Recombinant Cyanothece sp. Acyl carrier protein (acpP)

What distinguishes Cyanothece sp. acpP from other bacterial acyl carrier proteins?

Cyanothece sp. acpP belongs to the family of housekeeping acyl carrier proteins (ACPs) responsible for fatty acid biosynthesis in cyanobacteria. Unlike specialized ACPs (such as AcpR found in Rhodobacteria that are involved in sphingolipid biosynthesis), the housekeeping acpP in Cyanothece sp. primarily functions in the type II fatty acid synthase system . The distinguishing feature of Cyanothece sp. acpP is its 4'-phosphopantetheine prosthetic group which serves as the attachment site for growing fatty acyl chains during biosynthesis, similar to what has been observed in other bacterial ACPs like those in C. crescentus and E. coli .

How does the structure of Cyanothece sp. acpP compare with other cyanobacterial ACPs?

Cyanothece sp. acpP shares the characteristic structural features of bacterial ACPs:

The conserved serine residue in ACPs serves as the attachment site for the 4'-phosphopantetheine group, as demonstrated in ACP characterization studies . Mass spectrometry analysis of bacterial ACPs typically reveals distinct forms: apo-ACP (without the prosthetic group), holo-ACP (with the prosthetic group), and acyl-ACP (with attached fatty acid), similar to the patterns observed in C. crescentus where an average mass of approximately 9,020 Da was observed for the ACP .

What expression systems provide optimal yields for recombinant Cyanothece sp. acpP?

E. coli-based expression systems have proven highly effective for recombinant ACP production, as demonstrated in studies with ACPs from various bacterial sources . For optimal expression of Cyanothece sp. acpP:

• Recommended expression vectors: pET series vectors (particularly pET9a or pET28a) under the control of T7 promoter

• Host strains: E. coli BL21(DE3) or its derivatives

• Expression conditions:

  • Induction with 0.5-1.0 mM IPTG

  • Post-induction temperature: 25-30°C (rather than 37°C)

  • Expression duration: 4-6 hours or overnight at lower temperatures

This approach has successfully yielded properly folded ACPs from various bacteria, as evidenced by native PAGE analysis showing characteristic migration patterns of the overexpressed proteins .

What purification strategy yields the highest purity and activity of recombinant Cyanothece sp. acpP?

A multi-step purification approach is recommended for obtaining high-purity recombinant Cyanothece sp. acpP:

• Initial clarification:

  • Cell lysis by sonication in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM DTT

  • Centrifugation at 20,000×g for 30 minutes to remove cell debris

• First purification step (choose based on construct):

  • For His-tagged constructs: Ni-NTA affinity chromatography

  • For untagged constructs: Anion exchange chromatography (Q Sepharose)

• Second purification step:

  • Size exclusion chromatography using Superdex 75 column

• Quality assessment:

  • Purity evaluation by SDS-PAGE (should appear as a single band at ~10 kDa)

  • Native PAGE to assess proper folding (should show characteristic migration pattern)

  • Mass spectrometry to confirm molecular weight and post-translational modifications

This approach has been effective for purifying ACPs from various bacterial sources while maintaining their functional properties .

How can I confirm and quantify the phosphopantetheinylation status of recombinant Cyanothece sp. acpP?

The phosphopantetheinylation status (conversion from apo- to holo-form) is critical for ACP function. Multiple complementary methods should be employed:

• Mass spectrometry analysis:

  • MALDI-TOF or ESI-MS can distinguish between apo-ACP and holo-ACP forms

  • The mass difference is approximately 339 Da (the mass of the 4'-phosphopantetheine group)

  • As observed in C. crescentus ACP analysis, apo-ACP shows a mass of ~9,020 Da while holo-ACP shows a mass of ~9,360 Da

• Radioactive labeling:

  • In vivo labeling with [³H]β-alanine (precursor of 4'-PPT)

  • Analysis by native PAGE followed by fluorography

  • This approach successfully detected 4'-PPT groups in various ACPs

• Conformational analysis:

  • Urea-PAGE can separate apo- and holo-forms based on subtle conformational differences

  • Circular dichroism spectroscopy can detect structural changes upon phosphopantetheinylation

What enzymatic assays can demonstrate the functionality of recombinant Cyanothece sp. acpP?

