Recombinant Pseudomonas putida 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (ispG)

What is 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG) and its function in P. putida?

IspG, also known as 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, is a key enzyme in the methylerythritol phosphate (MEP) pathway of Pseudomonas putida. This enzyme catalyzes the conversion of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECPP) to 4-hydroxy-3-methyl-2-butene pyrophosphate (HMBPP) in the penultimate step of the MEP pathway .

The MEP pathway in Pseudomonas is also called the non-mevalonate pathway, which begins with pyruvate and glyceraldehyde 3-phosphate (GAP) as substrates. Through a series of enzymatic reactions, MECPP is formed and then converted to HMBPP by IspG. Subsequently, HMBPP is reduced to isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) by HMBPP reductase (IspH) . These isoprenoid precursors are essential for various cellular processes including cell membrane formation and secondary metabolite production.

In P. putida, IspG has been identified as one of the rate-limiting enzymes in the MEP pathway, making it a significant target for metabolic engineering efforts aimed at enhancing the production of isoprenoid-derived compounds .

How does the MEP pathway in P. putida differ from other biosynthetic pathways?

The MEP pathway in Pseudomonas putida represents an alternative route for isoprenoid biosynthesis compared to the more widely known mevalonate (MVA) pathway found in eukaryotes and some bacteria. A distinctive feature of Pseudomonas species is that they exclusively possess the MEP pathway for isoprenoid production, lacking the alternative MVA pathway .

The MEP pathway begins with the condensation of pyruvate and glyceraldehyde 3-phosphate, proceeding through several intermediates including MECPP, which is then converted to HMBPP by IspG. The final steps involve the formation of IPP and DMAPP, which can be reversibly interconverted by isopentenyl diphosphate isomerase (IDI) .

What makes this pathway particularly interesting in P. putida is its integration with the organism's remarkably versatile metabolism. This bacterium has evolved to thrive in harsh environments and can efficiently utilize various carbon sources, allowing for the development of bioprocesses that convert renewable feedstocks into valuable compounds through the MEP pathway .

Recent research has identified key rate-limiting steps in this pathway, with genes ispG and ispH playing crucial roles. Studies have demonstrated that these enzymes could be important control points for enhancing the production of secondary metabolites derived from the MEP pathway .

What is the genomic context of ispG in P. putida KT2440?

In Pseudomonas putida KT2440, the ispG gene exists within a complex genomic landscape characterized by the organism's adaptability to various environments. P. putida KT2440 has a genome that contains numerous genetic elements including insertion sequences such as ISPpu9, which are inserted into repetitive extragenic palindromic sequences (REPs) .

While the ispG gene itself is not directly associated with these insertion sequences, understanding the genomic context is important because genetic engineering approaches often need to account for the potential impact of mobile genetic elements on gene expression and stability. The presence of REPs, which are highly conserved sequences containing imperfect palindromes found mostly in non-coding regions, may influence the expression of nearby genes including those involved in the MEP pathway .

P. putida KT2440 contains more than 800 REPs with a conserved 35 bp sequence, which serves as targets for some insertion sequence elements . This genomic landscape must be considered when designing recombinant systems involving ispG, as these elements could potentially affect gene expression or lead to genomic instability if not properly addressed in genetic engineering strategies.

How does co-optimization of IspG and IspH enhance MEP pathway flux?

Recent research has revealed a surprising finding: P. putida IspG and IspH require co-optimization to effectively enhance flux through the methylerythritol phosphate pathway . This discovery challenges the conventional approach of optimizing individual enzymes separately and highlights the complex interactions between consecutive enzymes in metabolic pathways.

The co-dependence between IspG and IspH likely stems from their sequential roles in the MEP pathway, where IspG produces HMBPP, which is then immediately processed by IspH to form IPP and DMAPP. Studies involving the cloning of ispG and ispH from different organisms into expression vectors have demonstrated this interdependence . When both enzymes are optimized together, the efficiency of HMBPP conversion to final products significantly improves, preventing potential bottlenecks caused by imbalanced enzyme activities.

Metabolic engineering approaches that account for this co-optimization can achieve substantially higher yields of isoprenoid-derived compounds. This finding is particularly relevant for the development of P. putida as an industrial cell factory, as it provides a more effective strategy for enhancing the production of valuable bioproducts through the MEP pathway .

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

Developing effective expression systems for recombinant P. putida IspG requires careful consideration of multiple factors including promoter strength, induction conditions, and host compatibility. Based on experimental approaches described in recent literature, several effective strategies have emerged.

The broad host range, IPTG-inducible expression vector system has proven effective for functional expression of ispG genes. For instance, the pRL814 vector has been successfully used to clone and express ispG from various bacterial sources . This vector provides tight regulation of gene expression and can be used in different bacterial hosts, offering flexibility for comparative studies.

