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A kind of recombinant strain of dapb gene modification and its construction method and application

A technology of recombinant strain and construction method, applied in the fields of genetic engineering and microorganisms, can solve the problems of high production cost, low acid production by fermentation, small production scale, etc., and achieve the effects of reducing production cost, increasing yield and improving production efficiency.

Active Publication Date: 2021-04-09
HEILONGJIANG EPPEN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low level of fermented acid production and small production scale, the production cost is high and it is difficult to compete with imported products

Method used

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  • A kind of recombinant strain of dapb gene modification and its construction method and application
  • A kind of recombinant strain of dapb gene modification and its construction method and application
  • A kind of recombinant strain of dapb gene modification and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Construction of transformation vector pK18-PdapB comprising point-mutated dapB gene promoter region

[0045] (C(-49)A,G(-51)T,CTGCA(-54--58)GGTGT)

[0046] According to the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, two pairs of primers for amplifying the sequence of the dapB gene promoter region were designed and synthesized, and were used in strain YP97158 (preservation number: CGMCCNo.12856, date of preservation: On August 16, 2016, depository unit: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807355) the dapB gene promoter region (SEQ ID NO: 1) in the background Introducing point mutations, the -49bp position C in the nucleotide sequence of the dapB gene promoter region is changed to A, the G at -51bp position is changed to T, and the -54--58bpCTGCA is changed to GGTGT (SEQ ID NO: 2).

[0047] The primers were designed as follows (synthesize...

Embodiment 2

[0060] Example 2 Construction of pK18-PdapB comprising point mutations (C(-49)A,G(-51)T,CTGCA(-54--58)GGTGT) engineered strain

[0061] Allele replacement plasmid pK18-PdapB (C(-49)A,G(-51)T,CTGCA(-54--58)GGTGT) Transformed into the patented strain YP97158 of L-lysine producing bacteria by electric shock (for its construction method, please refer to WO2014121669A1; it was confirmed by sequencing that the wild-type dapB gene promoter was retained on the chromosome of the strain), and the single colonies produced by culture were respectively passed primers P1 and universal primer M13F were used for identification, and the strains that could amplify a band with a size of 1350 bp were positive strains. The positive strains were cultured on the medium containing 15% sucrose, and the single colonies produced by the culture were cultured on the medium containing kanamycin and kanamycin-free respectively.

[0062] The composition of the basal medium used in each of the above-mention...

Embodiment 3

[0071] Example 3 L-lysine fermentation experiment

[0072] The bacterial strain YPL-4-010 constructed in Example 2 and the original bacterial strain YP97158 were used in the fermenter of the BLBIO-5GC-4-H model (purchased from Shanghai Bailun Biotechnology Co., Ltd.) with the medium and table shown in Table 1. The control process shown in 2 was used for fermentation experiments. Each strain was repeated three times, and the results are shown in Table 3.

[0073] Table 1 Fermentation Medium Formula

[0074] Element formula starch hydrolyzed sugar 30g / L ammonium sulfate 12g / L magnesium sulfate 0.87g / L molasses 20g / L acidified corn steep liquor 3mL / L phosphoric acid 0.4mL / L potassium chloride 0.53g / L Defoamer (2% foam enemy) 4mL / L ferrous sulfate 120mg / L Manganese sulfate 120mg / L Nicotinamide 42mg / L thbrthdrexvbdr 6.3mg / L Vitamin B1 6.3mg / L Copper, zinc salt solution 0.6...

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Abstract

The invention discloses a nucleotide sequence and a recombinant strain comprising the polynucleotide sequence, which is formed by performing a point mutation on the promoter region sequence of the dapB gene in Corynebacterium glutamicum, and the mutated promoter region gene sequence As shown in SEQ ID NO:2. Compared with the non-mutated strain, the recombinant strain has improved L-lysine production and good strain stability, and can further reduce production cost as an L-lysine production strain.

Description

technical field [0001] The invention belongs to the field of genetic engineering and microorganism technology, and specifically relates to an improvement of a dapB gene promoter, a modified recombinant bacterial strain obtained therefrom, a construction method thereof and an application in L-lysine production. Background technique [0002] L-Lysine is the most important of the eight essential amino acids that cannot be synthesized by humans and animals. At present, lysine is the most widely used amino acid feed additive in the world. About 90% of L-lysine products are used as feed additives, about 5% are used as food additives, and the remaining 5% are used as pharmaceutical intermediates. For example, adding an appropriate amount of L-lysine to soybean meal can greatly improve the utilization rate of protein and promote the growth of poultry and livestock. Due to the astonishing development speed of the domestic feed industry, the market demand for L-lysine is increasing d...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/113C12N15/10C12N15/77C12N1/21C12P13/08C12R1/15
CPCC12N9/001C12N15/1031C12N15/77C12P13/08C12Y103/01026C12Q2531/113
Inventor 魏爱英孟刚贾慧萍高晓航马风勇周晓群
Owner HEILONGJIANG EPPEN BIOTECH CO LTD
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