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Corynebacterium glutamicum engineering bacterium and application thereof in preparation of L-tryptophan

A technology of Corynebacterium glutamicum and tryptophan, applied in the biological field, can solve the problem that the production strain cannot be used for bacterial protein application and the like

Active Publication Date: 2020-03-06
金河生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the L-tryptophan produced by fermentation is realized by engineering strains of E. coli, but in view of the endotoxin properties of E. coli, its safety has also attracted more attention. Not only that, but the production strains themselves Can not get more applications as bacterial protein

Method used

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  • Corynebacterium glutamicum engineering bacterium and application thereof in preparation of L-tryptophan

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1, construction of alanine transaminase knockout module

[0066] 1. Construction of alanine aminotransferase (AlaT) knockout module

[0067] In Corynebacterium glutamicum FNT112, the AlaT gene is shown in sequence 2 of the sequence listing, and encodes the AlaT protein shown in sequence 1 of the sequence listing.

[0068] 1. Take Corynebacterium glutamicum FNT112 and extract genomic DNA.

[0069] 2. Using the genomic DNA obtained in step 1 as a template, a primer pair composed of alaT-P1 and alaT-P2 is used for PCR amplification, and the amplified product is recovered.

[0070] alaT-P1: 5'- GAATTC GTGACTACAGACAAGCGCAAAACCTC-3';

[0071] alaT-P2: 5'-AACTACAGACCTAGAACCTATTGAGGAGTGCTTGGGTGGTCATG-3'.

[0072] 3. Using the genomic DNA obtained in step 1 as a template, a primer pair consisting of alaT-P3 and alaT-P4 is used for PCR amplification, and the amplified product is recovered.

[0073] alaT-P3: 5'-TAGGTTCTAGGTCTGTAGTTACTGGACCAAAGCAATACGCACGTGG-3';

...

Embodiment 2

[0092] Example 2, Construction of Leucine and Tryptophan Synthesis Regulator (LtbR) Knockout Module

[0093] In Corynebacterium glutamicum FNT112, the LtbR gene is shown in nucleotides 138-845 of Sequence 6 in the Sequence Listing, and encodes the LtbR protein shown in Sequence 5 in the Sequence Listing.

[0094] 1. Take Corynebacterium glutamicum FNT112 and extract genomic DNA.

[0095] 2. Using the genomic DNA obtained in step 1 as a template, perform PCR amplification with a primer pair composed of ltbR-P1 and ltbR-P2, and recover the amplified product.

[0096] ltbR-P1: 5'- GAATTC atgaccttgaaatacacggtgaag-3';

[0097]ltbR-P2: 5'-AACTACAGACCTAGAACCTAATGCAGGGTCAGCAGCGCGC-3'.

[0098] 3. Using the genomic DNA obtained in step 1 as a template, perform PCR amplification with a primer pair composed of ltbR-P3 and ltbR-P4, and recover the amplified product.

[0099] ltbR-P3: 5'-TAGGTTCTAGGTCTGTAGTTagcgccgcgtgcacccaatg-3';

[0100] ltbR-P4: 5'- GTC GAC ATATCGTTTCATGGGACAGTA...

Embodiment 3

[0105] Embodiment 3, the construction of Corynebacterium glutamicum engineering strain

[0106] 1. Construction of AlaT Gene Modified Strains

[0107] 1. Transform Corynebacterium glutamicum FNT112 with the recombinant plasmid pk18ΔAlaT by electric shock method. Immediately after the electric shock, add it to the SOC medium preheated at 45°C, and cultivate it at 42°C and 200rpm for 1 hour, then at 37°C and 120-150rpm Incubate for 3-5 hours. An Eppendorf electroporator was used. Shock conditions: 1800V, shock time 4-5ms.

[0108] 2. After completing step 1, take the bacterial liquid, spread it on the LBG medium plate containing 25 μg / mL kanamycin sulfate, and culture it upside down at 30°C for 24-30 hours. The clones grown on the plate were the ones that successfully underwent the first recombination. The first recombination, that is, the recombinant plasmid pk18ΔAlaT is inserted into the chromosome as a whole by means of single exchange.

[0109] 3. After completing step ...

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Abstract

The invention discloses a corynebacterium glutamicum engineering bacterium and an application of the corynebacterium glutamicum engineering bacterium in preparation of L-tryptophan. The invention provides the corynebacterium glutamicum FJR-025, and a preservation number of the corynebacterium glutamicum FJR-025 is CCTCC NO: M 2019868. The invention also discloses the application of corynebacteriumglutamicum FJR-025 in preparation of L-tryptophan. The invention further discloses application of corynebacterium glutamicum FJR-025 in preparation of a feed additive. The strain provided by the invention can be directly fermented to obtain L-tryptophan, the content of L-tryptophan in fermentation liquor is as high as 52.5 g / L after 48 days of fermentation, and a feed additive with the content ofL-tryptophan being 30% or above can be obtained through direct spray drying without L-tryptophan purification, so that the strain has extremely high application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a Corynebacterium glutamicum engineering bacterium and its application in preparing L-tryptophan. Background technique [0002] L-tryptophan (L-Ile) is one of the eight essential amino acids for the human body. It is widely used in the fields of medicine, food, feed and chemical industry. The increase has also further promoted the amount of amino acids used in feed, especially the continuous development of the functions of L-tryptophan and its derivatives in intestinal microorganisms, and its commercial application value is gradually being expanded. [0003] L-tryptophan has been transformed from the traditional hydrolysis method to the microbial production method, and the most mainstream production method is the direct microbial fermentation method. The two most important strains in the amino acid fermentation industry are Escherichia coli and Corynebacterium glutamicum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N1/21C12P13/22A23K20/142A23K10/18C12R1/15
CPCA23K10/18A23K20/142C07K14/34C12N9/1096C12P13/227C12Y206/01002C12N1/205C12R2001/15
Inventor 谢昌贤刘运添王鹏飞
Owner 金河生物科技股份有限公司
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