For expression in P. putida itself, several genetic tools have been developed that enable precise control of gene expression. The I-SceI-based system, which involves two rounds of recombination using suicide plasmids, has proven effective for chromosome modification in P. putida . This approach allows for stable integration of recombinant genes into the chromosome, which can be advantageous for long-term expression compared to plasmid-based systems.

Recent innovative developments have also introduced CRISPR/Cas9 technologies for genetic manipulation in P. putida . These systems offer precise genome editing capabilities and can be used for gene deletion, insertion, and replacement, providing versatile options for optimizing ispG expression.

What structural features affect the catalytic activity of P. putida IspG?

IspG belongs to a family of iron-sulfur cluster-containing enzymes that perform complex radical-mediated reactions. The catalytic activity of P. putida IspG is heavily influenced by several structural features, though specific crystallographic data for the P. putida enzyme remains limited.

The enzymatic mechanism of IspG involves a [4Fe-4S] cluster that plays a critical role in the reductive dehydroxylation of MECPP to form HMBPP. This iron-sulfur cluster must be properly formed and maintained for optimal enzyme activity, requiring specific growth and expression conditions that support iron-sulfur cluster biogenesis.

Comparative analysis with IspG from other organisms suggests that the enzyme contains conserved cysteine residues responsible for coordinating the iron-sulfur cluster, as well as specific amino acid residues that interact with the substrate and facilitate electron transfer. Mutations in these residues can significantly impact catalytic efficiency.

The optimization of expression conditions, including growth media composition (particularly iron supplementation), induction parameters, and purification strategies, is crucial for obtaining catalytically active recombinant P. putida IspG for structural and functional studies.

How can recombinant P. putida IspG be effectively expressed and purified?

The successful expression and purification of recombinant P. putida IspG requires specific methodological approaches that address the challenges associated with iron-sulfur proteins. The following protocol has been established based on current research:

Expression Strategy:

• Clone the ispG gene from P. putida into an appropriate expression vector such as pRL814, which provides IPTG-inducible expression .

• Transform the construct into a suitable expression host. While E. coli is commonly used, expression in P. putida itself may provide advantages due to compatible codon usage and chaperone systems.

• Culture the recombinant strain in iron-supplemented media to support iron-sulfur cluster formation. LB medium supplemented with ferric ammonium citrate (0.1-0.5 mM) is often effective.

• Induce expression at reduced temperatures (16-25°C) to enhance proper protein folding and iron-sulfur cluster incorporation.

• Include iron-sulfur cluster biogenesis helper proteins by co-expressing isc or suf operon genes if expression levels or activity are suboptimal.

Purification Protocol:

• Harvest cells and lyse under anaerobic conditions to protect the oxygen-sensitive iron-sulfur clusters.

• Clarify the lysate by centrifugation and proceed with initial capture using affinity chromatography (if using tagged protein) or ion exchange chromatography.

• Perform additional purification steps under anaerobic conditions, which may include size exclusion chromatography to achieve high purity.

• Confirm protein identity and purity using SDS-PAGE, Western blotting, and mass spectrometry.

• Verify iron-sulfur cluster formation using UV-visible spectroscopy, looking for characteristic absorption peaks around 410-420 nm.

This methodological approach addresses the specific challenges of expressing and purifying an iron-sulfur protein while maintaining its catalytic activity for subsequent functional studies.

What assays can be used to measure IspG enzymatic activity?

Several complementary assays have been developed to accurately measure the enzymatic activity of recombinant P. putida IspG, each with specific advantages and limitations:

HPLC-Based Substrate Consumption Assay:
This assay monitors the conversion of MECPP to HMBPP by quantifying substrate depletion and product formation using high-performance liquid chromatography (HPLC). MECPP can be detected by reverse-phase HPLC using appropriate ion-pairing reagents, while product formation can be measured either directly or after dephosphorylation. This assay provides direct quantitative measurements but requires specialized equipment and synthetic standards.

Coupled Spectrophotometric Assay:
The reductive nature of the IspG reaction allows for coupling to electron donor regeneration systems. The reaction can be coupled to the oxidation of NADPH via ferredoxin/flavodoxin and reductase, with activity measured as the decrease in NADPH absorbance at 340 nm. This assay provides continuous real-time monitoring but may be affected by the efficiency of the coupling system.

Functional Complementation Assay:
For studying IspG variants, functional complementation in bacterial strains with conditional ispG mutations offers a physiologically relevant approach. As demonstrated in the literature, ispG genes from different sources can be cloned into expression vectors like pRL814 for complementation assays . This approach assesses function in a cellular context but provides qualitative rather than quantitative data.

