Recombinant Methylobacterium sp. Acyl carrier protein (acpP)

What is Methylobacterium sp. acyl carrier protein (acpP) and what role does it play in bacterial metabolism?

Acyl carrier protein (acpP) in Methylobacterium sp. functions as a critical component of fatty acid biosynthesis (FAS), serving as a carrier for growing fatty acid chains during synthesis. The protein contains a conserved DSL motif where the serine residue serves as the active site for phosphopantetheinylation, the post-translational modification that converts inactive apo-ACP to active holo-ACP . The phosphopantetheinyl arm provides a flexible attachment point where acyl intermediates are linked during fatty acid elongation cycles. In most bacteria, including Methylobacterium sp., acpP is essential for survival as it participates in primary metabolism through type II fatty acid synthase systems. Like AcpP1 in Ralstonia solanacearum, Methylobacterium sp. acpP is likely encoded by a gene co-transcribed with other fatty acid biosynthesis genes in a conserved cluster .

How does recombinant production affect the structure and function of Methylobacterium sp. acpP?

When properly phosphopantetheinylated, recombinant acpP retains the structural features necessary for interaction with fatty acid synthase enzymes. The three-dimensional structure maintains the characteristic four-helix bundle typical of bacterial ACPs, with the conserved serine in the DSL motif positioned for modification . Using serum-free mammalian expression systems for recombinant production provides highly pure protein with minimal contaminants and low endotoxin levels, beneficial for downstream applications .

What experimental methods can verify the identity and purity of recombinant Methylobacterium sp. acpP?

Verification of recombinant Methylobacterium sp. acpP identity and purity requires a multi-method approach:

• SDS-PAGE Analysis: Demonstrates protein purity and apparent molecular weight, typically showing a band at approximately 9-10 kDa for acpP. Native PAGE can distinguish between apo- and holo-forms based on their differential migration patterns .

• Mass Spectrometry: Provides precise molecular weight determination and can confirm post-translational modifications. The mass increase of 340 Da confirms successful phosphopantetheinylation of the conserved serine residue.

• Western Blotting: Using antibodies against the acpP or attached epitope tags verifies protein identity.

• Circular Dichroism (CD) Spectroscopy: Confirms proper secondary structure formation, characterized by the distinctive alpha-helical content of acpP.

• Activity Assays: Functional verification through in vitro acylation assays using acyl-ACP synthetases (such as VhAasS) that demonstrate the ability of holo-acpP to be acylated with fatty acids in the presence of ATP .

What expression systems are most effective for producing functional recombinant Methylobacterium sp. acpP?

The most effective expression systems for producing functional recombinant Methylobacterium sp. acpP include:

For optimal results, co-expression with a compatible phosphopantetheinyl transferase (PPTase) such as Sfp from Bacillus subtilis or AcpS from E. coli is recommended to ensure conversion of apo-acpP to the active holo-form . Expression at lower temperatures (16-18°C) after induction can increase solubility and proper folding. Addition of 4-10% glycerol to lysis buffers helps stabilize the protein during purification. Affinity tags (particularly His6) positioned at the N-terminus rather than C-terminus generally preserve functionality better, as the C-terminal region of acpP can be involved in protein-protein interactions.

How can site-directed mutagenesis be used to investigate the function of conserved residues in Methylobacterium sp. acpP?

Site-directed mutagenesis represents a powerful approach for investigating the structure-function relationships in Methylobacterium sp. acpP. Based on findings from other bacterial ACPs, several strategic mutations can provide valuable insights:

• Active Site Serine Mutation (S36T or S36A): Modifying the conserved serine in the DSL motif to threonine or alanine prevents phosphopantetheinylation, as demonstrated in Ralstonia solanacearum ACPs . This mutation creates a negative control that cannot be converted to holo-acpP, useful for validating activity assays and interaction studies.

