Recombinant Synechococcus sp. Acyl carrier protein (acpP)

What is the functional role of acpP in Synechococcus sp.?

Acyl Carrier Protein (acpP) in Synechococcus sp. serves as an essential component of the fatty acid synthesis (FAS) pathway. It functions as the central cofactor that carries growing acyl chains during fatty acid biosynthesis. All bacteria possess a constitutively expressed ACP (acpP), which is needed for essential housekeeping functions and acts as an acyl group carrier and donor .

The protein contains a characteristic 4'-phosphopantetheine prosthetic group attached to a conserved serine residue, which forms the attachment site for acyl intermediates through thioester linkages. The protein plays a critical role in membrane lipid biosynthesis, which is particularly important for photosynthetic organisms like cyanobacteria that have extensive thylakoid membrane systems housing their photosynthetic apparatus.

How does acpP differ from specialized acyl carrier proteins in cyanobacteria?

The constitutively expressed housekeeping acpP differs from specialized acyl carrier proteins in several key aspects:

Research has shown that in certain bacteria, specialized ACPs like AcpR are specifically used for sphingolipid biosynthesis. For example, in the α-proteobacterium Caulobacter crescentus, a specialized ACP (CC_1163) works with a specialized acyl-ACP synthetase and serine palmitoyltransferase for sphingolipid biosynthesis, while the housekeeping AcpP (CC_1677) is involved in general fatty acid biosynthesis .

What is the significance of the 4'-phosphopantetheine prosthetic group in acpP?

The 4'-phosphopantetheine (4'-PPT) prosthetic group is absolutely essential for acpP function in all bacteria, including Synechococcus sp. This prosthetic group serves multiple critical roles:

• Acyl Carrier Function: The terminal thiol (-SH) group of the 4'-PPT forms thioester bonds with acyl intermediates.

• Molecular Swinging Arm: The 4'-PPT acts as a flexible arm that can reach into the active sites of various enzymes in the fatty acid synthase complex.

• Conversion to Active Form: The addition of the 4'-PPT converts the inactive apo-acpP to the active holo-acpP.

• Substrate Recognition: The 4'-PPT contributes to substrate recognition by various enzymes.

The presence of the 4'-PPT prosthetic group can be confirmed through in vivo labeling with radioactive β-alanine (a precursor of 4'-PPT), as demonstrated in research with ACPs from various bacteria .

What are the optimal expression systems for recombinant Synechococcus sp. acpP?

The expression of functional recombinant Synechococcus sp. acpP requires careful consideration of the expression system to ensure proper folding and post-translational modification:

Optimization considerations:

• Lower temperatures (16-25°C) after induction often improve solubility

• Lower IPTG concentrations (0.1-0.5 mM) for T7 systems

• Co-expression with a phosphopantetheinyl transferase (such as Sfp from Bacillus subtilis) is often necessary to ensure conversion to the holo form

How can I confirm proper post-translational modification of recombinant acpP?

Confirming proper post-translational modification of recombinant acpP, particularly the attachment of the 4'-phosphopantetheine prosthetic group, is crucial for ensuring functionally active protein:

1. Mass Spectrometry Analysis:

• MALDI-TOF MS can detect the 339 Da mass difference between apo and holo forms

• ESI-MS provides higher resolution for accurate mass determination

2. Gel-based Methods:

• Native PAGE: Holo-acpP typically migrates faster than apo-acpP due to the additional negative charge

• Radiolabeled bands with similar relative mobility as overexpressed ACPs can be detected after in vivo labeling with radioactive β-alanine

3. Functional Assays:

• Acylation assay: Incubation with acyl-CoA and acyl-ACP synthetase followed by analysis of acylation

• Fatty acid synthase reconstitution assays: Testing the ability of the recombinant acpP to support fatty acid synthesis in vitro

What are the common challenges in purifying functional recombinant acpP?

Purifying functional recombinant acpP presents several challenges that researchers need to address:

1. Maintaining the 4'-PPT Prosthetic Group:

• Ensure conversion from apo to holo form through co-expression with a phosphopantetheinyl transferase or perform in vitro modification after purification

2. Preventing Acyl Group Contamination:

• Endogenous E. coli acyltransferases can load fatty acids onto recombinant acpP

• Consider deacylation steps during purification

3. Protein Stability Issues:

• Include stabilizing agents (glycerol, low concentrations of reducing agents)

• Optimize buffer conditions (pH 6.5-7.5 is often optimal)

4. Co-purification of E. coli ACP:

• Use high-resolution chromatography steps

• Consider tag-specific purification methods

How can I assess the acylation state of recombinant acpP?

