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Method for producing L-threonine by adopting bacterial fermentation with weakened aconitase expression and/or reduced enzymatic activity

An aconitase and threonine-producing technology, which is applied in the field of amino acid fermentation, can solve the problems of long metabolic distance, difficult bacterial growth, and cannot be simply improved or knocked out, so as to achieve the effect of increasing production and facilitating popularization and application

Active Publication Date: 2014-07-23
NINGXIA EPPEN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is currently known that in Escherichia coli, the acnA gene (its nucleotide sequence is shown in SEQ ID No: 1) encodes aconitase A, but it may be due to its metabolic distance from the final The l-threonine product is too far away, the intermediate metabolic branches are too many and complicated, and it has not been paid attention to in l-threonine fermentation
[0004] After long-term research and practice, especially with some luck, the inventor accidentally found that the transformation of acnA gene can help to improve l- The production of threonine; however, the prior art either introduces beneficial enzyme genes with increased expression and / or enzymatic activity by increasing copies and / or site-directed mutations, or by knocking out unfavorable genes to make them enzymatically active and / or The expression level disappears, but the difference is that the inventors found that the acnA gene cannot be simply improved or knocked out, especially after knocking out, it makes bacterial growth difficult and difficult for practical application, so the developed A new method for acnA gene regulation to increase the production of L-threonine, and this method is compatible with the existing chromosomal modification sites of a large number of high-production L-threonine bacteria There is no conflict, and the improved effects can be superimposed, so that it can be practically used for bacterial fermentation to produce l-threonine

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Build Example 1 Replace acnA The start codon ATG is GTG

[0035] wild-type Escherichia coli E. coli The genome chromosome of K12 W3110 strain (available from NITE Biological Resource Center (NBRC)) was used as a template, and PCR amplification was performed with primers P1 and P2, P3 and P4 respectively, and the lengths were 510 bp and two DNA fragments of 620 bp (named Up1 and Down1 fragments, respectively). Among them, PCR was carried out as follows: denaturation at 94°C for 30 s (seconds), annealing at 52°C for 30 s (seconds), and extension at 72°C for 30 s (seconds) (30 cycles). Wherein, the primer sequence is as follows:

[0036] P1:5'-CGC GGATCC GGAGTCGTCACCATTATGCC-3' (underlined Bam H I restriction site)

[0037] P2:5'-TCTCGTAGGGTTGACGACA C AGCTCCTCCTTAATGACAGG-3’ (underline indicates point mutation)

[0038] P3:5'-CCTGTCATTAAGGAGGAGCT G TGTCGTCAACCCTACGAGA-3' (underline indicates point mutation)

[0039] P4:5'-ATT GCGGCCGC TCCATTCACCGTCCTGCAAT...

Embodiment 2

[0043] Build Example 2 acnA Gene sequence mutation reduces aconitase activity

[0044] The 90 bp base before the stop codon was deleted to reduce the activity of aconitase. Specifically, the extracted wild-type E. coli E. coli K12 W3110 genome chromosome was used as a template, PCR amplification was carried out with primers P5 and P6, P7 and P8 respectively, and two DNA fragments (Up3 and Down3 fragments) with lengths of 752 bp and 657 bp were obtained, respectively. Among them, PCR was carried out as follows: denaturation at 94°C for 30 s (seconds), annealing at 52°C for 30 s (seconds), and extension at 72°C for 30 s (seconds) (30 cycles). Wherein, the primer sequence is as follows:

[0045] P5:5'-CGC GGATCC CGTCACACGATCCGATACCT-3' (underlined Bam H I restriction site)

[0046] P6:5'- CGGCAAGCAAATAGTTGTTATACGACTTCCTGGCTACCAT -3' (underline indicates point mutation)

[0047] P7: 5'-ATGGTAGCCAGGAAGTCGTATAACAACTATTTGCTTGCCG-3' (underline indicates point mutation)

...

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Abstract

The invention provides a method for fermentatively producing l-threonine, which comprises transforming bacteria to reduce the expression level and / or enzyme activity of aconitase A but not disappearing; and, fermenting and producing l-threonine with the transformed bacteria acid. In addition, the present invention also provides methods and uses derived from this method, as well as polynucleotides, vectors and bacteria that can be used in these methods and applications.

Description

technical field [0001] The present invention belongs to the field of amino acid fermentation, in particular, the present invention relates to the fermentative production method of l-threonine and its derivation method and application, and polynucleotides, vectors and bacteria that can be used in these methods and applications. Background technique [0002] Production of l-threonine by fermentation of l-threonine-producing bacteria (eg, Escherichia coli of the genus Escherichia and rod-shaped bacteria of the genus Corynebacterium) has been industrially applied. These bacteria can be bacteria isolated from nature, bacteria obtained through mutagenesis or genetic engineering, or both. In the current literature reports, the attention through genetic engineering is mainly focused on AKIII, thrR In terms of isogenes (see Chinese patents 94102007 and 99121353, etc.), there is no focus on aconitase (eg, aconitase A) and its coding gene for the production of L-threonine. [0003] A...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/08C12N15/70C12N1/21C12R1/19
Inventor 马吉银温廷益陈金龙梁勇刘树文魏爱英杨立鹏孟刚任瑞
Owner NINGXIA EPPEN BIOTECH
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