• Conserved Acidic Residue Mutations (D35V, D38V, E41A): These residues typically participate in electrostatic interactions with AcpS during phosphopantetheinylation. Mutation of the aspartate at position 39 to valine in R. solanacearum AcpP1 significantly impaired growth , suggesting these residues are critical for function.

• Acyl Chain Interaction Mutations (I54A, F28A): These residues often form the hydrophobic pocket that accommodates the acyl chain. In R. solanacearum, mutation of Ile55 to alanine impaired growth , suggesting altered acyl chain binding or interactions with pathway enzymes.

The mutagenesis procedure should follow established protocols using overlap extension PCR with primers containing the desired mutations. Mutated genes can be cloned into expression vectors with appropriate tags for purification. After expression and purification, the mutant proteins should be assessed for:

• Ability to be phosphopantetheinylated by PPTases using gel shift assays

• Capacity to be acylated by acyl-ACP synthetases

• Interaction with fatty acid synthesis enzymes using pull-down assays

• Complementation of acpP-deficient bacterial strains to assess in vivo functionality

What techniques can be employed to study the interactions between recombinant Methylobacterium sp. acpP and fatty acid synthesis enzymes?

Several sophisticated techniques can be employed to characterize the interactions between recombinant Methylobacterium sp. acpP and fatty acid synthesis enzymes:

• Surface Plasmon Resonance (SPR): Provides real-time, label-free measurement of binding kinetics between acpP and FAS enzymes. By immobilizing either acpP or the enzyme partner on a sensor chip, association and dissociation rate constants (kon and koff) can be determined, yielding equilibrium dissociation constants (KD).

• Isothermal Titration Calorimetry (ITC): Measures the heat released or absorbed during binding, providing thermodynamic parameters (ΔH, ΔS, ΔG) in addition to binding constants.

• Nuclear Magnetic Resonance (NMR) Spectroscopy: Especially useful for acpP due to its relatively small size (~10 kDa). Chemical shift perturbation experiments can map interaction interfaces when 15N-labeled acpP is titrated with unlabeled enzyme partners.

• Crosslinking Mass Spectrometry: Uses bifunctional crosslinking reagents to capture transient protein-protein interactions, followed by mass spectrometric analysis to identify contact residues.

• Complementation Assays: Examines the ability of Methylobacterium sp. acpP to functionally replace acpP in heterologous systems. Similar to how R. solanacearum AcpP1 partially restored growth to an E. coli acpP mutant strain (CY1877) , Methylobacterium sp. acpP can be tested in various bacterial systems to assess functional compatibility with different FAS machineries.

• FRET-based Assays: Using fluorescently labeled acpP and enzyme partners to monitor real-time interactions in solution or in living cells.

Experimental data from these approaches should be integrated to develop comprehensive interaction models. For example, residues identified by NMR as participating in binding can be confirmed through site-directed mutagenesis followed by SPR or ITC to quantify the impact on binding affinity.

What are the optimal conditions for phosphopantetheinylation of recombinant Methylobacterium sp. acpP and how can this modification be verified?

Optimal conditions for phosphopantetheinylation of recombinant Methylobacterium sp. acpP depend on the PPTase used and require careful optimization:

Phosphopantetheinylation Protocol:

• PPTase Selection:

  • Broad-specificity PPTases like B. subtilis Sfp typically modify ACPs more efficiently than narrow-specificity enzymes like E. coli AcpS

  • Recommended ratio: 1:10 (PPTase:acpP) for Sfp; 1:5 for AcpS

• Reaction Buffer Composition:

  • 50 mM Tris-HCl (pH 8.0)

  • 10 mM MgCl₂ (essential for PPTase activity)

  • 5 mM DTT (maintains reduced state)

  • 125 μM CoA (substrate for phosphopantetheinyl transfer)

  • Optional: 10% glycerol to enhance stability

• Reaction Conditions:

  • Temperature: 30°C for Sfp; 37°C for AcpS

  • Duration: 1-4 hours (Sfp typically requires less time)

  • Protein concentration: 50-100 μM acpP

Verification Methods:

• Conformational Gel Shift Assay: Holo-acpP migrates differently from apo-acpP on native PAGE or urea-PAGE gels due to the addition of the phosphopantetheinyl group .

