Recombinant Pseudomonas putida Tryptophan--tRNA ligase (trpS)

What is the biochemical function of Tryptophan--tRNA ligase in Pseudomonas putida?

Tryptophan--tRNA ligase (EC 6.1.1.2) in P. putida catalyzes the ATP-dependent esterification of L-tryptophan to its cognate tRNA(Trp). The reaction follows the equation:

L-tryptophan + tRNA(Trp) + ATP → AMP + L-tryptophanyl-[tRNA(Trp)] + diphosphate

This aminoacylation reaction is essential for translation, as it provides the charged tRNA necessary for incorporating tryptophan into nascent polypeptide chains during protein synthesis. The enzyme belongs to the class I aminoacyl-tRNA synthetase family and shows high substrate specificity for tryptophan and tRNA(Trp).

How is the trpS gene organized in the P. putida KT2440 genome?

The trpS gene in P. putida KT2440 is located at chromosomal position 1,498,074 to 1,499,423 (24.23 centisomes, 87°). It encodes a protein of 449 amino acids (1350 bp) and is identified with accession numbers G1G01-1398 (Pput160488Cyc), PP_1311, and Q88NA1 (UniProt) . Unlike the trp operons involved in tryptophan biosynthesis (such as trpE and trpGDC), the trpS gene is not part of a larger operon structure and appears to be independently regulated. This genomic organization differs from the tryptophan biosynthesis pathway genes, which are arranged in operons regulated by attenuation mechanisms in response to tryptophan availability .

What are the key structural differences between P. putida TrpS and tryptophan--tRNA ligases from other bacterial species?

While the search results don't provide specific structural information about P. putida TrpS, comparative analyses of bacterial tryptophanyl-tRNA synthetases generally reveal:

• A highly conserved catalytic domain containing the HIGH and KMSKS motifs characteristic of class I aminoacyl-tRNA synthetases

• Species-specific variations in the anticodon-binding domain that affect tRNA recognition

• Differences in oligomeric state (most bacterial TrpS enzymes function as dimers)

• Variations in surface charge distribution that may affect interaction with other cellular components

These structural differences can be exploited for the development of species-specific inhibitors or for engineering TrpS variants with altered properties for research applications.

What are the optimal expression systems for producing recombinant P. putida TrpS?

For recombinant expression of P. putida TrpS, several host systems can be considered based on the research findings:

E. coli Expression Systems:

• Standard E. coli BL21(DE3) with T7 promoter-based vectors offers high expression levels

• Expression under control of the tac promoter with IPTG induction (1 mM) has been successfully employed for other P. putida proteins

P. putida Self-Expression:

• P. putida KT2440 itself can serve as an expression host, particularly when native folding and post-translational modifications are critical

• Integrating the gene into ribosomal RNA operons (rrn) can provide strong constitutive expression

• CRISPR-assisted base editing systems have been developed for P. putida, facilitating precise genomic manipulations for optimized expression

When selecting an expression system, consider the research goals—high yield (E. coli) versus native-like processing (P. putida self-expression).

How can chromosomal integration methods improve recombinant TrpS expression in P. putida?

Chromosomal integration offers several advantages over plasmid-based expression for TrpS in P. putida:

Integration into rrn Operons:
Studies have demonstrated that integration of heterologous genes into ribosomal RNA (rrn) operons in P. putida results in remarkably stable and high-level expression. For example, the prodigiosin biosynthetic gene cluster from Serratia marcescens was exclusively found to integrate into the seven rrn operons when random Tn5 transposition was employed . This suggests that:

• rrn operons provide strong constitutive promoters for gene expression

• The expression level depends on the specific rrn operon and the distance between the rrn promoter and the integrated gene

• Integration does not significantly impact cellular fitness despite modifying essential rRNA genes

Integration Protocol:

• Construct a Tn5-based transposon containing the trpS gene

• Perform random transposition into P. putida genome

• Screen for high-expressing clones

• Identify integration sites using PCR with rrn-specific primers and sequencing

The efficiency of this approach was demonstrated with the prodigiosin gene cluster, where titers reached 94 mg/L in optimized conditions .

What media components can optimize recombinant TrpS expression and activity in P. putida?

Medium composition significantly affects recombinant protein expression and activity in P. putida. Based on research findings:

Key Media Components:

Optimized Media Composition for TrpS Expression:

Cultivation at 20-30°C with high aeration (>70% dissolved oxygen) typically yields optimal results for recombinant protein expression in P. putida .

How can CRISPR-based systems be used to optimize trpS expression in P. putida?

