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Production strain of trans-L-hydroxyproline as well as construction method and application of production strain

A technology for producing hydroxyproline and bacterial strains, which is applied in the biological field and can solve the problems of further improvement in yield, cumbersome steps, and high price

Active Publication Date: 2020-08-04
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the production methods of trans-3-hydroxyl-L-proline are mainly chemical methods or enzymatic methods. The chemical methods have the disadvantages of chiral center selection, involving highly toxic substances, and cumbersome steps, while the enzymatic methods usually use arginine Acid is a substrate and expensive (Huang, 2004; Hara, 2015)
The inventors of the present invention reported proline 3-hydroxylase with better catalytic performance in CN201810886801.0, and realized the production of trans-3-hydroxyproline with proline or cheaper glucose as raw material, However, its output needs to be further improved, thereby reducing production costs and realizing the industrialized production of trans-3-hydroxyl-L-proline

Method used

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  • Production strain of trans-L-hydroxyproline as well as construction method and application of production strain
  • Production strain of trans-L-hydroxyproline as well as construction method and application of production strain
  • Production strain of trans-L-hydroxyproline as well as construction method and application of production strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1. Construction of the expression vector of Escherichia coli transporter gene

[0064] The transporter in Escherichia coli is as follows: the coding nucleotide sequence of ProP is shown in SEQ ID NO: 1; ProU is made up of 3 subunits, and the coding nucleotide sequence is shown in SEQ ID NO: 2; ProY's The coding nucleotide sequence is shown in SEQ ID NO:3. In order to improve the expression of the transporters ProP, ProU and ProY in Escherichia coli, it is necessary to introduce into the cells an expression vector that improves the expression of the proP, proU and proY genes. First, the E.coli MG1655 genome was used as a template, and proP-1 / prop-2, proU-1 / proU-2, proY-1 and proY-2 were used as primers to amplify proP, proU, proY gene. Then use the pSB4K5-I52002 (GenBank No.: EU496099.1) plasmid as a template, respectively use pSB4K5-1 / pSB4K5-2, pSB4K5-1 / pSB4K5-3, pSB4K5-1 / pSB4K5-4 as primers to amplify proP and proU , the pSB4K5 plasmid backbone corresponding...

Embodiment 2

[0067] Example 2. Construction of trans-4-hydroxy-L-proline-producing Escherichia coli that enhances the expression of transporters

[0068] According to the literature (Gene.1988 Apr 29; 64 (2): 199-205.Nucleotide sequence of amutation in the proB gene of Escherichia coli that confers proline overproduction and enhanced tolerance to osmotic stress) report, Escherichia coli glutamic acid kinase ProB ( Glutamate-5-kinase, GenBank number: NP_414777.1) Asp 107 is mutated to Asn, that is, the ProB74 mutant can relieve the feedback inhibition of L-proline on ProB. According to the literature (Shibasaki T, Hashimoto S, Mori H, etc., Construction of a novel hydroxyproline-producing recombinant Escherichia coli by introducing a proline 4-hydroxylase gene. [J]. J Biosci Bioeng. 2000, 90 5: 522-525) reported that in the large intestine Overexpression of glutamate kinase ProB74, glutamate-semialdehyde dehydrogenase ProA (Glutamate-semialdehyde dehydrogenase, GenBank number: NP_414778.1) ...

Embodiment 3

[0069] Example 3: E. coli production of trans-4-hydroxy-L-proline with enhanced transporter expression

[0070] Seed medium: yeast extract 5g / L; tryptone 10g / L; sodium chloride 10g / L. Fermentation medium: glucose 10g / L; fish meal peptone 8g / L; ammonium sulfate 5g / L; dipotassium hydrogen phosphate 1g / L; sodium chloride 2g / L; magnesium sulfate 0.5g / L; ferrous sulfate 0.278g / L ; Calcium chloride 0.015g / L; MOPS 40g / L, pH7.0. 250mL Erlenmeyer bottle with 30mL seed culture medium, add 15mg / L tetracycline and 25mg / L kanamycin, then insert appropriate amount of glycerin to preserve the bacterial liquid, 37 Cultivate overnight. The cultured seed liquid was transferred to a 250 mL Erlenmeyer flask containing 30 mL of fermentation medium according to 5% inoculation amount, and fermented at 33° C. and 220 rpm for 24 hours. The concentration of trans-4-hydroxy-L-proline was measured in the fermentation end liquid, and the detection method of trans-4-hydroxy-L-proline refers to the nation...

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Abstract

The invention discloses a construction method of a trans-L-hydroxyproline production strain. The method comprises the step of enhancing the activity of a transport protein encoded by a nucleotide sequence shown in SEQ ID NO: 1, 2 or 3 in the trans-L-hydroxyproline production strain. The invention also discloses a trans-L-hydroxyproline production strain constructed by using the method and a methodfor preparing the trans-L-hydroxyproline by using the strain. The trans-L-hydroxyproline yield of the constructed trans-L-hydroxyproline producing strain is remarkably increased, and a new thought isdeveloped for constructing the trans-L-hydroxyproline producing strain.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to a trans-L-hydroxyproline producing strain, a construction method and application of the producing strain. Background technique [0002] Trans-L-hydroxyproline is the main amino acid in collagen and widely exists in nature as a constituent of elastin. As a naturally occurring trans-L-hydroxyproline, according to the position of the hydroxyl group, it is divided into trans-4-hydroxy-L-proline and trans-3-hydroxy-L-proline. Trans-4-hydroxy-L-proline is ubiquitous in nature and is widely used in fields such as medicine, food additives, animal feed and beauty industry. Due to its high price, it is mainly used for the synthesis of carbapenems (third generation, the last line of defense of antibiotics) side chains of antibiotics. The third-generation antibiotics are a type of atypical β-lactam antibiotics with the broadest antibacterial spectrum. The...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/31C12N1/21C12P13/24C12R1/19
CPCC12N15/70C07K14/245C12P13/24
Inventor 孙际宾刘娇王兴初郭轩周文娟郑平马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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