• Mass Spectrometry:

  • MALDI-TOF MS: Shows mass increase of 340 Da (phosphopantetheinyl moiety)

  • LC-MS/MS: Can identify the modified serine residue in tryptic digests

• Fluorescent Labeling: Reaction of holo-acpP with thiol-reactive fluorescent probes that specifically bind to the terminal thiol of the phosphopantetheinyl arm.

• Functional Assay: Incubation with acyl-ACP synthetase (e.g., VhAasS), fatty acid, and ATP should result in acylation of only correctly modified holo-acpP, detectable by gel shift or mass spectrometry .

Data from R. solanacearum ACPs shows that mutating the conserved serine in the DSL motif prevents phosphopantetheinylation completely (as seen with AcpP1 S36T), providing a useful negative control for verification procedures .

How does the structure of Methylobacterium sp. acpP influence its specificity in various biosynthetic pathways?

The structure of Methylobacterium sp. acpP likely plays a crucial role in determining its pathway specificity through several key features:

• Surface Charge Distribution: The distribution of charged residues on the acpP surface creates an electrostatic recognition pattern for specific interaction partners. Similar to observations in other bacterial ACPs, Methylobacterium sp. acpP likely has a predominantly negatively charged surface that facilitates recognition by synthetases and other pathway enzymes.

• Acyl Chain Binding Pocket: The hydrophobic pocket that accommodates the growing acyl chain affects substrate specificity. The size and shape of this pocket determine the length and branching pattern of acyl chains the protein can efficiently carry. In R. solanacearum, residues like Ile55 are critical for this function, as mutations at this position impaired growth significantly .

• Helix II-Helix III Interface: This region often undergoes conformational changes upon acylation and is crucial for enzyme recognition. The specific amino acid sequence in this region affects which pathway enzymes can productively interact with the acpP.

Studies on ACPs from various bacteria have shown that even small variations in ACP structure can direct the protein toward different pathways. For example, in R. solanacearum, only AcpP1 functions in fatty acid synthesis, while AcpP3 appears capable of carrying acyl chains but likely participates in secondary metabolite production . Similar structural determinants likely exist in Methylobacterium sp. acpP.

To experimentally determine pathway specificity, complementation assays in different bacterial systems could be performed. For instance, testing whether Methylobacterium sp. acpP can:

• Restore growth in an E. coli acpP temperature-sensitive mutant (primary metabolism)

• Restore production of specific secondary metabolites in appropriate mutant strains

Additionally, domain-swapping experiments, where regions of Methylobacterium sp. acpP are exchanged with other bacterial ACPs of known function, could help identify which structural elements confer pathway specificity.

What are the challenges and solutions in scaling up production of functional recombinant Methylobacterium sp. acpP for structural studies?

Scaling up production of functional recombinant Methylobacterium sp. acpP presents several significant challenges with corresponding solutions:

For structural studies requiring isotope-labeled protein, a strategic approach is critical. High-cell-density fermentation in minimal media can increase yields of labeled protein. For NMR studies, perdeuteration (growing in D2O-based media) may be necessary for proteins larger than 15 kDa to improve spectral quality.

To ensure homogeneity of the sample for structural analysis, separation of apo- and holo-forms is essential. Anion exchange chromatography can often resolve these forms due to the slight charge difference introduced by the phosphopantetheinyl group. Alternatively, in vitro phosphopantetheinylation can be performed after purification to convert all protein to the holo-form.

For cryo-EM studies of acpP-enzyme complexes, GraFix (gradient fixation) protocols can stabilize transient interactions. Chemical crosslinking with isotope-labeled crosslinkers can provide distance constraints for computational modeling when crystallization proves challenging.

