Method for fermenting production of L-lysine through using aconitase expression weakened and/or enzymatic activity reduced bacteria

An aconitase and bacterial fermentation technology, which is applied in the field of amino acid fermentation, can solve the problems of difficult bacterial growth, long metabolic distance, many intermediate metabolic branches, etc., and achieves the effect of facilitating popularization and application and improving yield

Active Publication Date: 2014-06-18
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 because its metabolism is too far away from the final L-lysine product. Far, there are too many and complex branches of intermediate metabolism, but it has not attracted people's attention in the fermentation of L-lysine
[0004] After long-term research and practice, especially with some luck, the inventor accidentally found that the modification of the acnA gene can help to increase the production of L-lysine; Enzyme gene whose enzyme activity and / or expression level can be eliminated by knocking out unfavorable gene, but different from it, the inventors found that the acnA gene cannot be simply improved Or knockout, especially after knockout, makes bacterial growth difficult and difficult for practical application, so a new method for acnA gene regulation has been developed to improve the production of L-lysine, and this method is compatible with the existing modified There is no conflict in the chromosomal modification sites of a large number of high-yield L-lysine bacteria, and the improved effect can be superimposed, so it can be used for bacterial fermentation to produce L-lysine in practice

Method used

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  • Method for fermenting production of L-lysine through using aconitase expression weakened and/or enzymatic activity reduced bacteria
  • Method for fermenting production of L-lysine through using aconitase expression weakened and/or enzymatic activity reduced bacteria
  • Method for fermenting production of L-lysine through using aconitase expression weakened and/or enzymatic activity reduced bacteria

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Construction Example 1 replaces the start codon ATG of acnA with GTG

[0033] Using the extracted wild-type Escherichia coli K12W3110 strain (available from NITE Biological Resource Center (NBRC)) genome chromosome as a template, and primers P1 and P2, P3 and P4 respectively PCR amplification obtained two DNA fragments (respectively named Up1 and Down1 fragments) with lengths of 510 bp and 620 bp respectively. Wherein, PCR is carried out as follows: denaturation at 94°C for 30s (seconds), annealing at 52°C for 30s (seconds), and extension at 72°C for 30s (seconds) (30 cycles). Wherein, the primer sequence is as follows:

[0034] P1: 5'-CGC GGATCC GGAGTCGTCACCATTATGCC-3' (the underline indicates the restriction site of BamHI)

[0035] P2: 5'-TCTCGTAGGGTTGACGACA C AGCTCCTCCTTAATGACAGG-3' (underline indicates point mutation)

[0036] P3: 5'-CCTGTCATTAAGGAGGAGCT G TGTCGTCAACCCTACGAGA-3' (underline indicates point mutation)

[0037] P4: 5'-ATT GCGGCCGC CCATTCACCGTCC...

Embodiment 2

[0041] Construction Example 2 acnA gene sequence mutation reduces aconitase activity

[0042] The 90 bp base before the stop codon was deleted to reduce the activity of aconitase. Specifically, using the extracted genome chromosome of wild-type Escherichia coli K12W3110 as a template, PCR amplification was performed with primers P5 and P6, P7 and P8 respectively, and two DNA fragments (Up3 and Down3 fragment). Wherein, PCR is carried out as follows: denaturation at 94°C for 30s (seconds), annealing at 52°C for 30s (seconds), and extension at 72°C for 30s (seconds) (30 cycles). Wherein, the primer sequence is as follows:

[0043] P5: 5'-CGC GGATCC CGTCACACGATCCGATACCT-3' (the underline indicates the restriction site of BamHI)

[0044] P6: 5'-CGGCAAGCAAATAGTTGTTATACGACTTCCTGGCTACCAT-3' (underline indicates point mutation)

[0045] P7: 5'-ATGGTAGCCAGGAAGTCGTATAACAACTATTTGCTTGCCG-3' (underline indicates point mutation)

[0046] P8: 5'-ATT GCGGCCGC CATGGGGCGATTTCCTGATG-3' (...

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Abstract

The invention provides a fermenting production method of L-lysine. The method comprises a step of modifying bacteria to reduce not disappear the aconitase expression level or the enzymatic activity of the bacteria, and a step of fermenting the modified bacteria to produce L-lysine. The invention also provides processes derived from the method, applications thereof, and polynucleotides, vectors and bacteria used in the processes and the method and the applications.

Description

technical field [0001] The present invention belongs to the field of amino acid fermentation, in particular, the present invention relates to a method for fermentative production of L-lysine and its derivation method and application, as well as polynucleotides, vectors and bacteria that can be used in these methods and applications. Background technique [0002] Production of L-lysine by fermentation of L-lysine-producing bacteria (eg, Escherichia coli of the genus Escherichia and rod-shaped bacteria of the genus Corynebacterium) has been industrially used. 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 concentrated on genes such as pnt, dap and ppc, and no attention has been paid to aconitase (such as aconitase A) and its encoding gene. [0003] Aconitase is an enzyme in the tricarboxylic acid cycle that cataly...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/08C12N15/70C12N1/21C12N15/61C12R1/19
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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