Recombinant Enterococcus faecalis 3-oxoacyl-[acyl-carrier-protein] synthase 3 (fabH)

What is the role of FabH in Enterococcus faecalis fatty acid synthesis?

FabH (3-oxoacyl-[acyl-carrier-protein] synthase 3) serves as a critical short-chain ketoacyl-acyl carrier protein synthase in E. faecalis, catalyzing the initial condensation reaction in the fatty acid synthesis pathway. This enzyme initiates the fatty acid elongation cycle by condensing acetyl-CoA with malonyl-ACP to form acetoacetyl-ACP. Unlike some other bacteria that can function with alternative enzymes, E. faecalis appears to require FabH for normal growth and fatty acid synthesis, as evidenced by studies on related organisms like Lactococcus lactis where FabH deletion strains showed severely compromised growth that required fatty acid supplementation .

The importance of FabH becomes apparent when examining its position in the fatty acid synthesis pathway. E. faecalis can derive phospholipid acyl chains either through de novo synthesis (requiring FabH) or by incorporating exogenous fatty acids through alternative pathways . When functioning in the de novo pathway, FabH provides the critical initial substrate that will be further processed by downstream enzymes like FabF (elongation), FabG (reduction), FabZ/FabA (dehydration), and FabI/FabK (enoyl reduction) to produce the complete fatty acid chains necessary for membrane phospholipid synthesis.

What experimental approaches are recommended for expressing recombinant E. faecalis FabH?

For successful expression of recombinant E. faecalis FabH, researchers should consider a multi-stage approach:

• Expression System Selection: While E. coli remains the preferred expression host for bacterial proteins, specialized strains like BL21(DE3) or Rosetta that supply rare codons may optimize expression. A comparative expression analysis between different strains is recommended to determine optimal yield.

• Vector Design: Incorporate a strong inducible promoter (IPTG-inducible T7 or arabinose-inducible BAD) and include affinity tags (His6 or GST) for purification. When designing your construct, consider including a precision protease cleavage site for tag removal if the tag might interfere with activity assays.

• Expression Conditions: Optimize through small-scale expression trials varying:

  • Induction temperature (15-37°C)

  • Inducer concentration (0.1-1.0 mM IPTG)

  • Duration of induction (3-24 hours)

  • Media composition (LB, TB, or defined media)

• Purification Strategy: Implement a multi-step purification process:

  • Initial capture via affinity chromatography (Ni-NTA for His-tagged protein)

  • Ion exchange chromatography for removing contaminating proteins

  • Size exclusion chromatography for final polishing and buffer exchange

• Activity Verification: Validate the recombinant enzyme's functionality using:

  • Spectrophotometric assays tracking substrate consumption/product formation

  • Radioisotope-based assays incorporating labeled acetyl-CoA or malonyl-ACP

  • Mass spectrometry to confirm product formation

This methodological approach would parallel expression strategies used for similar enzymes, such as those used in studies with FabK and FabI from E. faecalis where controlled expression was achieved using inducible systems and activity was verified through functional complementation and biochemical assays .

How do the dual enoyl-ACP reductases (FabI and FabK) interact with the FabH-initiated fatty acid synthesis pathway in E. faecalis?

E. faecalis possesses a distinctive feature in its fatty acid synthesis pathway: two structurally different enoyl-ACP reductases—FabI and FabK. This dual system has significant implications for the FabH-initiated pathway and presents interesting research questions regarding pathway regulation and flux.

FabI is the dominant enoyl-ACP reductase in E. faecalis, while FabK is weakly expressed due to poor translational initiation despite being located within the main fatty acid synthesis operon . FabI catalyzes the NADH-dependent reduction of enoyl-ACP, while FabK functions as a flavoprotein with the same catalytic activity. The differential expression creates a regulatory node that affects the balance between saturated and unsaturated fatty acid synthesis.

Experimental evidence demonstrates that deletion of fabI results in UFA auxotrophy, indicating that the weakly expressed FabK cannot fully compensate for FabI's role in fatty acid synthesis . Interestingly, deleting fabK increases the proportion of unsaturated fatty acids in membrane phospholipids, suggesting that FabK preferentially processes saturated fatty acid intermediates .