To confirm that the recombinant Cyanothece sp. acpP is functional, several enzymatic assays can be performed:

• Acylation assay:

  • Incubate holo-acpP with acyl-ACP synthetase (AasR or similar enzyme), ATP, and a fatty acid (typically palmitate)

  • Detect acylation by:
    a) Native PAGE (acyl-ACP migrates differently than holo-ACP)
    b) Mass spectrometry (mass increase of ~238 Da for palmitoyl group)
    c) Radioactive assay using [³H]palmitate

• Integration into fatty acid synthesis:

  • Reconstituted in vitro fatty acid synthesis system with purified FAS components

  • Monitor fatty acid production by GC-MS or radioactive assays

• Phosphopantetheinyl transferase (PPTase) assay:

  • Conversion of apo-acpP to holo-acpP using a PPTase enzyme

  • Detection by mass spectrometry or conformational changes on native PAGE

How can I identify and characterize protein partners of Cyanothece sp. acpP?

Several complementary approaches are recommended for comprehensive identification of acpP protein interactions:

• Pull-down assays:

  • Immobilize His-tagged acpP on Ni-NTA resin

  • Incubate with Cyanothece sp. lysate

  • Elute and analyze binding partners by mass spectrometry

• Surface plasmon resonance (SPR):

  • Immobilize acpP on sensor chip

  • Measure binding kinetics with purified candidate partners

  • Determine association/dissociation constants

• Bacterial two-hybrid system:

  • Use acpP as bait against a Cyanothece sp. genomic library

  • Screen for positive interactions

  • Validate with secondary assays

• Crosslinking studies:

  • Chemical crosslinking of acpP in vivo or in cell lysates

  • Mass spectrometric analysis of crosslinked products

  • Identification of proximal proteins

Studies with ACPs from other bacteria have shown interactions with numerous proteins involved in fatty acid synthesis, including acyl-ACP synthetases that selectively acylate specific ACPs .

What methods are most effective for studying the interaction between Cyanothece sp. acpP and its cognate synthetase?

Based on studies of ACP-synthetase interactions in other bacteria, the following approaches are recommended:

• Enzymatic activity assays:

  • Measure synthetase activity with varying concentrations of acpP

  • Determine kinetic parameters (Km, Vmax, kcat)

  • Plot saturation curves to analyze binding characteristics

  • Similar to the approach used for AasR activity dependence on AcpR concentration

• Isothermal titration calorimetry (ITC):

  • Directly measure binding thermodynamics

  • Determine binding constants, enthalpy, and stoichiometry

• Specificity analysis:

  • Compare activity with Cyanothece sp. acpP versus ACPs from other organisms

  • Identify specificity determinants through mutagenesis

  • This approach revealed that AasR proteins preferentially acylate cognate AcpRs

• Structural analysis of the complex:

  • Co-crystallization of acpP with its synthetase

  • Alternative: Chemical crosslinking followed by structural analysis

How can recombinant Cyanothece sp. acpP be utilized for directed biosynthesis of fatty acid derivatives?

Recombinant Cyanothece sp. acpP can serve as a platform for engineering novel biosynthetic pathways:

• Heterologous expression systems:

  • Co-express Cyanothece sp. acpP with enzymes from other organisms

  • Engineer E. coli or other hosts to produce desired compounds

  • This approach has been used successfully with other bacterial ACPs

• Substrate specificity engineering:

  • Mutate key residues in acpP to alter fatty acid specificity

  • Screen for variants with desired properties

  • Analyze lipid profiles by TLC or mass spectrometry

• Integration with non-native pathways:

  • Similar to how specialized ACPs like AcpR are used for sphingolipid biosynthesis

  • Target production of biofuels, pharmaceutical precursors, or specialty chemicals

• Pathway optimization strategies:

  • Balance expression levels of acpP and partner enzymes

  • Optimize growth conditions for maximum product yield

  • Use metabolic flux analysis to identify bottlenecks

What considerations are important when using recombinant Cyanothece sp. acpP in heterologous expression systems?

Several factors must be carefully considered:

• Post-translational modification:

  • Ensure proper phosphopantetheinylation by co-expressing appropriate PPTase

  • Verify holo-acpP formation by mass spectrometry

  • Consider native versus heterologous PPTases for optimal modification

• Protein-protein interaction specificity:

  • Assess compatibility with fatty acid synthesis enzymes in the host

  • Consider co-expressing cognate enzymes from Cyanothece sp.

