Recombinant Phaeodactylum tricornutum Acyl carrier protein (acpP)

Basic Research Questions

• What is the acyl carrier protein (acpP) in Phaeodactylum tricornutum and what is its role in diatom metabolism?

  The acyl carrier protein (acpP) in Phaeodactylum tricornutum is a critical component of fatty acid biosynthesis located in the plastid. It functions as a key enzyme in fatty acid modification and plays a significant role in eicosapentaenoic acid (EPA) synthesis and triacylglycerol production . In P. tricornutum, acpP contains predicted N-terminal bipartite targeting sequences with the conserved amino acid sequence motif "ASAFAP" surrounding the signal peptide cleavage site, consistent with its stromal location .

  Functionally, acpP serves as a carrier for growing acyl chains during fatty acid synthesis, with acyl intermediates covalently attached to its phosphopantetheine prosthetic group. Research has shown that beyond basic fatty acid synthesis, acpP influences EPA biosynthesis, a valuable omega-3 polyunsaturated fatty acid with significant biotechnological applications. This makes it a potential target for metabolic engineering to enhance production of valuable lipid compounds.

• How is the acyl carrier protein gene identified and characterized in P. tricornutum?

  The acyl carrier protein Δ9-desaturase gene (Phat3_J9316) in P. tricornutum was identified through bioinformatic analysis as the ortholog of the Arabidopsis soluble stearoyl-ACP desaturase (SAD) gene . The identification process typically involves:

  • Genome sequence analysis referencing the P. tricornutum genome database

  • Prediction of targeting sequences for subcellular localization

  • Analysis of conserved motifs, particularly the "ASAFAP" sequence

  For experimental characterization, researchers employ multiple approaches:

  Method	Purpose	Key Techniques
  Gene cloning	Expression studies	Chemical synthesis, vector construction, sequencing verification
  Functional analysis	Phenotypic assessment	Overexpression, knockouts, lipid profiling
  Protein characterization	Biochemical properties	Recombinant expression, activity assays, structural studies

  Confirmation of gene function often involves comparative analysis with homologs from other organisms, complementation studies, and detailed biochemical characterization of the purified protein.

• What expression systems are commonly used for producing recombinant acpP from P. tricornutum?

  Several expression systems can be utilized for producing recombinant proteins from P. tricornutum, including acpP:

  Homologous expression in P. tricornutum:

  • The HASP1 promoter has been identified as a strong endogenous promoter that drives high expression during all growth phases of P. tricornutum, outperforming the commonly used fcpA promoter which is less active during stationary phase .

  • The HASP1 signal peptide facilitates efficient secretion of recombinant proteins, as demonstrated using green fluorescent protein (GFP) as a reporter .

  Heterologous expression in E. coli:

  • While not specifically mentioned for acpP in the search results, prokaryotic expression systems using E. coli strains like BL21(DE3) are standard for recombinant protein production .

  • Typical vectors include pET system vectors with IPTG-inducible promoters.

  Transformation methodology for P. tricornutum:

  • Biolistic transformation (gene gun) is commonly used

  • Selection using antibiotic resistance markers

  • Screening for positive transformants using reporter genes like GFP

  The choice between homologous and heterologous expression depends on research goals, with homologous expression preferred when studying native function and post-translational modifications.

Advanced Research Questions

• How can the HASP1 promoter and signal peptide be optimized for enhancing recombinant acpP expression in P. tricornutum?

  Based on research findings, the HASP1 promoter and signal peptide have significant potential for recombinant protein expression and secretion in P. tricornutum . To optimize this system for acpP expression:

  Promoter optimization strategies:

  • Ensure full-length inclusion of the HASP1 promoter sequence

  • Create synthetic promoter variants with enhanced regulatory elements

  • Analyze promoter activity during different growth phases using reporter assays

  • Compare with other promoters like fcpA under various conditions

  Signal peptide enhancement:

  • Verify correct signal peptide cleavage sites

  • Consider codon optimization for the signal sequence

  • Test chimeric signal peptides combining elements from HASP1 and other efficient secretion signals

  • Monitor secretion efficiency using reporter proteins like GFP

  Expression construct design considerations:

  • Include appropriate introns if they enhance expression

  • Optimize the Kozak sequence for efficient translation initiation

  • Consider adding purification tags that don't interfere with protein folding

  • Include transcription terminators that enhance mRNA stability

  Experimental validation should involve quantitative PCR for transcript levels, Western blotting for protein expression, functional assays for purified protein, and microscopy for localization studies if using fluorescent tags.

