Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A kind of L-proline-4-hydroxylase and its genetic engineering bacteria, construction method and application

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

Active Publication Date: 2021-08-06
TIANJIN UNIV OF SCI & TECH
View PDF11 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of L-proline-4-hydroxylase and its genetic engineering bacteria, construction method and application
  • A kind of L-proline-4-hydroxylase and its genetic engineering bacteria, construction method and application
  • A kind of L-proline-4-hydroxylase and its genetic engineering bacteria, construction method and application

Examples

Experimental program
Comparison scheme
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 the elimination system of gRNA expression plasmid pGRB, Red recombination system of λ phage 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 constru...

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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present invention provides a novel L-proline-4-hydroxylase, the amino acid sequence of which is shown in SEQ ID No.2, derived from Micromonospora sp. CNB394. The present invention also provides a genetically engineered bacterium E.coli HYP that efficiently expresses the above-mentioned L-proline-4-hydroxylase and synthesizes high-yield trans-4-hydroxyl-L-proline de novo. The genetically engineered bacterium has The advantages of good genetic stability and strong fermentative acid production ability, no plasmid, using the genome to express the above-mentioned novel L-proline-4-hydroxylase, using glucose and other cheap carbon sources as substrates to efficiently synthesize trans- 4-Hydroxy-L-Proline, the yield of trans-4-Hydroxy-L-Proline after 42 hours of fermentation is as high as 51g / L, which 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
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 UNIV OF SCI & TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com