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Total biosynthesis method of glutaric acid

A technology for producing glutaric acid and genes, applied in the field of bioengineering, can solve the problems of unsuitability for large-scale production, high cost, low yield, etc., and achieves the effects of low cost, convenient operation and reduced pollution degree

Active Publication Date: 2019-01-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

like Figure 9 A. Figure 9 According to B, there are two main methods for the biosynthesis of glutaric acid currently reported: one is to synthesize glutaric acid using L-lysine as a substrate, and the recombinant E. davA, davB, gabT, gabD genes, finally use 20g / L glucose, 10g / L lysine, 10g / L a-ketoglutaric acid to produce 1.7g / L glutaric acid, this method requires exogenous addition of L- Lysine, the cost is relatively high; Another method is to use glucose as carbon source in Escherichia coli BW25113, still overexpress the davA of Pseudomonas putida, davB, gabT, gabD gene, finally produce 0.85g / L Diacid, although this method does not require exogenous addition of L-lysine, but the yield is low and is not suitable for large-scale production

Method used

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  • Total biosynthesis method of glutaric acid
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  • Total biosynthesis method of glutaric acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Construction of recombinant plasmid pAD-1 and acquisition of recombinant Escherichia coli.

[0033] The sequences of Tfu_0875, Tfu_2399, Tfu_0068, Tfu_1648, Tfu_2576, Tfu_2577 are described in NCBI.

[0034] Plasmid pRSFDuet-1 was double digested with EcoR I and HindIII, 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 purified with 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 then separate the two ...

Embodiment 2

[0037] Example 2: Shake flask fermentation and result analysis of recombinant Escherichia coli.

[0038] Fermentation medium:

[0039] 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.

[0040] Seed solution preparation: Streak the strains preserved in glycerol on a plate, pick a single colony and inoculate it in a 250ml Erlenmeyer flask filled with 50ml of LB liquid medium, shake the flask overnight at 37°C and 250rpm / min.

[0041] Fermentation conditions: 2% inoculum size (1ml), inoculated in shake flask fermentation medium to make initial OD 600 0.1. Cultivate to OD at 37℃, 250r / min 600 Add 1.0mM IPTG to induce the recombinant bacteria at 0.6, respectively, and change to 30°C, 250rpm / min culture.

[0042] Result analysis: take a sample every 4 hours during the fermentation process, centrifuge at 10,000r / min for 2 minutes to separate the fe...

Embodiment 3

[0043] Example 3: Gas-phase mass spectrometry detection and result analysis of glutaric acid.

[0044] The fermented samples were first frozen in a -80°C refrigerator for 2-3 hours to ensure complete freezing, and then placed in a freeze dryer for 2 days until completely dry. Take out the completely dry sample, add 3ml of distilled water, and ultrasonically dissolve the insoluble matter. After complete dissolution, add 3ml of ethyl acetate to extract for 2 minutes. After static layering, transfer the ethyl acetate layer to another glass reaction test tube. If the emulsification phenomenon is serious and the layers cannot be separated, transfer it to a glass centrifuge tube and centrifuge at 10,000r / min. 2 min; the lower layer was continuously extracted twice with 5 ml of ethyl acetate, all the ethyl acetate layers were collected, and dried under nitrogen at 30°C. Add 0.5ml of acetonitrile to the reaction tube of the dried extract, ultrasonically dissolve, then add 0.5ml of si...

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Abstract

The invention discloses a total biosynthesis method of glutaric acid, and belongs to the field of bioengineering. According to the present invention, beta-ketothiolase gene, 3-hydroxyacyl-CoA dehydrogenase gene, 3-hydroxyadipyl dehydrogenase gene, 5-carboxy-2-pentenoyl CoA reductase gene and adipyl Coenzyme A are subjected to over-expression in modules by using Escherichia coli as a host, and theyield of glutaric acid can reach 3.61 g / L; and with the recombinant strain, the new method for total biosynthesis of glutaric acid in Escherichia coli is provided, and the new method for detecting glutaric acid is provided.

Description

technical field [0001] The invention relates to a method for the full biosynthesis of glutaric acid, which belongs to the field of bioengineering. Background technique [0002] Glutaric acid (Glutaric acid, 1,3-Propanedicarboxylic acid; 1,5-Pentanedioic acid; Pentanedioic acid; Pentanedioate), also known as glutaric acid, a, γ-propanedicarboxylic acid, 1,3-propanedicarboxylic acid, is a It is an important organic chemical raw material and intermediate. It and its derivatives have extensive and important uses in chemistry, medicine, construction, agriculture, etc. [0003] At present, the industrial production of glutaric acid is mainly to recover glutaric acid from the by-product of adipic acid production, but this method depends on the production of adipic acid, and the yield cannot be guaranteed. The laboratory preparation method of glutaric acid also mainly adopts the multi-step oxidation method of strong oxidant with high toxicity and high cost. These methods have compl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/44C12N15/70C12N15/66C12R1/19
CPCC12N9/0006C12N9/001C12N9/1025C12N9/1029C12P7/44C12Y101/01035
Inventor 邓禹赵梅张熙黄荻萱毛银
Owner JIANGNAN UNIV
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