Recombinant Bacillus licheniformis Acylphosphatase (acyP)

What is Bacillus licheniformis acylphosphatase (acyP) and why is it significant for research?

Bacillus licheniformis acylphosphatase (acyP) is an enzyme that catalyzes the hydrolysis of acyl phosphates, releasing inorganic phosphate. This enzyme belongs to the acylphosphatase family and is of particular interest in research due to B. licheniformis' exceptional status as an expression platform in biomanufacturing. B. licheniformis is recognized for its ability to produce high-value products and secrete proteins into the extracellular medium, making its acylphosphatase a valuable target for recombinant expression studies . The significance of acyP extends beyond its catalytic function, as it serves as a model system for studying protein folding, stability, and function in thermophilic bacteria.

Which promoter systems are most effective for expressing recombinant acyP in B. licheniformis?

Several promoter systems have demonstrated effectiveness for recombinant protein expression in B. licheniformis, with their selection depending on experimental requirements:

• Constitutive promoters: The P43 promoter derived from B. subtilis is widely used in B. licheniformis and provides consistent expression without requiring induction . This is suitable for basic acyP expression when controlled induction is not necessary.

• Inducible promoters: For controlled acyP expression, several options exist:

  • The P-bacA promoter derived from the bacitracin synthase operon provides strong endogenous expression in B. licheniformis .

  • The P-alsSD promoter, which regulates the alsSD operon, has shown significantly higher activity than many other promoters in B. licheniformis .

  • The APase I gene promoter functions as a very strong inducible promoter that is tightly regulated by phosphate concentration .

• Substrate-specific inducible promoters:

  • Xylose-inducible promoter (P-xyl)

  • Rhamnose-inducible promoter (P-rha)

  • Mannose-inducible promoter (P-man)

The phosphate-regulated APase I promoter may be particularly relevant for acyP expression, as both enzymes are involved in phosphate metabolism, potentially allowing for coordinated expression in response to cellular phosphate levels .

What expression vectors are recommended for cloning and expressing acyP in B. licheniformis?

For effective cloning and expression of acyP in B. licheniformis, researchers should consider the following vector characteristics:

• Temperature-sensitive vectors: These facilitate genome integration and are useful for stable expression. The pHY-amyL system has demonstrated successful high-yield recombinant protein production in B. licheniformis .

• Shuttle vectors: Vectors that can replicate in both E. coli and B. licheniformis facilitate easier cloning processes.

• Signal peptide incorporation: Including the appropriate signal peptide, such as the aprE signal peptide, can enhance secretion of recombinant acyP, enabling simpler purification processes .

• Selection markers: Vectors should contain appropriate selection markers compatible with B. licheniformis.

• Promoter flexibility: Vectors that allow for easy exchange of promoters provide versatility in expression strategies. For acyP expression, considering vectors that accommodate the phosphate-regulated APase I promoter might be beneficial due to functional relationships between these enzymes .

When constructing expression vectors, include appropriate regulatory elements such as ribosome binding sites (RBS) and terminators to optimize expression levels .

What growth conditions optimize recombinant acyP expression in B. licheniformis?

Optimizing growth conditions for recombinant acyP expression in B. licheniformis requires attention to several parameters:

• Media composition:

  • Low-cost fermentation media containing soybean meal and cottonseed meal have demonstrated success for high-level recombinant protein production in B. licheniformis .

  • For regulated expression, media composition should be adjusted based on the selected promoter system. For instance, when using P-rha, the presence of rhamnose at 1.5% concentration for 8 hours has shown optimal induction .

• Temperature and pH:

  • B. licheniformis is thermotolerant, with optimal growth at 30-50°C.

  • An alkaline pH typically enhances protein production in B. licheniformis .

• Induction conditions:

  • For phosphate-regulated promoters like APase I, low phosphate concentrations trigger induction .

  • For substrate-specific promoters, induction timing and concentration significantly influence expression efficiency. For example, with the rhamnose-inducible system, an additional 24-hour cultivation period (approximately three generations) following induction has shown improved results .

• Aeration and agitation:

  • B. licheniformis requires oxygen for optimal growth and protein expression.

  • Appropriate agitation rates in shake-flask cultures or controlled dissolved oxygen levels in bioreactors are essential.

• Growth phase for induction:

  • Timing induction to correspond with specific growth phases can significantly impact recombinant protein yields.

A systematic optimization approach testing various combinations of these parameters is recommended to achieve maximum acyP expression levels.

What strategies can improve recombinant acyP solubility and prevent inclusion body formation in B. licheniformis?

Improving recombinant acyP solubility in B. licheniformis requires a multifaceted approach targeting both expression conditions and protein characteristics:

• Promoter strength modulation:

  • Using moderate-strength promoters instead of very strong ones can reduce protein accumulation rate, allowing proper folding.

  • Consider employing the phosphate-regulated APase I promoter with carefully controlled phosphate levels to fine-tune expression rates .

• Co-expression strategies:

  • Co-express molecular chaperones to assist in protein folding.

  • Implement sequential induction systems where chaperones are expressed before acyP.

• Signal peptide optimization:

  • Engineer the signal peptide to improve protein secretion efficiency. The aprE signal peptide has shown success in B. licheniformis for other recombinant proteins .

  • Consider overexpressing appropriate signal peptidases (like SipV) to enhance secretion efficiency .

• Fusion tags approach:

  • N-terminal fusions with solubility-enhancing partners like thioredoxin or SUMO.

  • Include appropriate protease cleavage sites for tag removal post-purification.

• Growth conditions adjustment:

  • Lower cultivation temperature (25-30°C) during induction phase to slow protein synthesis.

  • Optimize media composition to provide precursors needed for proper folding.

• Genetic modifications:

  • Consider codon optimization based on B. licheniformis preference.

