Recombinant Pueraria montana var. lobata Isoprene synthase, chloroplastic (ISPS), partial
Description
Introduction
Pueraria montana var. lobata, commonly known as kudzu, is a plant species native to East Asia but has become an invasive species in many other regions . It is known for its rapid growth and ability to outcompete native flora . P. montana var. lobata impacts carbon and nitrogen concentrations in soil and can lead to economic losses in forestry and agriculture . Isoprene synthase (IspS) is an enzyme responsible for the synthesis of isoprene, a volatile hydrocarbon . Understanding the properties and function of isoprene synthase in P. montana var. lobata may provide insights into the plant's adaptability and invasiveness.
Isoprene and Isoprene Synthase (IspS)
Isoprene is a five-carbon molecule that serves as a building block for all cellular isoprenoids and has various industrial applications . Plants synthesize isoprene using isoprene synthase (IspS), which uses dimethylallyl diphosphate (DMADP) as a substrate . The reaction is typically most efficient at temperatures around 40–45°C and a pH between 7.0 and 10.5, and in the presence of $$Mg^{2+}$$ .
Pueraria montana var. lobata
Pueraria montana var. lobata is used in traditional medicine and food products, precise identification is crucial because similar species like P. montana var. thomsonii have different properties and uses . DNA barcoding using ITS2 sequences can effectively distinguish between these varieties, even in processed materials where morphological characteristics are lost .
3.1. Identification Using ITS2 Sequences
ITS2 sequences can differentiate P. montana var. lobata from P. montana var. thomsonii due to stable single nucleotide polymorphism (SNP) sites . The ITS2 sequence length is 246 bp for P. montana var. lobata and 247 bp for P. montana var. thomsonii .
Table 1: Key Sequence Differences between P. montana var. lobata and P. montana var. thomsonii
| Feature | P. montana var. lobata | P. montana var. thomsonii |
|---|---|---|
| ITS2 Sequence Length | 246 bp | 247 bp |
| SNP Site (at 244 bp) | T/G | None |
| Insertion/Deletion (at 29bp) | T/- | None |
| SNP (at 52 bp) | A/G | None |
| SNP (at 201 bp) | C/T | None |
Recombinant Production of Isoprene Synthase
Recombinant isoprene synthase refers to the enzyme produced using genetic engineering techniques, where the gene encoding the isoprene synthase from P. montana var. lobata is inserted into a host organism (e.g., bacteria, yeast, or algae) to produce the enzyme in large quantities .
4.1. Expression in Chlamydomonas reinhardtii
Chlamydomonas reinhardtii, a green microalga, can be engineered to produce isoprene by introducing isoprene synthase genes from other plants such as Ipomoea batatas . The Ipomoea batatas IspS showed high activity in algae .
4.2. Factors Affecting Isoprene Production
Several factors influence isoprene production in engineered organisms:
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Temperature: Higher cultivation temperatures can increase isoprene yield .
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Metabolic Perturbations: Modifying the carotenoid pathway or co-expressing isopentenyl-diphosphate delta-isomerase (IDI) can enhance isoprene production .
Chloroplastic Localization
In plants, isoprene synthase is typically targeted to the chloroplast, where the substrate DMADP is produced via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway . Targeting the recombinant isoprene synthase to the chloroplast in engineered organisms can enhance its activity by ensuring proximity to its substrate.
Potential Applications and Further Research
Understanding the characteristics of isoprene synthase in P. montana var. lobata, especially through recombinant production and detailed enzymatic studies, could offer insights into managing this invasive species. Further research could focus on:
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Enzyme Kinetics: Detailed analysis of the enzyme's activity, including its $$K_m$$ and $$V_{max}$$ values, under different conditions.
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Structural Analysis: Determining the crystal structure of the enzyme to understand its catalytic mechanism.
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Regulation: Investigating the factors that regulate the expression and activity of isoprene synthase in P. montana var. lobata.
Product Specs
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Target Background
Lyase catalyzing isoprene formation from dimethylallyl diphosphate.
