Recombinant Synechocystis sp. Enoyl-[acyl-carrier-protein] reductase [NADH] FabI (fabI)

What is the enzymatic function of FabI in Synechocystis sp.?

FabI in Synechocystis sp., like in other organisms, functions as an enoyl-ACP reductase that catalyzes the reduction of trans-2-acyl-ACP substrates (enoyl-ACP) to acyl-ACP using either NADH or NADPH as the reductant. This reaction represents the final and rate-determining step in the elongation cycle of fatty acid synthesis (FAS II) in bacteria. The reaction involves a nucleophilic conjugate addition of a hydride ion from the 4S hydrogen position of the nicotinamide ring of either NADH or NADPH to the C3 position (Cβ) of the α,β-unsaturated thioester of the enoyl-ACP substrate, followed by protonation at C2 (Cα) of the enzyme-stabilized enolate intermediate .

Does Synechocystis FabI demonstrate cofactor preference between NADH and NADPH?

Based on research with E. coli FabI, which was previously thought to have separate NADH-dependent and NADPH-dependent enoyl-ACP reductase activities attributed to different proteins, it was later demonstrated that both activities reside in a single FabI protein. When FabI is purified in pH 6.5 buffers, the protein exhibits both NADH and NADPH-dependent activities . Similar dual cofactor utilization may exist in Synechocystis FabI, though specific biochemical characterization would be necessary to confirm this property for the Synechocystis enzyme.

How does the mechanism of inhibition by FabI-targeting compounds differ between Synechocystis and other bacterial species?

The mechanism of inhibition varies based on both the specific inhibitor and the bacterial species. For instance, fabimycin demonstrates nanomolar potency against FabI in Gram-negative bacteria like E. coli and demonstrates enhanced stability of the enzyme-inhibitor complex, as confirmed by differential scanning fluorimetry (DSF) experiments. The molecular interactions involved in fabimycin binding include hydrogen-bond interactions with critical residues such as Ala95 and Tyr156 .

For Synechocystis FabI specifically, the inhibition mechanism may share similarities with that observed in E. coli but could also possess unique features due to potential structural differences. Comparative analysis of crystal structures of inhibitor-bound FabI from different species would be necessary to fully elucidate these differences.

How does photosynthetic activity in Synechocystis influence FabI expression and function compared to heterotrophic bacteria?

In Synechocystis sp. PCC 6803, a photoautotrophic cyanobacterium, metabolic processes are tightly linked to photosynthetic activity. The CRISPR interference (CRISPRi) screening of Synechocystis revealed that there are far fewer diurnal-specific fitness genes compared to previously published data from Synechococcus PCC 7942 . This suggests that Synechocystis may have different regulatory mechanisms governing metabolic enzyme expression under varying light conditions.

For fatty acid synthesis specifically, key enzymes in central carbon metabolism that supply precursors for fatty acid synthesis (such as pyruvate) are regulated differently in Synechocystis compared to heterotrophic bacteria. For example, pyruvate production in Synechocystis has been predicted to occur mainly through decarboxylation of oxaloacetate by NADP-dependent malic enzyme rather than dephosphorylation of phosphoenolpyruvate by pyruvate kinase . This distinct central carbon metabolism likely influences the regulation and function of FabI in Synechocystis.

What role does FabI play in engineered Synechocystis strains designed for biofuel production?

In engineered Synechocystis strains designed for biofuel production, particularly those producing fatty acid-derived products, FabI represents a critical control point due to its role in the rate-determining step of fatty acid elongation. CRISPRi screening of Synechocystis has revealed that targeting certain genes involved in nutrient assimilation, central carbon metabolism, and cyclic electron flow can enhance productivity, even when targeting essential genes that are difficult to access by transposon insertion .

For L-lactate production specifically, a study transformed a CRISPRi library into an L-lactate-secreting Synechocystis strain and identified that repression of genes related to cyclic electron flow and central carbon metabolism enhanced lactate production . While FabI was not specifically mentioned as a target in this context, its role in fatty acid metabolism makes it a potential target for similar approaches aimed at redirecting carbon flux toward desired bioproducts.

What are the optimal conditions for expressing recombinant Synechocystis FabI in heterologous systems?

For optimal expression of recombinant Synechocystis FabI, researchers should consider:

• Expression System Selection: E. coli BL21(DE3) is commonly used for expressing cyanobacterial proteins due to its lack of key proteases and compatibility with T7 expression systems.

• Codon Optimization: Synechocystis sp. PCC 6803 has different codon usage preferences compared to E. coli. Codon optimization of the fabI gene for the expression host can significantly improve protein yields.

• Induction Conditions: For IPTG-inducible systems, lower temperatures (16-20°C) during induction often result in better folding of cyanobacterial proteins. Typical induction conditions would include 0.1-0.5 mM IPTG at OD600 of 0.6-0.8, followed by expression at 18°C for 16-20 hours.

• Buffer Composition: Based on studies with E. coli FabI, purification in pH 6.5 buffers helps maintain both NADH and NADPH-dependent activities . A typical buffer might contain 50 mM sodium phosphate (pH 6.5), 300 mM NaCl, and 10% glycerol.