This complex interaction provides a metabolic control point where:

• Products from FabH enter both saturated and unsaturated fatty acid synthesis pathways

• FabI predominantly supports unsaturated fatty acid synthesis

• FabK competition potentially diverts intermediates away from the unsaturated pathway

Researchers studying FabH should consider designing experiments that track metabolic flux through these competing pathways, perhaps using isotope labeling combined with mass spectrometry to quantify how FabH-generated products are distributed between the FabI and FabK processing routes. This would illuminate how the initial condensation reaction catalyzed by FabH influences downstream pathway selection.

What role does FabH play in the regulatory network governed by the FabT transcription factor?

The FabT transcription factor in E. faecalis plays a central role in regulating fatty acid synthesis by binding to specific promoter regions and repressing transcription, particularly in response to exogenous fatty acids. Understanding how FabH fits into this regulatory network provides insights into metabolic control mechanisms.

FabT binds to the promoter regions of fabT (autoregulation), the fabI/fabO operon, as well as the promoter region controlling the large fatty acid synthesis operon that includes fabH . Electrophoretic mobility shift assays (EMSA) confirm that FabT binding to these promoters is enhanced by acyl-ACPs, particularly oleoyl-AcpB and to a lesser extent palmitoyl-AcpB .

The regulatory circuit functions as follows:

• Exogenous fatty acids (e.g., oleic acid) enter the cell and are activated by the Fak system to form acyl-phosphates

• Acyl-phosphates are converted to acyl-ACPs by PlsX

• These acyl-ACPs, particularly when bound to AcpB, enhance FabT binding to promoter regions

• Enhanced FabT binding represses transcription of fatty acid synthesis genes, including fabH

• Reduced FabH expression decreases de novo fatty acid synthesis when exogenous fatty acids are available

Experimental data demonstrate that the presence of oleic acid leads to a decrease in β-galactosidase expression from the fabT promoter and other FabT-regulated promoters, confirming this regulatory mechanism . This creates a feedback loop where products of the pathway (acyl-ACPs) regulate the expression of the enzymes that produce them, including FabH.

This regulatory network presents opportunities for metabolic engineering approaches that might target FabH expression to modulate fatty acid composition in E. faecalis membranes or potentially develop novel antimicrobial strategies that disrupt this regulatory circuit.

How does FabH activity influence the balance between saturated and unsaturated fatty acid synthesis in E. faecalis?

The balance between saturated and unsaturated fatty acids (SFA and UFA) in bacterial membranes is crucial for maintaining appropriate membrane fluidity and function. In E. faecalis, this balance involves complex interactions between multiple enzymes, with FabH playing a pivotal role as the initiator of fatty acid synthesis.

E. faecalis lacks the classical FabA/FabB system for UFA synthesis found in E. coli but instead employs FabN (a FabZ isozyme with additional isomerase activity) and FabO (a specialized β-ketoacyl-ACP synthase) for unsaturated fatty acid synthesis . The fabI gene is co-located with fabN and fabO, suggesting coordinated regulation of these UFA synthesis components .

The influence of FabH on SFA/UFA balance can be understood through several mechanisms:

• Substrate supply: FabH provides the initial β-ketoacyl-ACP substrate that enters both saturated and unsaturated pathways, making its activity a potential rate-limiting step for total fatty acid synthesis.

• Competition with specialized synthases: The products of FabH-catalyzed reactions can be channeled toward either SFA or UFA synthesis pathways. The relative activity of FabH compared to FabO likely influences this partitioning.

• Regulatory coupling: FabH expression is regulated by FabT alongside other fatty acid synthesis genes, creating coordinated responses to environmental conditions.

Experimental evidence from related organisms provides insights: in L. lactis, which has a single long-chain ketoacyl-ACP synthase (FabF) rather than the dual FabF/FabO system found in E. faecalis, overexpression of FabF can compensate for FabH deletion . This suggests flexibility in the initiating steps of fatty acid synthesis that might be exploited for metabolic engineering.

Researchers interested in FabH's role in SFA/UFA balance should consider designing experiments that:

• Manipulate FabH expression levels and assess changes in membrane fatty acid composition

• Examine FabH substrate specificity and its influence on downstream pathway selection

• Investigate potential protein-protein interactions between FabH and the specialized UFA synthesis enzymes

What molecular dynamics insights can inform structure-based inhibitor design for E. faecalis FabH?

Molecular dynamics (MD) simulations provide valuable insights into protein flexibility and conformational changes that cannot be captured by static crystal structures alone. For E. faecalis FabH, these approaches can reveal dynamic properties crucial for structure-based inhibitor design.

