A kind of Escherichia coli mutant strain with high cytidine production and method for producing cytidine by fermentation

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

Active Publication Date: 2021-12-31
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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  • A kind of Escherichia coli mutant strain with high cytidine production and method for producing cytidine by fermentation
  • A kind of Escherichia coli mutant strain with high cytidine production and method for producing cytidine by fermentation

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 a high cytidine-producing Escherichia coli mutant strain and a method for fermenting and producing cytidine, which belong to the field of fermentation technology. The Escherichia coli mutant strain is obtained through mutagenesis screening and has the following characteristics: ① synthesis of carbamoyl phosphate The alanine at position 182 of the enzyme is mutated to valine, and the serine at position 948 is mutated to phenylalanine; ② Isoleucine at position 86, glycine at position 93, and Cysteine ​​at position 109 is missing; ③ Asparagine at position 72 of uridine kinase is mutated to alanine, and aspartic acid at position 159 is mutated to asparagine; ④ Asparagine at position 147 of cytidine triphosphate synthase Amino acid was mutated to glutamic acid, and glutamic acid at position 149 was mutated to alanine. After the mutant strain was fermented in a 50 L fermenter, the yield of cytidine reached 90±2 g / L, and the sugar-acid conversion rate reached 30±1%, which were the highest values ​​reported so far.

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 Patents(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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