• What methodological approaches can be used for studying the role of acpP in EPA biosynthesis in P. tricornutum?

  Research has shown that acyl carrier protein Δ9-desaturase plays a previously unknown role in EPA synthesis . Methodological approaches to study this include:

  Genetic manipulation techniques:

  • Overexpression using strong promoters like HASP1

  • Gene knockout using CRISPR-Cas9

  • RNAi-mediated gene silencing

  • Site-directed mutagenesis of catalytic residues

  Lipid analysis methods:

  • Gas chromatography-mass spectrometry (GC-MS) for fatty acid profiling

  • Liquid chromatography-mass spectrometry (LC-MS) for complex lipid analysis

  • Radiolabeling studies with 14C-acetate to track fatty acid flux

  • Lipidomics approaches for comprehensive lipid profile changes

  Recommended experimental workflow:

  1. Generate acpP overexpression and knockout strains

  2. Cultivate under standardized conditions

  3. Extract lipids using chloroform-methanol extraction

  4. Analyze fatty acid profiles by GC-MS

  5. Quantify EPA and precursors

  6. Perform statistical analysis to correlate acpP expression with EPA levels

  Strain Type	Expected Outcome	Analytical Method
  Wild-type	Baseline EPA levels	GC-MS, LC-MS
  acpP overexpression	Increased EPA production	GC-MS, LC-MS
  acpP knockout	Reduced EPA synthesis	GC-MS, LC-MS
  acpP site-directed mutants	Altered substrate specificity	Enzyme assays, GC-MS

  These approaches collectively provide a comprehensive understanding of how acpP influences EPA biosynthesis in P. tricornutum.

• What are the key considerations for purifying recombinant acpP from P. tricornutum for structural and functional studies?

  Purification of recombinant acpP requires careful consideration of protein properties and expression systems:

  Expression system selection:

  • Homologous expression in P. tricornutum using the HASP1 promoter and signal peptide for native-like protein

  • Heterologous expression in E. coli with appropriate tags for high yield

  • Consider secretion-based approaches using the HASP1 signal peptide for simplified purification

  Purification strategy design:

  • Affinity chromatography (His-tag, GST-tag, or other fusion partners)

  • Ion exchange chromatography based on theoretical pI

  • Size exclusion chromatography for final polishing

  • Consider native purification conditions to maintain activity

  Quality control assessments:

  • SDS-PAGE for purity evaluation

  • Western blotting for identity confirmation

  • Mass spectrometry for accurate mass determination and PTM analysis

  • Circular dichroism for secondary structure confirmation

  • Activity assays to verify functional integrity

  Storage considerations:

  • Buffer optimization (pH, salt concentration, additives)

  • Stability testing at different temperatures

  • Flash-freezing protocols to maintain activity

  • Addition of glycerol or other stabilizing agents

  For structural studies, additional purification steps may be necessary to achieve >95% purity, and special attention should be paid to removing aggregates and ensuring homogeneity of the sample for crystallization or NMR studies.

• How can metabolic flux analysis be applied to understand the impact of acpP modification on lipid biosynthesis in P. tricornutum?

  Metabolic flux analysis (MFA) provides quantitative insights into how acpP modifications affect lipid biosynthesis pathways:

  Stable isotope labeling approaches:

  • 13C-acetate or 13C-glucose feeding experiments

  • 2H (deuterium) labeling for hydrogen transfer reactions

  • 15N-labeling for tracking nitrogen metabolism

  • Multiple isotope labeling for comprehensive pathway analysis

  Analytical platforms:

  • GC-MS for fatty acid isotopomer distribution

  • LC-MS/MS for complex lipid analysis

  • NMR for positional isotopomer analysis

  • High-resolution MS for elemental composition confirmation

  Experimental design considerations:

  • Steady-state vs. non-steady-state approaches

  • Pulse-chase experiments for temporal resolution

  • Parallel labeling experiments with different tracers

  • Compartment-specific labeling strategies

  Sample experimental protocol:

  1. Cultivate wild-type and acpP-modified P. tricornutum in media with 13C-labeled substrate

  2. Sample at multiple time points

  3. Extract metabolites and perform GC-MS and LC-MS analysis

  4. Determine isotopomer distributions

  5. Calculate flux ratios and absolute fluxes

  6. Map changes onto the metabolic network to identify altered pathways

  Analysis Type	Information Provided	Relevance to acpP Function
  Isotopomer analysis	Pathway utilization	Determines acpP's role in directing carbon flow
  Flux ratio analysis	Relative pathway activities	Identifies bottlenecks in EPA synthesis
  Absolute flux calculation	Quantitative pathway rates	Measures impact of acpP modifications
  Time-course analysis	Dynamic responses	Reveals regulatory effects of acpP

  This approach provides quantitative insights into how acpP modifications specifically affect the flow of carbon through lipid biosynthesis pathways, particularly for EPA production .

