Genetically engineered bacterium producing tetrahydropyrimidine and structuring method and application thereof

A technology of genetically engineered bacteria and tetrahydropyrimidine, which is applied in the field of genetically engineered bacteria producing tetrahydropyrimidine and its construction, can solve problems such as being unsuitable for large-scale industrial production, corrosion of fermentation equipment, long fermentation period, etc., and achieve production efficiency High, low equipment loss, short fermentation period

Active Publication Date: 2015-11-04
合肥和晨生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the above two methods, the deficiency of halophilic bacteria fermentation to produce ectoine is that a high NaCl concentration is required to stimulate the bacteria to accumulate more products during the cultivation process, the fermentation cycle is longer, and the high concentration of NaCl solution is harmful to the fermentation equipment. Severe corrosion, not suitable for large-scale industrial production, and high-salt fermentation waste liquid has caused great pressure on the environment; the substrate for the production of ectoine by enzyme catalysis is sodium aspartate, and the enzyme needs to be induced, expressed and extracted. Therefore, the operation is more complicated and the production cost is higher

Method used

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  • Genetically engineered bacterium producing tetrahydropyrimidine and structuring method and application thereof
  • Genetically engineered bacterium producing tetrahydropyrimidine and structuring method and application thereof
  • Genetically engineered bacterium producing tetrahydropyrimidine and structuring method and application thereof

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Embodiment 1

[0054] Construction of Genetically Engineered Bacteria E.coli ECT06 Producing ectoine

[0055] (1) Construction of the metabolic pathway from L-aspartic acid-β-semialdehyde to ectoine.

[0056] ①A pair of primers were designed according to the ectABC gene sequence by using the genome of Halomonas elongatus (CGMCC 1.6329) as a template by PCR technology, and the ectABC fragment was amplified. The pair of primers respectively comprise enzyme cutting sites EcoR I and BamH I.

[0057] ②Use Takara restriction endonucleases EcoR I and BamH I to double digest step ① to obtain the target fragment and pTrc99a vector plasmid, and obtain ectABC and pTrc99a linear fragments with the same cohesive ends.

[0058] ③ Use Takara T4 DNA ligase to connect the two target fragments obtained in step ② to obtain the target vector pTrc99a-ectABC.

[0059] ④ Transform the vector obtained in step ③ into E.coli W3110 (ATCC 27325) to obtain E.coliECT01

[0060] (2) Knockout of thrA gene

[0061] ① Th...

Embodiment 2

[0088] The specific steps for the fermentation, cultivation and detection of the genetically engineered bacterium E.coliECT06 producing ectoine are as follows:

[0089] (1) Activate the genetically engineered bacterium (E.coli ECT06 engineered bacterium) producing ectoine by using the complete bacterial medium, and culture at a constant temperature of 37°C for 12 hours;

[0090] (2) Transfer the above-mentioned activated slant to the second-generation activated slant, and incubate at a constant temperature of 37° C. for 10 h;

[0091] (3) Use an inoculation loop to scrape a ring of strains into a 500mL round-bottomed conical flask with a liquid volume of 30mL, and incubate at 37°C and 200rpm for 7h;

[0092](4) Use a pipette to inoculate the fermentation shaker flask according to 10% inoculum amount. The fermentation bottle is a 500mL baffled Erlenmeyer flask with a liquid volume of 30mL, cultivated at 37°C and 200rpm for 28h; Add ammonia water to maintain the pH at 7.2, add ...

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Abstract

The invention relates to a genetically engineered bacterium producing tetrahydropyrimidine and a structuring method and application thereof. The genetically engineered bacterium is Escherichia coli with a specific genotype, comprises Halomonas-elogata-derived ectABC gene, has three gene deficiency variants including lysA, thrA and iclR and has corynebacterium glutamicum lysC gene controlled by a lac promoter and ppc gene controlled by a trc promoter. The defect that chemical synthesis methods have harsh reaction conditions and high energy consumption can be overcome by using the genetically engineered bacterium and utilizing glucose fermentation to produce tetrahydropyrimidine. The defect that Halomonas-elogata fermentation or enzyme-catalyzed methods are complex in process and high in production cost can be overcome. After fermentation for 20-28h, yield of tetrahydropyrimidine reaches 12-18g/L, and the genetically engineered bacterium has high industrial application value.

Description

technical field [0001] The invention relates to the field of compound biotechnology production, in particular to a genetically engineered bacterium producing ectoine and its construction method and application. Background technique [0002] The current production methods of ectoine include fermentation and enzyme catalysis. in, [0003] There is a synthetic pathway of ectoine in halophilic microorganisms, so it is widely used in the production of ectoine by fermentation. Sauer T et al. used the "bacterial milking method" to obtain high-yield ectoine by high-density fermentation, that is, to cultivate bacteria under high osmotic pressure, then release solute by hypotonic shock, and then re-culture the bacteria with high osmotic shock, then hypotonic shock The solute is released and the product is obtained by successive cycles up to 8-9 times. Zhu Wanyi (201310416404.4) and others disclosed a new Halomonas sp.HS-2255 and its mutants that can be used to produce ectoine, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P17/12C12R1/19
Inventor 谢希贤陈宁宁义科范晓光吴雪娇徐庆阳张成林
Owner 合肥和晨生物科技有限公司
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