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Method for synthesizing alpha-pinene or beta-pinene by adopting biological process

A biological method, pinene technology, applied in the field of preparing isoprene derivatives

Inactive Publication Date: 2014-10-29
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

[0006] Previously, there was no report on the use of microbial fermentation to obtain pinene

Method used

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  • Method for synthesizing alpha-pinene or beta-pinene by adopting biological process
  • Method for synthesizing alpha-pinene or beta-pinene by adopting biological process
  • Method for synthesizing alpha-pinene or beta-pinene by adopting biological process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] By co-expressing the acetyl-CoA acyltransferase gene / hydroxymethylglutaryl-CoA reductase gene (mvaE, SEQ ID NO: 1) from Enterococcus faecalis in Escherichia coli, 3-hydroxy- 3-methylglutaryl-CoA synthase gene (mvaS, SEQ ID NO: 2); mevalonate kinase gene (ERG12, SEQ ID NO: 5) derived from Saccharomyces cerevisiae, mevalonate-5- Phosphokinase gene (ERG8, SEQ ID NO:6), mevalonate-5-diphosphate decarboxylase gene (ERG19, SEQ ID NO:7), isopentenyl pyrophosphate isomerase gene (IDI1, SEQ ID NO : 8); the geranyl diphosphate synthase gene (GPPS2, SEQ ID NO: 3) derived from North American fir (Abiesgrandis) and the α-pinene synthase gene (Pt30, SEQ ID NO: 3) derived from loblolly pine (Pinustaeda) NO: 4), the biosynthesis of isoprene derivative - α-pinene, using the intermediate product of glucose degradation, acetyl-CoA.

[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 genes in the ...

Embodiment 2

[0086] The constructed plasmid was transformed into Escherichia coli competent cells, and the recombinant bacteria were fermented and cultured by shaking flask fermentation and fermenter fermentation, and the fermentation products were detected qualitatively and quantitatively by gas chromatography.

[0087] 2.1 Construction of E.coli recombinant strain

[0088] Transform E.coli BL21 (DE3) competent cells with pYJM27 (pACY-mvaE-mvaS-GPPS2-Pt30) and pYJM14 (pTrc-low) recombinant plasmids together by heat shock, and apply them to the antibiotics added with chloramphenicol and ampicillin The LB solid plate was obtained by PCR screening to obtain positive clones, thereby obtaining engineering Escherichia coli containing pYJM27 and pYJM14.

[0089] 2.2 Cultivation of engineering Escherichia coli

[0090] Inoculate the activated engineering Escherichia coli into the LB liquid culture solution containing chloramphenicol and ampicillin at a ratio of 1:100, and culture it with shaking...

Embodiment 3

[0099] Different fermentation conditions, such as induction temperature, rotation speed, inducer concentration, nitrogen source, substrate concentration, medium pH value and composition ratio, etc., will affect the yield of the fermentation product pinene. The present invention detects the effects of different induction temperatures, inducer concentrations and nitrogen sources on pinene output.

[0100] 3.1 Construction of E.coli recombinant strain

[0101] Transform E.coli BL21 (DE3) competent cells with pYJM27 (pACY-mvaE-mvaS-GPPS2-Pt30) and pYJM14 (pTrc-low) recombinant plasmids together by heat shock, and apply them to the antibiotics added with chloramphenicol and ampicillin The LB solid plate was obtained by PCR screening to obtain positive clones, thereby obtaining engineering Escherichia coli containing pYJM27 and pYJM14.

[0102] 3.2 Study the effect of different induction temperatures on the yield of pinene

[0103] Pick a single clone and culture it in a 5ml LB vi...

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Abstract

The invention provides a biological method for synthesizing alpha-pinene or beta-pinene from acetyl coenzyme A and a reconstitution cell capable of synthesizing the alpha-pinene or beta-pinene, wherein the acetyl coenzyme A is obtained from a simple starting material like glucose. The method for synthesizing the alpha-pinene or beta-pinene by adopting a biological process comprises the following steps: transferring acetyl coenzyme A acyltransferase, 3-hydroxy-3-methyl glutaryl coenzyme A synthase, hydroxymethyl glutaryl coenzyme A reductase, mevalonic acid kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-diphosphonic acid decarboxylase, isopentenylpyrophosphate isomerase, geranyldiphosphate synthetase and pinene synthase into an appropriate host cell by adopting metabolic engineering technology, and culturing the obtained reconstitution cell, so that the target product, namely alpha-pinene or beta-pinene can be obtained.

Description

technical field [0001] The invention relates to a method for preparing isoprene derivatives by a biological method. Specifically, the present invention relates to a method for biologically synthesizing α- or β-pinene and recombinant cells capable of synthesizing α- or β-pinene, wherein the acetyl-CoA used is obtained from a simple starting material such as glucose of. Background technique [0002] Isoprene derivatives - pinene is an important platform compound, due to the high energy density and high net calorific value of combustion of pinene dimer (energy density: 0.938g / cm 3 ; Combustion net calorific value: 39.5MJ / L), is widely used in the synthesis of high-density fuels. In addition, it can also be used to synthesize pharmacologically active substances, agricultural and household biologically active substances, functional materials, spices, terpineol, aromatic alcohols, terpineol acetate, dihydroterpineol, dihydromyrcenol and High Density Jet Fuel, etc. [0003] At ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P5/00C12N15/70C12N1/21C12R1/19
Inventor 咸漠杨建明冯红茹张海波刘炜
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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