  • Introduce site-directed mutations in acyP to enhance thermostability and folding without affecting catalytic activity.

Implementing a combination of these strategies with systematic evaluation will likely yield the best results for improving acyP solubility in this expression system.

How can gene editing tools be optimized for modifying the native acyP gene in B. licheniformis?

Advanced gene editing of the native acyP gene in B. licheniformis can be accomplished through several optimized approaches:

• RecT-based recombination system:

  • Implement the bacteriophage-derived RecT recombinase system under the control of a rhamnose-inducible promoter (P-rha).

  • This system has demonstrated a remarkable 10^5-fold enhancement in recombination efficiency in B. licheniformis .

  • Optimal conditions include transformation with the genome editing plasmid, followed by cultivation and induction with 1.5% rhamnose for 8 hours, with subsequent cultivation for an additional 24 hours (approximately three generations) .

• CRISPR/Cas9n system:

  • Utilize the CRISPR/Cas9n gene editing system with optimized expression of Cas9n protein under the P43 promoter.

  • This system has achieved 100% editing efficiency for single gene modifications in B. licheniformis .

  • Following Cas9n-mediated DNA single-strand cleavage, homologous recombination with complementary fragments enables precise integration or modification of the acyP gene .

• Homologous recombination optimization:

  • Design extended homology arms (>500 bp) flanking the target locus to enhance recombination efficiency.

  • Implement counter-selection markers to facilitate the isolation of successful recombinants.

• Temperature-sensitive plasmid strategy:

  • Employ temperature-sensitive plasmids as vectors for homologous recombination components.

  • This approach facilitates plasmid integration at permissive temperatures and resolution at non-permissive temperatures.

For optimal results, researchers should consider combining the RecT-based recombination system with CRISPR/Cas9n technology, as the efficiency of homologous recombination is often the rate-limiting step in B. licheniformis genome editing .

What analytical methods are most effective for characterizing recombinant acyP activity and structural integrity?

Comprehensive characterization of recombinant acyP requires multiple analytical approaches targeting enzyme activity, structural integrity, and biophysical properties:

• Enzymatic activity assays:

  • Spectrophotometric monitoring of phosphate release using malachite green or molybdate-based detection methods.

  • Continuous assays tracking the hydrolysis of model substrates like benzoyl phosphate.

  • Determination of kinetic parameters (K_m, k_cat, v_max) under varying conditions of pH, temperature, and ionic strength.

• Structural analysis:

  • Circular dichroism (CD) spectroscopy to assess secondary structure elements and thermal stability.

  • Intrinsic fluorescence spectroscopy to evaluate tertiary structure integrity.

  • Differential scanning calorimetry (DSC) to determine transition temperatures and thermodynamic parameters.

  • X-ray crystallography or NMR spectroscopy for high-resolution structural determination.

• Mass spectrometry approaches:

  • Intact protein mass analysis to confirm correct processing and absence of modifications.

  • Peptide mapping after proteolytic digestion to verify sequence integrity.

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe structural dynamics and solvent accessibility.

• Stability assessments:

  • Accelerated stability studies under various storage conditions.

  • Thermostability analysis using activity retention assays after heat treatment.

  • Resistance to proteolysis as a measure of structural compactness.

• Comparative analysis:

  • Direct comparison with native acyP purified from B. licheniformis.

  • Benchmarking against acylphosphatases from other bacterial sources.

These analytical methods should be employed in combination to provide a comprehensive profile of the recombinant acyP, ensuring that the expressed protein maintains both structural integrity and functional activity compared to the native enzyme.

How can transcriptomic and proteomic analyses be used to optimize acyP expression in B. licheniformis?

Implementing multi-omics approaches can significantly enhance acyP expression optimization in B. licheniformis:

• Transcriptomic analysis strategies:

  • RNA-seq to identify strongest native promoters under various conditions. For example, Wu et al. used transcriptome data to select the top 10 promoters with the largest upregulation fold for study, identifying P-alsSD as a highly active promoter .

  • Analysis of transcript stability and degradation kinetics for acyP mRNA.

  • Identification of transcription factor binding sites affecting acyP expression.

  • Assessment of global transcriptional changes in response to acyP overexpression to identify potential bottlenecks.

• Proteomic approaches:

  • Quantitative proteomics to measure actual acyP protein levels and correlate with transcriptomic data.

  • Pulse-chase experiments to determine protein half-life and degradation rates.

  • Secretome analysis to evaluate secretion efficiency and identify potential proteases affecting yield.

  • Systematic analysis of chaperone and folding factor expression levels during acyP production.

• Integrated analysis workflow:

  • Comparative analysis of wild-type and engineered strains.

  • Time-course sampling during batch fermentation.

  • Correlation of transcriptomic and proteomic data to identify post-transcriptional bottlenecks.

  • Metabolomic integration to assess metabolic burden and substrate availability.

• Data-driven strain engineering:

  • Use multi-omics data to identify limiting factors (e.g., tRNA availability, chaperone levels).

  • Design rational engineering strategies targeting identified bottlenecks.

  • Implement iterative design-build-test-learn cycles with multi-omics analysis at each iteration.

Based on such analyses, researchers have successfully enhanced protein production in B. licheniformis, achieving up to 17.6 mg/ml protein concentration in optimized systems . Similar approaches can be applied specifically to acyP expression optimization.

What are the challenges and solutions for scaling up recombinant acyP production from shake flasks to bioreactors?

Scaling up recombinant acyP production in B. licheniformis presents several challenges that require systematic solutions:

This systematic approach to bioreactor scale-up has enabled researchers to achieve protein concentrations of up to 17.6 mg/ml in B. licheniformis cultivation , suggesting similar success could be achieved for recombinant acyP production with appropriate optimization.