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Isoprene synthase and gene encoding same and method for producing isoprene monomer

A technology of isoprene and residues, applied in the field of isoprene synthase and its encoding genes, can solve the problem of isoprene reduction and other problems

Active Publication Date: 2018-06-22
BRIDGESTONE CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in recent years, with the use of light oil crackers, the amount of isoprene produced tends to decrease, and its supply becomes a problem

Method used

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  • Isoprene synthase and gene encoding same and method for producing isoprene monomer
  • Isoprene synthase and gene encoding same and method for producing isoprene monomer
  • Isoprene synthase and gene encoding same and method for producing isoprene monomer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0151] Example 1: Evaluation of the ability to produce isoprene in plants

[0152] 1-1) Measurement of the amount of isoprene formed per unit weight of dry leaves

[0153] First, the amount of isoprene formed per 1 g of dry leaves in plants was measured to evaluate the ability to produce isoprene in plants. Mucuna (Mucuna bracteata), weeping willow (Salix babylonica), sweetgum (Liquidambar styraciflua), myrtle (Myrtus communis) and kudzu (Puerarialobata) were used as plants.

[0154] In the measurement of the amount of isoprene formed, a gas replaceable desiccator (trade name: Vacuum Desiccator, manufactured by AS ONE Corporation) was placed in an incubator (trade name: Growth Chamber MLR-351H, manufactured by SANYO), And set the incubator to high temperature induction conditions (100 μmol E / m at 40 °C 2 / s lighting), and at the same time drive the fan for stirring the air arranged in the gas replaceable dryer to stir the air in the space in the gas replaceable dryer. After...

Embodiment 2

[0168] Example 2: Cloning of the isoprene synthase gene from Mucuna

[0169] 2-1) Evaluation of sampling time

[0170] The isoprene gas released from the leaves of Mucuna irradiated with light for 1 hour, 2 hours, 3 hours and 5 hours at a temperature of 40°C was sampled and the amount of isoprene produced was quantified by gas chromatography as described below. , and it was determined that 4, 8, 12 and 10 μg isoprene / g dry weight leaves were produced. Therefore, it was determined that the optimum light exposure was 3 hours.

[0171] 2-2) Extraction of total RNA lysate

[0172] Total RNA was extracted from Mucuna leaves using total RNA lysate according to the following procedure.

[0173] (1) The leaves of Mucuna pruriens subjected to light at a temperature of 40° C. for 3 hours were sampled.

[0174] (2) Under quick-freezing of the leaf tissue with liquid nitrogen, 100 mg of the leaf tissue was ground in a mortar, then the leaf tissue was dispensed into RNA-free 2 mL Eppendo...

Embodiment 3

[0188] Example 3: Preparation of expression plasmids derived from isoprene synthase from different plants

[0189] 3-1) Chemical analysis of isoprene synthase derived from Pueraria montana var. lobata (Kudzu)

[0190] The nucleotide and amino acid sequences of isoprene synthase derived from Australian kudzu are known (Accession Number: AAQ84170: P. montana var. lobata isoprene synthase (IspS)). SEQ ID NO:3 and SEQ ID NO:4 represent the amino acid sequence of the IspS protein derived from Pueraria mirifica (P.montana) and the nucleotide sequence of its gene, respectively. The IspS gene was optimized with respect to codon usage frequency in E. coli to efficiently express the IspS gene in E. coli and further designed to cut off the chloroplast localization signal. The designed gene was named IspSK. SEQ ID NO:5 represents the nucleotide sequence of IspSK. The IspSK gene was chemically synthesized, and then cloned into pUC57 (manufactured by GenScript), and the resulting plasmid...

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Abstract

The present invention provides methods for establishing superior isoprene monomer production systems. Specifically, the present invention provides polynucleotides and analogs thereof of the following (a), (b) or (c): (a) polynucleotides comprising (i) nucleosides represented by SEQ ID NO:1 acid sequence, or (ii) a nucleotide sequence consisting of the 133rd to 1785th nucleotide residues in the nucleotide sequence represented by SEQ ID NO:1; (b) a polynucleotide comprising A nucleotide sequence having 90% or more identity to the nucleotide sequence of (i) or (ii) above, and the polynucleotide encodes a protein having isoprene synthase activity; or (c) A polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of (i) or (ii) above, and the polynucleotide encodes a polynucleotide having isoprene synthesis Enzymatically active protein.

Description

technical field [0001] The present invention relates to isoprene synthase and genes encoding the same, and the like. Background technique [0002] Natural rubber is a very important raw material in the tire and rubber industry. While the demand for it will increase in the future mainly due to motorization in emerging countries, increasing farms is not easy given regulations on deforestation and competition from palms, and it is difficult to expect an increase in natural rubber production. As a result, the supply-demand balance will become tight. Synthetic polyisoprene is available as an alternative to natural rubber. Its raw material monomer (isoprene (2-methyl-1,3-butadiene)) is mainly obtained by extraction from C5 components obtained from naphtha cracking. However, in recent years, with the use of light oil crackers, the amount of isoprene produced tends to decrease, and its supply has become a problem. And in recent years, it is necessary to establish a system for ec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C08F36/08C12N1/21C12N9/88C12P5/02
CPCC12N9/88C12Y402/03027C12P5/007C08F36/08C12N15/09
Inventor 林泰行原田美奈子高冈纱彩福岛靖王横山敬一西尾阳介田岛义教三原洋子中田国夫
Owner BRIDGESTONE CORP
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