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Biosynthesis method for increasing yield of trans-4-hydroxyproline by knocking out other metabolic pathway

A technology of hydroxyproline and metabolic pathways, applied in the field of microbial genetic engineering, can solve the problems of waste of raw materials, high cost, and many synthesis steps

Inactive Publication Date: 2016-05-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical synthesis method has many synthesis steps and high cost, so it is not suitable for industrial production
The biological tissue extraction method uses animal protein sources such as gelatin and pig skin as raw materials, and extracts hydroxyproline after being hydrolyzed by acid, alkali or proteolytic enzymes. This method has long purification steps, high cost, waste of a large amount of raw materials, and waste pollution. Serious, easily eliminated by the market

Method used

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  • Biosynthesis method for increasing yield of trans-4-hydroxyproline by knocking out other metabolic pathway
  • Biosynthesis method for increasing yield of trans-4-hydroxyproline by knocking out other metabolic pathway
  • Biosynthesis method for increasing yield of trans-4-hydroxyproline by knocking out other metabolic pathway

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: argB gene knockout

[0027] The gene sequence of argB obtained from NCBI (GenBank: AM946981.2): argB gene (SEQ ID NO: 1)

[0028] Using short homology arms, design primer pairs: primer 5'-3': P1 (SEQIDNO: 2), P2 (SEQIDNO: 3)

[0029] Using pKD4 as a template, a target fragment containing the upstream and downstream homology arms of argB and the resistance gene (Kan) was obtained. The E. coli BL21(DE3) △putA containing plasmid pKD46 was obtained by chemical transformation, and then it was prepared as electro-competent, and the target fragment was introduced into the electro-competent by electroporator. The effect of the three proteins expressed in plasmid pKD46 Next, use the Red recombination system to complete gene recombination. After electroporation, resuscitate at 30°C for 3 hours, and spread a plate containing kanamycin. After that, the plasmid pCP20 was transferred to eliminate the resistance gene. The strains were spotted on non-resistant, Amp resistant, an...

Embodiment 2

[0030] Example 2: Obtaining the recombinant plasmid (pUC19-ptrp2-Hyp-VHb)

[0031] Activate the strain containing the target plasmid stored in the laboratory and incubate at 37°C and 220 rpm for 12-16 hours. Take the cultured bacterial liquid to extract the plasmid, and all operations are carried out in strict accordance with the instructions.

Embodiment 3

[0032] Example 3: Construction of recombinant strains

[0033] Take out the E. coli BL21(DE3) △putA / △argB competent, place it on ice and thaw, add 37μL of sterile water, 10μL KCM, 3μL plasmid. After mixing, place on ice for 30min, heat shock at 42°C for 90s, then place on ice for 5min, add 600μL of fresh LB medium, culture with shaking at 37°C for 1h, take 100μL and spread on ampicillin resistant plate. After culturing at 37°C for 8-16 hours, when a single colony grows, select a single colony for verification.

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Abstract

The invention discloses a biosynthesis method for increasing the yield of trans-4-hydroxyproline by knocking out other metabolic pathways. A system for producing hydroxyl-L-proline by using the biosynthesis method refers to a production method that free L-proline is converted into trans-4-hydroxyl-L-proline from a living body by virtue of proline hydroxylase in biological cells in the presence of alpha-oxoglutarate and ferrous ions. The method for increasing the yield of hydroxyproline comprises a step of cutting off other metabolic pathways of a precursor substrate glutamic acid which competes with L-proline in biological cells, wherein other metabolic pathways refer to an arginine metabolic pathway. By using the method, the yield of the trans-4-hydroxyl-L-proline can be up to 921 mg / L.

Description

Technical field [0001] A biosynthetic method that improves the production of trans-4-hydroxyproline by knocking out other metabolic pathways belongs to the field of microbial genetic engineering. Background technique [0002] Hydroxy-L-proline (abbreviation: Hydroxyproline, Hyp) is an imino acid, which is the product of the hydroxylation of L-proline, and can be divided into 3-hydroxyproline according to the different hydroxylation positions. Acid (3-Hyp) or 4-hydroxyproline (4-Hyp). Hydroxyproline (Hydroxyproline, Hyp) is the main component of collagen and does not belong to the 20 common amino acids. [0003] In recent years, the research and development of Hyp has attracted widespread attention in the fields of medicine, biochemistry, food and beauty. It can be used as a cosmetic additive with anti-oxidation and anti-radiation effects; it has a weight-loss effect and is expected to become an ideal weight-loss drug; it has a variety of physiological functions and unique biologi...

Claims

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Application Information

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IPC IPC(8): C12P13/24
CPCC12P13/24
Inventor 张震宇王晓姣姚动邦黄建华魏照辉
Owner JIANGNAN UNIV
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