Method for producing isoprene

A technology of isoprene and synthetase, applied in the field of genetic engineering, can solve the problems of high pollution, low production efficiency, high energy consumption, etc., achieve good activity, simple production method, and increase production

Active Publication Date: 2014-06-18
QINGDAO VLAND BIOTECH GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the current production method of isoprene mainly relies on non-renewable fossil fuel oil, although the synthesis and separation technology continues to mature, the raw material will eventually become a key factor restricting the development of the isoprene industry. There are also problems of high energy consumption and high pollution
Therefore, the use of clean methods such as biology to produce isoprene has become a research hotspot, but the existing production methods still have problems such as low production efficiency

Method used

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  • Method for producing isoprene
  • Method for producing isoprene
  • Method for producing isoprene

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1 Construction of isoprene synthase mutant MIsps and pPIC3.5-MIsps expression vector

[0024] The amino acid sequence of isoprene synthase (Isps) from Pueraria montana var. lobata is SEQ ID NO:3, and its coding nucleotide sequence is SEQ ID NO:4. Through the analysis of the amino acid sequence and molecular structure of the enzyme, it was found that the replacement of individual amino acids in the active center of isoprene synthase can effectively improve its enzyme activity, thereby increasing the production of isoprene. The applicant found that replacing Ser at position 408 with Asp can enhance the enzyme activity. The amino acid sequence of the mutated isoprene synthase is SEQ ID NO: 1, and its encoded nucleotide sequence is SEQ ID NO: 2 (MIsps ).

[0025] Primers were designed, and MIsps were constructed by fusion PCR method, and restriction enzyme cutting sites EcoRI and NotI were added at both ends.

[0026] MIsps primers were designed as follows:

[00...

Embodiment 2

[0034] Embodiment 2pPIC3.5-MIsps transforms Pichia pastoris and fermentation culture

[0035] Digest pPIC3.5-MIsps with restriction endonuclease SacI or SalI to linearize it, which is more conducive to the homologous recombination of the target gene into the chromosome of Pichia pastoris. After the yeast cells were treated with 1M sorbitol, they were mixed with the purified linear plasmid containing the MIsps gene, and transformed with an electrotransformer. The revived yeast cells were spread on MD plate and cultured at 30°C for 3-4d. Pick the single colony grown on the MD plate, inoculate into 25mL BMGY (250ml shake flask), shake at 30°C, 250-300rpm until OD600=2-6 (logarithmic growth, about 16-18h). Centrifuge at room temperature at 1500-3000g for 5min, collect the cells, remove the supernatant, resuspend the cells with BMMY to OD600=1.0 (about 100-200ml), add 0.5% methanol to induce expression. Add the above-mentioned culture into a 1L shaker flask, cover with two layers...

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Abstract

The invention aims at providing a method for producing isoprene. An isoprene synthetase mutant gene is transformed into pichia pastoris to build a pichia pastoris engineering strain capable of recombining and expressing heterogenous isoprene synthetase mutant, so as to produce the isoprene in an efficient fermentation manner. The isoprene synthetase mutant obtained by the method has better activity than that of untransformed enzyme. Furthermore, the method for producing isoprene provided by the invention is simple, reliable, and environmentally-friendly; the built pichia pastoris engineering strain can largely produce the isoprene, and can be widely applied to fermentation production of the isoprene; and the yield of the isoprene is improved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a production method of isoprene. Background technique [0002] Isoprene (2-methyl-1,3-butadiene) is a highly volatile conjugated diene, the main monomer of synthetic rubber, and its consumption accounts for 95% of the total output of isoprene , It is mainly used to synthesize isoprene rubber, and its output is second only to styrene-butadiene rubber and butadiene rubber, ranking third in synthetic rubber. It can also be used for synthetic resin, liquid polyisoprene rubber, etc. In recent years, people use isoprene to synthesize linalool, squalene, etc., thereby further synthesizing spices, medicines, pesticides, etc. Isoprene is also a precursor of natural products terpenoids. [0003] At present, there are many ways to produce isoprene in industry, except C 5 In addition to isoprene obtained from the separation of distillates, it can also be produced b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12P5/02C12N15/81C12N1/19C12R1/84
Inventor 王华明黄亦钧李宾
Owner QINGDAO VLAND BIOTECH GRP
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