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A high-yielding l-leucine engineering bacterium and its application

A technology of leucine and Corynebacterium glutamicum, applied in application, genetic engineering, bacteria and other directions, can solve the problems of long cycle, difficulty and increase the yield of target products

Active Publication Date: 2017-07-21
FUJIAN NORMAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, industrialized L-leucine producing bacteria are mainly obtained through traditional mutagenesis screening. Although this classic breeding method can obtain excellent strains with relatively high L-leucine production, this method will also introduce harmful mutations, making the target The strain grows slowly and the heteroacids increase
In addition, this method has a large workload and a long cycle, and it is difficult to further increase the output of the target product in a short period of time.

Method used

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  • A high-yielding l-leucine engineering bacterium and its application
  • A high-yielding l-leucine engineering bacterium and its application
  • A high-yielding l-leucine engineering bacterium and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1, the construction of engineering bacteria

[0032] 1. Exogenous high-level expression of leuA * Construction of recombinant vector

[0033] 1. Cloning and site-directed mutation of leuA gene

[0034] 1), using the genomic DNA of Corynebacterium glutamicum 13032 as a template, carrying out PCR amplification with primers composed of F1 and R1, to obtain a PCR amplification product (leuA gene fragment).

[0035] F1: 5'- GAATTC ATGCCAGTTAACCGCTACATGCCT-3';

[0036] R1: 5'- GTC GAC TTAAACGCCGCCAGCCAGGAC-3'.

[0037] Among them, F1 has an EcoR I restriction site, and R1 has a Sal I restriction site (the underlined partial sequence of the above primer).

[0038] PCR amplification conditions: pre-denaturation at 95°C for 5 minutes; denaturation at 94°C for 40 seconds, annealing at 59°C for 1 minute, extension at 72°C for 2 minutes, 30 cycles; reaction at 72°C for 10 minutes, and incubation at 4°C.

[0039] 2) Recovering the PCR amplified product of step 1)...

Embodiment 2

[0119] Embodiment 2, Corynebacterium glutamicum MD0032 produces L-leucine

[0120] 1. Preparation of culture medium

[0121] Seed medium (pH=6.5-7.0): Take 100g glucose, 5g corn steep liquor, 10g brown sugar, 50g molasses, 100ml soybean meal hydrolyzate (purchased from Shandong Yangcheng Biotechnology Co., Ltd., catalog number DP001), 3g yeast extract , 25g (NH 4 ) 2 SO 4 , 0.2g MgSO 4 ·7H 2 O, 0.05g FeSO 4 ·7H 2 O, 0.3g KH 2 PO 4 ·3H 2 O. Dissolve 0.025g of kanamycin monosulfate and 10g of calcium carbonate in deionized water and adjust the volume to 1L; sterilize at 125°C for 25min.

[0122] Fermentation medium (pH=6.5-7.5): take 120g glucose, 150ml soybean meal hydrolyzate, 27g (NH 4 ) 2 SO 4 , 5.5gKH 2 PO 4 ·3H 2 O, 2.5g K 2 HPO 4 ·3H 2 O, 0.2g MgSO 4 ·7H 2 O, 0.05g FeSO 4 ·7H 2 O, 0.02gMnSO 4 ·H 2 O, 200 μg vitamin H, 200 μg vitamin B1, 100 μg vitamin B2, 100 μg vitamin B6, 0.025 g kanamycin monosulfate, 30 g CaCO 3 Dissolve in deionized water an...

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Abstract

The invention discloses a high yield L-leucine engineering bacterium and an application of the bacterium.The invention provides a method for preparing a recombinant bacterium. The method comprises the following step of importing an alpha-isopropylmolic acid synthetase mutant encoding gene into a target bacterium to form the recombinant bacterium, wherein an alpha-isopropylmolic acid synthetase mutant is enzyme obtained by mutating 494th arginine of alpha-isopropylmolic acid synthetase into histidine, 497th glycine into aspartic acid and 499th leucine into valine.The high yield L- leucine engineering bacterium, particularly corynebacterium glutamicum MD0032, has higher L-leucine yield than bacterial strains produced at home at present, has a unique identification label and is a L-leucine production strain with a high production and application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a high-yielding L-leucine engineering bacterium and its application. Background technique [0002] The molecular structure of L-leucine, L-isoleucine (L-isoleucine) and L-valine (L-valine) contains a methyl side chain, so it is called branched chain amino acid (branched chain amino acid). acids, BCAA). As one of the eight essential amino acids for the human body, L-leucine is a raw material for the synthesis of human hormones and enzymes. It has the effect of promoting protein synthesis and inhibiting decomposition, but it cannot be synthesized in the body. Therefore, L-leucine has a wide range of applications and commercial value in the fields of food, medicine, feed and sports health care, and with the continuous development of the function of L-leucine, its application fields are gradually expanding, and the market is also continuing rising. [0003] At present, the production ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/54C12P13/06C12R1/15
CPCC07K14/34C12N9/0006C12N9/1025C12N9/1096C12N9/88C12N9/93C12P13/06C12Y203/03013C12Y206/01002C12Y403/01019
Inventor 黄钦耿黄建忠梁玲翁雪清吴伟斌黄祥峰施巧琴吴松刚
Owner FUJIAN NORMAL UNIV
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