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Method for synthesizing propionic acid by using threonine and recombinant bacterium used in method

A technology of recombinant bacteria and threonine, applied in the field of recombinant bacteria, can solve problems such as global overcapacity

Pending Publication Date: 2022-02-08
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] L-threonine is an essential amino acid, which is mainly prepared by microbial fermentation. At present, there is a mature production process, and the global production capacity is facing the risk of excess

Method used

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  • Method for synthesizing propionic acid by using threonine and recombinant bacterium used in method
  • Method for synthesizing propionic acid by using threonine and recombinant bacterium used in method
  • Method for synthesizing propionic acid by using threonine and recombinant bacterium used in method

Examples

Experimental program
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Effect test

Embodiment 1

[0128] Embodiment 1, the preparation of recombinant bacteria PS30

[0129] 1.1 Knockout of threonine aldolase ltaE

[0130] Starting from Pseudomonas putida KT2440 (ATCC 47054), the threonine aldolase gene ltaE was knocked out by homologous recombination (Gene ID: 1044018, discontinued on 2-Apr-2020; Genome: NC_002947.4:c386535 -385495), which blocks the threonine to glycine pathway pathway. The amino acid sequence of threonine aldolase is shown in WP_010951681.1 (2017-5-14) in NCBI. The specific steps are as follows:

[0131] 1.1.1. Construction of knockout plasmid pK18-ltaE

[0132] Using the pK18 plasmid (ATCC 87097) as a template, it was cut with two restriction enzymes, BamHI and HindIII, to obtain a 6022bp DNA linearized fragment.

[0133] Select 500bp upstream and downstream of the ltaE gene (SEQ ID No.2 1-500 and SEQ ID No.2 2163-2662), and design primers ltaE-up-F / ltaE-up-R and ltaE-down-F / ltaE-down-R, using the genomic DNA of Pseudomonas putida KT2440 as a temp...

Embodiment 2

[0162] Embodiment 2, the preparation of recombinant bacterium PS31

[0163] 2.1. Genome overexpression of threonine deaminase gene ilvA derived from Escherichia coli

[0164] The amino acid sequence of threonine deaminase derived from Escherichia coli is shown in EEV5737228.1 (20200406) in NCBI. The preparation steps are as follows:

[0165] 2.1.1. Construction of overexpression ilvA plasmid

[0166] Extract genomic DNA from Escherichia coli (ATCC 700926), amplify ilvA gene with primer ilvA-F / ilvA-R, obtain the DNA fragment of 1545bp (the nucleotide sequence of coding chain is as SEQ ID No.2 the 617th-2161st position shown). Plasmid pUCP18 (BioVector, Cloning vector pUCP18) was cut with restriction endonucleases BamHI and HindIII to obtain a 5465bp DNA linearized fragment. The 1545bp DNA fragment ilvA and the 5465bp pUCP18 plasmid DNA linear fragment were ligated together by the Gibson method, and transformed into Escherichia coli DH5α (transgen biotech, CD201-01), identif...

Embodiment 3

[0188] Embodiment 3, the preparation of recombinant bacterium PS32

[0189] 3.1. Overexpression of 2-ketoisovalerate deacidase derived from Lactococcus lactis

[0190] The amino acid sequence of 2-ketoisovalerate decarboxylase (kivD) derived from Lactococcus lactis is as WP_012897921.1 (20190728) in NCBI, and the preparation method is as follows:

[0191] Genomic DNA was extracted from Lactococcus lactis (NCDO 2118), and the kivD gene was amplified with primers kivD-F / kivD-R to obtain a 1647bp DNA fragment (the coding sequence of the coding strand is shown in SEQ ID No.3). Plasmid pUCP18 (BioVector, Cloning vector pUCP18) was cut with restriction endonucleases BamHI and HindIII to obtain a 5465bp DNA linearized fragment. The 1647bp DNA fragment kivD and the 5465bp pUCP18 plasmid DNA linear fragment were ligated together by the Gibson method, and transformed into Escherichia coli DH5α (transgen biotech, CD201-01), identified with primers pucp18-F / pucp18-R, and selected the tar...

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Abstract

The invention discloses a recombinant bacterium. The recombinant bacterium is used for expressing 2-ketoisovalerate deacidification enzyme (also called ketoacid decarboxylase). The recombinant bacterium is obtained by modifying a genome of a recipient bacterium, wherein the modification comprises the step of enabling the recipient bacterium to express the 2-ketoisovalerate deacidification enzyme, and the recipient bacterium is pseudomonas or escherichia coli. The method for synthesizing the propionic acid by using the recombinant bacterium is characterized in that the propionic acid is synthesized from 2-ketobutyric acid under the catalysis of 2-ketoisovalerate deacidilase. A novel metabolic pathway for synthesis of propionic acid is provided. The transformation rate of propionic acid is 96.67% when the recombinant pseudomonas PS32 obtained after transformation in the invention is used for catalyzing threonine synthesis for 24 hours; and after material supplementation, the conversion rate of the propionic acid can be 98.47% during catalysis of threonine synthesis by the PS32. The recombinant escherichia coli obtained after transformation in the invention can be used for transforming L-threonine with a concentration of 400 mM into propionate with a concentration of 393 mM within 24 hours, and a conversion rate is 98.25%.

Description

technical field [0001] The invention relates to the fields of microorganisms and genetic engineering, in particular to a method for synthesizing propionic acid from threonine and the recombinant bacteria used therein. Background technique [0002] Pseudomonas putida was the first Gram-negative bacterium recognized as environmentally safe by the U.S. Department of Health's Recombinant DNA Advisory Committee (RAC) (1982) and licensed KT2440 as a genetically engineered host bacteria. Pseudomonas putida KT2440 (P.putida KT2440) can decompose some organic matter in the environment, has the functions of biocatalysis and bio-pollution, and is a microorganism with extremely high industrial catalytic value. [0003] Escherichia coli is currently one of the most common biocatalytic hosts, and a variety of biochemical products have been mass-produced, but it is rare to use Escherichia coli to produce propionate. [0004] Propionic acid and its derivatives are widely used in industry,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/78C12N15/60C12N15/54C12N15/53C12N15/52C12N15/31C12N9/88C12P7/52C12R1/38C12R1/19
CPCC12N9/88C12N15/70C12N15/78C12N15/52C12N9/1025C07K14/21C12N9/93C12N9/0008C12P7/52C12Y401/01C12Y401/02005C12Y401/02042C12Y203/03005C12Y403/01019C12Y602/01C12Y102/01003C12Y102/01004C12Y102/01005
Inventor 牟庆璇陶勇于波
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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