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Colibacillus recombinant bacterium for producing adipic acid and application of colibacillus recombinant bacterium

A technology for recombining Escherichia coli and hydroxyadipoyl dehydrogenase, which is applied in the field of bioengineering, can solve problems such as restricting the application of the method for total biosynthesis of adipic acid, and achieve the effects of saving costs, reducing usage and reducing pollution.

Active Publication Date: 2018-05-08
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above reactions require the addition of inducers in the process of producing adipic acid, and the price of expensive inducers limits the application of the method of full biosynthesis of adipic acid

Method used

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  • Colibacillus recombinant bacterium for producing adipic acid and application of colibacillus recombinant bacterium
  • Colibacillus recombinant bacterium for producing adipic acid and application of colibacillus recombinant bacterium
  • Colibacillus recombinant bacterium for producing adipic acid and application of colibacillus recombinant bacterium

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1: Construction of recombinant plasmid pAD-12 and acquisition of recombinant Escherichia coli

[0037]The sequences of Tfu_0875, Tfu_2399, Tfu_0068, Tfu_1648, Tfu_2576, Tfu_2577 have been published in NCBI before the filing date.

[0038] Nco I and Hind III double-digested plasmid pTrc99a, cut the gel to recover the plasmid fragment, used primers to PCR out Tfu_2576 and Tfu_2577, cut the gel to recover the target gene fragment, and then connected these three fragments to the plasmid pTrc99a with Gibson JABSBAN, transformed into JM109, The positive transformant was picked by colony PCR, and the plasmid was digested for verification. The verified plasmid was named pAD-12. The target gene fragments of plasmid pAD-12 and PUTRlpp were digested with NcoⅠ and EcoRⅤ enzymes, the target gene fragments were recovered by cutting the gel, and then the two target gene fragments were purified by T 4 DNA ligase ligation, transformation of JM109, positive transformants were pi...

Embodiment 2

[0041] Example 2: Recombinant Escherichia coli preliminary shake flask fermentation and result analysis

[0042] Fermentation medium: SOB medium, the composition is 2% tryptone + 0.5% yeast powder + 0.05% NaCl + 2.5mMKCl + 10mM MgCl 2 +8g / L glucose+50μg / ml kanamycin sulfate+50μg / ml ampicillin.

[0043] Seed solution preparation: Streak the bacterial species preserved in glycerol on the plate, pick a single colony and inoculate it in a 250ml Erlenmeyer flask filled with 50ml of LB liquid medium, shake the flask at 37°C and 250r / min overnight.

[0044] Fermentation conditions: 2% inoculum size, inoculated in shake flask fermentation medium SOB to make initial OD 600 0.1. Cultivate to OD at 37℃, 200r / min 600 When it is about 0.6-0.8, change to 30°C and 200rpm / min for cultivation.

[0045] Result analysis: take a sample every 4 hours during the fermentation process, centrifuge at 10,000r / min for 2 minutes to separate the fermentation broth from the bacteria, and process the fer...

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Abstract

The invention discloses a colibacillus recombinant bacterium for producing adipic acid and application of the colibacillus recombinant bacterium. Colibacillus BL21(DE3) is taken as a host, a beta-ketothiolase gene, a 3-hydroxyacyl-CoA dehydrogenase gene, a 3-hydroxyadipyl dehydrogenase gene, a 5-carboxyl-2-pentenyl CoA reductase gene and an adipic CoA are overexpressed by modules, a promoter is replaced, the flask shaking yield of adipic acid can reach 2.23 g / L, and the productivity reaches 51.6%. As the promoter of the recombinant bacterium is replaced, and no inducer needs to be additionallyadded, the cost is lowered.

Description

technical field [0001] The invention relates to an E. coli recombinant bacterium producing adipic acid and an application thereof, belonging to the field of bioengineering. Background technique [0002] Adipic acid (adipate), also known as fatty acid, is an important organic dibasic acid, widely used in chemical production, organic synthesis industry, medicine, lubricant manufacturing, etc. At present, the main production method of adipic acid is chemical synthesis, but the product yield of this method is not high. In addition, in the chemical synthesis process of adipic acid, benzene is mainly used as raw material, and the raw materials and intermediate products are highly toxic through chemical synthesis, and a large amount of N is produced in the process. 2 O and other greenhouse gases, the environmental pollution is serious and unsustainable. [0003] In order to solve the above problems, people have focused on the biosynthesis of adipic acid, and a lot of basic work h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/44C12N15/70C12N1/21C12R1/19
CPCC12N9/0004C12N9/0006C12N9/1029C12N9/88C12P7/44C12Y101/01034C12Y101/01035
Inventor 邓禹赵梅赵运英毛银周宇隋雪黄荻萱
Owner JIANGNAN UNIV
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