• Protein Solubility: Addition of solubility-enhancing tags such as MBP (maltose-binding protein) or SUMO can improve the soluble expression of FabI if initial attempts yield primarily insoluble protein.

What are the recommended protocols for measuring Synechocystis FabI activity in vitro?

For measuring Synechocystis FabI activity:

• Spectrophotometric Assay: The standard assay monitors the oxidation of NAD(P)H at 340 nm as it donates a hydride ion to the enoyl-ACP substrate. The reaction mixture typically contains:

  • 100 mM sodium phosphate buffer (pH 7.0)

  • 200 μM NAD(P)H

  • 100-200 μM crotonyl-CoA (a model substrate)

  • 1-5 μg purified FabI enzyme

  Activity is calculated using the extinction coefficient of NAD(P)H (6,220 M⁻¹cm⁻¹).

• Substrate Considerations: While crotonyl-CoA is commonly used as a model substrate, physiologically relevant enoyl-ACP substrates of varying chain lengths can provide insights into chain-length specificity.

• Cofactor Preference: Testing both NADH and NADPH at various concentrations (typically 50-500 μM) is essential to determine cofactor preference and kinetic parameters for each.

• Inhibition Studies: IC₅₀ determinations for potential inhibitors typically involve pre-incubation of the enzyme with the inhibitor for 10-30 minutes before initiating the reaction with substrate and cofactor.

How can natural transformation be used to generate Synechocystis fabI mutants?

Natural transformation is an effective method for generating Synechocystis sp. PCC 6803 mutants, including those affecting the fabI gene. The protocol involves:

• Plasmid Construction: Design a plasmid containing homology arms flanking the fabI gene (approximately 500-1000 bp each) with the desired modification (point mutation, deletion, or insertion of a resistance cassette). The plasmid can be constructed by amplifying the homology regions using PCR and ligating them into a suitable vector such as pJET1.2/blunt .

• Transformation Protocol:

  • Grow Synechocystis cells to mid-logarithmic phase (OD₇₃₀ = 0.5-0.8)

  • Harvest cells by centrifugation and resuspend in fresh BG11 medium at approximately 10⁹ cells/ml

  • Add 1-5 μg of plasmid DNA to 200 μl of cell suspension

  • Incubate cells with DNA at 30°C under standard light conditions for 4-6 hours

  • Plate cells on BG11 agar containing the appropriate antibiotic for selection

  • Re-streak colonies on selective media 3-4 times to ensure complete segregation

• Verification: Verify integration and complete segregation using colony PCR with primers flanking the targeted region. Complete segregation is crucial for phenotypic analysis since Synechocystis contains multiple genome copies .

How should researchers address contradictory findings regarding FabI cofactor specificity in Synechocystis?

When faced with contradictory findings regarding FabI cofactor specificity:

• Buffer Conditions: As demonstrated with E. coli FabI, buffer pH significantly influences cofactor preference. Purification and assays conducted at pH 6.5 may reveal dual NADH/NADPH activity that might be obscured at different pH values .

• Protein Purity: Ensure high protein purity (>95% by SDS-PAGE) to rule out contamination by other reductases that might contribute to observed activities.

• Site-Directed Mutagenesis: Generate mutants targeting residues in the cofactor binding pocket predicted to interact specifically with either NADH or NADPH. This approach can help determine which residues are responsible for cofactor discrimination.

• Structural Analysis: If contradictory findings persist, X-ray crystallography of Synechocystis FabI with both cofactors can provide definitive evidence of binding modes and potential dual specificity.

• Kinetic Characterization: Comprehensive kinetic analysis including determination of kcat and Km values for both cofactors under identical conditions can quantitatively assess true cofactor preferences.

What are the common pitfalls in interpreting phenotypic data from Synechocystis fabI mutants?

When interpreting phenotypic data from Synechocystis fabI mutants, researchers should be aware of:

• Incomplete Segregation: Synechocystis contains multiple genome copies, and incomplete segregation can result in mixed genotypes that obscure phenotypic effects. Always verify complete segregation through multiple rounds of selection and PCR verification .

• Polar Effects in Operons: Modification of fabI may have unintended consequences on downstream genes if it resides in an operon, similar to how interpretation of pyk2 (sll1275) fitness in Synechocystis is complicated by its position in an operon .

• Metabolic Redundancy: Alternative pathways might compensate for reduced FabI activity. For example, in the Calvin cycle of Synechocystis, two energetically equivalent routes for synthesis of fructose-6-phosphate exist through different enzymes . Similar redundancy might exist for aspects of fatty acid metabolism.

• Crosstalk with Other Metabolic Systems: Changes in fatty acid metabolism due to fabI mutations may affect seemingly unrelated processes. The CRISPRi screening of Synechocystis revealed that targeting genes in amino acid metabolism and protein biosynthesis improved tolerance to L-lactate , highlighting the complex interplay between different metabolic systems.

• Growth Condition Specificity: Phenotypes may manifest differently under various growth conditions (light intensity, carbon source, temperature). A comprehensive phenotypic analysis should include multiple growth conditions to fully characterize the impact of fabI mutations.

