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Method for enhancing adipic acid yield in Escherichia coli

A technology of Escherichia coli and recombinant Escherichia coli, which is applied in the field of bioengineering, can solve the problems of long routes, low adipic acid yield, etc., and achieves the effects of reducing the degree of pollution and convenient and simple recovery.

Active Publication Date: 2017-06-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the full biosynthesis of adipate using glucose has been realized in Escherichia coli, this pathway requires more than 10 steps of enzymatic reactions, the pathway is lengthy and there are reversible reactions, and the final yield of adipate is extremely low (less than 1 mg / L), the highest output of the currently reported adipic acid after optimization is 2.5g / L

Method used

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  • Method for enhancing adipic acid yield in Escherichia coli
  • Method for enhancing adipic acid yield in Escherichia coli
  • Method for enhancing adipic acid yield in Escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of recombinant plasmid pAD-1 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] Plasmid pRSFDuet-1 was double-digested with EcoR I and Hind III, the target gene fragment (3798bp) was recovered by cutting the gel, and the plasmid pUC57-Tfu_0875 was digested with the same enzyme, the target gene fragment Tfu_0875 was recovered by cutting the gel, and then the two target fragments were used T 4 DNA ligase ligation, transformation of JM109, positive transformants were picked by colony PCR, and the extracted plasmid was digested for verification. The verified plasmid was named pRSF-Tfu_0875. Digest the plasmid pRSF-Tfu_0875 with Bgl II and Kpn I, cut the gel to recover the 4936bp target gene fragment, digest the plasmid pUC57-Tfu_2399 with the same enzyme, cut the gel to recover the target gene fragment, and ...

Embodiment 2

[0041] Example 2: Optimization of preliminary shake flask fermentation conditions for recombinant Escherichia coli Mad1.

[0042] Fermentation medium:

[0043] SOB medium, the composition is 2% tryptone + 0.5% yeast powder + 0.05% NaCl + 2.5mM KCl + 10mM MgCl 2 +8g / L glucose+50μg / ml kanamycin sulfate+50μg / ml ampicillin+50μg / ml streptomycin.

[0044] M9 medium: M9 salt solution + 8g / L glucose + 2mM MgSO 4 +0.1mM CaCl 2 + 50 μg / ml kanamycin sulfate + 50 μg / ml ampicillin + 50 μg / ml streptomycin.

[0045] LB medium: 1% tryptone + 0.5% yeast powder + 1% NaCl + 8g / L glucose + 50μg / ml kanamycin sulfate + 50μg / ml ampicillin + 50μg / ml streptomycin.

[0046] MOPS medium: 40mM MOPS+0.3%NH 4 Cl+0.1%K 2 HPO 4 +2mM MgSO 4 +0.1mM CaCl 2 +50mM NaCl+100mM Bis-Tris+134μM EDTA+31μM FeCl 3 +6.2 μM ZnCl 3 +0.76 μM CuCl 2 +0.42 μM H 3 BO 3 +0.081 μM MnCl 2 + 50 μg / ml kanamycin sulfate + 50 μg / ml ampicillin + 50 μg / ml streptomycin.

[0047] TB medium: 1.2% tryptone + 2.4% yeast powde...

Embodiment 3

[0051] Embodiment 3: Mad1, Mad2 shake flask fermentation and result analysis.

[0052] 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+50μg / ml streptomycin.

[0053] Seed solution preparation: Streak the bacterial species preserved in glycerol on the plate, pick a single colony and inoculate it into a 250ml Erlenmeyer flask filled with 50ml of LB liquid medium, shake the flask at 37°C and 250r / min overnight.

[0054] Fermentation conditions: 2% inoculum size, inoculated in shake flask fermentation medium SOB to make initial OD 600 0.1. Cultivate to OD at 37℃, 250r / min 600 When it is about 0.6-0.8, add the corresponding inducer to induce the expression of 1mM IPTG to induce Mad1 and Mad2, and change to 30°C, 250rpm / min for culture.

[0055] Result analysis: take a sample every 4 hours during the fermentation process, centrifuge at 10,000r / min f...

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Abstract

The invention discloses a method for enhancing adipic acid yield in Escherichia coli, belonging to the field of bioengineering. The method is implemented by overexpressing beta-ketothiolase gene, 3-hydroxyacyl-coenzyme A dehydrogenase gene, 3-hydroxyadipyl dehydrogenase gene, 5-carboxyl-2-pentene acylcoenzymea A reductase gene and adipyl coenzyme A in different modules by using atoB-gene-knock-out Escherichia coli BL21 (DE3) as a host; and the adipic acid yield can reach 25.57 g / L. The recombinant bacterium can also perform cyclic fermentation; and after ten cycles, the adipic acid accumulated yield is up to 18.94 g / L. The method has important functions on industrial continuous mass production, can shorten the thallus growth time, and has the advantages of time saving, high speed and cost saving since the cyclic thallus fermentation is directly utilized.

Description

technical field [0001] The invention relates to a method for increasing the yield of adipic acid in Escherichia coli, 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. [0003] 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. [0004] In order to solve the above problems, people have focused on the biosynthesis of adipic acid, and a lot of basic work has b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/44C12R1/19
CPCC12N9/0006C12N9/1029C12N9/93C12N15/70C12P7/44C12Y101/01035C12Y203/01009C12Y602/01001
Inventor 邓禹赵梅毛银张晓娟黄荻萱
Owner JIANGNAN UNIV
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