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L-proline-4-hydroxylase and genetic engineering bacterium thereof, construction method and application thereof

A technology of genetically engineered bacteria and proline, applied in the field of genetic engineering, can solve problems such as low enzyme activity, unused industrial application, and limited strain application, and achieve good genetic stability, strong fermentation acid production ability, and good industrial production. The effect of applying the foreground

Active Publication Date: 2018-12-07
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In 2003, Petersen et al. isolated L-proline-4-hydroxylase from the fungus Glarea lozoyensis, while L-proline-4-hydroxylase isolated from the Low enzyme activity, has not been used in industrial applications
The key enzyme L-proline-4-hydroxylase gene in these bacterial strains all derives from the L-proline-4-hydroxylase gene in the cyst fungus RH1, and various plasmids are used as expression vectors, The use of antibiotics and the existence of problems such as easy loss of plasmids limit the application of strains in large-scale production

Method used

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  • L-proline-4-hydroxylase and genetic engineering bacterium thereof, construction method and application thereof
  • L-proline-4-hydroxylase and genetic engineering bacterium thereof, construction method and application thereof
  • L-proline-4-hydroxylase and genetic engineering bacterium thereof, construction method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0041] The present invention provides a novel high-efficiency L-proline-4-hydroxylase, the amino acid sequence of which is shown in SEQ ID No.2 and derived from Micromonospora sp. CNB394. The screening work of this enzyme is as follows: According to the amino acid sequence (GenBank: BAA20094.1) of L-proline-4-hydroxylase (GenBank: BAA20094.1) that is derived from the L-proline-4-hydroxylase reported at present, compares by BLAST in NCBI database, selects Some genes derived from other microorganisms with a certain degree of similarity in amino acid sequence were identified. Then, these genes were codon-optimized according to the codon preference of Escherichia coli, and the codon-optimized genes were connected to the pTrc99a expression vector through gene synthesis. The constructed vector was introduced into E.coli W3110 which had already accumulated a certain amount of L-proline by electrotransformation. Then the above-mentioned constructed strains are subjected to shake flas...

Embodiment 2

[0043] Construction of strain E.coli HYP:

[0044] 1 Methods of gene editing

[0045] The gene editing method used in the present invention is carried out with reference to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR–Cas9 edited genome editing [J]. Metabolic engineering, 2015, 31:13-21.) , the two plasmid maps used in this method are shown in the attached figure 1 . Among them, pREDCas9 carries gRNA expression plasmid pGRB elimination system, bacteriophage λ Red recombination system and Cas9 protein expression system, spectinomycin resistance (working concentration: 100mg / L), cultured at 32°C; pGRB uses pUC18 as the backbone, including the promoter J23100, gRNA-Cas9 binding region sequence and terminator sequence, ampicillin resistance (working concentration: 100mg / L), cultured at 37°C.

[0046] The concrete steps of this method are as follows:

[0047] 1.1. pGRB plasmid construction

[0048] The purpose of construct...

Embodiment 3

[0110] Shake flask fermentation experiment of strain E.coli HYP:

[0111] Slant culture: Streak inoculation of -80°C preserved strains on the activated slant, culture at 37°C for 12 hours, and passage once;

[0112] Shake flask seed culture: Scrape a ring of slant seeds with an inoculation loop and inoculate in a 500mL Erlenmeyer flask containing 30mL of seed medium, seal with nine layers of gauze, and incubate at 37°C and 200rpm for 7-10h;

[0113] Shake flask fermentation culture: Inoculate 10-15% inoculum into a 500mL Erlenmeyer flask (final volume is 30mL), seal with nine layers of gauze, 37°C, 200r / min shaking culture, maintain pH by adding ammonia water during fermentation At 7.0-7.2; add 60% (m / v) glucose solution to maintain fermentation; fermentation cycle 24-30h;

[0114] The composition of the slant medium is: glucose 1-5g / L, peptone 5-10g / L, beef extract 5-10g / L, yeast powder 1-5g / L, NaCl 1-2.5g / L, agar 15-20g / L , the rest is water, pH 7.0-7.2;

[0115] The comp...

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Abstract

The invention provides novel L-proline-4-hydroxylase, amino acid sequence of which is as shown in the SEQ ID No.2. The L-proline-4-hydroxylase is derived from Micromonospora sp. CNB394. The inventionalso provides genetic engineering bacterium E. coli HYP for high-efficiency expression of the above L-proline-4-hydroxylase and denovo synthesis of high-yield trans-4-hydroxy-L-proline. The genetic engineering bacterium has advantages of good genetic stability and strong fermentation and acidogenesis capability, and contains no plasmid. By genome expression of the above novel L-proline-4-hydroxylase and using a cheap carbon source such as glucose and the like as a substrate, efficient denovo synthesis of trans-4-hydroxy-L-proline is achieved. After fermentation for 42 h, yield of the trans-4-hydroxy-L-proline reaches up to 51 g / L. The invention has a good industrial application prospect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a novel high-efficiency L-proline-4-hydroxylase and a genetic engineering bacterium without plasmid and high-yield trans-4-hydroxyl-L-proline and its construction Methods and applications. Background technique [0002] Trans-4-hydroxy-L-proline (trans-4-hydroxy-L-proline, H-Hyp-OH, CAS: 51-35-4) is a rare imino acid, widely used in medicine, Food, chemical and cosmetic industries, especially as the precursor of angiotensin-converting enzyme preparations, carbapenem antibiotics, anti-inflammatory drugs and other drugs, have extremely important medical value; in the food field, trans-4-hydroxy- L-proline can be used as a nutrient to supplement the glucose, glycine, pyruvate, etc. needed by newborns. In the field of chemical industry, trans-4-hydroxy-L-proline can be used as a chiral raw material for the synthesis of various high value-added products, such a...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12P13/24C12R1/19
CPCC12N9/0071C12N15/70C12P13/24C12Y114/11002
Inventor 谢希贤李强蒋帅吴鹤云徐庆阳陈宁马倩范晓光张成林李燕军
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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