Mass Spectrometry-Based Assay:
LC-MS/MS can be used to detect and quantify MECPP and HMBPP with high sensitivity and specificity. This approach is particularly valuable for kinetic studies and can detect potential reaction intermediates, though it requires specialized equipment and expertise.

How can the MEP pathway be engineered to enhance ispG-dependent product formation?

Engineering the MEP pathway to enhance ispG-dependent product formation requires a systems biology approach that addresses multiple aspects of pathway regulation and enzyme function. Several strategies have proven effective in recent research:

Co-optimization of IspG and IspH:
Research has demonstrated that IspG and IspH need to be co-optimized to improve flux through the MEP pathway . This can be achieved by:

• Balancing expression levels of both enzymes

• Engineering protein scaffolds that facilitate substrate channeling between IspG and IspH

• Selecting enzyme variants with complementary kinetic properties

Increasing Precursor Availability:
Enhancing the supply of MECPP, the substrate for IspG, can increase pathway flux. This has been accomplished by:

• Overexpressing upstream MEP pathway enzymes

• Engineering central carbon metabolism to increase pyruvate and GAP availability

• Implementing dynamic regulatory systems that respond to precursor levels

Overcoming Rate-Limiting Steps:
Research has identified ispG and ispH as important rate-limiting steps in isoprenoid biosynthesis . Strategies to address these limitations include:

• Overexpression of key enzymes using strong, inducible promoters

• Expression of isopentenyl diphosphate isomerase (IDI) to enhance the interconversion between IPP and DMAPP

• Implementing feedback-resistant enzyme variants to prevent pathway inhibition

Chassis Optimization:
P. putida has emerged as a valuable chassis for industrial biotechnology due to its versatile metabolism and stress tolerance . Optimizing this chassis for enhanced MEP pathway function involves:

• Deletion of competing pathways that drain precursors or cofactors

• Engineering cofactor regeneration systems to maintain reducing power

• Implementing novel genetic tools including CRISPR/Cas9 technologies for precise genome modifications

These engineering approaches have facilitated the development of P. putida strains with enhanced production of various valuable compounds through the MEP pathway, establishing this organism as a promising industrial cell factory for sustainable bioproduction .

How is recombinant P. putida IspG utilized in terpenoid production?

Recombinant P. putida IspG plays a crucial role in metabolic engineering efforts aimed at producing high-value terpenoids. The strategic manipulation of IspG activity, along with other MEP pathway enzymes, has enabled significant advances in this field:

P. putida has emerged as an excellent chassis for terpenoid production due to its metabolic versatility and stress tolerance. Unlike many other bacterial hosts, Pseudomonas exclusively relies on the MEP pathway for isoprenoid precursor synthesis , making IspG optimization particularly impactful in these organisms.

One noteworthy application is the production of terpenoid phenazines, which exhibit potential antitumor and antibacterial activities. Researchers have successfully constructed artificial biosynthetic pathways in Pseudomonas species by engineering the MEP pathway in conjunction with downstream enzymes . For instance, the introduction of prenyltransferase PpzP from Streptomyces anulatus into P. chlororaphis P3 enabled the synthesis of endophenazine A and endophenazine A1, with yields reaching 279.43 mg/L and 189.2 mg/L respectively after metabolic engineering and medium optimization .

The engineering of these pathways involves careful consideration of IspG activity, as this enzyme catalyzes the conversion of MECPP to HMBPP, which can either continue through the canonical pathway to form IPP and DMAPP, or potentially be directly utilized as a substrate for certain prenyltransferases. In the case of endophenazine A1 production, researchers discovered that this compound was produced through a "leakage" of the intermediate HMBPP, highlighting the importance of understanding IspG activity and product fate in designing effective production systems .

What are the challenges in scaling up processes involving recombinant P. putida IspG?

Scaling up processes involving recombinant P. putida IspG presents several significant challenges that researchers must address to achieve efficient and economically viable production:

Maintaining Optimal Enzyme Activity:
IspG contains oxygen-sensitive iron-sulfur clusters that can be challenging to maintain in large-scale fermentation systems. Strategies to address this include:

• Implementing controlled dissolved oxygen levels in bioreactors

• Engineering more oxygen-tolerant IspG variants

• Developing fed-batch processes that balance growth and product formation phases

Metabolic Burden and Genetic Stability:
The expression of recombinant proteins can impose a significant metabolic burden on the host, potentially leading to genetic instability and reduced productivity during scale-up. This challenge can be addressed through:

• Integration of expression cassettes into the chromosome rather than using plasmid-based systems