While specific MD studies on E. faecalis FabH are not mentioned in the search results, research on the E. coli FabH enzyme demonstrates the importance of MD simulations in understanding this enzyme class . The E. coli study revealed high conformational flexibility that significantly impacts inhibitor binding, a characteristic likely shared by E. faecalis FabH.

Key considerations for applying MD simulation to E. faecalis FabH include:

• Conformational space exploration: MD simulations over extended timescales (100+ ns) can reveal conformational states not captured in crystal structures. This comprehensive sampling is essential as the study on E. coli FabH showed that "they cannot be correctly modeled in any available X-ray structure, while by using our set of conformations extracted from the MD simulations, this task can be accomplished."

• Water molecule dynamics: The presence of water molecules in catalytic sites, particularly the oxyanion hole, correlates with conformational stability of structurally important loops . These interactions should be carefully analyzed in E. faecalis FabH simulations.

• Principal component analysis: Extracting concerted motions through principal component analysis can identify allosteric sites and communication pathways within the enzyme that might offer alternative targeting strategies.

• Cluster analysis for representative conformations: Selecting diverse enzyme conformations through cluster analysis provides a more robust ensemble for virtual screening campaigns than single structures .

• Refinement of docking poses: Short MD simulations (10-20 ns) can be employed to refine initial docking poses and assess the stability of protein-inhibitor complexes, followed by MM-PBSA calculations to predict binding energies .

For E. faecalis FabH inhibitor development, researchers should implement a workflow that includes:

• Extended MD simulations of the apo enzyme

• Clustering to identify diverse conformational states

• Ensemble docking against these states

• MD refinement of promising complexes

• Binding free energy calculations to prioritize candidates

This approach would address the limitations of rigid receptor docking and increase the likelihood of discovering inhibitors with actual binding potential, as demonstrated in the E. coli FabH study .

How does the dual acyl carrier protein (ACP) system in E. faecalis influence FabH function and fatty acid synthesis?

The functional separation between these ACPs is clear: "AcpA is thought to play the central carrier role in fatty acid synthesis whereas AcpB functions in incorporation of exogenous fatty acids" . This specialization affects how FabH interacts with the fatty acid synthesis machinery, particularly in environments where both de novo synthesis and exogenous fatty acid incorporation occur simultaneously.

The dual ACP system influences FabH function through several mechanisms:

• Substrate availability: FabH likely preferentially interacts with AcpA for de novo fatty acid synthesis, utilizing malonyl-AcpA as a substrate for the initial condensation reaction.

• Regulatory feedback: Acyl-AcpB formation via the Fak-PlsX system enhances FabT binding to promoter regions, including those controlling fabH expression . This creates a regulatory circuit where exogenous fatty acid incorporation through AcpB downregulates FabH expression and consequently de novo synthesis.

• Metabolic channeling: The dual ACP system may create distinct pools of intermediates that are channeled toward different metabolic fates, with FabH-initiated pathways primarily flowing through AcpA-bound intermediates.

Experimental evidence shows that both acyl-AcpA and acyl-AcpB can enhance FabT binding to promoter regions, but with different efficiencies . EMSA analysis demonstrates that oleoyl-AcpB and palmitoyl-AcpB enhance FabT binding to the fabT promoter, with oleoyl-AcpB showing stronger effects . Additionally, data from an E. faecalis ΔacpB strain indicates that acyl-AcpA can also function in this regulatory capacity, though potentially through different mechanisms .

The dual ACP system presents an interesting experimental target for researchers studying FabH. Investigation approaches might include:

What are the implications of FabH inhibition for developing novel antimicrobials against E. faecalis?

As an opportunistic pathogen associated with hospital-acquired infections that often display high levels of antibiotic resistance , E. faecalis presents a significant clinical challenge. The essential role of FabH in bacterial fatty acid synthesis makes it an attractive target for novel antimicrobial development.

The potential of FabH as an antimicrobial target against E. faecalis is supported by several factors:

• Essentiality: Studies in related organisms suggest FabH is essential for normal growth, as demonstrated by the growth defects in fabH deletion strains that require fatty acid supplementation .

• Lack of mammalian homologs: Mammals use a fundamentally different fatty acid synthesis system (type I FAS), reducing the likelihood of target-based toxicity.

• Structural distinctiveness: FabH has a unique catalytic mechanism involving a cysteine residue in the active site, offering opportunities for selective inhibition.

• Integration with membrane homeostasis: Inhibiting FabH would disrupt membrane phospholipid composition, potentially affecting multiple cellular processes simultaneously.

