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Method for improving production capacity of L-histidine producing bacteria

A technology of production capacity and histidine, applied in the field of genetic engineering, can solve the problem that the production of microbial fermentation method has not yet been realized.

Active Publication Date: 2020-11-27
ZHEJIANG HUARUI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the domestic medicinal L-histidine is mainly imported, and the production of microbial fermentation method has not yet been realized.

Method used

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  • Method for improving production capacity of L-histidine producing bacteria
  • Method for improving production capacity of L-histidine producing bacteria
  • Method for improving production capacity of L-histidine producing bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: Construction of wild-type hisG genetically engineered bacteria

[0049] Synthetic primer pair 99A-HisG-F and 99A-HisG-R sequences SEQ ID NO: 1 and SEQ ID NO: 2, using the Escherichia coli W3110 strain genome as a template for PCR amplification (the following PCR reagents were purchased from KOD of Toyobo TOYOBO FX series).

[0050] The 50 μL PCR amplification system is: KOD FX 1 μL, KOD FX buffer 25 μL, dNTP 3 μL, genome template 0.5 μL, upstream and downstream primers 2 μL, ddH 2 O to make up 50 μL.

[0051] The PCR program was: 105°C hot lid, 95°C pre-denaturation for 5 min; 98°C denaturation for 30 s, 57°C annealing for 30 s, 68°C extension for 60 s, 30 cycles; finally 68°C extension for 10 min; 16°C cooling for 10 min.

[0052] The gene SEQ ID NO: 19 of the wild-type hisG was obtained by PCR amplification. The pTrc99a plasmid was double-digested with restriction endonucleases NcoI and BamHI (see figure 1 , the plasmid was donated by researcher Yang...

Embodiment 2

[0053] Example 2: Construction of hisG random mutation point library by error-prone PCR method

[0054] Mutation of wild-type ATP phosphoribosyltransferase was performed by error-prone PCR.

[0055] The 50 μL error-prone PCR reaction system includes: 50ng of the pTrc99a-hisG plasmid template constructed in Example 1, 30pmol HISG-EP-F and HISG-EP-R primer pair SEQ ID NO:14 and SEQ ID NO:15, 1×Taq buffer, 0.2mM dGTP, 0.2mM dATP, 1mM dCTP, 1mM dTTP, 7mM MgCl2, (0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM) MnCl 2 , 2.5 units of Taq enzyme (fermentas).

[0056] The PCR reaction conditions were: 95°C for 5 minutes; 30 cycles of 94°C for 30s, 55°C for 30s, 72°C for 2min / kbp; 72°C for 10min.

[0057] Restriction endonuclease DpnI was digested at 50°C for 1 hour, and the 0.9kb mutant fragment was recovered from the gel, and the pTrc99a linearized vector constructed in Example 1 was digested with NcoI and BamHI for homologous recombination, and then transformed into the large intestine by the c...

Embodiment 3

[0058] Embodiment 3: High-throughput screening hisG mutant library

[0059] 3.1 Select the transformant in the mutant library, inoculate it into a 96-well deep-well culture plate containing 700 μL LB medium, the medium contains 100 μg / mL ampicillin, culture at 37°C for 6 hours, and add a final concentration of 0.1mM IPTG , cooled to 25°C, and incubated overnight. Centrifuge at 5000rpm for 10min, discard the supernatant, freeze at -70°C for 1h, and thaw at room temperature for 30min. Add 200 μL of 0.1M potassium phosphate buffer (pH 8.0) to resuspend the bacteria for the determination of hisG enzyme activity.

[0060] 3.2 Determination of HisG enzyme activity

[0061] For the determination of HisG enzyme activity, refer to the method provided by (Biochimie, 94, 2012, P829-838). Enzyme activity assay system: the reaction mixture contains 100mM Tris-HCl (pH 8.5), 150mM KCl, 10mM MgCl 2 , 5mM ATP, 0.5mM RPP, 1U yeast pyrophosphatase, the volume is 0.1mL. Performed in 96-well ...

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Abstract

The invention discloses a method for improving the production capacity of L-histidine producing bacteria. The method includes the following steps that mutating is carried out on ATP phosphoribosyl transferase derived from escherichia coli to obtain a mutant with improved enzyme activity; the gene of the mutant is designed and encoded; and the coded gene is integrated into a genome of escherichia coli to obtain genetically-engineered bacteria. The method can increase the L-histidine production capacity of L-histidine producing bacteria by more than 3.5 times.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a method for improving the production capacity of L-histidine producing bacteria, an L-histidine genetic engineering producing bacteria and applications thereof. Background technique [0002] L-histidine (L-histidine) is a semi-essential amino acid, which is especially important for the growth of infants and animals. The human body can synthesize it with age. In addition, it can also be used as a biochemical reagent or a pharmaceutical intermediate. for the production of drugs. According to industry research reports, in 2017, the global consumption of histidine was roughly 559.5 tons for food, 1516.2 tons for medicines, and 393.1 tons for feed. It is estimated that by 2023, the consumption of L-histidine will reach 3000-4000 tons. The amount is getting bigger and bigger. [0003] At present, there are mainly two methods for the preparation of L-histidine: domestically, it is ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/90C12N9/10C12N15/54C12P13/24C12R1/19
CPCC12N9/1077C12N15/70C12N15/902C12P13/24C12Y204/02017
Inventor 范文超刘映淼王金刚高书良梁岩任亮
Owner ZHEJIANG HUARUI BIOTECHNOLOGY CO LTD
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