• What are the challenges and solutions in resolving contradictory data regarding acpP function in lipid metabolism of P. tricornutum?

  When facing contradictory data regarding acpP function in P. tricornutum:

  Common sources of contradictions:

  • Strain differences between laboratories

  • Variations in culture conditions

  • Different analytical methods with varying sensitivities

  • Differences in genetic manipulation approaches

  • Influence of environmental factors on lipid metabolism

  Standardization approaches:

  • Establish standardized P. tricornutum reference strains

  • Develop detailed protocols for culture conditions

  • Create shared analytical standards and methods

  • Use multiple complementary analytical techniques

  • Report detailed metadata with experimental results

  Experimental design to resolve contradictions:

  • Side-by-side comparison of strains under identical conditions

  • Systematic variation of individual parameters

  • Collaboration between laboratories to replicate findings

  • Blind sample analysis by multiple research groups

  • Round-robin testing of analytical methods

  Methodological triangulation strategy:

  Approach	Method	Advantage	Limitation
  Genetic	Multiple manipulation techniques (overexpression, knockout, RNAi)	Direct causality	Potential compensation
  Analytical	Multiple platforms (GC-MS, LC-MS, NMR)	Comprehensive data	Method-specific biases
  Environmental	Testing under various conditions	Reveals context-dependence	Complex interactions
  Temporal	Multiple time points throughout growth	Captures dynamics	Resource intensive

  A comprehensive approach using multiple methods and rigorous standardization ensures that the true function of acpP in P. tricornutum lipid metabolism can be accurately determined despite initial conflicting results.

• How can CRISPR-Cas9 gene editing be optimized for studying acpP function in P. tricornutum?

  CRISPR-Cas9 gene editing provides powerful tools for precise genetic manipulation of acpP in P. tricornutum:

  CRISPR-Cas9 design considerations:

  • Codon-optimization of Cas9 for P. tricornutum

  • Selection of appropriate promoters for Cas9 expression (e.g., HASP1 )

  • Design of specific gRNAs targeting acpP coding regions

  • Inclusion of appropriate selection markers

  Delivery methods:

  • Biolistic transformation (gene gun)

  • Electroporation

  • Conjugation from bacteria

  • Chemical transformation methods

  Screening and validation protocols:

  • PCR-based screening for mutations

  • Sequencing of target regions

  • T7 endonuclease I assay for mutation detection

  • Protein expression analysis by Western blot

  Target selection strategies:

  • Target catalytic domains for complete loss of function

  • Target specific regions for domain-specific effects

  • Design homology-directed repair templates for precise modifications

  • Create conditional knockouts using inducible systems

  Off-target effect minimization:

  • Computational prediction of potential off-target sites

  • Whole-genome sequencing to detect off-target mutations

  • Use high-fidelity Cas9 variants

  • Titrate Cas9 and gRNA expression levels

  Editing Strategy	Application	Expected Outcome	Validation Method
  Complete knockout	Function elimination	Loss of EPA synthesis	Lipid profiling
  Domain-specific edits	Structure-function	Altered substrate specificity	Enzyme assays
  Promoter replacement	Expression control	Conditional phenotypes	qPCR, Western blot
  Tag insertion	Localization studies	Visualization of acpP	Fluorescence microscopy

  This approach provides precise insights into acpP function by creating defined genetic modifications and observing the resulting phenotypes.

• How can systems biology approaches integrate multi-omics data to elucidate acpP regulatory networks in P. tricornutum?

  Systems biology offers powerful approaches to integrate multi-omics data for understanding acpP regulatory networks:

  Data generation workflow:

  • RNA-seq for transcriptome profiling of wild-type vs. acpP-modified strains

  • Proteomics using LC-MS/MS for protein abundance changes

  • Targeted and untargeted metabolomics for metabolite profiling

  • Lipid profiling focusing on fatty acids and complex lipids

  • Chromatin immunoprecipitation sequencing (ChIP-seq) for transcription factor binding

  Integration methods:

  • Correlation network analysis across omics layers

  • Pathway enrichment analysis from multi-omics data

  • Genome-scale metabolic models incorporating expression data

  • Bayesian network inference of causal relationships

  • Machine learning approaches for pattern recognition

  Example experimental design:

