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Pyrimidine nucleoside high-yielding strain and carbamyl phosphate synthetase adjusting site thereof

A carbamoyl phosphate and synthase technology, which is applied in the field of enzyme engineering, can solve the problems of application limitation, the release effect is not too obvious, and the regulatory site of carbamoyl phosphate synthase is not clear, so as to achieve improved tolerance and improved tolerance. Ability-enhancing effect

Inactive Publication Date: 2016-06-15
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0009] The difficulty in the breeding of pyrimidine nucleoside high-yielding strains is to release the feedback repression and feedback inhibition of the final product, especially the release of feedback inhibition. Although researchers have revealed the specific regulation mechanism of pyrimidine nucleoside synthesis and metabolism, and from the molecular level The mechanism by which the pyrimidine operon is repressed by the end product is elucidated, but the specific regulatory site of the carbamoyl phosphate synthase that is repressed by the end product feedback is not clear
[0010] With the application of molecular biology in microbial breeding, relevant researchers have also tried to use PCR to introduce point mutations to release the feedback inhibition of carbamoyl phosphate synthase, but due to the structure and function of carbamoyl phosphate synthase The research is not too in-depth. The method of introducing point mutations by PCR is limited in the application of releasing the feedback inhibition of carbamoyl phosphate synthase. The effect of the reported site on the release of feedback inhibition is not too obvious.

Method used

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  • Pyrimidine nucleoside high-yielding strain and carbamyl phosphate synthetase adjusting site thereof
  • Pyrimidine nucleoside high-yielding strain and carbamyl phosphate synthetase adjusting site thereof
  • Pyrimidine nucleoside high-yielding strain and carbamyl phosphate synthetase adjusting site thereof

Examples

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Embodiment 1

[0034] Example 1. Screening of Bacillus subtilis A219

[0035] In the present invention, the initial strain TD131 is subjected to plasma mutagenesis at normal pressure and room temperature-resistance screening of structural analogs-high-throughput screening-re-screening of shake flasks to obtain strain A219, and the specific process is as follows:

[0036] 1. Starting strain: B.subtilisTD131

[0037] 2. Atmospheric room temperature plasma mutagenesis

[0038] (1) Bacterial culture method: the bacteria preservation tube is connected to the LB plate, three-section line is drawn, and cultured at 37°C for 12 hours; a single colony is picked from the plate and connected to the LB shaker tube, 37°C, 200rpm, and cultured for 12h; the shaker tube is transferred to the LB shaker flask , the inoculum size was 1%, 37°C, 200rpm, and cultured for 4-6h.

[0039] (2) Preparation method of bacterial suspension: centrifuge to collect cultured bacteria, wash 2-3 times with sterile normal sali...

Embodiment 2

[0061] Example 2 Fermentative production of uridine by mutant strain A219

[0062] (1) Medium used in the present embodiment:

[0063] Activated slant medium (g / L): glucose 1, peptone 10, beef extract 10, yeast powder 5, NaCl2.5, agar 20, pH6.7~7.0, 0.1MPa, 20min.

[0064] Seed medium (g / L): glucose 40, yeast powder 5, peptone 10, sodium nitrate 15, potassium dihydrogen phosphate 1, dipotassium hydrogen phosphate 5.2, magnesium sulfate 1, sodium citrate 1, pH6.7~7.0, 0.075MPa, 15min. Glucose is sterilized separately.

[0065] 5L fermenter fermentation medium (g / L): glucose 50, yeast powder 5, corn steep liquor 20, sodium nitrate 15, potassium dihydrogen phosphate 1, dipotassium hydrogen phosphate 5.2, magnesium sulfate 5, ammonium sulfate 5, sodium citrate 10. Sodium glutamate 10, calcium chloride 1, manganese sulfate 0.02, zinc sulfate 0.02, pH6.7~7.0, corn steep liquor is sterilized separately, calcium salt is sterilized separately, magnesium, manganese and zinc salts are...

Embodiment 3

[0077] Example 3 Detection of tolerance of mutant strain A219 to different structural analogues

[0078] (1) Detection method: Take 10 microliters of bacterial liquid from the bacterial preservation tube and connect it to an LB shaker tube, culture at 37°C and 200 rpm for 10-12 hours, collect the cultured bacteria by centrifugation, wash with sterile normal saline for 2-3 times 100,000-fold gradient dilution with sterile physiological saline, and then get 100 microliters of bacterial solution and spread it on the basic medium plate containing different concentrations of structural analogs, each concentration is three parallel, and the contrast is the basic medium plate ( without structural analogues). After culturing at 37°C for 24 hours, count the number of colonies on the plate, and the number of colonies reflects the level of tolerance. A large number of colonies can grow on high-concentration resistant plates within a certain period of time, indicating that the strain has s...

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Abstract

The invention belongs to the technical field of enzyme engineering, and concretely relates to a pyrimidine nucleoside high-yielding strain and a carbamyl phosphate synthetase adjusting site thereof. The invention provides a pyrimidine nucleoside production Bacillus subtilis mutant strain and a carbamyl phosphate synthetase encoding gene. A key regulation site related to carbamyl phosphate synthetase end product feedback inhibition is known, and provides reference for breeding of later pyrimidine nucleoside high-yielding strains. The Bacillus subtilis mutant strain allows the output of fermentation process produced nucleoside pyrimidine uridine to reach 12.5-14.5g / L, and has substantially improved tolerance to different pyrimidine nucleoside structure analogs.

Description

Technical field: [0001] The invention belongs to the technical field of enzyme engineering, and specifically relates to a high-yield pyrimidine nucleoside strain and its carbamoyl phosphate synthetase regulatory site. Background technique: [0002] Pyrimidine nucleosides include uridine and cytidine, which are important components of all organisms. They are widely used, especially as intermediates of antiviral and antitumor drugs, and occupy an important position in the field of medicine. The production methods of pyrimidine nucleosides include chemical synthesis, RNA hydrolysis and microbial fermentation. Based on the advantages and disadvantages of several methods, microbial fermentation has the advantages of short cycle, easy control, high yield and low pollution. The potential of industrialization is therefore increasingly valued by people. [0003] The breeding of pyrimidine nucleoside-producing strains began in the 1960s. The breeding of pyrimidine nucleoside high-yie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/52C12N1/20C12P19/38C12R1/125
CPCC12N9/93C12P19/385C12Y603/05005
Inventor 谢希贤陈宁吴鹤云徐庆阳张成林范晓光袁辉
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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