How does Methylobacterium sp. acpP integrate with the methylotrophic metabolism characteristic of this bacterial genus?

Methylobacterium sp. acpP likely plays specialized roles in supporting the unique C1 metabolism of these bacteria. As methylotrophic organisms capable of growing on single-carbon compounds like methanol, Methylobacterium species require specialized lipid compositions to maintain cellular function in their distinctive ecological niches.

The acpP protein likely participates in both primary fatty acid synthesis and specialized lipid production pathways specific to methylotrophs. In Methylobacterium, acpP would interact with the fatty acid synthase machinery to produce membrane phospholipids, but may also channel intermediates toward:

• Hopanoid Biosynthesis: These sterol-like molecules strengthen bacterial membranes and may be particularly important in methylotrophs living in plant-associated environments.

• Production of Specialized Phospholipids: Methylobacterium species often contain unique phospholipid compositions that support methanol oxidation enzymes in the membrane.

• Biosynthesis of Unique Storage Compounds: Some methylotrophs accumulate specialized carbon storage compounds during growth on C1 substrates.

The integration of acpP with methylotrophic metabolism could be studied by comparing expression levels and post-translational modifications of acpP when Methylobacterium is grown on methanol versus multi-carbon substrates. Additionally, analyzing the acyl chain composition carried by acpP under different growth conditions would provide insights into its metabolic integration.

Complementation experiments similar to those performed with R. solanacearum ACPs could determine whether Methylobacterium sp. acpP can functionally replace ACPs in non-methylotrophic bacteria, or if it has acquired specialized features for functioning in C1 metabolism.

What techniques can be used to track the flux of acyl chains through recombinant Methylobacterium sp. acpP in metabolic studies?

Tracking acyl chain flux through recombinant Methylobacterium sp. acpP requires sophisticated analytical approaches that can capture the dynamic nature of these interactions:

• Stable Isotope Labeling: Growing Methylobacterium with 13C-labeled substrates (e.g., [13C]-methanol, [13C]-acetate) followed by analysis of acpP-bound intermediates can reveal the origin and fate of acyl chains. Mass spectrometric analysis can determine incorporation patterns and metabolic routing.

• ACP Immunoprecipitation (ACP-IP): Using antibodies against recombinant acpP or epitope tags to pull down the protein from cell lysates, followed by extraction and analysis of bound acyl chains. This technique can capture the profile of intermediates attached to acpP at different growth stages or conditions.

• Activity-Based Protein Profiling (ABPP): Using chemical probes that react specifically with the phosphopantetheine arm of acpP to tag and isolate the protein along with its bound intermediates.

• Targeted Metabolomics: Liquid chromatography-mass spectrometry (LC-MS) can identify and quantify acyl-ACP species extracted from cells expressing recombinant acpP.

• Pulse-Chase Experiments: Introducing labeled substrates for a short period followed by unlabeled substrates to track the progression of labeled acyl chains through metabolic pathways.

A particularly informative experimental design would involve expressing recombinant Methylobacterium sp. acpP with an affinity tag in the native organism, then performing parallel ACP-IP experiments under different growth conditions (e.g., methanol vs. succinate as carbon source). The acyl chain profiles could reveal how acpP participates in different metabolic modes and how flux changes in response to environmental conditions.

How can heterologous expression systems be optimized to study interactions between Methylobacterium sp. acpP and fatty acid synthesis enzymes?

Optimizing heterologous expression systems for studying Methylobacterium sp. acpP interactions requires careful consideration of several factors:

• Selection of Host Organism:

  • E. coli BL21(DE3): Most common, but may lack compatibility with Methylobacterium enzymes

  • Pseudomonas species: Closer phylogenetic relationship to Methylobacterium may preserve native-like interactions

  • Cell-free expression systems: Allow precise control of reaction components for reconstituting specific interactions

• Co-expression Strategies:

  • Polycistronic constructs: Encode acpP and interacting enzymes under control of a single promoter to ensure stoichiometric expression