  1. Generate acpP overexpression and knockout strains

  2. Culture under standard and stress conditions

  3. Collect samples for parallel omics analyses

  4. Process and normalize data from each platform

  5. Perform integrated analysis using multiple computational approaches

  6. Identify key regulatory hubs and interactions

  7. Validate predictions experimentally

  Omics Level	Technique	Information Provided	Relevance to acpP
  Genomics	Whole genome sequencing	Genetic background	Strain verification
  Transcriptomics	RNA-seq	Gene expression patterns	Co-regulated genes
  Proteomics	LC-MS/MS	Protein abundance	Post-transcriptional regulation
  Metabolomics	GC-MS, LC-MS	Metabolite levels	Pathway activities
  Lipidomics	Targeted LC-MS	Lipid profiles	acpP functional impact

  This systems biology approach provides a comprehensive understanding of how acpP functions within the broader regulatory and metabolic networks of P. tricornutum, particularly in relation to lipid metabolism and EPA production .

• What experimental approaches can resolve contradictory data regarding the effect of acpP overexpression on growth and lipid accumulation in P. tricornutum?

  To resolve contradictions regarding acpP effects on growth and lipid accumulation:

  Standardized strain development:

  • Create acpP overexpression strains using the same genetic background

  • Use multiple expression levels (low, medium, high) using different promoters

  • Include proper controls with empty vectors

  • Verify expression levels using qPCR and Western blotting

  Controlled cultivation conditions:

  • Use standardized media composition

  • Maintain consistent light intensity, photoperiod, and temperature

  • Monitor and control pH and dissolved CO2

  • Use bioreactors for precise parameter control

  Comprehensive phenotypic analysis:

  • Growth rates under multiple conditions

  • Cell size and morphology assessment

  • Detailed lipid profiling by class and fatty acid composition

  • Photosynthetic efficiency measurements

  Multi-laboratory validation:

  • Distribute identical strains to multiple laboratories

  • Implement standardized protocols

  • Perform blind analysis of samples

  • Pool data for meta-analysis

  Growth Phase	Parameter to Measure	Expected Outcome in acpP Overexpression	Method
  Lag phase	Cell viability	Possible decreased viability	Flow cytometry
  Exponential	Growth rate	Altered division rate	Cell counting, OD measurements
  Early stationary	Lipid accumulation onset	Earlier lipid accumulation	Nile red staining, GC-MS
  Late stationary	Total lipid content	Increased EPA content	Lipid extraction, GC-MS

  By systematically controlling variables and using multiple analytical approaches, contradictory data can be resolved to establish the true relationship between acpP expression and phenotypic outcomes.

• What are the best experimental designs for studying the impact of environmental factors on recombinant acpP expression and function in P. tricornutum?

  To study environmental impacts on recombinant acpP expression and function:

  Factorial experimental design approach:

  • Test multiple environmental factors simultaneously

  • Include appropriate controls for each condition

  • Use statistical design of experiments (DoE) to optimize testing efficiency

  • Apply response surface methodology to identify optimal conditions

  Key environmental variables to test:

  • Light intensity and spectral quality

  • Temperature ranges

  • Nutrient concentrations (nitrogen, phosphorus, silicon, iron)

  • Carbon dioxide levels

  • Salinity gradients

  • pH variations

  • Culture density effects

  Expression analysis methods:

  • qRT-PCR for transcript levels

  • Western blotting for protein abundance

  • Reporter gene assays (e.g., GFP fusion constructs)

  • Enzymatic activity assays for functional protein

  • Subcellular localization studies under different conditions

  Environmental factor testing matrix:

  Environmental Factor	Levels to Test	Measurement Endpoints
  Light intensity	50, 100, 200 μmol photons m⁻² s⁻¹	acpP expression, EPA content, growth rate
  Temperature	15°C, 20°C, 25°C	Protein activity, lipid profile, cell morphology
  Nitrogen concentration	N-limited, N-replete, N-excess	Gene expression, protein abundance, fatty acid composition
  CO₂ concentration	Ambient, 2x ambient, 5x ambient	Carbon fixation rate, lipid accumulation, EPA synthesis
  Salinity	25‰, 35‰, 45‰	Osmotic stress response, acpP localization, enzyme activity

  Time-course considerations:

  • Short-term responses (hours)

  • Acclimation responses (days)

  • Adaptation responses (weeks)

  • Transgenerational effects (multiple transfers)

  This comprehensive experimental design provides detailed insights into how environmental factors influence recombinant acpP expression and function, helping to optimize conditions for both research and potential biotechnological applications.