Vector, engineering strain and method for producing l-2-aminobutyric acid

An amino acid, recombinant vector technology, applied in genetic engineering, biochemical equipment and methods, and introduction of foreign genetic material using vectors, etc., can solve the problems of low yield, high production cost and high cost

Active Publication Date: 2015-11-18
SHANGHAI PUYI CHEM CO LTD
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the early stage, ketoacid and glutamic acid were used as substrates to generate L-2-aminobutyric acid under the action of amino acid transaminase. -2-aminobutyric acid, the method has low yield and by-products affect the purification of the product; the amino acid oxidase method uses D-amino acid oxidase to prepare L-2-aminobutyric acid under the action of a metal catalyst, and the method costs High, not suitable for large-scale industrial applications
[0004] In addition, there are dehydrogenase methods, aminoacylase methods, etc., but there are problems of high production costs and enzyme activity being inhibited by substrates, so the industrial application effect is not good.

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
  • Vector, engineering strain and method for producing l-2-aminobutyric acid
  • Vector, engineering strain and method for producing l-2-aminobutyric acid
  • Vector, engineering strain and method for producing l-2-aminobutyric acid

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0088] For the preparation and transformation methods of competent cells, refer to Scheme 25 in Chapter 1 of "Molecular Cloning Experiment Guide (Third Edition)" (Science Press, 2002).

[0089] For the measurement method of glucose content, refer to "Zhou Yaxuan et al., Enzymatic Determination of Glucose Content in Wine, Chinese Journal of Health Inspection, 2005, 12(5): 194-221".

[0090] Advantages of the invention

[0091] 1. Fermentation and cultivation The genetically engineered bacterial strain constructed by the present invention is used to produce L-2-aminobutyric acid, and the content of L-2-aminobutyric acid in the obtained fermentation liquid is high, and the fermentation property is stable;

[0092] 2. The cost of the method of the present invention is greatly reduced, and the process is stable;

[0093] 3. The conversion rate of the method of the present invention is high, there is no influence of by-products, the product is easy to purify, and is very suitable f...

Embodiment 1

[0096] Construction of L-threonine high-yielding strain and fermentation of L-threonine

[0097] 1. Construction of high-yield L-threonine metabolic engineering bacteria

[0098] 1.1 Genetic modification of gene cluster thrABC in Escherichia coli W3110

[0099] With reference to the method disclosed in the literature of LeeKH et al. (LeeKHetal.SystemsmetabolicengineeringofEscherichiacoliforL-threonineproduction.Molecularsystemsbiology.2007,3:149), genes ThrABC, rhtC, rhtA, rhtB were amplified from the Escherichia coli W3110 genome; thrA encodes aspartokinase I, rhtA , rhtB encodes a threonine and homoserine transporter, transports threonine out of the cell, rhtC encodes a threonine transporter, transports threonine out of the cell. The 1034th base C of gene thrA was site-directedly mutated into base T (Ser345→Phe) by overlapping PCR to obtain ThrA*BC'.

[0100] ThrA*BC was connected to the plasmid pKK223-3 with a tac promoter, and then the rop gene and rhtC, rhtA, rhtB genes...

Embodiment 2

[0120] Construction of Genetic Engineering Strains THR / pSUGAP-leuDH-ilvA and THR / pSUGAP-ilvA-leuDH

[0121] Threonine deaminase gene ilvA is from Escherichia coli W3110 (NCBI accession number: AP009048); leucine dehydrogenase gene leuDH is from Bacillus cereus (NCBI accession number: AE016877).

[0122] 1. Construction of pSUGAP plasmid

[0123] The plasmid pSU2718 (Martinez et al., 1988) was extracted with a plasmid extraction kit, and the plasmid was digested with SacI / SmaI double enzymes at 37°C for 2 hours. The enzyme digestion system was: 76 μl of plasmid, 20 μl of 10×Tangobuffer, 2 μl of SacI, and 2 μl of SmaI, and recovered by electrophoresis 2.3Kb pSU2718 fragment, such as figure 1 .

[0124] A 0.2 Kb fragment containing the GAP promoter was isolated from Escherichia coli (NCBI accession number: CP001509) by PCR using the ecgapup and ecgapdn primers in Table 3.

[0125] PCR conditions: ddH 2 O3 3μl, 10×KODbuffer 5μl, template 2μl, 25mMMgCl 2 3 μl, 1 μl of each pri...

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 relates to a vector, an engineering strain and a method for producing L(+)-2-aminobutyric acid. The vector is a recombinant vector containing a threonine dehydratase coding gene, an L-amino acid dehydrogenase gene and appropriate vector segments. The engineering strain is obtained by transforming host bacteria by the recombinant vector. The L(+)-2-aminobutyric acid is prepared by fermentation culture of the engineering strain. The engineering strain is fermentation-cultured by glucose as a raw material to produce L(+)-2-aminobutyric acid. The method has the advantages of low cost, high product concentration, no by-product, easy product purification, and good industrial application feasibility.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a carrier, an engineering strain and a method for producing L-2-aminobutyric acid. Background technique [0002] L-2-aminobutyric acid (L(+)-2-Aminobutyric acid), is a non-natural chiral α-amino acid, the molecular formula is C 4 h 9 NO 2 , which is mainly used for the synthesis and treatment of localized and secondary generalized epilepsy diseases, and is also the key chiral precursor for the synthesis of the antibacterial and anti-tuberculosis drug ethambutol. [0003] The transaminase method is currently a widely used method for producing L-2-aminobutyric acid. In the early stage, ketoacid and glutamic acid were used as substrates to generate L-2-aminobutyric acid under the action of amino acid transaminase. -2-aminobutyric acid, the method has low yield and by-products affect the purification of the product; the amino acid oxidase method uses D-amino acid oxidase ...

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): C12N15/63C12N15/53C12N15/60C12N1/15C12N1/19C12N1/21C12N5/10C12P13/04
Inventor 杨晟陶荣盛朱傅赟赵丽丽蒋宇杨俊杰孙周通沈正权黄鹤孙梁栋董枫刘映淼
Owner SHANGHAI PUYI CHEM CO LTD
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