• Employment of inducible promoters with tunable expression levels

• Utilization of advanced genetic tools such as CRISPR/Cas9 for stable genome modifications

Balancing Pathway Flux:
The need for co-optimization of IspG and IspH to improve pathway flux becomes even more critical at scale, where imbalances can lead to:

Process Economics:
The economic viability of processes involving recombinant P. putida IspG depends on:

• Developing efficient downstream processing methods for product recovery

• Utilizing inexpensive renewable feedstocks and waste streams as carbon sources

• Optimizing media composition to minimize production costs while maintaining productivity

The advancement of genetic tools specifically developed for P. putida, including various recombineering methods and CRISPR/Cas9 technologies , has significantly improved our ability to address these challenges. These tools enable precise genetic modifications that enhance stability and productivity during scale-up, supporting the continued development of P. putida as an industrial cell factory for sustainable bioproduction.

What emerging technologies might enhance our understanding of P. putida IspG?

Several cutting-edge technologies are poised to significantly advance our understanding of P. putida IspG structure, function, and application:

Cryo-Electron Microscopy (Cryo-EM):
The application of cryo-EM to study the structure of IspG, particularly in complex with IspH, could provide unprecedented insights into:

• The atomic-level structure of the enzyme

• Potential protein-protein interactions between IspG and IspH

• Conformational changes during catalysis
  This technique is particularly valuable for studying iron-sulfur proteins like IspG that have proven challenging for traditional crystallography.

Systems and Synthetic Biology Approaches:
The integration of multi-omics data with genome-scale metabolic models of P. putida could enable:

• Prediction of metabolic flux through the MEP pathway under various conditions

• Identification of non-obvious genetic targets for enhancing IspG function

• Design of synthetic regulatory circuits for dynamic control of IspG expression
  These approaches benefit from the steady progress in systems biology understanding of P. putida .

Advanced Genome Editing Technologies:
The continued development of precise genome editing tools for P. putida will facilitate:

• Creation of libraries of IspG variants for structure-function studies

• Generation of reporter strains for high-throughput screening of IspG activity

• Implementation of multiplexed genetic modifications to optimize the entire MEP pathway
  Recent innovations such as CRISPR interference-mediated gene regulation offer powerful new capabilities for these studies.

Artificial Intelligence and Machine Learning:
The application of AI/ML approaches to protein engineering could accelerate:

• Design of IspG variants with enhanced catalytic properties

• Prediction of optimal expression conditions for recombinant IspG

• Identification of novel inhibitors or activators of IspG for metabolic engineering

These emerging technologies, combined with the growing toolkit for genetic manipulation of P. putida, position researchers to make significant advances in understanding and utilizing IspG for biotechnological applications.

How might recombinant P. putida IspG contribute to sustainable bioeconomy?

Recombinant P. putida IspG plays a pivotal role in the development of sustainable bioeconomy processes by enabling the efficient conversion of renewable resources into high-value products:

Bioproduction from Renewable Feedstocks:
P. putida has emerged as a promising industrial cell factory due to its remarkable versatility in metabolizing various carbon sources . By optimizing IspG activity within the MEP pathway, researchers can enhance the conversion of renewable feedstocks such as agricultural residues, lignocellulosic materials, and industrial byproducts into valuable isoprenoid-derived compounds. This approach reduces dependence on petrochemical feedstocks while providing an economic incentive for biomass utilization.

Valorization of Lignin:
Lignin, a complex aromatic polymer in plant cell walls, represents an abundant yet underutilized renewable resource. P. putida has shown promise in metabolizing lignin-derived compounds . Strategic engineering of the MEP pathway, including IspG optimization, could enable the conversion of lignin breakdown products into high-value terpenoids, creating new opportunities for biorefinery operations.

Production of Biopharmaceuticals:
The applications of P. putida as a cell factory range from bioeconomy chemicals to biosynthetic drugs . Optimized IspG activity could facilitate the production of complex terpenoid pharmaceuticals, including anticancer compounds, antimicrobials, and other bioactive molecules. The successful synthesis of endophenazines in Pseudomonas species demonstrates the potential for producing compounds with potential antitumor and antibacterial activities .

Biodegradable Materials Production:
P. putida is known for producing polyhydroxyalkanoates (PHAs), biodegradable biopolymers with applications in sustainable materials . Engineering the MEP pathway in conjunction with PHA biosynthesis could lead to the development of novel biopolymers with enhanced properties, contributing to efforts to replace conventional plastics with biodegradable alternatives.

These applications highlight the significant potential of recombinant P. putida IspG in advancing a sustainable bioeconomy through the efficient conversion of renewable resources into high-value products using environmentally friendly bioprocesses.