniques. Based on published research, the following methodological pipeline has proven effective:

• Primer design based on consensus sequences:

  • Align known pepc genes from related cyanobacteria (e.g., Anabaena variabilis, Anacystis nidulans, Synechocystis PCC 6803)

  • Generate consensus sequences from the alignment

  • Design primers targeting conserved regions

• PCR amplification of gene fragments:

  • Use high-fidelity thermostable DNA polymerase such as PfuUltra High Fidelity polymerase

  • Optimize PCR conditions: initial denaturation at 95°C, 30-35 cycles of amplification, and final extension at 72°C

  • Target smaller fragments initially if full-length amplification is unsuccessful

• Genomic database mining:

  • Use sequences of PCR products to search genomic databases

  • Even with incomplete genome data, fragments can guide further isolation efforts

• Gap filling and complete gene assembly:

  • Design new primers based on fragment sequences

  • Use additional PCR reactions to fill gaps and assemble the complete gene

  • Verify sequence integrity through multiple sequencing reactions

• Cloning considerations:

  • Include directional cloning adapters (e.g., NdeI and XhoI sites) in primers for efficient subcloning

  • Use vectors appropriate for subsequent expression (e.g., pET-15b for E. coli expression)

This strategic approach successfully yielded the complete 2988 bp pepc gene from Synechococcus PCC 7002, despite initial challenges with direct amplification of the full-length gene .

What expression systems are most effective for producing soluble, active Synechococcus PEPC?

Selecting the optimal expression system for Synechococcus PEPC requires balancing protein yield with solubility and activity. The following table compares expression strategies based on research findings:

For Synechococcus PEPC, a promising approach may be to combine codon optimization with strategies to enhance solubility in E. coli, such as:

• Using specialized E. coli strains designed for expressing difficult proteins (e.g., ArcticExpress, Rosetta-gami)

• Co-expressing molecular chaperones to assist proper folding

• Using fusion tags that enhance solubility (e.g., MBP, SUMO, TrxA)

• Optimizing induction conditions (lower IPTG concentration, reduced temperature during induction)

• Exploring native cyanobacterial hosts for expression when authentic post-translational modifications are required

How can site-directed mutagenesis be used to study PEPC function in Synechococcus?

Site-directed mutagenesis provides powerful insights into structure-function relationships of Synechococcus PEPC through targeted alterations of specific amino acids. Based on current knowledge of PEPC domains, the following strategic approach can be implemented:

• Targeting functionally critical residues:

  • Mutate invariant arginine residues in the GRGG repeats, which are associated with catalytic activity

  • The R683 residue in Synechococcus PCC 7002 PEPC (corresponding to R587 in E. coli) is particularly important for both catalysis and aspartate-mediated inhibition

  • Modify residues in the glycine-rich loop (FHGRGGXXGRGG) that positions substrates and protects reaction intermediates

• Investigating regulatory mechanisms:

  • Mutate amino acids in the aspartate-binding domains (EM(T/V)(L/F)(S/A)K, LRN(G/I)(T/Y), and MRNTG)

  • Compare effects with Anacystis nidulans PEPC, which contains the aspartate-binding site but is not inhibited by aspartate

  • Explore the molecular basis for differential regulation among cyanobacterial PEPCs

• Optimizing catalytic efficiency:

  • Design mutations based on comparison with more efficient PEPC variants

  • Target regions that differ between marine and freshwater cyanobacterial PEPCs

  • Create chimeric enzymes combining domains from different cyanobacterial PEPCs

• Experimental validation protocol:

  • Generate mutations using overlap extension PCR or commercial site-directed mutagenesis kits

  • Express wild-type and mutant proteins under identical conditions

  • Assess kinetic parameters (Km, Vmax, kcat) to quantify effects on enzyme function

  • Conduct inhibition studies to evaluate regulatory properties

  • Perform structural analyses (if possible) to correlate functional changes with structural alterations

Systematic application of this approach can elucidate the molecular basis for PEPC function in Synechococcus and potentially guide enzyme engineering efforts to enhance carbon fixation efficiency for biotechnology applications .

How can PEPC be engineered to enhance biofuel production in cyanobacteria?

• Optimizing PEPC expression levels:

  • Balance PEPC overexpression to enhance carbon fixation while preventing excessive diversion from pathways leading to biofuel precursors

  • Use tunable or inducible promoters (such as Ptrc) to fine-tune expression levels

  • Consider temporal regulation strategies to coordinate PEPC activity with growth phase and biofuel production

• Engineering PEPC regulatory properties:

  • Modify allosteric regulation sites to reduce inhibition by metabolites that accumulate during biofuel production

  • Create feedback-resistant variants that maintain activity under high product concentrations

  • Adjust sensitivity to activators to enhance activity under photosynthetic conditions

• Coordinated pathway engineering:

  • Combine PEPC engineering with enhanced expression of RuBisCO to maximize carbon fixation

  • Integrate PEPC modifications with downstream pathway engineering for specific biofuels

