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L-tryptophan gene engineering bacterium, method for constructing same and method for fermenting and producing L-tryptophan by using same

A tryptophan and gene technology, applied in the field of high-yield L-tryptophan genetically engineered strains, can solve problems such as complex regulatory mechanism, weak metabolic flow, and long biosynthetic pathway

Active Publication Date: 2014-08-27
GLOBAL BIO CHEM TECH SONGYUAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main reason is that the biosynthetic pathway from glucose to L-tryptophan is relatively long and its metabolic flow is relatively weak. On the other hand, the regulatory mechanism in the biosynthetic pathway of L-tryptophan is also relatively complicated

Method used

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  • L-tryptophan gene engineering bacterium, method for constructing same and method for fermenting and producing L-tryptophan by using same
  • L-tryptophan gene engineering bacterium, method for constructing same and method for fermenting and producing L-tryptophan by using same
  • L-tryptophan gene engineering bacterium, method for constructing same and method for fermenting and producing L-tryptophan by using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 : Preparation of strain CGSC7692 (ΔpheA) with knockout pheA gene

[0045] 1. Preparation of mutant strain CGSC7692 (ΔpheA::Kan)

[0046] Using the CGSC10048 (ΔpheA::Kan) genome as a template, primers pheA-V-F and pheA-V-R were used to amplify the pheA mutant gene fragment by PCR. The primer sequences are as follows:

[0047] Forward primer pheA-V-F: 5'-GGAGGCGTTTCGTCGTGTGA-3' (SEQ ID NO: 1)

[0048] Reverse primer pheA-V-R: 5'-GCAAGGTGGAGCACTGGTTC-3' (SEQ ID NO: 2)

[0049] Amplification reaction system: 10×KOD buffer (without MgSO 4 )5μl, MgSO 4 (25mM) 3μl, dNTP (2.5mM) 4μl, KOD polymerase 1μl, template 1μl, forward primer (10μM) 2μl, reverse primer (10μM) 2μl, add ddH O to 50μl (the PCR reaction reagents used in the present invention are all purchased from TOYOBO Corporation).

[0050] The amplification conditions are: 94°C, 2min, 94°C, 45sec, 55°C, 45sec, 68°C, 90sec, 30 cycles.

[0051] The 1.5kb PCR product of the pheA mutant gene fragment was rec...

Embodiment 2

[0064] Example 2 Preparation of bacterial strain CGSC7692 (ΔtyrA) with knockout of tyrA gene

[0065] 1. Preparation of mutant strain CGSC7692 (ΔtyrA::Kan)

[0066] Using the CGSC10049 (ΔtyrA::Kan) genome as a template, primers tyrA-V-F and tyrA-V-R were used to amplify the tyrA mutant gene fragment by PCR. increase method):

[0067] Forward primer tyrA-V-F: 5'-TATCCGTAACCGATGCCTGC-3' (SEQ ID NO: 4)

[0068] Reverse primer tyrA-V-R: 5'-GGGAAATCACCCGTTCAATG-3' (SEQ ID NO: 5)

[0069] The 1.5kb PCR product of the tyrA mutant gene was recovered by gel (refer to the recovery method of the Hangzhou Aisijin Kit for gel recovery), and the PCR product was transformed into E. coli CGSC7692 / pKD46 by electric shock transformation (electric transformation conditions: 2.5kV, 200Ω, 25μF). In state cells (the method is the same as described in Example 1), culture in LB medium at 37°C for 1 to 2 hours, spread the bacterial solution on a solid LB plate containing kanamycin (100 μg / ml), 37 ...

Embodiment 3

[0080] Example 3 Preparation of strain CGSC7692 (ΔtrpR) with knockout trpR gene

[0081] 1. Preparation of mutant strain CGSC7692 (ΔtrpR::Kan)

[0082] Using the CGSC11110 (ΔtrpR::Kan) genome as a template, using primers trpR-V-F and trpR-V-R, PCR amplifies the trpR mutant gene fragment, and the primer sequence is as follows (the reagents and reaction system used for PCR are the same as the gene amplification method in Example 1) :

[0083] Forward primer trpR-V-F: 5'-ATGGGGGATAAACCGACGTT-3' (SEQ ID NO: 7)

[0084] Reverse primer trpR-V-R: 5'-ATGCCGTGTATTAAGCGCCT-3' (SEQ ID NO: 8)

[0085] The 1.5kb PCR product of the trpR mutant gene was recovered by gel (refer to the recovery method of the Hangzhou Aisin Kit for gel recovery), and the PCR product was transformed into E. coli CGSC7692 / pKD46 by electric shock transformation (electric transformation conditions: 2.5kV, 200Ω, 25μF). In state cells (the method is the same as that in Example 1), culture in LB medium at 37°C for ...

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Abstract

The invention discloses an L-tryptophan producing bacterial strain, a gene engineering construction method for the L-tryptophan producing bacterial strain and a method for producing and preparing L-tryptophan by a fermentation method. Genes relevant to an L-tryptophan metabolic pathway are mutated and deleted by the gene engineering measure to promote metabolism disorder of the L-tryptophan, so that the L-tryptophan has high yield; simultaneously, the metabolic flow of the L-tryptophan is reinforced by gene integration or by overexpressing the genes relevant to the L-tryptophan metabolic pathway through a proper expression vector; finally, a large quantity of L-tryptophan can be accumulated in the fermentation solution by fermenting.

Description

technical field [0001] The present invention relates to an L-tryptophan fermentative production strain, its construction method and a method for using it to ferment and produce L-tryptophan. And a method for producing L-tryptophan by fermentation thereof. Background technique [0002] There are four main production methods of L-tryptophan: proteolytic extraction, chemical synthesis, enzymatic conversion and direct fermentation. The first two methods have the disadvantages of limited material sources, multi-step synthesis process and optical resolution, low yield, and long cycle. The enzymatic conversion method has the advantages of high accumulation of end products, short reaction cycle, and easy separation and purification. , which is a more effective method for producing L-tryptophan. At present, many enterprises in Japan and my country adopt enzymatic production. Yu Zhihua, Institute of Microbiology, Chinese Academy of Sciences, etc. used recombinant DNA technology to ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/63C12P13/22
Inventor 孙周通刘映淼孙兵兵王祎吴辉杨俊杰黄鹤杨晟李成玉于丽王德辉
Owner GLOBAL BIO CHEM TECH SONGYUAN
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