  • Dual-plasmid systems: Use plasmids with compatible origins and different antibiotic selection markers

  • Sequential induction: Use orthogonal inducible promoters (e.g., T7/lac and araBAD) to control timing of protein expression

• Protein Tagging Approaches:

  • Split-tag complementation: Fusion of complementary fragments of a reporter protein to acpP and its partner enzyme

  • FRET pairs: Fusion of fluorescent proteins optimized for FRET (e.g., mTurquoise2/SYFP2) to study interactions in live cells

  • Affinity tags positioned to minimize interference with interaction surfaces

• Optimization of Expression Conditions:

  • Temperature: Lower temperatures (16-20°C) often favor proper folding and complex formation

  • Induction timing: Induce at higher cell densities (OD600 > 0.8) for membrane-associated FAS enzymes

  • Media composition: Addition of glycerol (0.5-1%) can enhance production of membrane proteins

For systems with multiple components, a stepwise reconstitution approach is often effective. This involves purifying individual components and systematically adding them to in vitro assays to identify minimal required components and potential regulatory factors.

To verify physiological relevance, complementation assays in R. solanacearum or E. coli acpP conditional mutants can be performed . Successful complementation by Methylobacterium sp. acpP would indicate conservation of critical interaction interfaces.

How does Methylobacterium sp. acpP compare structurally and functionally to acyl carrier proteins from other bacterial species?

Comparative analysis of Methylobacterium sp. acpP with other bacterial ACPs reveals important structural and functional relationships:

The specificity of Methylobacterium sp. acpP can be assessed through complementation studies similar to those performed with R. solanacearum ACPs . For example, testing whether it can functionally replace E. coli AcpP in the temperature-sensitive mutant CY1877 or restore production of quorum sensing signals in P. aeruginosa PA-A1 would indicate functional conservation across species boundaries.

Structural predictions based on homology modeling would likely show the characteristic four-helix bundle structure seen in other bacterial ACPs, with possible specialized features in the acyl chain binding pocket to accommodate the specific fatty acid profile of Methylobacterium species. These predictions could be experimentally verified through CD spectroscopy and NMR structural studies of the recombinant protein.

How can evolutionary analysis of acpP sequences inform optimization of recombinant Methylobacterium sp. acpP expression?

Evolutionary analysis of acpP sequences across bacterial species can significantly inform recombinant expression strategies for Methylobacterium sp. acpP:

• Codon Optimization Strategies:

  • Analysis of codon usage bias in highly expressed Methylobacterium genes compared to acpP can identify rare codons that might limit expression

  • Phylogenetic comparison of acpP across alphaproteobacteria can distinguish conserved rare codons that might be functionally important from those that can be safely optimized

• Identification of Critical Residues:

  • Residues conserved across all bacterial acpP homologs (like the serine in the DSL motif) are essential and should not be modified

  • Residues conserved only among methylotrophic bacteria might be specialized for their unique metabolism and should be preserved

  • Variable regions may tolerate insertions of affinity tags or reporter proteins

• Protein Stability Enhancement:

  • Thermostable acpP homologs from related species can suggest stabilizing mutations

  • Identifying natural sequence variations that correlate with expression level or stability in other bacteria

• Optimization of Protein-Protein Interfaces:

  • Co-evolutionary analysis of acpP and its partner enzymes can identify residue pairs that maintain interaction specificity

  • These insights can guide expression of compatible enzyme partners from the same organism

• Signal Peptide and Localization Analysis:

  • Identification of any N-terminal signal sequences that might affect recombinant expression

  • Analysis of potential membrane association domains that could impact solubility

What are the most common challenges in purifying functional recombinant Methylobacterium sp. acpP and how can they be addressed?

Purification of functional recombinant Methylobacterium sp. acpP presents several common challenges with specific solutions:

• Low Solubility:

  • Challenge: Small ACPs (9-10 kDa) often express well but can aggregate during purification.

