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A kind of isoprene-producing genetically engineered bacteria and its application

A genetically engineered bacteria and isoprene-producing technology, applied in the field of genetic engineering, can solve the problems of self-metabolism disorder of cells, affect the normal growth and metabolism of cells, etc., and achieve the effect of shortening the reaction process

Active Publication Date: 2016-08-17
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

When obtaining the exogenous MVA pathway in the engineered bacteria, up to 8 heterologous genes need to be transferred. Excessive expression of heterologous genes may cause the disorder of the cell's own metabolism and affect the normal growth and metabolism of the cell.

Method used

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  • A kind of isoprene-producing genetically engineered bacteria and its application
  • A kind of isoprene-producing genetically engineered bacteria and its application
  • A kind of isoprene-producing genetically engineered bacteria and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Cloning of foreign gene and construction of expression vector

[0039] 1. Cloning of foreign genes

[0040] 1.1 Cloning of MVA upstream metabolic pathway genes of Enterococcus faecalis

[0041] The mvaS gene (GenBank No. AAG02439) and the mvaE gene (GenBank No. AAG02438) from Enterococcus faecalis were obtained by chemical synthesis from Shanghai Jierui Company. Afterwards, it was respectively connected with the vector pGH (purchased from Shanghai Jierui Bioengineering Co., Ltd.) to obtain pGH / mvaS and pGH / mvaE.

[0042] 1.2 Cloning of OleT, OhydEM gene

[0043] The OleT gene (GenBank No. ADW41779.1) from Jeotgalicoccus sp. ATCC8456 and the OhydEM gene (GenBank No. ACT54545.1) of Elizabethkingia meningoseptica were chemically synthesized by Shanghai Jierui Company, and were respectively linked into pGH vector to form pGH / OleT, pGH / OhydEM vector.

[0044] 2 Construction of expression vector

[0045] 2.1 pFHR-1 vector construction

[0046] The pGH-OleT and ...

Embodiment 2

[0051] The construction and fermentation culture of embodiment 2 recombinant strains

[0052] The constructed plasmids were transformed into E. coli competent cells, and the recombinant bacteria were fermented and cultured by shake flask fermentation. The fermentation products were characterized by gas chromatography-mass spectrometry (GC-MS) and gas chromatography (GC), respectively. and quantitative detection.

[0053] 2.1 Construction of recombinant strain of E.coli

[0054] The recombinant plasmids pFHR-2 (pCOLA-OleT-OhydEM) and pFHR-3 (pACY-mvaE-mvaS) were co-heat-shocked to transform E. coli BL21 (DE3) competent cells, and plated on chloramphenicol and kana The LB solid plate of mycin was screened by PCR to obtain positive clones, thereby obtaining the engineered Escherichia coli containing pFHR-2 and pFHR-3.

[0055] 2.2 Cultivation of engineered Escherichia coli

[0056] The activated engineered Escherichia coli were inoculated into the LB liquid culture medium cont...

Embodiment 3

[0059] The influence of embodiment 3 different fermentation conditions on the yield of recombinant bacteria

[0060] 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 fermentation product isoprene. The present invention detects the effects of different induction temperatures and inducer concentrations on the production of isoprene. The cultivation method was the same as that in Example 2, and GC was used to quantify the fermentation product.

[0061] GC detection conditions: The GC system adopts Shandong Lunan Ruihong SP-6890 gas chromatograph, the chromatographic column is HP-INNOWAXcolumn (25m×250μm×0.2μm), and the detector is FID detector; the temperature of the gasification chamber is 200 °C, and the detection The temperature of the apparatus was 230 °C, and the flow rate of the carrier gas was 1 ml / min. Co...

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Abstract

The invention discloses an isoprene-producing genetic engineering bacterium and an application thereof, belonging to the technical field of genetic engineering. The present invention comprises acetyl-CoA acyltransferase, 3-hydroxy-3-methylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase, terminal alkene-forming fatty acid decarboxylase and oleic acid hydratase. The gene is transformed into the host bacteria to obtain recombinant bacteria, and the recombinant bacteria are used for fermentation to produce isoprene. The method provided by the invention greatly shortens the reaction process of Escherichia coli synthesizing isoprene by using the exogenous MVA pathway, and the isoprene can be synthesized from acetyl-CoA in only 5 steps, avoiding the need for excessive exogenous genes. effects on the cell's own metabolism. At the same time, by optimizing the fermentation conditions, the yield of fermentation product isoprene can reach 39.49 μg / L.

Description

technical field [0001] The invention relates to an isoprene-producing genetic engineering bacterium and its application, and belongs to the technical field of genetic engineering. technical background [0002] Isoprene is an important chemical platform compound, 95% of which is used in synthetic rubber; it is also the second monomer of butyl rubber. In addition, isoprene is also widely used in the fields of pesticides, medicines, fragrances and binders. [0003] At present, the source of isoprene is mainly through petroleum-based raw material isopentane, isopentene dehydrogenation method, chemical synthesis method (including isobutene-formaldehyde method, acetylene-acetone method, propylene dimerization method) and extraction of cracked C5 fraction Distillation. However, with the increasing depletion of fossil resources, the source of raw materials is an important bottleneck for the production of isoprene from petroleum-based raw materials. [0004] There are mainly two n...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12P5/02C12R1/19
Inventor 咸漠杨建明邹慧斌冯红茹
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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