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Genetically engineered bacterium for producing orotic acid as well as construction method and application of genetically engineered bacterium

A technology of genetically engineered bacteria and orotic acid, applied in the directions of genetic engineering, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of difficult to control the production process, heavy growth and metabolism burden, and decreased product yield, etc. The method is simple, the fermentation yield is high, and the sugar-acid conversion rate is high.

Pending Publication Date: 2022-07-22
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in industrial production, engineering bacteria carrying plasmids have disadvantages such as poor genetic stability and heavy growth and metabolism burdens, which will lead to industrial problems such as extremely reduced product yields and difficult control of the production process.

Method used

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  • Genetically engineered bacterium for producing orotic acid as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing orotic acid as well as construction method and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing orotic acid as well as construction method and application of genetically engineered bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] like figure 2 Construction of strain E. coli Ora6 as shown:

[0044] 1. Methods of gene editing

[0045] The gene editing method adopted in the present invention refers to literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21.), the method involves See the map of the engineered plasmids pRED / Cas9 and pGRB figure 1 . Among them, pRED / Cas9 carries the elimination system of the gRNA expression plasmid pGRB, the Red recombination system of λ phage, the Cas9 protein expression system and the spectinomycin resistance (working concentration: 100 mg / L), cultured at 32 °C; pGRB uses pUC18 as the backbone, including Promoter J23100, gRNA-Cas9 binding region sequence and terminator sequence and ampicillin resistance (working concentration: 100 mg / L), cultured at 37°C.

[0046] The specific steps of this method are as follows:

[0047] 1.1 pGRB plasmid construction ...

Embodiment 2

[0103] The Escherichia coli engineering bacteria described in Example 1 were used to produce orotic acid by shaking flask fermentation.

[0104] 1. Culture medium

[0105] 1.1 Slant medium

[0106] Glucose 2g / L, Peptone 10g / L, Yeast Extract Powder 5g / L, Sodium Chloride 2.5g / L, KH 2 PO 4 1.0g / L, MgSO 4 0.2g / L, 25% agar powder, dissolve in water and dilute to the required volume, adjust the pH to 7.0-7.2 with sodium hydroxide, sterilize in a high-pressure steam cooker at 121°C for 20min, and then dispense into test tubes.

[0107] 1.2 Seed medium

[0108] Glucose 30g / L, Yeast Powder 8g / L, Peptone 2.0g / L, MgSO 4 ·7H 2 O 0.5g / L, KH 2 PO 4 3.0g / L, methionine 2g / L, (NH 4 ) 2 SO 4 2.0g / L, V B1 2mg / L, V B3 2mg / L, V B5 2mg / L, V B12 2mg / L, V H 1mg / L, trace element mixture 1mL / L, phenol red: 2% of the constant volume, adjust the pH to 7.0-7.2 with NaOH, 1 drop of defoamer, the rest are water, autoclave sterilization at 115°C, 15min. The component contents of the t...

Embodiment 3

[0120] Utilize the Escherichia coli engineering bacteria 5L fermentation tank described in Example 1 to ferment and produce orotic acid.

[0121] 1. Culture medium

[0122] 1.1 Slant medium

[0123] Glucose 2g / L, Peptone 10g / L, Yeast Extract Powder 5g / L, Sodium Chloride 2.5g / L, KH 2 PO 4 1.0g / L, MgSO 4 0.2g / L, 25% agar powder, dissolve in water and dilute to the required volume, adjust the pH to 7.0-7.2 with sodium hydroxide, sterilize in a high-pressure steam cooker at 121°C for 20min, and then dispense into test tubes and eggplant-shaped bottles.

[0124] 1.2 Seed medium

[0125] Glucose 30g / L, Yeast Powder 8g / L, Peptone 2.0g / L, MgSO 4 ·7H 2 O 0.5g / L, KH 2 PO 4 3.0g / L, methionine 2g / L, (NH 4 ) 2 SO 4 2.0g / L, V B1 2mg / L, V B3 2mg / L, V B5 2mg / L, V B12 2mg / L, V H 1mg / L, trace element mixture 1mL / L, phenol red: 2% of the constant volume, adjust the pH to 7.0-7.2 with NaOH, 2 drops of defoamer, and the rest is water. The component contents of the trace el...

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Abstract

The invention provides a genetically engineered bacterium for producing orotic acid and a construction method and application of the genetically engineered bacterium, the genetically engineered bacterium is a genetically engineered bacterium E.coli Ora which does not contain plasmids and is used for synthesizing high-yield orotic acid from the beginning, and compared with an existing orotic acid producing bacterium, the genetically engineered bacterium has the advantages of being good in genetic stability, high in fermentation yield, high in saccharic acid conversion rate and the like, and has a good application prospect. The genetically engineered bacterium takes glucose and other cheap carbon sources as substrates to efficiently synthesize the orotic acid from the beginning, the yield of the orotic acid can reach up to 135.6 g / L, and the genetically engineered bacterium has a good industrial application prospect; the genetically engineered bacterium analyzes and reconstructs metabolic flux related to orotic acid in a pyrimidine nucleotide metabolic network in escherichia coli, and enhances synthesis and accumulation of orotic acid; then, dihydrolactate dehydrogenase in wild type bacillus subtilis pyrimidine nucleoside operon is introduced, so that the synthesis of orotic acid is further enhanced, and the requirement of large-scale industrial production is met.

Description

technical field [0001] The invention relates to the production field of compound biotechnology and fermentation engineering technology, in particular to a genetically engineered bacterium for producing orotic acid and a construction method and application thereof. Background technique [0002] Orotic acid (OA), chemical name uracil-6-carboxylic acid. Exist in the milk of various mammals, also known as VB 13 . Orotic acid and its derivatives are widely used in the fields of medicine, food, feed and cosmetics. Nowadays, with the gradual deepening of the research on the function of orotic acid, its market demand is still expanding. [0003] The current production method of orotic acid is mainly chemical production, but the process of chemical preparation of orotic acid has the disadvantages of using dangerous reagents, complicated operation, high cost of raw materials, and many "three wastes", which do not meet the requirements of green environmental protection. Therefore, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/52C12N15/53C12N15/54C12N15/55C12N15/90C12P17/12C12R1/19
CPCC12N9/1018C12N9/1077C12N9/001C12N9/93C12N9/86C12N15/902C12P17/12C12Y201/03003C12Y204/0201C12Y201/03002C12Y603/04016C12Y305/02003C12Y103/05002C12N2800/22C12N2800/101
Inventor 徐庆阳李长庚孙鹏杰
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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