  • Solution: Add 10-15% glycerol to all buffers; maintain pH between 7.0-8.0; include 1-5 mM DTT or TCEP to prevent disulfide formation; consider expressing as fusion with solubility-enhancing partners like MBP or SUMO .

• Heterogeneous Post-translational Modification:

  • Challenge: Mixture of apo- and holo-forms complicates structural and functional studies.

  • Solution: Either co-express with PPTase (e.g., Sfp) or perform complete in vitro phosphopantetheinylation after purification; verify completion by mass spectrometry .

• Proteolytic Degradation:

  • Challenge: Despite small size, flexible regions of acpP can be susceptible to proteolysis.

  • Solution: Include protease inhibitors during lysis; perform purification at 4°C; minimize purification time with streamlined protocols.

• Acyl Chain Heterogeneity:

  • Challenge: Recombinant acpP may be partially acylated with fatty acids from expression host.

  • Solution: Treatment with acyl-ACP thioesterase can generate homogeneous holo-acpP; alternatively, strong anion exchange chromatography can separate different acylated forms.

• Loss of Activity During Storage:

  • Challenge: Freeze-thaw cycles can reduce activity of purified acpP.

  • Solution: Store as single-use aliquots; include 10% glycerol and flash-freeze in liquid nitrogen; for long-term storage, lyophilization may be superior to freezing.

For the most efficient purification strategy, a two-step approach is recommended:

• Initial capture via nickel affinity chromatography using an N-terminal His6 tag

• Followed by anion exchange chromatography to separate apo- and holo-forms

Quality control should include native PAGE to verify phosphopantetheinylation status and functional assays such as in vitro acylation with VhAasS to confirm activity . For acpP mutants, parallel analysis with wild-type protein provides essential comparative controls.

How can isotope labeling be optimized for structural studies of recombinant Methylobacterium sp. acpP?

Optimizing isotope labeling for structural studies of recombinant Methylobacterium sp. acpP requires careful consideration of expression systems and protocols:

• Expression System Selection:

  • E. coli BL21(DE3) remains the most cost-effective system for isotope labeling

  • Cell-free protein synthesis offers rapid production but at higher cost

  • P. pastoris can be considered for selective methyl labeling in a deuterated background

• Media Optimization for Different Labeling Schemes:

• Expression Protocol Optimization:

  • Use high-density fermentation to maximize yield from expensive labeled media

  • Extend expression time (16-24h) at lower temperatures (16-18°C)

  • For deuterated media, gradually adapt cells through increasing D2O concentrations

• Efficient Use of Labeled Material:

  • Establish non-labeled test expressions to optimize conditions before using isotope-labeled media

  • Recover unincorporated isotope-labeled compounds from spent media for reuse

  • Consider REDPRO approach (reduced volume expression) for economical labeling

• Post-translational Modification Considerations:

  • Perform phosphopantetheinylation in vitro after purification to ensure homogeneity

  • Use non-labeled CoA for modification to avoid introducing unlabeled regions

For NMR studies specifically, expression in 100% D2O with selective protonation of methyl groups provides excellent results for studying acpP-enzyme interactions, as the methyl probes remain visible even in large complexes. Sample conditions should be optimized with 5-10% D2O, 50-100 mM NaCl, 20 mM phosphate buffer at pH 6.5-7.0, and 5 mM DTT to maintain sample stability during extended data collection.

What quality control measures are critical for ensuring functional integrity of recombinant Methylobacterium sp. acpP?

