Recombinant microorganisms for producing orotic acid and method thereof

A technology of recombinant microorganisms and orotic acid, applied in the biological field, can solve the problems of complex process conditions, high cost, low yield and the like

Active Publication Date: 2019-12-13
SUZHOU BIOSYNTHETICA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]At present, orotic acid is mainly produced chemically, and there are two reported routes: one is to obtain 5-ethoxy by condensation and ring closure of diethyl oxalate Orotic acid is obtained through ring expansion and acid analysis. The process conditions are complicated, the cost is high, and the yield is low. Therefore, this route has been eliminated by industrial production.

Method used

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  • Recombinant microorganisms for producing orotic acid and method thereof
  • Recombinant microorganisms for producing orotic acid and method thereof
  • Recombinant microorganisms for producing orotic acid and method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Example 1 The method for gene knockout in Escherichia coli

[0020] The present invention adopts Datsenko's method to carry out gene knockout in Escherichia coli (Datsenko 2000.ProcNatl Acad Sci USA, 97 (12): 6640-6645), corresponding gene knockout primer sees Baba 2006.Mol SystBiol, 2 (1) 0008.

Embodiment 2

[0021] Embodiment 2 shaking flask fermentation verifies the method for recombinant bacterial strain

[0022] The fermentation medium for verifying that the recombinant strain produces orotic acid in shake flask fermentation is specifically 100ml of YC solution per liter of medium, 200ml of 5 times salt solution, 1ml of TM2 solution, 10mg of iron citrate, 120mg of anhydrous magnesium sulfate, Calcium chloride 111mg, thiamine 1ug, constant volume with deionized water, wherein YC solution is glycerin 2g, peptone 0.6g, yeast powder 0.2g, deionized water 100ml; 5 times the salt solution contains 30g of disodium hydrogen phosphate, Potassium dihydrogen phosphate 15g, sodium chloride 2.5g, ammonium chloride 5.0g, dilute to 1L with deionized water; TM2 solution is zinc chloride tetrahydrate 2.0g, calcium chloride hexahydrate 2.0g, molybdic acid dihydrate Sodium 2.0g, copper sulfate pentahydrate 1.9g, boric acid 0.5g, hydrochloric acid 100ml, dilute to 1L with ionized water. The above...

Embodiment 35

[0024] Embodiment 35L fermenter verifies the method that recombinant bacterial strain produces orotic acid

[0025] Verification of the fermentation medium for the production of orotic acid by fermentation of the recombinant strain in the fermenter, specifically containing 10 g of ammonium sulfate, 10 g of sodium chloride, 5 g of potassium dihydrogen phosphate, 1 g of magnesium sulfate heptahydrate, 50 g of glucose, and chlorine Calcium chloride 105mg, zinc chloride 10mg, TM3 trace element solution 1mL, ferric citrate 94mg, yeast extract 5g, corn steep liquor 20g, cytidine 0.04g, dilute to volume with deionized water. The TM3 trace element solution is zinc chloride 1.31g, calcium chloride 1.01g, ammonium molybdate tetrahydrate 1.18g, copper sulfate 3.9g, manganese sulfate monohydrate 7.5g hydrochloric acid 10mL, and deionized water to volume. The feed medium contained 500 g of glucose per liter.

[0026] The fermentation process is as follows: first prepare the seeds, pick a ...

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Abstract

The invention provides a plurality of recombinant microorganisms for producing orotic acid and a method thereof. The recombinant microbial strains have one or more or all of the following characteristics: (1) the synthesis of phosphoribosyltransferase or orotic acid nucleoside monophosphate decarboxylase in an orotic acid metabolic pathway is weakened or completely lost; (2) a corresponding enzymeamount is increased through overexpression of five genes of enzymes participating in the orotic acid synthesis route; and (3) the corresponding transport protein is increased by overexpression of a transport protein coding gene for intracellular transport of orotic acid.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to transforming microorganisms by means of genetic engineering and the like, and the transformed microorganism strains can accumulate a large amount of orotic acid. Background technique [0002] Orotic acid, chemical name uracil-6-carboxylic acid, molecular formula C 5 h 5 N 2 o 4 , molecular weight 156.10. Its shape is white shiny needle-like crystal white crystal or crystalline powder, odorless and sour. Melting point 345-346°C, hardly soluble in cold water, ethanol and organic solvents, slightly soluble in hot water, insoluble in ether. Orotic acid is a nutritional medicine, also known as vitamin B13, orotate salts, such as calcium orotate, magnesium orotate and lithium orotate are good nutrients. [0003] At present, orotic acid is mainly produced chemically, and there are two reported routes: one is to obtain 5-ethoxymethylene lactourea by condensation and ring cl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P17/12C12R1/19
CPCC12N15/52C12N9/93C12N9/86C12N9/1018C12N9/001C12N9/1077C12N9/88C12Y603/05005C12Y201/03002C12Y305/02003C12Y103/05002C12Y204/0201C12Y401/01023C07K14/245C12P17/12
Inventor 江君君胡志浩田锋王欣彤刘鹏
Owner SUZHOU BIOSYNTHETICA CO LTD
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