Determining the acylation state of recombinant acpP is crucial for many functional studies:

Mass Spectrometry-Based Methods:

• Direct ESI-MS analysis can detect mass increases corresponding to specific acyl chains

• Intact protein MS can distinguish unacylated, mono-acylated, and poly-acylated species

Chromatographic Methods:

• Reverse-phase HPLC: Acylated ACPs elute later than unacylated forms

• Can separate ACPs bearing different acyl chains based on hydrophobicity

Gel-Based Methods:

• Urea-PAGE: Acylated ACPs typically migrate faster than unacylated forms

• Can resolve ACPs with different acyl chain lengths

What experimental approaches determine acpP-protein interactions in Synechococcus sp.?

Understanding the protein interaction network of acpP requires complementary experimental approaches:

In Vitro Interaction Methods:

• Pull-down assays using immobilized His-tagged acpP

• Surface Plasmon Resonance (SPR) to measure association and dissociation kinetics

• Isothermal Titration Calorimetry (ITC) for binding thermodynamics

In Vivo Interaction Methods:

• Bacterial Two-Hybrid System for identifying protein partners

• Crosslinking coupled to Mass Spectrometry to capture transient interactions

• Proximity-based labeling (BioID or TurboID) for mapping protein interaction neighborhoods

Functional Validation Methods:

• Mutagenesis of predicted interaction interfaces

• Competition assays using synthetic peptides corresponding to predicted interaction interfaces

How does acpP from Synechococcus sp. PCC 11901 compare to other cyanobacterial strains?

Synechococcus sp. PCC 11901 is a newly discovered cyanobacterial strain with promising features for green biotechnology. It is naturally transformable, has a short doubling time of ≈2 hours, grows at high light intensities and in a wide range of salinities .

When comparing acpP across different Synechococcus strains, several factors should be considered:

• Sequence conservation of the core functional regions, particularly the serine residue for 4'-PPT attachment

• Variations in the N- and C-terminal regions that might affect protein-protein interactions

• Expression levels under different growth conditions

• Genetic context and potential specialized functions

The high biomass accumulation capability of PCC 11901 (up to ≈33 g dry cell weight per litre) makes it particularly interesting for metabolic engineering applications involving acpP, such as enhanced fatty acid production .

What are the recommended protocols for site-directed mutagenesis of acpP in Synechococcus sp.?

Site-directed mutagenesis of acpP requires careful planning, especially considering the essential nature of this gene:

For Heterologous Expression Studies:

• PCR-based site-directed mutagenesis using high-fidelity DNA polymerase

• Design mutagenic primers with desired mutations flanked by 15-20 nucleotides on each side

• Digest template DNA with DpnI to remove methylated parental DNA

For Genomic Modifications in Synechococcus:

• Consider a markerless gene replacement strategy using counter-selection

• The serine residue that serves as the attachment site for the 4'-phosphopantetheine prosthetic group is a common mutagenesis target

• Since acpP is essential, consider complementation strategies or conditional mutations

How can I develop a markerless gene modification system for acpP in Synechococcus sp.?

A markerless gene modification system for acpP can be developed using the PCPA-based counter-selection system:

1. Vector Construction:

• Assemble: Upstream homology region - modified acpP gene - antibiotic resistance gene - mutated pheS gene - downstream homology region

• Clone into a vector that cannot replicate in Synechococcus

2. First Recombination (Integration):

• Transform Synechococcus sp. with the constructed vector

• Select transformants on media containing the appropriate antibiotic

• Verify integration by PCR

3. Second Recombination (Marker Removal):

• Culture verified first recombinants without antibiotic selection

• Plate on media containing p-chlorophenylalanine (PCPA)

• Screen PCPA-resistant colonies by PCR to identify clones that have lost the markers

Special Considerations for acpP:

• Since acpP is essential, introduce a wild-type copy at a neutral site before attempting modification

• Place this copy under an inducible or constitutive promoter to provide a functional backup

What methods are available for visualizing acpP localization in Synechococcus cells?

Visualizing acpP localization requires approaches that overcome the challenges of the small size of both the protein and cyanobacterial cells:

Fluorescent Protein Fusion Approaches:

• Create a genetic construct with acpP fused to a fluorescent protein (preferably with emission spectra distinct from chlorophyll autofluorescence)

• Integrate at the native locus using markerless recombination

• Include a flexible linker between acpP and the fluorescent protein

Immunofluorescence Microscopy:

• Develop specific antibodies against acpP or use epitope tagging

• Fix Synechococcus cells with paraformaldehyde

• Permeabilize cell wall with appropriate treatments

• Detect with fluorescently labeled secondary antibodies

Super-Resolution Microscopy Techniques:

• PALM/STORM achieves resolution down to ~20 nm

• Structured Illumination Microscopy (SIM) achieves resolution of ~100 nm

• Particularly valuable for precise localization within small cyanobacterial cells

How can I quantitatively measure acpP expression levels under different growth conditions?

