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Genetically engineered bacterium used for combined production of succinic acid and isoprene, and construction method thereof

A technology of genetically engineered bacteria and isoprene, applied in the field of genetic engineering, can solve the problem of no co-production of isoprene and succinic acid, etc., and achieve the effects of improving the total yield and being easy to separate.

Inactive Publication Date: 2017-05-10
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

However, based on the complexity of cell metabolism, there is great unpredictability and uncertainty in the use of carbon dioxide produced in the synthesis of isoprene for the synthesis of succinic acid through genetic engineering technology. Therefore, there is no co-production at present. Reports of isoprene and succinic acid

Method used

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  • Genetically engineered bacterium used for combined production of succinic acid and isoprene, and construction method thereof
  • Genetically engineered bacterium used for combined production of succinic acid and isoprene, and construction method thereof

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Experimental program
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Effect test

Embodiment 1

[0038] (1) Cloning and expression of pyruvate carboxylase gene

[0039] Using the genome of Bacillus subtilis (Bacillus subtilis subsp.subtilis str.168, NC_000964.3) as a template, the gene fragment was amplified, and the restriction sites AatⅡ and XhoI were connected at the same time. Take PYC-F-AatⅡ: 5’-ATCG GACGTC ATGTTTGGTGACAAGGTAAAAG-3’

[0040] PYC-R-XhoI:5’-CCG CTCGAG TTATGCTTTTTCAATTTCAAGG-3' was used as a primer, and Bacillus subtilis DNA was used as a template for amplification. The amplification system is shown in Table 1:

[0041] Table 1 PCR amplification system

[0042]

[0043] PCR amplification program: 95°C pre-denaturation for 5min; 95°C denaturation for 30s, 53°C annealing for 30s, 72°C extension for 2min, 35 cycles; 72°C extension for 10min. After the PCR product was purified, it was double digested with endonucleases AatⅡ and XhoI, and then ligated to the pCOLADUet-1 vector that was also double digested to form plasmid pLWPYC1.

[0044] The ligation product w...

Embodiment 2

[0055] (1) Construction of E.coli recombinant strain

[0056] The codon optimized isoprene synthase gene ispS (GenBankaccession No.HQ684728) derived from poplar (Populus nigra) and 1-deoxy-D-xylulose derived from Bacillus subtilis The 5-phosphate synthase gene dxs (Gene ID: 938609) and the 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene dxr (GeneID: 939636) were linked to the plasmid pACYCDuet-1 to construct a plasmid pAdxs2 / dxr2 / ispS, the specific connection method is included in the literature (Applied Microbiology and Biotechnology (2011) 90:1915–1922). The plasmid pAdxs2 / dxr2 / ispS was transformed into E.coli BL21(DE3) cells to construct an engineered strain ZYR4 containing MEP pathway. Prepare ZYR4 into competent cells, then transfer plasmid pLWPYC1 into competent cells, spread on LB solid plates with chloramphenicol and kanamycin, and obtain positive clones by PCR screening, thereby obtaining engineered E. coli LWPYC2 . The engineered strain ZYR4 was used as the cont...

Embodiment 3

[0063] M9 seed medium ( / L): 20g glucose, 6g Na 2 HPO 4 , 3g KH 2 PO 4 , 1g NH 4 Cl, 0.5gNaCl, 0.24g MgSO 4 , 121 ℃ high pressure steam sterilization 15min.

[0064] Fermentation medium ( / 2L): 19.6g K 2 HPO 4 ·3H 2 O, 4.2g citric acid·H 2 O, 0.6g ferric ammonium citrate, 0.8ml concentrated sulfuric acid, 40g glucose, 0.123mg (NH 4 ) 6 Mo 7 O 2 ·4H2O, 0.097mg ZnSO 4 ·7H 2 O, 0.823mg H 3 BO 4 , 0.083mg CuSO 4 ·5H 2 O, 0.527mg MnCl 2 ·4H 2 O, 4ml 1M MgSO 4 , 1900ml distilled water.

[0065] Pick the single clone of strain LWPYC1 into 50ml M9 seed medium and activate it overnight (18-24h) at 37°C and 180rpm. The seeds were inoculated into a 5L small fermentor containing 2L of fermentation medium according to 10% of the inoculum, with aeration volume of 1.3VVM, rotation speed of 400rpm, and cultivation at 37°C to OD 600 At about 20, add IPTG with a final concentration of 0.1-1mM, induce expression at 25°C-37°C, introduce carbon dioxide, adjust the pH with potassium carbonate and potassium...

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Abstract

The invention discloses a genetically engineered bacterium used for combined production of succinic acid and isoprene, and a construction method thereof, and belongs to the technical field of gene engineering. The genetically engineered bacterium contains the gene of an enzyme needed in simultaneous overexpression of isoprene synthetic route in escherichia coli and pyruvate carboxylase gene. The invention also provides the construction method and the application method of the genetically engineered bacterium. The genetically engineered bacterium is capable of realizing combined production of succinic acid and isoprene, avoiding carbon loss in isoprene biosynthesis process, improving atom economy, reducing emission of carbon dioxide, and possesses high environmental benefits.

Description

Technical field [0001] The invention relates to a genetic engineering bacterium for co-producing succinic acid and isoprene and a construction method thereof, belonging to the technical field of genetic engineering. Background technique [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, it can also be widely used in fields such as pesticides, medicines, perfumes and adhesives. 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 cracked C5 fraction extraction Distillation method. 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. The ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/46C12P5/02C12R1/19
Inventor 咸漠刘炜刘会洲
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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