Comprehensive quality control is essential for ensuring the functional integrity of recombinant Methylobacterium sp. acpP, requiring multiple analytical approaches:

• Purity Assessment:

  • SDS-PAGE: Should show >95% purity with a single band at approximately 9-10 kDa

  • Size exclusion chromatography: Single symmetrical peak confirming monodispersity

  • Mass spectrometry: Intact mass within 1 Da of theoretical value

• Structural Integrity Verification:

  • Circular dichroism: Strong alpha-helical signature (negative peaks at 208 and 222 nm)

  • Thermal shift assay: Stable melting curve with Tm typically between 50-70°C

  • 1D 1H NMR: Well-dispersed signals indicating properly folded protein

• Post-translational Modification Analysis:

  • Native PAGE: Distinct migration pattern between apo- and holo-forms

  • Mass spectrometry: +340 Da mass shift confirming phosphopantetheinylation

  • HPLC analysis: Separation of apo- and holo-forms on reverse phase or ion exchange

• Functional Activity Tests:

  • In vitro acylation: Incubation with VhAasS, ATP, and fatty acids should produce acylated acpP

  • PPTase modification kinetics: Time-course of phosphopantetheinylation

  • Protein-protein interaction assays: Pull-down or SPR with cognate FAS enzymes

• Stability Assessment:

  • Time-course analysis at different temperatures (4°C, 25°C, 37°C)

  • Freeze-thaw stability: Activity measurement after multiple freeze-thaw cycles

  • Long-term storage test: Activity retention after different storage conditions

A critical control experiment involves parallel analysis of site-directed mutants where the conserved serine in the DSL motif is replaced with alanine or threonine. These mutants should be identical in all physicochemical properties except their inability to be phosphopantetheinylated, providing an excellent negative control .

For quantitative assessment of holo-acpP content, a standardized protocol using Ellman's reagent (DTNB) can measure the free thiol content of the phosphopantetheinyl arm, allowing precise determination of functional protein concentration.

How can recombinant Methylobacterium sp. acpP be utilized in drug discovery targeting bacterial fatty acid synthesis?

Recombinant Methylobacterium sp. acpP offers several strategic applications in drug discovery targeting bacterial fatty acid synthesis:

• High-Throughput Screening Platform:

  • FRET-based assays using labeled acpP and FAS enzymes can detect compounds that disrupt protein-protein interactions

  • Displacement assays using fluorescently labeled inhibitors can identify compounds competing for binding sites

  • Thermal shift assays can identify compounds that stabilize or destabilize acpP structure

• Structure-Based Drug Design:

  • Crystallographic or NMR studies of acpP alone or in complex with FAS enzymes provide atomic-level detail for rational inhibitor design

  • Computational docking against acpP or acpP-enzyme interfaces can identify promising chemical scaffolds

  • Fragment-based approaches can discover building blocks for novel inhibitors

• Mechanism of Action Studies:

  • In vitro reconstitution of fatty acid synthesis using recombinant components allows precise determination of where inhibitors act in the pathway

  • Comparative studies with acpP from pathogens and human host can identify species-specific inhibition properties

  • Resistance mutation mapping by expressing mutant acpP variants

• Target Validation:

  • As demonstrated in R. solanacearum, acpP1 is essential for bacterial viability , confirming it as a potential antibiotic target

  • Site-directed mutants similar to R. solanacearum AcpP1 D39V and I55A can reveal critical functional sites for drug targeting

• Bacterial Specificity Profiling:

  • Parallel testing of inhibitors against acpP from multiple bacterial species can establish spectrum of activity

  • Comparisons with human ACP can identify compounds with selectivity for bacterial targets

A particularly powerful approach would combine structural data from labeled recombinant acpP with in silico screening to identify compounds that target the interaction surfaces between acpP and specific FAS enzymes. These compounds could then be optimized based on structure-activity relationships determined through biochemical assays using the recombinant protein. The ability to generate site-specific mutants of acpP provides crucial tools for validating the mechanism of action of promising inhibitors.

What experimental designs can elucidate the role of Methylobacterium sp. acpP in plant-microbe interactions?

