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Escherichia coli mutant strain with high cytidine production and method for fermentation production of cytidine

A technology of Escherichia coli and mutant strains, applied in the field of fermentation, can solve problems such as unclear amino acid positions, and achieve the effect of improving tolerance

Active Publication Date: 2020-06-23
HENAN JULONG BIOLOGICAL ENG CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the binding sites of metabolites and enzymes can be predicted by advanced structural analysis of enzymes, the amino acid sites related to feedback regulation are not clear

Method used

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  • Escherichia coli mutant strain with high cytidine production and method for fermentation production of cytidine
  • Escherichia coli mutant strain with high cytidine production and method for fermentation production of cytidine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Breeding of Escherichia coli mutant strain E.coli JL027 for cytidine production

[0029] 1. ARTP mutagenesis

[0030] The starting strain E.coli W3110ΔcddΔthrA was inoculated into a shaking tube containing LB medium, and cultured in a shaker at 37°C and 200r / min for 8h. Dilute the cultivated seed liquid with saline to OD 600 =0.8, then use a pipette to draw 10 μL of bacterial solution and apply it evenly on the circular metal sheet. Place the metal piece in the ARTP breeding machine (Beijing Siqingyuan Biotechnology Co., Ltd.), use helium as the working gas, set the gas flow rate to 10L / min, the distance between the gas injection port and the metal piece is 2mm, and the working power is 100W. The time is 30S.

[0031] 2. Initial screening of resistance plate

[0032] The treated bacterium was resuspended in 1mL of normal saline, and spread on the 5-fluoroorotic acid / 200mg / L of 6-azauracil / 150mg / L containing nucleoside structural analog 5-azacytosine / 200m...

Embodiment 2

[0040] Example 2 Fermentative production of cytidine by Escherichia coli mutant strain E.coli JL027

[0041] Use E.coli JL027 as the production strain, inoculate into a 10L fermenter containing 6L seed medium for seed culture, the inoculum size is 8%, the culture temperature is 37°C, pH 7.0, dissolved oxygen is controlled at 30%, residual sugar Control at 2%, and the culture period is 12h. After the seed culture is completed, inoculate into a 50L fermenter containing 20L fermentation medium for cytidine fermentation, the inoculum size is 9%, the culture temperature is 37°C, pH 7.0, the dissolved oxygen is controlled at 25%, and the residual sugar is controlled at 1%. , The fermentation period is 40h. After the fermentation is completed, the concentration of cytidine in the fermentation broth can reach 80g / L, and the sugar-acid conversion rate can reach 30%.

[0042] The fermenter seed medium is: glucose 30g / L, yeast powder 6g / L, citric acid 1.5g / L, peptone 2g / L, potassium di...

Embodiment 3

[0044] Example 3 Escherichia coli mutant strain E.coli JL027 and starting strain E.coli W3110ΔcddΔthrA tolerance test to different nucleoside structural analogues

[0045] The mutant strain E.coli JL027 and the starting strain E.coli W3110ΔcddΔthrA were inoculated in shake tubes containing LB medium, and cultured in a shaker at 37°C and 200r / min for 8h. The cultured seed solution was diluted 108 times with physiological saline, and then 10 μL of the bacterial solution was pipetted and evenly spread into LB solid medium containing different concentrations of nucleoside structural analogs. Three parallels were performed for each concentration, and the control was LB solid medium. Put all the solid medium into the incubator at 37°C for 10 h at the same time. Plate colony counting was used to calculate the lethal rate and lethal concentration, and the results are shown in Table 1.

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Abstract

The present invention relates to an escherichia coli mutant strain with high cytidine production and a method for fermentation production of cytidine, and belongs to the technical field of fermentation. The escherichia coli mutant strain is obtained by mutagenic screening and has the following characteristics: 1, alanine at 182nd position of carbamyl phosphate synthetase is mutated to valine, andserine at 948th position is mutated to phenylalanine; 2, isoleucine at 86th position, glycine at 93rd position and cysteine at 109th position of aspartic acid carbamoyl transferase regulatory subunitare deleted; 3, asparagine at 72nd position of uridine kinase is mutated to alanine and aspartic acid at 159th position is mutated to asparagine; and 4, aspartic acid at 147th position of cytidine triphosphate synthase is mutated to glutamic acid, and glutamic acid at 149th position is mutated to alanine. The mutant strain is fermented in a 50-L fermentation tank, cytidine yield reaches (90 plus or minus 2) g / L, sugar-acid conversion rate reaches (30 plus or minus 1)%, and the cytidine yield and sugar-acid conversion rate are both the highest values reported.

Description

technical field [0001] The invention belongs to the technical field of fermentation, and in particular relates to an Escherichia coli mutant strain with high cytidine production and a method for producing cytidine by fermentation. Background technique [0002] Cytidine is a precursor substance for the synthesis of ribonucleic acid and deoxyribonucleic acid, and participates in many reactions in living organisms. Cytidine is an important pharmaceutical synthesis intermediate, which can be used to synthesize zalcitabine, capecitabine, cyclocitidine, cytarabine, azacitidine, cytarabine hydrochloride and other antiviral and antitumor drugs . Therefore, it is an important issue to develop a large-scale, industrialized, low-cost and high-efficiency production method of cytidine to meet the demand for cytidine raw materials in the pharmaceutical field. [0003] The synthesis of cytidine by biological fermentation mainly depends on the fermentation of microorganisms, so the type o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P19/38C12R1/19
CPCC12N9/93C12N9/14C12Y603/04016C12Y201/03002C12Y207/01048C12N9/1018C12N9/1205C12P19/385C12N1/20
Inventor 李静李国栋刘晓东张孟涛滕佳佳张兆昆宋会文
Owner HENAN JULONG BIOLOGICAL ENG CO LTD
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