When designing FabH inhibitors against E. faecalis, researchers should consider:

• Structural flexibility challenges: As demonstrated in E. coli FabH studies, "high flexibility... makes difficult the structure-based design of FabH inhibitors" . MD simulations reveal conformational diversity not captured in crystal structures, necessitating an ensemble-based approach to virtual screening.

• Potential for exogenous fatty acid bypass: E. faecalis can incorporate exogenous fatty acids for membrane phospholipid synthesis , potentially bypassing FabH inhibition in fatty acid-rich environments like the gastrointestinal tract. Combination strategies targeting both de novo synthesis and exogenous incorporation pathways might be necessary.

• Specificity considerations: Targeting features unique to E. faecalis FabH rather than conserved features could enhance specificity and reduce impacts on beneficial microbiota.

• Resistance development: Potential mechanisms of resistance might include mutations in FabH that maintain function while preventing inhibitor binding, or upregulation of exogenous fatty acid incorporation pathways. These possibilities should inform inhibitor design strategies.

For experimental evaluation of potential FabH inhibitors, researchers should implement a tiered approach:

• Biochemical assays with recombinant enzyme

• Whole-cell activity testing against E. faecalis

• Specificity testing against mammalian cells and commensal bacteria

• Efficacy in infection models that mimic relevant physiological environments

What experimental approaches can distinguish between FabH activity and other ketoacyl-ACP synthases in E. faecalis?

Distinguishing the activity of FabH from other ketoacyl-ACP synthases (particularly FabF and FabO) in E. faecalis requires specialized experimental approaches that exploit the unique characteristics of each enzyme. This distinction is crucial for accurately assessing FabH's contribution to fatty acid synthesis.

FabH (KAS III) differs from FabF (KAS II) and FabO in several key aspects:

• FabH initiates fatty acid synthesis using acetyl-CoA and malonyl-ACP

• FabF and FabO elongate existing acyl-ACPs using malonyl-ACP

These functional differences enable several experimental strategies:

Substrate Specificity Assays:

• Pure enzyme assays: Using recombinant purified enzymes, compare activity with:

  • Acetyl-CoA + malonyl-ACP (FabH-specific initial condensation)

  • Acyl-ACP + malonyl-ACP (preferred by FabF/FabO for elongation)

• Selective inhibition: Cerulenin preferentially inhibits FabF/FabO at low concentrations while having minimal effect on FabH, allowing selective measurement of FabH activity in mixed systems.

• Chain-length specificity: FabH typically shows preference for short-chain substrates, while FabF/FabO prefer longer-chain substrates. Assays using acyl-ACPs of varying chain lengths can differentiate these activities.

Genetic Approaches:

• Conditional expression systems: Developing strains with controlled expression of fabH, fabF, or fabO allows selective manipulation of each enzyme's levels and observation of resulting metabolic effects.

• Reporter fusions: Creating transcriptional or translational fusions with reporter genes enables monitoring of expression patterns under various conditions, helping distinguish regulatory differences.

• Complementation studies: As demonstrated with L. lactis FabF, which "can functionally replace both FabB and FabF in E. coli" , complementation experiments can reveal functional overlaps and distinctions between these enzymes.

Analytical Methods:

• Isotope labeling: Using labeled acetyl-CoA or acyl-ACPs combined with mass spectrometry can trace the flow of substrates through different synthesis routes.

• Product profile analysis: Gas chromatography-mass spectrometry (GC-MS) analysis of fatty acid profiles under conditions where different synthases are selectively inhibited or depleted can reveal their distinct contributions.

These approaches would help researchers precisely characterize FabH's role in relation to other ketoacyl-ACP synthases in E. faecalis, providing insights into potential compensatory mechanisms and regulatory interactions among these enzymes.

What are the optimal conditions for assaying recombinant E. faecalis FabH activity in vitro?