Plant-associated Methylobacterium species establish important symbiotic relationships, and acpP likely plays crucial roles in these interactions. The following experimental designs can elucidate these functions:

• Conditional Mutant Analysis:

  • Create IPTG-inducible or tetracycline-regulated acpP expression systems in Methylobacterium

  • Assess plant colonization efficiency under varying acpP expression levels

  • Monitor changes in bacterial metabolites during plant association

• Domain Swapping Experiments:

  • Replace Methylobacterium sp. acpP with homologs from non-plant-associated bacteria

  • Create chimeric acpP proteins with domains from different bacterial species

  • Test colonization efficiency and metabolite production of engineered strains

• Metabolomics Approaches:

  • Compare acyl-ACP profiles in bacteria grown in plant-associated versus free-living conditions

  • Use stable isotope probing to track carbon flow from plant exudates through acpP

  • Identify specialized lipids or secondary metabolites that require acpP and affect plant interactions

• In planta Imaging:

  • Express fluorescently-tagged acpP in Methylobacterium to track localization during plant colonization

  • Use BiFC (Bimolecular Fluorescence Complementation) to visualize acpP interactions with partner proteins in planta

  • Apply FRET microscopy to study dynamics of acpP interactions during symbiosis establishment

• Interspecies Complementation:

  • Test whether Methylobacterium sp. acpP can complement mutants of plant-associated bacteria like Sinorhizobium or Rhizobium

  • Assess if Methylobacterium sp. acpP supports production of plant-interaction metabolites in heterologous hosts

• Transcriptomics Integration:

  • Correlate acpP expression levels with expression of plant-interaction genes

  • Compare transcriptomes of wild-type and acpP-modified strains during plant colonization

These approaches would benefit from using recombinant acpP with site-specific mutations at the conserved serine residue in the DSL motif as a negative control, providing clear evidence for acpP-dependent phenotypes. The experimental designs should consider the specificity observed in other bacterial species; for example, in R. solanacearum, only AcpP1 functions in primary metabolism while other ACPs have specialized roles .

How can synthetic biology approaches incorporate recombinant Methylobacterium sp. acpP for production of novel compounds?

Synthetic biology offers powerful approaches to harness recombinant Methylobacterium sp. acpP for novel compound production:

• Pathway Engineering Strategies:

  • Express Methylobacterium sp. acpP alongside heterologous polyketide synthases (PKS) to produce novel polyketides

  • Create fusion proteins linking acpP to specific biosynthetic enzymes to increase pathway efficiency

  • Engineer substrate specificity by directed evolution of acpP to accept non-native acyl substrates

• Multi-modular Synthetic Systems:

  • Design synthetic operons containing Methylobacterium sp. acpP with combinations of FAS, PKS, and NRPS components

  • Incorporate orthogonal PPTases to enable simultaneous operation of multiple acpP-dependent pathways

  • Create metabolic branching points by varying acpP concentrations or affinities

• Protein Engineering Approaches:

  • Create acpP variants with altered surface properties to enhance interactions with specific synthases

  • Develop "loading module" acpP variants optimized for specific starter units

  • Engineer acpP-enzyme interfaces based on structural data to improve catalytic efficiency

• Novel Production Platforms:

  • Incorporate Methylobacterium sp. acpP into cell-free systems for toxic compound production

  • Develop Methylobacterium as a production platform leveraging its methylotrophic metabolism

  • Create synthetic minimal cells with streamlined metabolism centered around acpP function

• Biosensor Development:

  • Engineer acpP-based FRET biosensors for real-time monitoring of fatty acid synthesis

  • Develop reporter systems linked to acpP loading state to optimize production

A particularly promising approach would be to leverage Methylobacterium's natural methylotrophic capabilities by engineering pathways that connect C1 metabolism to specialized acpP-dependent synthesis routes. This could enable conversion of methanol (a cheap, renewable substrate) into high-value compounds through acpP-dependent pathways.

For implementation, a modular cloning strategy using standardized parts would facilitate rapid testing of different combinations. Initial proof-of-concept could focus on producing colored compounds (like pigments or fluorescent molecules) to enable visual screening of successful designs. Progressively more complex targets could then be attempted, building on successful modules and optimizing rate-limiting steps identified through metabolic flux analysis.