CRISPR-based systems offer precise genetic manipulation capabilities for optimizing trpS expression in P. putida:

Cytosine Base Editing Approach:
The development of CRISPR-assisted multiplex base editing systems for P. putida KT2440 provides powerful tools for trpS manipulation :

• Promoter Engineering: Cytosine base editors can be used to modify the trpS promoter region to enhance transcription, without creating double-strand breaks.

• Codon Optimization: Silent mutations can be introduced to optimize codon usage for improved translation efficiency.

• Multiplexed Engineering: The system supports simultaneous editing at multiple genomic loci, allowing coordinated modification of trpS and related genes.

Optimal CRISPR System Configuration:
Research has identified that the most efficient base editing system for P. putida consists of:

• APOBEC1 (rat cytosine deaminase)

• eSpCas9pp D10A (enhanced specificity Cas9 nickase codon-optimized for Pseudomonas)

• UGI (uracil DNA glycosylase inhibitor)

• sgRNA(s) targeting the desired sequence(s)

The one-plasmid system (pSEVA6BE) demonstrated 25-35% editing efficiency for multi-locus targeting, making it suitable for sophisticated trpS engineering approaches .

What are the regulatory mechanisms controlling native trpS expression in P. putida, and how can they be manipulated?

Understanding and manipulating the regulatory mechanisms of trpS expression requires insight into its transcriptional and post-transcriptional control:

Native Regulatory Mechanisms:
Unlike the trpE and trpGDC operons involved in tryptophan biosynthesis (which are regulated by attenuation mechanisms) , trpS likely employs different regulatory strategies:

• Potential Attenuation: Studies on P. putida tryptophan operons suggest that attenuation mechanisms involving tRNA(Trp) modification by MiaA might also influence trpS expression. Tn5 insertion mutations in miaA affected regulation of trp operons , potentially impacting trpS as well.

• Aminoacyl-tRNA Synthetase Regulation: In many bacteria, aminoacyl-tRNA synthetase genes are autoregulated through binding of the enzyme to its own mRNA, creating a feedback loop.

Manipulation Approaches:

• Promoter Replacement: Substituting the native promoter with constitutive or inducible promoters (e.g., P_tac) allows control over expression levels.

• 5′UTR Engineering: Modifying the 5′ untranslated region can alter translation efficiency and regulatory responsiveness.

• Attenuation Bypass: Based on the findings about attenuation in trp operons , engineering the leader peptide sequence or removing attenuation elements could enhance expression.

• Integration into rrn Operons: As demonstrated with heterologous genes , integration of trpS into ribosomal RNA operons places it under control of strong constitutive promoters, bypassing native regulation.

What are the most effective purification strategies for recombinant P. putida TrpS?

Purification of recombinant P. putida TrpS requires a multi-step approach to achieve high purity and preserved enzymatic activity:

Affinity Chromatography:

• His-tagged TrpS can be purified using Ni-NTA or TALON resins with imidazole gradients for elution

• Activity-based purification using tRNA(Trp)-coupled resins provides higher specificity

Chromatographic Separation Protocol:

• Cell lysis in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT, and protease inhibitors

• Clarification by centrifugation at 20,000 × g for 30 minutes

• Initial capture using affinity chromatography

• Intermediate purification by ion exchange chromatography (typically Q-Sepharose)

• Polishing step using size exclusion chromatography in 25 mM HEPES pH 7.5, 100 mM KCl, 10 mM MgCl₂, and 5 mM β-mercaptoethanol

Purification Challenges:
Based on protocols developed for related heterologous proteins in P. putida , special considerations include:

• Maintaining cold temperatures (4°C) throughout purification to preserve activity

• Including stabilizing agents such as glycerol (10-20%) and reducing agents

• Testing multiple buffer systems as TrpS activity is pH-dependent

What assays can be used to evaluate the aminoacylation activity of recombinant P. putida TrpS?

Several complementary assays can be employed to assess the aminoacylation activity of recombinant P. putida TrpS:

Radioactive Aminoacylation Assay:

• Incubate purified TrpS with ATP, [³H]- or [¹⁴C]-labeled L-tryptophan, and total tRNA or purified tRNA(Trp)

• At timed intervals, precipitate aminoacyl-tRNA using trichloroacetic acid

• Collect precipitates on filter discs and wash to remove unincorporated labeled amino acid

• Measure radioactivity by scintillation counting

Pyrophosphate Release Assay:

• Couple the TrpS reaction to enzymatic detection of released pyrophosphate (PPi)

• Use a continuous spectrophotometric assay with inorganic pyrophosphatase and other coupling enzymes

• Monitor NADH oxidation at 340 nm, which is proportional to PPi release

tRNA Charging Level Analysis:

• Perform the aminoacylation reaction with unlabeled tryptophan

• Use acid-urea PAGE to separate charged from uncharged tRNA species

• Visualize with methylene blue staining or Northern blotting

Kinetic Parameter Determination:
For comprehensive characterization, determine the following parameters:

How can structural studies of P. putida TrpS inform protein engineering efforts?

