Pyrethrin Biosynthetic Enzyme Gene Isolation and Vector Construction
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Solution Overview
Problem
Current methods fail to effectively isolate and utilize the pyrethrin biosynthetic enzyme gene, hindering efficient pyrethrin production, as the enzyme's amino acid sequence and gene encoding it remain unknown, limiting genetic engineering approaches for enhanced biosynthesis.
Innovation Solution
Purification and sequencing of the pyrethrin biosynthetic enzyme protein from pyrethrum flowers, followed by constructing vectors bearing the determined gene sequence, allowing for the expression of the enzyme in plants with faster growth rates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If pyrethrin is extracted from pyrethrum flowers through traditional methods, then pyrethrin can be obtained, but the production efficiency is low due to the long growth duration of pyrethrum (over three years)
Solution Approach 1:
The patent extracts and isolates the specific gene (CYP75A13) encoding pyrethrin biosynthetic enzyme from pyrethrum, separating the key biosynthetic capability from the entire plant system. This enables targeted production through genetic engineering rather than waiting for complete plant growth and flowering.
Solution Approach 2:
The patent performs preliminary identification and characterization of the pyrethrin biosynthetic enzyme gene before attempting large-scale production. By first isolating and sequencing the CYP75A13 gene, the invention prepares the genetic material in advance for subsequent transformation into fast-growing plant species.
2Productivity
If selection and breeding of high-producing pyrethrum strains is performed, then pyrethrin production may increase, but the process requires extensive time and genetic resources
Solution Approach 1:
The patent copies the identified CYP75A13 gene into fast-growing plant species through genetic transformation. Instead of breeding pyrethrum for higher production, the invention replicates the key biosynthetic gene in alternative host plants that have inherently faster growth cycles.
Solution Approach 2:
The patent changes the host plant parameter from slow-growing pyrethrum to fast-growing alternative species while maintaining the pyrethrin biosynthetic capability through gene transfer. This parameter change in plant growth rate directly accelerates production without compromising pyrethrin synthesis ability.
3Productivity
If genetic engineering approaches are used to enhance pyrethrin biosynthesis, then production efficiency can be improved, but the specific gene encoding pyrethrin biosynthetic enzyme was unknown and could not be isolated
Solution Approach 1:
The patent segments the complex pyrethrin biosynthesis pathway to identify and isolate the specific CYP75A13 gene responsible for the rate-limiting step. By focusing on this single gene rather than attempting to modify the entire pathway simultaneously, the invention makes genetic engineering feasible and effective.
Solution Approach 2:
The patent uses molecular biology techniques as intermediaries to bridge the gap between unknown gene identity and usable genetic information. Through RNA extraction, cDNA synthesis, and sequence analysis, the invention transforms inaccessible genetic material into identifiable and transferable gene sequences.
4Loss of time
If fast-growing plants are used for pyrethrin production through genetic transformation, then production time can be reduced, but vectors bearing the gene must be constructed and transformation efficiency must be ensured
Solution Approach 1:
The patent uses universal plant expression vectors that can accommodate the CYP75A13 gene and function across different plant species. These vectors contain universal regulatory elements (promoters, terminators) and selection markers that work broadly, reducing the need for species-specific vector development.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables effective and inexpensive production of pyrethrin, addressing the decline in pyrethrin demand by utilizing fast-growing plants for environmentally friendly insecticides, thus meeting social and industrial demands for safe and efficient insecticides.
Implementation Method 1
Pyrethrin has an ester-bonded structure between chrysanthemic acid that is a monoterpene carboxylic acid and rethrolones (alcohols), which is a metabolite of fatty acid oxidation. It is known that in biosynthesis of pyrethrin, chrysanthemic acid and rethrolones are biosynthesized in different metabolic pathways and an ester binding is eventually formed therebetween.
Data Source
Figure 1(a)~1(d)
Figure 2
Figure 3~5
AI summary
The present invention relates to an enzyme determining amino acid sequences of an enzyme involved in pyrethrin biosynthesis and a base sequence of the gene thereof; constructing vectors bearing the gene and transformants; and extractable from plant bodies producing pyrethrin by applying such creative techniques to plant bodies with faster growth aiming to provide a method to efficiently produce pyrethrin; and the enzyme is a gene encoding a protein of the following (i) or (ii): (i) a protein consisting of an amino acid sequence shown in Sequence No. 1; or (ii) a protein consisting of an amino acid sequence including one ormore of a substitution, deletion, insertion, and/or addition of amino acid in the amino acid sequence shown in Sequence No. 1, in which the protein exhibits activity of pyrethrin biosynthetic enzyme.