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High yield L-leucine engineering bacterium and application thereof
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A technology of Leucine and Corynebacterium glutamicum, applied in application, genetic engineering, bacteria, etc., can solve the problems of increased miscellaneous acids, long cycle, heavy workload, etc.
Active Publication Date: 2015-04-01
FUJIAN NORMAL UNIV +1
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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.
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Embodiment 1
[0031] Embodiment 1, the construction of engineering bacteria
[0032] 1. Exogenous high-level expression of leuA * Construction of recombinant vector
[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).
[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)...
[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 calciumcarbonate 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 , 5.5g KH 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 a...
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Abstract
The invention discloses a high yield L-leucineengineering 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- leucineengineering 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 ...
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