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Engineering bacteria producing guanidinoacetic acid and its construction method and application

A technology of guanidinoacetic acid and a construction method, applied in the biological field, can solve the problems of low conversion rate and yield of guanidinoacetic acid, difficult to apply to industrial production, and high cost of substrates

Active Publication Date: 2022-07-05
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have found that the reaction of AGAT catalyzing arginine to guanidinoacetic acid is inhibited by the by-product ornithine feedback, so the conversion rate and yield of one-step synthesis of guanidinoacetic acid by enzymatic method are relatively low, and the substrate cost is high, which is difficult to apply to industrialization Production

Method used

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  • Engineering bacteria producing guanidinoacetic acid and its construction method and application
  • Engineering bacteria producing guanidinoacetic acid and its construction method and application
  • Engineering bacteria producing guanidinoacetic acid and its construction method and application

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Effect test

Embodiment 1

[0088] Embodiment 1, construct the genetically engineered bacteria that produces guanidinoacetic acid

[0089]1. Construction of a recombinant plasmid for co-expressing glutamine synthase and L-arginine: glycine amidinotransferase and co-expressing ornithine carbamoyl transferase, arginyl succinate synthase, and arginyl succinate cleavage Enzyme, aspartate ammonia lyase recombinant plasmid

[0090] 1. Construction of recombinant plasmid pL01 expressing glutamine synthase

[0091] According to the nucleic acid sequence GenBank: AF005635.2 (amino acid sequence GenBank: WP_003859638.1) of the glutamine synthase (GS) of Corynebacterium glutamicum, primers (P1 and P2) were designed and amplified to obtain glutamine The coding gene of amide synthase (glnA), the fragment size is about 1200bp, which is consistent with the target fragment. The sequencing results show that the amplified fragment sequence is correct, and the fragment is used to construct the recombinant plasmid pL01.

...

Embodiment 2

[0165] Embodiment 2. Utilize guanidinoacetic acid-producing genetically engineered bacteria to prepare guanidinoacetic acid

[0166] 1. Induction of genetically engineered bacteria producing citrulline, arginine and guanidinoacetic acid respectively

[0167] Step-by-step induction of 2YT medium: Streak guanidinoacetic acid-producing metabolic genetically engineered bacteria CG24 onto LB plates containing 1.5% mass percent agar powder, 50 μg / mL ampicillin and 50 μg / mL streptomycin 37°C overnight shaking culture at 200 rpm; the overnight culture was inoculated into 2YT medium with a volume percentage of 1%, and shake cultured at 37°C for 2-3h to OD 600 After 0.5-0.8, add L-arabinose with a final concentration of 0.02% mass fraction, 30°C, 200rpm, and culture for 12h. After induction of the cells, according to the growth of the bacterial liquid, take a certain amount of bacterial cells, centrifuge at 4°C, 8000 rpm for 10 min, and the cells collected by centrifugation are broken ...

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Abstract

The invention discloses a guanidinoacetic acid-producing genetically engineered bacteria and a construction method and application thereof. The invention provides a method for constructing a guanidinoacetic acid-producing engineering bacterium, comprising the following steps: introducing a gene L-arginine:glycine amidinotransferase encoding gene related to guanidinoacetic acid synthesis into a host bacterium to obtain guanidinoacetic acid producing Engineering bacteria; the host bacteria is Escherichia coli or mutant Escherichia coli. The present invention constructs the AGAT catalytic reaction that does not exist in the background in Escherichia coli by expressing the exogenous protein. At the same time, by overexpressing proteins on plasmids, replacing promoters on chromosomes, and knocking out genes, the flow of E. coli ornithine cycle metabolic pathway is enhanced, the inhibition of ornithine on AGAT catalytic reaction is relieved, and the whole-cell catalytic method is used for recycling. The production of guanidinoacetic acid from arginine has good environmental and economic prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a genetically engineered bacteria producing guanidinoacetic acid and a construction method and application thereof. Background technique [0002] Guanidinoacetic acid (Guanidinoacetate, GAA) is a white or slightly yellow crystalline powder, or flaky crystal, and the solubility in water at room temperature is 4g / L. Guanidinoacetic acid is the main endogenous substance for the synthesis of creatine in humans and animals, and it plays an irreplaceable role in the energy metabolism of muscle and nerve tissue. Creatine can be obtained from the diet or synthesized by the organism itself, so it is called a semi-essential nutrient. Creatine is an important molecule in intracellular energy metabolism, where energy is temporarily stored. Creatine is phosphorylated to form phosphocreatine, which is the main energy supply material in animal muscle tissue. Adding guanidinoacetic aci...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P13/10C12P13/04C12R1/19
CPCC12N9/1003C12P13/10C12P13/04C12Y201/04001
Inventor 林白雪张译文周航陶勇
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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