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Method for producing CIS-4-hydroxyproline

A technology for hydroxyproline and proline, which is applied in the field of cis-4-hydroxyproline production, can solve problems such as environmental pollution and affect the rapid growth of bacterial cells, so as to improve the transformation efficiency, reduce the reaction cost and reduce the The effect of consumption

Active Publication Date: 2017-02-22
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problems in the prior art, the object of the present invention is to provide a method for producing cis-4-hydroxyproline, which is simple and easy, improves the utilization rate of raw materials, saves production costs, and can utilize proline The production of cis-4-hydroxyproline from amino acid not only solves the problem of environmental pollution in the production process, but also solves the problem that knocking out multiple genes will affect the rapid growth of bacteria, and can increase the concentration of substrates and coenzymes to increase the concentration of products. produce

Method used

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  • Method for producing CIS-4-hydroxyproline
  • Method for producing CIS-4-hydroxyproline
  • Method for producing CIS-4-hydroxyproline

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1 Construction of recombinant plasmids

[0034] 1. The proline hydroxylase gene synthesized and optimized by Jinweizhi Gene Synthesis Co., Ltd. was constructed on the in vivo pET-28a vector, and the restriction sites were NdeI / XhoI. The recombinant plasmid pET-28a-Ecp4H was constructed.

[0035] 2. The pACYC-CAS9 protein is provided for the laboratory

[0036] 3. pCDF-303-CriRNA-putA-sucA-sucB-aceA-aceK

[0037] Inhibition of metabolic pathway proline degradation gene putA, succinate dehydrogenase genes sucA and sucB, isocitrate lyase gene aceA, isocitrate kinase aceK, transformed pET-28a-Ecp4H, pACYC-CAS9 and plasmid pCDF-putA- sucA-sucB-aceA-aceK.

Embodiment 2

[0039] A method for producing cis-4-hydroxyproline, comprising the following steps:

[0040] Step 1, preparing seed culture medium, fermentation medium, reaction solution, soc medium, bacterial body washing liquid, and sterilizing them together with culture medium, Erlenmeyer flask, and centrifuge tube for future use, wherein:

[0041] Fermentation medium LB: peptone 10g / L, yeast powder 0.5 g / L, NaCl 0.5 g / L, ampicillin 0.02%, chloramphenicol 0.01%, streptomycin 0.01% pH7.0.

[0042] Reaction solution: 10 g / L proline, 10 g / L α-ketoglutaric acid, 0.2 mol / L potassium dihydrogen phosphate, 0.2 mol / L dipotassium hydrogen phosphate, 0.8 mmol / L vitamin C, 0.4 mmol / L Ferrous sulfate, wherein ferrous sulfate is configured with pure water.

[0043] Bacteria washing solution: PBS 7.0 solution, 11.09g / LNaH 2 PO 4 , 2.96g / LNa 2 HPO 4 .

[0044] soc medium: peptone 4g / L, yeast powder 1g / L, NaCl 10mm / L, KCl 2.5mm / L, MgSO 4 10mm / L, MgCl 2 20mm / L, glucose 20 g / L.

[0045] The formula...

Embodiment 3

[0055] A method for producing cis-4-hydroxyproline, comprising the following steps:

[0056] Step 1, preparing seed medium, fermentation medium, reaction solution, soc medium, bacterial body washing solution, and sterilizing with petri dish and centrifuge tube for standby, wherein:

[0057] Soc medium: peptone 4g / L, yeast powder 1 g / L, NaCl 10mm / L, KCl 2.5mm / L, MgSO4 10mm / L, MgCl2 20mm / L, glucose 20 g / L.

[0058] Fermentation medium M9: Glucose 10g / L, NH4Cl 5g / L, NaCl 5g / L, MgSO4 1mol / L, CaCl21mol / L, FeSO4•7H2O 0.005mol / L;

[0059] Reaction solution: 10 g / L proline, 10 g / L α-ketoglutaric acid, 0.2mol / LKH2PO4, 0.2mol / LK2HPO4, 0.8mmol / L vitamin C, 0.4 mmol / L FeSO4, of which ferrous sulfate was used pure water configuration;

[0060] Bacteria washing liquid: PBS 7.0 solution, 11.09g / LNaH2PO4, 2.96g / LNa2HPO4.

[0061] Step 2 to step 3 are the same as in the embodiment.

[0062] Step 4: Introduce the fermentation medium and the fermentation strain into the Erlenmeyer flask in s...

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Abstract

The invention discloses a method for producing CIS-4-hydroxyproline. The method comprises the steps of obtaining a recombinant strain by constructing an overexpression prolyl hydroxylase gene Ecp4H in a host bacteria cell and inhibiting expressions of a prolyl hydroxylase gene putA during degradation pathway of proline and succinate dehydrogenase genes sucA and sucB, an isocitrate lyase gene aceA and an isocitrate kinase gene aceK during degradation pathway of alpha-ketoglutaric acid by utilizing a CRISPR-Cas9 technology, and conducting whole-cell transformation and production on the CIS -4- hydroxyproline after conducting fermentation and cultivation on the recombinant strain. The method for the CIS -4- hydroxyproline is simple in cultivation mode and high in strain vigor and product yield.

Description

technical field [0001] The invention relates to the field of microbial fermentation, in particular to a method for producing cis-4-hydroxyproline. Background technique [0002] Hydroxyproline (hydroxyproline), one of the imino acids, usually has a hydroxyl group in the fourth position, but sometimes it is also in the third position. Since there are two asymmetric carbon atoms, there are 4 stereoisomers. Contains L-hydroxyproline in animal glue and bone collagen. D-Hydroxyproline does not exist in nature. It is a biologically active nitrogenous base that exists widely in organisms and can be generated by proline hydroxylase. It has a wide range of applications in food, medicine and industry. In food, hydroxyproline can be used as a source of nutrients for infants to supplement glycine, alanine and glucose. Hydroxyproline also has a unique sweetness that can improve the flavor of beverages and is often used as an additive for beverages; Adding food and beverages can pre...

Claims

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Application Information

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IPC IPC(8): C12P13/24C12N15/70C12R1/19
CPCC12N9/001C12N9/0071C12N9/1205C12N9/88C12P13/24C12Y103/05001C12Y114/11002C12Y401/03001
Inventor 陈可泉张博文王昕钱娟蔡沛沛欧阳平凯
Owner NANJING UNIV OF TECH
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