Accurately measuring acpP expression requires complementary approaches:

Transcriptional Analysis Methods:

• Quantitative Real-Time PCR (qRT-PCR) with acpP-specific primers

• RNA-Seq analysis for genome-wide expression comparison

• Normalize to appropriate reference genes stable under your experimental conditions

Protein-Level Quantification:

• Western blot analysis using antibodies against Synechococcus sp. acpP

• Targeted proteomics (SRM/MRM) for absolute quantification

• Translational reporter fusions for real-time monitoring

Growth Condition Variables to Consider:

• Light intensity affects photosynthesis rate and carbon fixation

• Carbon source availability impacts metabolic flux through fatty acid synthesis

• Temperature affects membrane fluidity requirements

• Growth phase changes lipid synthesis needs

What bioinformatic tools are effective for analyzing acpP homologs across cyanobacterial species?

Analyzing acpP homologs requires a comprehensive bioinformatic approach:

Homology Identification and Retrieval:

• NCBI BLAST using Synechococcus sp. acpP as query against cyanobacterial genomes

• HMMER for detecting distant homologs using position-specific scoring matrices

• Specialized databases like CyanoBase and UniProt

Multiple Sequence Alignment Tools:

• MAFFT for large datasets with multiple alignment strategies

• Structure-informed alignments using tools like PROMALS3D

Phylogenetic Analysis:

• RAxML or IQ-TREE for maximum likelihood methods

• ProtTest to determine the best evolutionary model for protein sequences

• iTOL for interactive visualization with extensive annotation options

Genomic Context Analysis:

• Analyze gene neighborhood of acpP in different cyanobacteria

• Identify co-occurring genes that might indicate specialized functions

• Compare synteny patterns across evolutionary lineages

How can I use recombinant Synechococcus sp. PCC 11901 acpP for enhancing biofuel production?

Synechococcus sp. PCC 11901 has promising features for biotechnology applications, including biofuel production:

Advantages of PCC 11901:

• Short doubling time of ≈2 hours

• Growth at high light intensities

• High biomass accumulation (up to ≈33 g dry cell weight per litre)

• Natural transformability for genetic engineering

Strategies for enhancing biofuel production through acpP engineering:

• Overexpression of acpP to increase fatty acid biosynthesis capacity

• Engineering acpP to alter substrate specificity for producing specific fatty acid profiles

• Co-expression with thioesterases to release free fatty acids

• Modification of acpP-protein interactions to direct metabolic flux

Proof of Concept Results:

• PCC 11901 engineered to produce free fatty acids yielded over 6 mM (1.5 g L⁻¹), comparable to similarly engineered heterotrophic organisms

• Targeting specific acpP interactions could further enhance production

What ethical considerations should be addressed when working with genetically modified Synechococcus sp.?

Research with genetically modified cyanobacteria requires adherence to ethical guidelines:

Biosafety Considerations:

• Follow appropriate biosafety level guidelines for recombinant organisms

• Implement proper containment measures to prevent environmental release

• Obtain necessary institutional approvals before beginning work

Research Ethics Training:

• Complete required ethics and compliance training programs

• Ritsumeikan Asia Pacific University, for example, requires all doctoral students and master's students receiving research funds to take Research Ethics and Compliance Training programs before beginning research

Reporting and Transparency:

• Accurately document all methods and results

• Properly acknowledge prior work and contributions

• Ensure research methods are clearly described to enable replication

What funding opportunities exist for research on cyanobacterial acpP?

Several funding sources support research on cyanobacterial proteins like acpP:

Academic Institution Support:

• The Research Support Subsidy at institutions like Ritsumeikan Asia Pacific University provides funding up to JPY 75,000 for Master's students and JPY 100,000 for PhD students for research activities

• Institutional seed grants for preliminary studies

Government Funding Agencies:

• National Science Foundation (NSF) programs in molecular biosciences

• Department of Energy (DOE) funding for bioenergy research

• National Institutes of Health (NIH) for basic biochemical studies

Industry Partnerships:

• Biotechnology companies interested in sustainable production platforms

• Energy companies exploring biofuel alternatives

• Agricultural companies interested in photosynthetic improvement