Structural studies of P. putida TrpS can provide crucial insights for rational engineering approaches:

Structural Analysis Methods:

• X-ray Crystallography: Determine high-resolution structures of:

  • Apo-enzyme

  • Enzyme-tryptophan complex

  • Enzyme-ATP complex

  • Enzyme-tRNA complex

• Cryo-Electron Microscopy: Visualize conformational changes during the aminoacylation reaction

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Map protein dynamics and solvent-accessible regions

Engineering Applications Based on Structural Insights:

• Substrate Specificity Engineering:

  • Identify residues in the tryptophan-binding pocket to modify specificity for tryptophan analogs

  • Engineer the anticodon recognition domain for altered tRNA specificity

• Thermostability Enhancement:

  • Target surface loops and introduce stabilizing interactions based on structural comparison with thermophilic homologs

  • Identify and eliminate conformationally strained regions

• Activity Optimization:

  • Modify catalytic residues to enhance aminoacylation rate

  • Engineer enzyme dynamics based on HDX-MS data

Drawing from approaches used in other P. putida proteins, CRISPR-assisted multiplex base editing can be employed to introduce multiple simultaneous mutations guided by structural insights .

How can recombinant P. putida TrpS be used in tryptophan metabolism studies?

Recombinant P. putida TrpS serves as a valuable tool for investigating tryptophan metabolism in several research contexts:

Metabolic Engineering Applications:

• Tryptophan-Dependent Pathway Enhancement:

  • Overexpression of TrpS can increase the availability of charged tRNA(Trp) for production of tryptophan-rich proteins

  • This approach has been applied in similar contexts where targeted overexpression of aminoacyl-tRNA synthetases alleviated translation bottlenecks

• Isotope Labeling Studies:

  • TrpS can be used in vitro to specifically charge tRNA(Trp) with isotopically labeled tryptophan

  • The charged tRNA can then be used in cell-free protein synthesis systems for site-specific labeling of proteins

• Biosensor Development:

  • TrpS activity-based biosensors can be engineered to detect tryptophan availability in vivo

  • These systems can be integrated with the base editing tools developed for P. putida to create feedback-responsive systems

Integration with Tryptophan Biosynthesis Pathways:
P. putida contains well-characterized tryptophan biosynthesis pathway genes organized in operons (trpE and trpGDC) that are regulated by attenuation mechanisms . Coordinated engineering of these pathways along with trpS can create strains with optimized tryptophan metabolism for various applications.

What are the common challenges in working with recombinant P. putida TrpS and how can they be addressed?

Research with recombinant P. putida TrpS presents several challenges that require specific troubleshooting approaches:

Expression Challenges:

Activity and Stability Issues:

Experimental Design Considerations:

• For in vivo studies, consider the effect of media components—particularly tryptone, which has been shown to significantly affect P. putida physiology beyond simply providing nutrients

• When engineering TrpS variants, note that CRISPR-based cytidine deaminase systems in P. putida work most efficiently at TC and CC motifs, with moderate efficiency at AC motifs and poor efficiency at GC motifs

How does TrpS function integrate with broader regulatory networks in P. putida?

TrpS function is interconnected with several regulatory networks in P. putida, with implications for research applications:

Integration with Tryptophan Regulation:
Studies on P. putida trp operons revealed that their regulation occurs through attenuation mechanisms involving tRNA(Trp) . This suggests a regulatory circuit where:

• TrpS activity directly affects the pool of charged tRNA(Trp)

• The charged/uncharged tRNA(Trp) ratio influences attenuation-based regulation of trp operons

• This creates a feedback loop connecting translation efficiency, tryptophan biosynthesis, and TrpS activity

Stress Response Connections:
Research on P. putida has shown that:

• Media components like tryptone affect biofilm formation and stress responses

• Integration of heterologous genes into rRNA operons affects their expression but shows minimal impact on cellular fitness

• These observations suggest TrpS activity may be maintained even under stress conditions to ensure translation fidelity

Metabolic Network Position:
As demonstrated in studies with other P. putida genes, TrpS occupies a critical position between:

• Primary metabolism (tryptophan biosynthesis and utilization)

• Translation processes (providing charged tRNA)

• Secondary metabolism (tryptophan-derived specialized metabolites)

This network position makes TrpS an attractive target for metabolic engineering approaches, particularly when coordinated with modifications to other pathways using multiplex genome editing techniques developed for P. putida .