  • Research with various bacterial ACPs indicates that AcpR/AasR pairs from the same organism work most efficiently together

• Metabolic burden and toxicity:

  • Optimize expression levels to minimize growth inhibition

  • Use inducible promoters for controlled expression

  • Monitor growth curves to assess metabolic burden

• Product extraction and analysis:

  • Develop appropriate extraction protocols for target compounds

  • Use TLC, HPLC, or mass spectrometry for product characterization

  • Similar to approaches used for analyzing sphingolipid intermediates

Why might recombinant Cyanothece sp. acpP show reduced activity compared to the native protein?

Several factors can contribute to reduced activity of recombinant acpP:

• Incomplete post-translational modification:

  • Insufficient conversion to holo-form

  • Solution: Co-express with appropriate phosphopantetheinyl transferase

• Improper folding:

  • Rapid overexpression can lead to misfolding

  • Solutions:
    a) Lower induction temperature (16-25°C)
    b) Reduce inducer concentration
    c) Use slower expression systems
    d) Co-express with chaperones

• Presence of inhibitory factors:

  • Metal ions or oxidizing agents may inactivate acpP

  • Solution: Include DTT or other reducing agents in buffers

• Lack of appropriate partner proteins:

  • ACPs function in complex with other enzymes

  • Solution: Include necessary partner proteins in assays

• Degradation or truncation:

  • Proteolytic cleavage during expression or purification

  • Solution: Add protease inhibitors and verify intact protein by mass spectrometry

What are the best approaches for resolving solubility issues with recombinant Cyanothece sp. acpP?

ACPs are generally soluble proteins, but recombinant expression can sometimes lead to inclusion body formation. Strategies to enhance solubility include:

• Expression optimization:

  • Reduce temperature to 16-20°C during expression

  • Use lower inducer concentrations

  • Extend expression time (overnight)

  • These conditions have been successful for expressing various bacterial ACPs

• Fusion partners:

  • MBP (maltose-binding protein) fusion

  • SUMO fusion

  • Thioredoxin fusion

  • Include a cleavable linker for post-purification removal

• Buffer optimization:

  • Include glycerol (5-10%)

  • Add low concentrations of non-ionic detergents (0.05-0.1% Triton X-100)

  • Test different pH ranges (typically pH 7.0-8.0)

  • Include stabilizing agents like arginine or proline

• Refolding protocols (if inclusion bodies form):

  • Solubilize in 6M guanidine-HCl or 8M urea

  • Refold by gradual dialysis into native buffer

  • Monitor refolding by circular dichroism spectroscopy

How do structural variations in Cyanothece sp. acpP impact its interaction network compared to other cyanobacterial ACPs?

Current research suggests that subtle structural differences between ACPs significantly impact their functional interactions:

• Electrostatic surface mapping:

  • The distribution of charged residues on ACP surfaces influences partner recognition

  • Computational analysis of surface charge distribution can predict interaction specificity

  • Similar analyses have revealed why specialized ACPs like AcpR interact preferentially with their cognate synthetases

• Helical dynamics:

  • NMR studies of ACP dynamics reveal conformational shifts upon acylation

  • These shifts may differ between Cyanothece sp. acpP and other ACPs

  • The dynamics influence recognition by partner enzymes

• Acyl chain binding pocket:

  • Variations in the acyl chain binding pocket affect substrate specificity

  • Molecular dynamics simulations can predict these differences

  • Experimental validation through mutagenesis studies

• Structural determinants for specific interactions:

  • Phylogenetic analysis of ACP sequences correlates with functional specialization

  • Similar patterns have been observed in the specialized AcpRs from various bacterial species

What emerging technologies are advancing our understanding of ACP-dependent pathways in cyanobacteria?

Several cutting-edge technologies are transforming research on ACPs and their role in cyanobacterial metabolism:

• Cryo-electron microscopy:

  • Visualization of ACP-enzyme complexes in near-native states

  • Resolution of transient interaction states

  • Insights into the dynamic nature of ACP associations

• Activity-based protein profiling:

  • Chemical probes targeting the 4'-phosphopantetheine arm

  • In situ identification of ACP-interacting proteins

  • Mapping of the complete ACP interactome

• Systems biology approaches:

  • Multi-omics integration (proteomics, metabolomics, transcriptomics)

  • Flux analysis of ACP-dependent pathways

  • Network modeling of fatty acid metabolism

• Engineered biosensors:

  • FRET-based sensors for monitoring ACP-protein interactions in real-time

  • Live-cell imaging of ACP trafficking and dynamics

  • Quantification of acylation states in vivo

• CRISPR-based techniques:

  • Precise genome editing to study ACP function in native contexts

  • CRISPRi for controlled knockdown experiments

  • Base editing for structure-function studies without complete gene disruption