Establishing optimal conditions for assaying recombinant E. faecalis FabH activity is essential for accurate enzyme characterization and inhibitor screening. The following parameters should be considered and systematically optimized:

Buffer Composition:

• pH range: 7.0-8.0 (typical optimum for FabH enzymes)

• Buffer systems: Phosphate, HEPES, or Tris-HCl (50-100 mM)

• Ionic strength: 50-150 mM NaCl or KCl

• Divalent cations: 5-10 mM MgCl₂ (often required for optimal activity)

• Reducing agents: 1-5 mM DTT or β-mercaptoethanol (to maintain active site cysteine)

Substrate Considerations:

• Acetyl-CoA concentration: 10-500 μM

• Malonyl-ACP concentration: 10-100 μM

• Source of ACP: Consider using E. faecalis AcpA rather than heterologous ACPs for authentic activity assessment

Detection Methods:

• Spectrophotometric coupled assays: Monitor NADH oxidation coupled to β-ketoacyl-ACP reduction by FabG

• Radioactive assays: Use [¹⁴C]-labeled substrates to track product formation

• HPLC-based assays: Separate and quantify reaction products

• Mass spectrometry: Identify products and determine kinetic parameters with high precision

Optimization Strategy:

• Perform initial activity screens across broad ranges of pH (6.0-9.0) and temperature (25-45°C)

• Conduct factorial experiments to identify interactions between buffer components

• Determine Michaelis-Menten parameters (Km, Vmax) for each substrate under optimized conditions

• Assess product inhibition and develop appropriate assay timeframes to maintain initial rate conditions

Practical Considerations:

• Enzyme stability may be enhanced by addition of glycerol (10-20%)

• BSA (0.1-1.0 mg/mL) may prevent surface adsorption and activity loss

• Pre-incubation times and temperatures should be evaluated for optimal activity

• Appropriate negative controls (heat-inactivated enzyme, catalytic site mutants) are essential

The optimized assay conditions should be validated by demonstrating reproducibility, linearity with enzyme concentration, and sensitivity to known inhibitors or active-site modifications. These validated conditions can then serve as the foundation for inhibitor screening and mechanistic studies of E. faecalis FabH.

How can structural comparisons between E. faecalis FabH and other bacterial FabH enzymes inform targeted inhibitor design?

Structural comparison between E. faecalis FabH and other bacterial FabH enzymes can reveal both conserved features essential for function and species-specific variations that might be exploited for selective inhibition. This comparative approach is particularly valuable given the challenges of FabH flexibility noted in previous studies .

Key aspects for structural comparison include:

Conserved Features:

Variable Features:

• Substrate specificity determinants: Residues lining the acyl-binding pocket vary between species, influencing chain-length preference and branched-chain capability. E. faecalis-specific residues might offer targeting opportunities.

• Surface loops: Regions involved in protein dynamics and possibly protein-protein interactions show significant variation between species. MD simulations have demonstrated that "the conformational stability of structural important loops" is critical for function .

• Allosteric sites: Potential regulatory sites that influence conformational changes or dimer interface stability may differ between species.

Methodological Approach for Comparative Analysis:

• Sequence alignment and homology modeling: Generate accurate structural models of E. faecalis FabH based on existing bacterial FabH crystal structures.

• MD simulation of species-specific dynamics: Apply the approach described for E. coli FabH where "a 100 ns MD simulation of the unliganded enzyme" revealed "concerted motions that take place along the principal components of displacement" .

• Binding site mapping: Identify species-specific pockets or conformational states unique to E. faecalis FabH that could be targeted for selective inhibition.

• Fragment-based screening: Using representative conformations from MD trajectories, conduct virtual screening with diverse fragment libraries to identify moieties with preferential binding to E. faecalis FabH.

• Molecular interaction fingerprinting: Analyze interaction patterns of known inhibitors across different bacterial FabH enzymes to identify specificity determinants.

This comparative structural approach would enable the design of inhibitors that exploit unique features of E. faecalis FabH while maintaining activity against the core catalytic machinery, potentially leading to new antimicrobial agents with reduced propensity for resistance development.

What are the emerging technologies for studying FabH function in the context of whole-cell fatty acid synthesis?

Emerging technologies are transforming our ability to study FabH function within the context of intact cellular fatty acid synthesis pathways. These approaches move beyond traditional in vitro enzyme assays to capture the complexity of pathway interactions and regulation in living cells.

Advanced Genetic Tools:

• CRISPR interference (CRISPRi): Enables titratable repression of fabH expression without complete gene deletion, allowing investigation of partial loss-of-function phenotypes and dose-dependent effects on fatty acid synthesis.

• Inducible degradation systems: Protein degradation tags (e.g., AID, mDHFR) permit rapid depletion of existing FabH protein, revealing immediate consequences for pathway flux without allowing compensatory mechanisms to develop.

• Optogenetic control: Light-responsive gene expression or protein interaction systems provide spatial and temporal control over FabH activity, enabling precise perturbation experiments.

