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Method for synthesizing isopentenol by biological methods

A technology of isopentenyl pyrophosphate and synthase, applied in the directions of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problem of no isoprenol and isoprenol production route optimization, equipment corrosion Large, no substrate specificity research and other problems, to solve the unsustainable effect of raw materials

Active Publication Date: 2014-01-29
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Present three kinds of synthetic routes each have advantages and disadvantages: (1) take isobutylene as the method for raw material, reaction is carried out under high temperature and high pressure, harsh condition, and equipment investment is bigger; (2) take isoprene as the method for raw material, Hydrogen chloride participates in the reaction, and the reaction is relatively easy, but it is more corrosive to the equipment, and there is a problem that the reactant sodium acetate is difficult to separate; (3) the method with acetone as raw material originates from acetylene chemical industry, and the cost of raw material is higher than that of petrochemical industry. my country is a big country of acetylene, so this law is more suitable for national conditions
In order to produce prenol with high specificity, it is necessary to find a pyrophosphatase that can hydrolyze DMAPP with high specificity. pyrophosphatase
Wither et al identified a pyrophosphatase gene derived from Bacillus subtilis, and carried out its function through in vivo and in vitro reactions, and found that it can hydrolyze IPP and DMAPP to generate prenol and isoprenol (Wither et al, 2007), but did not investigate its substrate specificity nor optimize the prenol and prenol production pathways

Method used

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  • Method for synthesizing isopentenol by biological methods
  • Method for synthesizing isopentenol by biological methods
  • Method for synthesizing isopentenol by biological methods

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preparation example Construction

[0040] In the method for preparing recombinant E. coli strains of the present invention, the construction of plasmids or vectors and the method of transforming competent cells are not limited, and conventional methods known in the art can be used.

[0041] The antibiotic resistance genes in the above recombinant plasmids A, B, C and D may be the same or different. The antibiotic resistance gene can be selected from antibiotic resistance genes known in the art, for example, chloramphenicol, ampicillin, kanamycin, tetracycline and the like. The engineering Escherichia coli containing the above three recombinant plasmids or expression vectors can be screened out more efficiently and accurately through different combinations of antibiotic resistance genes.

[0042] In the method for preparing recombinant Escherichia coli strains of the present invention, the selection is carried out by using the antibiotic resistance of various recombinant plasmids, that is, according to different...

Embodiment 1

[0059] By co-overexpressing the HMG-CoA synthetase gene (mvaS) (SEQ ID NO: 1) and the HMG-CoA reductase gene (mvaE) derived from Enterococcus faecalis in Escherichia coli (E.coli) ( SEQ ID NO: 2), derived from the mevalonate kinase gene (ERG12), mevalonate phosphokinase (ERG8), mevalonate pyrophosphate decarboxylase gene (MVD1) and large intestine of Saccharomyces cerevisiae Bacillus (Escherichia coli) pyrophosphatase gene (EcNudF) (SEQ ID NO: 3), the obtained engineering Escherichia coli can use sugar to biosynthesize prenol.

[0060] 1.1 Cloning of foreign genes and construction of expression vectors

[0061] 1.1.1 Cloning of foreign genes

[0062] 1.1.1.1 Cloning of HMG-CoA synthetase gene from Enterococcus faecalis

[0063] According to the operating steps provided by the bacterial genome extraction kit (purchased from OMEGA, Cat.No.D3350-01), the genomic DNA of Enterococcus faecalis (purchased from ATCC, No.29212) was extracted, primers were designed according to the ...

Embodiment 2

[0097] The synthesis of embodiment 2 isopentenols

[0098] 2.1 Separation of isopentenol products (the following determination results take CGMCC No.5745 strain as an example)

[0099] During the fermentation process, air is continuously introduced, and the tail gas will continuously take out the isopentenol in the fermentation broth, and the tail gas will be cooled at a low temperature, and the water and prenol in the tail gas will be condensed to obtain concentrated isopentenol The enol product, the final prenol, is further isolated and purified by distillation.

[0100] 2.2 Determination of isopentenol product

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Abstract

The invention provides a recombinant Escherichia coli strain, a preparation method thereof and a method for synthesizing isopentenol therefrom by biological methods. According to the method, through an (MVA (Mevalonic Acid) way) for constructing and transforming mevalonic acid in an Escherichia coli body and pyrophosphatase for over-expressing Escherichia coli, a product, of which the composition contains over 80% of isopentenol, can be synthesized from engineering Escherichia coli strains in a high specificity manner.

Description

technical field [0001] The invention relates to the biosynthesis of prenol compounds, in particular to a recombinant Escherichia coli engineering strain, a preparation method of the strain and a method for synthesizing prenol compounds using the recombinant Escherichia coli engineering strain. Background technique [0002] Prenol, also known as 3-methyl-2-butene-1-ol (3-Methyl-2-butene-1-ol), has a relative molecular mass of 86.13, a density of 0.848g / CB, a boiling point of 140°C, and a flash It is a colorless transparent liquid at 43°C, and its solubility in water is 170g / L (20°C). Isopentenol is mainly used in the synthesis of pyrethroids, the intermediates of pyrethroids, which are high-efficiency and low-toxicity pesticides, and their downstream products, permethrin and DV chrysanthemum chloride. (Permethrin), etc., can also be used as an intermediate for synthetic fragrances. Present three kinds of synthetic routes each have advantages and disadvantages: (1) take isob...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/63C12P7/04C12R1/19
Inventor 咸漠郑艳宁刘炜
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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