  • For example, coupling PEPC enhancement with fatty acid thioesterase expression (e.g., 'tesA) has shown promise for free fatty acid production

• Strain-specific optimization:

  • Tailor PEPC engineering strategies to the specific cyanobacterial strain, as demonstrated by the different responses to RuBisCO overexpression in Synechococcus sp. PCC 7002 versus S. elongatus PCC 7942

  • Consider native metabolic network architecture when designing PEPC interventions

• Novel carbon fixation pathways:

  • Incorporate PEPC into synthetic carbon fixation pathways such as the malonyl-CoA–oxaloacetate–glyoxylate (MOG) pathways, which theoretically offer higher efficiency than native pathways

  • Design hybrid pathways leveraging PEPC's superior carbon fixation efficiency compared to RuBisCO

Research has demonstrated that enhancing carbon fixation through complementary approaches (e.g., RuBisCO overexpression with its own promoter) can lead to more than threefold improvement in biofuel precursor production in Synechococcus sp. PCC 7002 . Similar strategies focusing on PEPC hold promise for further improving carbon flux toward biofuel synthesis.

What are the regulatory mechanisms affecting PEPC activity in Synechococcus sp.?

PEPC activity in Synechococcus sp. is regulated through multiple interconnected mechanisms that together fine-tune carbon fixation in response to environmental and metabolic conditions:

• Allosteric regulation:

  • Synechococcus PCC 7002 PEPC contains the aspartate-binding sites that typically mediate feedback inhibition in bacterial PEPCs

  • The conserved arginine residue (R683) plays a crucial role in this inhibition mechanism, with aspartate binding trapping the glycine-rich loop and preventing its contribution to catalysis

  • Other potential allosteric effectors include acetyl-CoA (activator) and malate (inhibitor), though their effects may vary among cyanobacterial PEPCs

• Transcriptional regulation:

  • The pepc gene lacks obvious -10 and -35 promoter sequences in the region upstream of the start codon, suggesting complex transcriptional regulation mechanisms

  • Expression likely responds to carbon availability and energy status of the cell

  • Possible coordination with other carbon fixation genes through shared regulatory elements

• Post-translational modifications:

  • While plant PEPCs are regulated by phosphorylation, evidence for similar modifications in cyanobacterial PEPCs is limited

  • Potential protein-protein interactions with regulatory partners may modulate activity

  • Oligomerization state changes could affect catalytic properties

• Environmental response mechanisms:

  • Light/dark transitions likely influence PEPC activity through indirect metabolic effects

  • Carbon dioxide concentration affects the balance between PEPC and RuBisCO pathways

  • Nutrient limitation (particularly nitrogen) may alter PEPC expression and activity to rebalance carbon and nitrogen metabolism

Understanding these regulatory mechanisms is essential for effective metabolic engineering of Synechococcus sp. for enhanced carbon fixation and biofuel production. The presence of regulatory elements shared with other bacteria (like the aspartate-binding site) alongside potentially unique cyanobacterial features highlights the evolutionary adaptation of PEPC regulation to the photosynthetic lifestyle .

How does the genomic context of the pepc gene compare across different cyanobacterial species?

Comparative genomic analysis of the pepc gene across cyanobacterial species reveals important insights about evolutionary conservation and potential functional differences:

Key observations from genomic context analysis:

• Evolutionary relationships: Synechococcus PCC 7002 PEPC shares higher sequence identity with freshwater cyanobacterial PEPCs despite being a marine organism, suggesting complex evolutionary history .

• Conserved functional domains: Despite sequence divergence, essential catalytic and regulatory domains are conserved across species, indicating strong selective pressure on functional regions .

• Regulatory divergence: The absence of obvious promoter elements in Synechococcus PCC 7002 compared to potential differences in other species suggests species-specific regulation mechanisms .

• Habitat-specific adaptations: Differences between marine and freshwater cyanobacterial PEPC sequences may reflect adaptation to specific environmental conditions, particularly carbon availability and salinity .

This comparative genomic context provides essential insights for designing heterologous expression strategies and understanding the functional evolution of PEPC across cyanobacterial lineages .

What factors affect the solubility of recombinant Synechococcus PEPC in heterologous expression systems?

The solubility challenges encountered with recombinant Synechococcus PEPC in heterologous expression systems stem from multiple factors that can be systematically addressed:

Potential solutions to address solubility issues include:

• Reducing expression rate through lower temperature (16-20°C) and reduced inducer concentration

• Co-expression with molecular chaperones (GroEL/ES, DnaK/J)

• Use of solubility-enhancing fusion partners (MBP, SUMO, thioredoxin)

• Exploration of alternative expression hosts with physiology more similar to cyanobacteria

• Development of specialized refolding protocols tailored to cyanobacterial PEPC

Systematic optimization of these parameters could potentially overcome the solubility challenges and yield functional recombinant Synechococcus PEPC for detailed biochemical characterization and biotechnological applications .