Metabolic Flux Analysis:

• Stable isotope labeling: Using ¹³C-labeled glucose or acetate combined with mass spectrometry to trace carbon flow through the fatty acid synthesis pathway before and after FabH perturbation.

• Flux balance analysis: Computational modeling of fatty acid synthesis incorporating experimental data to predict systemic responses to FabH modulation.

• Metabolic flux ratio analysis: Determining the relative contributions of de novo synthesis versus exogenous fatty acid incorporation under various conditions, similar to studies showing "the fabH bypass strain that overproduced FabF and the wild type strain incorporated much less exogenous octanoate into long chain phospholipid fatty acids" .

Systems Biology Approaches:

• Multi-omics integration: Combining transcriptomics, proteomics, and lipidomics to build comprehensive models of how FabH activity influences global cellular processes.

• Protein-protein interaction mapping: Techniques like proximity labeling (BioID, APEX) can identify proteins that physically interact with FabH in living cells, revealing potential regulatory partners.

• Single-cell analyses: Microfluidic approaches combined with fluorescent reporters to monitor cell-to-cell variability in fatty acid synthesis and responses to FabH perturbation.

Advanced Analytical Methods:

• Lipidomics with ion mobility-mass spectrometry: Provides enhanced separation and identification of closely related lipid species, enabling detailed characterization of how FabH perturbation affects membrane composition.

• In vivo activity-based protein profiling: Chemical probes that selectively label active FabH in living cells, allowing quantification of functionally active enzyme rather than total protein.

• Cryo-electron tomography: Visualizing the spatial organization of fatty acid synthesis machinery within cells to understand how FabH integrates with other pathway components.

These emerging technologies will help researchers unravel the complex role of FabH within the cellular context, potentially revealing new regulatory mechanisms and interactions that could be exploited for antimicrobial development or metabolic engineering applications.

How might synthetic biology approaches leverage E. faecalis FabH for production of novel fatty acid derivatives?

Synthetic biology approaches offer exciting possibilities for repurposing E. faecalis FabH to produce novel fatty acid derivatives with applications in biofuels, specialty chemicals, pharmaceuticals, and biomaterials. The unique properties of this enzyme can be exploited through various engineering strategies.

Engineering FabH Substrate Specificity:

• Active site mutagenesis: Targeted modifications of the substrate binding pocket could alter chain-length specificity or enable acceptance of non-natural substrates, similar to approaches used with other ketoacyl-ACP synthases.

• Domain swapping: Creating chimeric enzymes by swapping domains between E. faecalis FabH and other bacterial synthases to generate novel substrate preferences.

• Directed evolution: Implementing high-throughput screening systems to select for FabH variants with desired catalytic properties for producing specific fatty acid derivatives.

Pathway Engineering Strategies:

• Modular pathway assembly: Combining engineered E. faecalis FabH with other fatty acid synthesis enzymes from diverse sources to create synthetic pathways for novel products. This could leverage the finding that "L. lactis FabF can functionally replace both FabB and FabF in E. coli" , suggesting flexibility in enzyme compatibility across species.

• Dynamic pathway regulation: Implementing synthetic regulatory circuits to control expression of FabH and downstream enzymes, optimizing production of target compounds.

• Precursor pool manipulation: Engineering precursor supply pathways to provide novel starter units for FabH, expanding the diversity of possible products.

Specific Applications:

• Branched-chain fatty acids: Engineering E. faecalis FabH to accept branched-chain acyl-CoA substrates derived from amino acid metabolism could produce branched-chain fatty acids with improved biofuel properties.

• Hydroxylated or functionalized fatty acids: Combining engineered FabH with downstream modifying enzymes to introduce specific functional groups at defined positions.

• Odd-chain fatty acids: Optimizing FabH to utilize propionyl-CoA as a starter unit would lead to odd-chain fatty acids with unique properties for specialty chemical applications.

• Medium-chain fatty acids: Engineering FabH and pathway regulation to terminate fatty acid elongation at medium chain lengths (C8-C12) for applications in surfactants and cosmetics.

Implementation Considerations:

• Host selection: Choosing appropriate expression hosts that can supply necessary cofactors and tolerate altered membrane compositions. E. faecalis itself might be suitable given its ability to incorporate diverse fatty acids into membrane phospholipids .

• Product toxicity management: Implementing export systems or sequestration strategies to mitigate potential toxicity of novel fatty acid derivatives.

• Process optimization: Developing fermentation strategies that balance growth with product formation, potentially using two-phase systems to capture hydrophobic products.