What emerging technologies could enhance recombinant TrpS applications in P. putida research?

Several cutting-edge technologies show promise for advancing recombinant TrpS research in P. putida:

Cell-Free Expression Systems:
Developing P. putida-based cell-free protein synthesis systems optimized for TrpS expression would allow:

• Rapid protein engineering without cellular transformation

• Direct incorporation of unnatural amino acids into TrpS

• High-throughput screening of TrpS variants

Non-Canonical Amino Acid Incorporation:
Engineering TrpS to recognize and charge tRNA(Trp) with tryptophan analogs could enable:

• Site-specific incorporation of fluorescent tryptophan analogs into proteins

• Creation of novel bioactive peptides with enhanced properties

• Development of orthogonal translation systems in P. putida

Systems Biology Integration:
The application of multi-omics approaches to study TrpS in the context of P. putida metabolism can:

• Reveal unexpected regulatory connections

• Identify optimal engineering targets for enhanced tryptophan metabolism

• Guide the development of P. putida as a chassis for specialized metabolite production

Advanced CRISPR Technologies:
Building upon the CRISPR-assisted multiplex base editing system developed for P. putida , future approaches could include:

• Prime editing for precise insertions and replacements in the trpS gene

• CRISPR interference (CRISPRi) for tunable repression of trpS

• CRISPR activation (CRISPRa) for enhanced expression without genetic modification

How might engineered TrpS variants contribute to expanding the genetic code in P. putida?

Engineered TrpS variants represent powerful tools for expanding the genetic code in P. putida through several mechanisms:

Site-Specific Incorporation of Non-Canonical Amino Acids:
By engineering the tryptophan-binding pocket of TrpS, researchers can develop variants that:

• Recognize and activate tryptophan analogs with novel chemical functionalities

• Charge these analogs onto tRNA(Trp)

• Enable incorporation at specific positions in response to amber stop codons or quadruplet codons

Orthogonal Translation Systems:
Drawing from research on heterologous expression in P. putida , engineered TrpS-tRNA pairs could:

• Function independently from the native translation machinery

• Support incorporation of multiple different non-canonical amino acids simultaneously

• Be integrated into chromosomal locations (like rrn operons) for stable expression

Applications in Synthetic Biology:
The expansion of the genetic code in P. putida through engineered TrpS variants would enable:

• Production of proteins with novel chemistries for biocatalysis

• Creation of biocontainment strategies through dependence on non-canonical amino acids

• Development of protein-based biosensors with enhanced capabilities

Integration with Existing P. putida Engineering Tools:
The CRISPR-assisted multiplex base editing systems developed for P. putida provide an ideal platform for engineering TrpS variants, allowing multiple precise modifications to be introduced simultaneously.

What potential exists for using P. putida TrpS in comparative studies across Pseudomonas species?

Comparative studies of TrpS across Pseudomonas species offer valuable insights for both fundamental research and biotechnological applications:

Evolutionary Analysis:
Comparing P. putida TrpS with homologs from other Pseudomonas species (P. aeruginosa, P. fluorescens, P. entomophila) can reveal:

• Adaptive changes related to different ecological niches

• Conservation patterns indicating functional constraints

• Species-specific regulatory mechanisms

Cross-Species Functionality:
The development of CRISPR-based genetic tools across multiple Pseudomonas species enables testing of TrpS interchangeability:

• Does P. putida TrpS function efficiently in other Pseudomonas hosts?

• Are there species-specific interactions with translation machinery?

• How do regulatory mechanisms differ across species?

Biotechnological Implications:
Comparative studies can guide the selection of optimal TrpS variants for specific applications:

• Identify naturally occurring TrpS variants with enhanced properties

• Develop chimeric enzymes combining beneficial features from different species

• Optimize heterologous expression systems based on species-specific characteristics

Methodological Approach:
Building on established techniques for Pseudomonas species , a comprehensive comparative study would:

• Express and characterize TrpS from multiple Pseudomonas species under identical conditions

• Analyze kinetic parameters, substrate specificity, and stability

• Perform cross-complementation studies in TrpS-deficient strains

• Identify species-specific differences in regulation and interaction partners