Recombinant bacteria with improved alpha-phenylpyruvic acid transformation production efficiency

A technology of phenylpyruvate and recombinant bacteria, applied in the field of recombinant bacteria, can solve the problems of low product yield, increased difficulty of separation and purification, toxic and harmful products, etc., achieve high yield, solve cumbersome steps, and increase production

Active Publication Date: 2015-08-26
JIANGNAN UNIV +1
View PDF3 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods all need to go through multi-step reactions, and have higher requirements on reaction conditions, such as high pressure, high temperature, etc., and the product yield is low, which increases the difficulty of later separation and purification, and easily produces toxic and harmful products.

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
  • Recombinant bacteria with improved alpha-phenylpyruvic acid transformation production efficiency
  • Recombinant bacteria with improved alpha-phenylpyruvic acid transformation production efficiency
  • Recombinant bacteria with improved alpha-phenylpyruvic acid transformation production efficiency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Knockout of aspC, tyrB and ilvE genes

[0022] Previously, we expressed the L-amino acid deaminase gene through E. coli, and obtained a recombinant E. coli strain expressing L-amino acid deaminase. This recombinant E. coli can be used for whole-cell transformation of L-phenylalanine to produce PPA. See The title of the invention is "A method for efficient production of α-phenylpyruvate by whole cell transformation", and the patent application number is 201310392427.6. The recombinant Escherichia coli genomic DNA is used as a template, and aspC-Left-S and aspC-Left-A are used , tyrB-Left-S and tyrB-Left-A, ilvE-Left-S and ilvE-Left-A as primers, PCR amplified about 1000bp upstream of aspC, tyrB and ilvE genes, and aspC-Right-S and aspC-Right -A, tyrB-Right-S and tyrB-Right-A, ilvE-Right-S and ilvE-Right-A were used as primers, and about 1000 bp downstream of aspC, tyrB and ilvE genes were amplified by PCR.

[0023] Using pKD13 as template, using aspC-Middle-S ...

Embodiment 2

[0024] Example 2 Preparation of Whole Cell Catalyst and Whole Cell Transformation Process

[0025] The recombinant Escherichia coli in Example 1 after aspC, tyrB and ilvE knockouts were inoculated into seed medium (containing ampicillin 10 mg / L), and cultivated overnight at 37° C. and 200 rpm. Fermentation was carried out in a 3L NBS fermenter, 1% inoculum was added to 1.8L fermentation medium, the stirring speed, ventilation and temperature were 400rpm, 1.0vvm and 28°C, respectively, when OD 600 When 0.6 was reached, 0.4 mM IPTG was added to induce the expression of L-amino acid deaminase. After 5 hours of induction, centrifuge at 8,000 rpm for 10 minutes at low temperature, collect the bacterial cells, and wash the bacterial cells twice with 20 mM Tris-HCl (pH 8.0) buffer. The whole cell transformation system is: L-phenylalanine 4g / L, whole cell catalyst 1.2g / L, the reaction is carried out in 20mM Tris-HCl (pH 8.0), 37°C, 200rpm transformation for 24h.

[0026] See Table 1...

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 invention discloses recombinant bacteria with improved alpha-phenylpyruvic acid transformation production efficiency, and belongs to the field of biotechnology. According to the invention, the knockout of aspC, tyrB and ilvE genes from Escherichia coli is achieved successfully, so that the degradation of the product PPA by cells is blocked, the yield of extracellularly transformed L-phenylalanine is further improved, and the yield of PPA can reach 3.9 g / L; the establishment of a whole cell transformation system solves the problems that the chemical synthesis of PPA is tedious in step and low in yield and results in environmental pollution and the enzymatic transformation production of PPA is low in transformation rate, realizes the pollution-free high-yield and one-step production of PPA, and lays a certain theoretical foundation for the subsequent industrial production.

Description

technical field [0001] The invention relates to a recombinant bacterium with improved transformation and production efficiency of α-phenylpyruvate, which belongs to the field of biotechnology. Background technique [0002] α-Phenylpyruvate (PPA) has many applications. It can be used to synthesize heterocyclic compounds of antineoplastic drugs, as an antioxidant and to promote wound healing. It is the raw material for the synthesis of D-phenylalanine, which is a chiral drug intermediate. PPA can also be used to prepare phenyllactic acid, which can be used as an antibacterial substance. At present, the production of PPA is mainly chemical synthesis, mainly including: acetamidocinnamic acid hydrolysis method, hydantoin and benzaldehyde synthesis method and hydantoin method. These methods all require multi-step reactions and have high requirements for reaction conditions, such as high pressure, high temperature, etc., and the product yield is low, which increases the difficult...

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
IPC IPC(8): C12N1/21C12N15/87C12P7/40C12R1/19
Inventor 陈坚刘龙李江华堵国成侯颖顾汉章徐堃
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap