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Engineering bacterium for producing guanidinoacetic acid as well as construction method and application thereof

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: 2020-10-09
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 bacterium for producing guanidinoacetic acid as well as construction method and application thereof
  • Engineering bacterium for producing guanidinoacetic acid as well as construction method and application thereof
  • Engineering bacterium for producing guanidinoacetic acid as well as construction method and application thereof

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Embodiment 1

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

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

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

[0091] According to the nucleotide sequence GenBank: AF005635.2 (amino acid sequence GenBank: WP_003859638.1) of glutamine synthetase (GS) of Corynebacterium glutamicum (Corynebacterium glutamicum), primers (P1 and P2) were designed to amplify glutamine The gene encoding amide synthetase (glnA) has a fragment size of about 1200bp, which is consistent with the target fragment. The sequencing results show that the sequence of the amplified fragment is correct, and the fragment is used to construct the reco...

Embodiment 2

[0165] Embodiment 2, utilize the guanidinoacetic acid gene engineering bacterium that produces guanidinoacetic acid to prepare guanidinoacetic acid

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

[0167] Step-by-step induction in 2YT medium: Streak the metabolic genetically engineered bacteria CG24 producing guanidinoacetic acid onto an LB plate containing 1.5% agar powder, 50 μg / mL ampicillin and 50 μg / mL streptomycin On, shake overnight at 37°C, rotate at 200rpm; inoculate the overnight culture into 2YT medium with an inoculation volume of 1% by volume, and shake 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, culture at 30° C. and 200 rpm for 12 hours. After the induced cells, according to the growth of the bacterial liquid, take a certain amount of bacterial cells, centrifuge at 4°C, 8000rpm for 10 minutes, and use ultrasonic waves to...

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Abstract

The invention discloses a genetically engineered bacterium for producing guanidinoacetic acid as well as a construction method and an application of the genetically engineered bacterium. The inventionprovides the construction method of guanidinoacetic acid-producing engineering bacteria, which comprises the following steps: introducing guanidinoacetic acid synthesis-related gene L-arginine: glycine amidinyl transferase encoding gene into host bacteria to obtain guanidinoacetic acid-producing engineering bacteria; wherein the host bacteria are escherichia coli or mutant escherichia coli. According to the invention, an AGAT catalytic reaction which does not exist in the background is constructed in escherichia coli by a method of expressing foreign protein. Meanwhile, by means of plasmid overexpression of protein, replacement of promoters on chromosomes, gene knockout and the like, the flow of an ornithine circulating metabolic pathway of escherichia coli is increased, inhibition of ornithine on AGAT catalytic reaction is relieved, arginine is recycled through a whole-cell catalysis method to produce guanidinoacetic acid, and good environmental and economic prospects are achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a genetically engineered bacterium producing guanidinoacetic acid and its construction method and application. Background technique [0002] Guanidinoacetate (GAA) is white or slightly yellow crystalline powder, or flaky crystal, and its 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 energy metabolism in cells, where energy is temporarily stored. Creatine is phosphorylated to form creatine phosphate, which is the main energy supply substance in animal muscle tissue. Adding guanidinoacetic acid can strengthen the body's en...

Claims

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Application Information

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Patent Type & Authority Applications(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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