How can researchers differentiate between the direct effects of FabI inhibition and indirect metabolic consequences in Synechocystis?

To differentiate between direct effects of FabI inhibition and indirect metabolic consequences:

• Time-Course Analysis: Rapid changes (minutes to hours) following inhibitor addition likely represent direct effects on FabI, while changes occurring later (hours to days) may reflect downstream metabolic adjustments.

• Metabolomic Profiling: Comprehensive metabolomic analysis can reveal the accumulation of specific intermediates. Direct FabI inhibition should cause accumulation of trans-2-enoyl-ACP intermediates, while changes in distal metabolites indicate indirect effects.

• Complementation Studies: Express a heterologous FabI (e.g., from E. coli) that is resistant to the specific inhibitor being studied. If the phenotype is rescued, the effects can be attributed specifically to FabI inhibition.

• Isotope Labeling: Use isotope-labeled precursors (e.g., ¹³C-bicarbonate) to track carbon flux through fatty acid synthesis and related pathways following FabI inhibition, distinguishing direct blocks in fatty acid elongation from indirect metabolic rerouting.

• Comparative Analysis with Known Inhibitors: Compare the phenotypic and metabolic profiles induced by new potential FabI inhibitors with those caused by well-characterized FabI inhibitors like diazaborines , which are known to target FabI specifically.

How might CRISPR interference approaches be used to study FabI function in Synechocystis?

CRISPR interference (CRISPRi) provides powerful approaches for studying FabI function in Synechocystis:

• Tunable Repression: Unlike knockout approaches that may be lethal for essential genes like fabI, CRISPRi allows tunable repression levels, enabling the study of essential gene functions. This approach has been successfully implemented in a pooled CRISPRi screening of Synechocystis sp. PCC 6803 .

• Temporal Control: By using inducible promoters to control dCas9 expression, researchers can implement temporal control of fabI repression, allowing the study of fatty acid synthesis dynamics during different growth phases.

• Multi-Gene Targeting: CRISPRi libraries targeting multiple genes simultaneously can reveal genetic interactions between fabI and other metabolic genes, potentially uncovering novel regulatory relationships .

• Fitness Profiling: By tracking the composition of a CRISPRi library during growth under different conditions (e.g., different light regimes, presence of fatty acid synthesis inhibitors), researchers can quantify the fitness contribution of fabI under various physiological states .

• Production Strain Engineering: For biotechnology applications, CRISPRi targeting of fabI could be used to redirect carbon flux from fatty acid synthesis to desired products, as demonstrated with L-lactate production in Synechocystis .

What opportunities exist for developing FabI-targeted antimicrobials specific to cyanobacteria?

Developing FabI-targeted antimicrobials specific to cyanobacteria presents several opportunities:

• Structural Divergence Exploitation: While FabI is conserved across many bacteria, cyanobacterial FabI may possess unique structural features that could be targeted by selective inhibitors, similar to how fabimycin was designed to target Gram-negative bacterial FabI .

• Photosynthesis-Metabolism Interface: Cyanobacteria like Synechocystis have unique metabolic interfaces between photosynthesis and fatty acid synthesis that are absent in heterotrophic bacteria. Compounds targeting these interfaces could provide selectivity.

• Cofactor Preference Differences: If Synechocystis FabI demonstrates different cofactor preferences compared to FabI from heterotrophic bacteria, this could be exploited for selective inhibition.

• Combination Approaches: Targeting FabI in combination with inhibitors of cyanobacteria-specific processes could provide synergistic effects specific to these organisms.

• Environmental Applications: Selective inhibition of cyanobacterial FabI could have applications in controlling harmful algal blooms without disrupting beneficial heterotrophic bacterial communities.

How does FabI activity integrate with the unique photoautotrophic metabolism of Synechocystis?

FabI activity in Synechocystis integrates with photoautotrophic metabolism through several mechanisms:

• Redox Balance: The choice between NADH and NADPH as cofactors for FabI may play a role in maintaining cellular redox balance, which is particularly important in photosynthetic organisms where light-dependent reactions generate NADPH.

• Membrane Lipid Remodeling: Synechocystis modifies its membrane lipid composition in response to environmental conditions, particularly light intensity and temperature. FabI, as a key enzyme in fatty acid synthesis, likely plays a critical role in this adaptive response.

• Carbon Partitioning: In photoautotrophic growth, carbon fixed through photosynthesis must be partitioned between different metabolic pathways. The regulation of FabI activity would influence how much carbon is directed toward fatty acid synthesis versus other pathways.

• Cyclic Electron Flow: CRISPRi screening in Synechocystis has revealed that several clones with increased growth rates have a common upregulation of genes related to cyclic electron flow . This process generates ATP without producing NADPH, potentially affecting the NADPH/NADH ratio available for FabI activity.

• Diurnal Regulation: Unlike many heterotrophic bacteria, cyanobacteria experience diurnal cycles that affect their metabolism. The study of Synechocystis found fewer diurnal-specific fitness genes compared to Synechococcus , suggesting that Synechocystis may have evolved different regulatory mechanisms for enzymes like FabI to maintain metabolic homeostasis across light-dark transitions.