Q&A
Basic Research Questions
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What is the molecular structure and functional characterization of Pueraria montana ISPS?
Pueraria montana (kudzu) isoprene synthase is a chloroplastic enzyme that converts dimethylallyl diphosphate (DMAPP) to isoprene. The kudzu ISPS gene encodes a protein of 608 amino acids with a predicted molecular mass of 70 kD, with the first 44 amino acids functioning as a transit peptide directing the protein to the chloroplast . The gene structure consists of seven exons and six introns, similar to Class III terpene synthase genes .
For functional characterization, expression of recombinant ISPS without the transit peptide sequence in E. coli confirms its activity as an isoprene synthase. Enzymatic analysis reveals that the kudzu ISPS exhibits sigmoidal kinetics with respect to substrate concentration, indicating cooperativity with a Hill coefficient of approximately 4.1 and an estimated Km of 7.7 mM . For proper characterization, purified enzyme should be assayed using various DMAPP concentrations to generate accurate kinetic parameters.
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How does the MEP pathway contribute to isoprene biosynthesis in Pueraria montana?
The methylerythritol 4-phosphate (MEP) pathway provides the DMAPP substrate required for isoprene synthesis in kudzu chloroplasts. The MEP pathway genes in kudzu show high similarity to corresponding genes in Arabidopsis, despite Arabidopsis not being an isoprene emitter .
Research methodology: To investigate the relationship between the MEP pathway and isoprene production, researchers should consider:
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Cloning and characterizing key regulatory enzymes of the MEP pathway, including DXP synthase (DXS), DXP reductoisomerase (DXR), and IPP isomerase (Idi)
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Measuring the correlation between enzyme activities and isoprene emission rates
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Using isotope labeling to track carbon flow from photosynthesis through the MEP pathway to isoprene
The supply of DXP can limit isoprene emission rates, and DXS may be highly regulated during isoprene emission . IPP isomerase activity also shows strong correlation with isoprene emission, confirming the importance of the MEP pathway in controlling isoprene production .
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What expression systems are most effective for producing recombinant kudzu ISPS?
Heterologous expression in E. coli has been successfully employed for kudzu ISPS production, with the transit peptide sequence removed to enhance solubility and functionality . For optimal expression, the following methodological approaches are recommended:
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Clone the mature ISPS sequence (minus transit peptide) into an expression vector with an inducible promoter
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Transform into an E. coli strain optimized for protein expression (BL21(DE3) or similar)
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Culture at lower temperatures (16-25°C) after induction to enhance protein folding
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Include 1-5% glycerol in the culture medium to stabilize the protein
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Purify using affinity chromatography followed by size exclusion chromatography
Additionally, cyanobacterial expression systems show promise, as Synechocystis PCC 6803 heterologously expressing the kudzu ISPS gene has demonstrated isoprene production .
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How does evolutionary analysis inform our understanding of isoprene synthase function across species?
Phylogenetic analysis reveals that isoprene synthases are either within the monoterpene synthase clade or sister to it . The aspen isoprene synthase nucleotide coding sequence shares 65% identity with the kudzu gene, while the protein sequences show 54% identity .
Methodology for evolutionary analysis:
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Multiple sequence alignment of isoprene synthases from various plant species
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Construction of phylogenetic trees using maximum likelihood methods
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Analysis of conserved domains and active site residues
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Comparison with other terpene synthase family members
Two phenylalanine residues found exclusively in isoprene synthases make the active site smaller than in other terpene synthase enzymes, likely conferring specificity for the five-carbon DMAPP substrate . In Arabidopsis, which doesn't emit isoprene, the gene most similar to isoprene synthase encodes a myrcene/ocimene synthase that produces acyclic monoterpenes .
Species ISPS Amino Acid Identity with Kudzu ISPS Gene Structure Isoprene Emission Kudzu (P. montana) 100% 7 exons, 6 introns High Aspen (P. tremuloides) 54% (57% without transit peptide) 7 exons, 6 introns High Arabidopsis N/A (closest is myrcene/ocimene synthase) Similar structure None -
