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Genetic engineering bacterium producing isoprene and application thereof

A genetically engineered bacteria and isoprene-producing technology, applied in the field of genetic engineering, can solve the problems of cells' own metabolic disorders and affect the normal growth and metabolism of cells, and achieve the effect of shortening the reaction process

Active Publication Date: 2014-09-10
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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  • Genetic engineering bacterium producing isoprene and application thereof
  • Genetic engineering bacterium producing isoprene and application thereof
  • Genetic engineering bacterium producing isoprene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The cloning of embodiment 1 exogenous gene and the construction of expression vector

[0039] 1. Cloning of foreign genes

[0040] 1.1 Cloning of genes in the upper metabolic pathway of Enterococcus faecalis MVA

[0041] The mvaS gene (GenBank No.AAG02439) and mvaE gene (GenBank No.AAG02438) from Enterococcus faecalis were obtained by chemical synthesis from Shanghai Jierui Company. Afterwards, they were 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 genes

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

[0044] 2 Construction of expression vector

[0045] 2.1 pFHR-1 vector construction

[0046] The pGH-OleT and pCOLADuet-1 vectors (Novagen) were digested with ...

Embodiment 2

[0051] Construction and fermentation culture of embodiment 2 recombinant bacterial strains

[0052] The constructed plasmid was transformed into Escherichia coli competent cells, the recombinant bacteria were fermented and cultured by shake flask fermentation, and the fermentation products were qualitatively analyzed by gas chromatography-mass spectrometry (GC-MS) and gas chromatography (GC). and quantitative detection.

[0053] 2.1 Construction of E.coli recombinant strain

[0054] Transform E.coli BL21 (DE3) competent cells with pFHR-2 (pCOLA-OleT-OhydEM) and pFHR-3 (pACY-mvaE-mvaS) recombinant plasmids together by heat shock, and apply to the cells added with chloramphenicol and kana Mycin LB solid plate, positive clones were obtained by PCR screening, thus obtaining engineering Escherichia coli containing pFHR-2 and pFHR-3.

[0055] 2.2 Cultivation of engineering Escherichia coli

[0056] Inoculate the activated engineering Escherichia coli into the LB liquid culture so...

Embodiment 3

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

[0060] Different fermentation conditions, such as induction temperature, rotation speed, inducer concentration, nitrogen source, substrate concentration, medium pH value and composition ratio, will affect the yield of the fermentation product isoprene. The present invention detects the influence of different induction temperatures and inducer concentrations on isoprene production. The cultivation method is the same as in Example 2, and the fermentation product is quantified by GC.

[0061] GC detection conditions: The GC system adopts Shandong Lunan Ruihong SP-6890 gas chromatograph, the chromatographic column is HP-INNOWAX column (25m×250μm×0.2μm), the detector is FID detector; the temperature of the gasification chamber is 200°C, Detector temperature 230°C, carrier gas flow rate: 1ml / min. Column temperature: constant temperature at 50°C.

[0062] 3.1 Construction of E.c...

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Abstract

The invention discloses a genetic engineering bacterium producing isoprene and application thereof, belonging to the field of genetic engineering technology. According to the invention, a recombinant bacterium is obtained by transforming a gene containing acetyl-CoA acyltransferase, 3-hydroxy-3-methylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase, terminal alkene formed fatty acid decarboxylase and oleic acid hydratase into a host bacterium, and is used for fermentation production of isoprene. Such a method substantially shortens reaction process for synthesis of isoprene through an exogenous MVA approach, only five reaction steps are needed for synthesis of isoprene of acetyl-CoA, so influence of excess exogenous gene expression on metabolism of cells is avoided. Through optimization of fermentation conditions, the yield of the fermentation product isoprene can reach 39.49 mu g / L.

Description

technical field [0001] The invention relates to an isoprene-producing genetically engineered bacterium and an application thereof, belonging 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, spices and adhesives. [0003] At present, the source of isoprene is mainly through petroleum-based raw material isopentane, isopentene dehydrogenation method, chemical synthesis method (including isobutylene-formaldehyde method, acetylene-acetone method, propylene dimerization method) and cracking C5 fraction extraction Distillation. However, with the increasing depletion of fossil resources, the source of raw materials is an important bottleneck issue for the preparation of isoprene from petroleum-based raw materials. [0004] There are...

Claims

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Application Information

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Patent Type & Authority Applications(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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