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Full-biosynthesis method of malonic acid

A technology of malonate and phosphoenolpyruvate carboxylase, which is applied in the field of bioengineering, can solve the problems of difficult transformation and slow progress in the biological production of malonate, and achieve the effects of reducing pollution, low cost and simple method

Active Publication Date: 2021-07-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Malonate semialdehyde is an important precursor of malonate, but due to the lack of malonate semialdehyde dehydrogenase, this step of conversion is difficult, which makes the progress of malonate bioproduction slow

Method used

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  • Full-biosynthesis method of malonic acid
  • Full-biosynthesis method of malonic acid
  • Full-biosynthesis method of malonic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Construction of recombinant plasmids and acquisition of recombinant Escherichia coli.

[0038]The nucleotide sequences of ppc, sdhC, aspA, panD, pa0123 and yne1 are respectively shown in SEQ ID NO.1-6.

[0039] Plasmid pRSFDuet-1 was double-digested with Nco I and Hind III, and the target gene fragment pRSF-1 (3761bp) was recovered by gel cutting. The plasmid pUC57-pa0123 synthesized by Jinweizhi Company was used as a template, and primer pa0123-F / R was used to amplify by PCR. The target gene fragment pa0123 (sequence shown in SEQ ID NO.5) was obtained by increasing, and then the two target fragments pRSF-1 and pa0123 were combined with T 4 DNA ligase ligation, transfection JM109, positive transformants were picked by colony PCR, and extracted plasmids were digested and verified by PCR. The verified primers were veri-pRSF-F / R, and the verified plasmid was named pRSF-pa0123.

[0040] Plasmid pRSF-pa0123 was digested with Xho I and Nde I, and the target gene f...

Embodiment 2

[0046] Example 2: Shake flask fermentation and result analysis of recombinant Escherichia coli.

[0047] Fermentation medium: SOB medium, the composition is 20g / L tryptone, 5g / L yeast powder, 0.5g / LNaCl, 2.5mMKCl, 10mM MgCl 2 , 4g / L glucose, 50μg / ml kanamycin sulfate, 50μg / ml ampicillin, 50μg / ml streptomycin.

[0048] Seed solution preparation: Streak the strains preserved in glycerol on a plate, pick a single colony and inoculate it in a 250ml Erlenmeyer flask filled with 50ml of LB liquid medium, shake the flask overnight at 37°C and 250rpm / min.

[0049] Fermentation conditions: 2% inoculum size (1ml), inoculated in shake flask fermentation medium to make initial OD 600 0.1. Cultivate to OD at 37℃, 250r / min 600 Add 1.0mM IPTG to induce recombinant bacteria at 0.8~1.0, respectively, and change to 30°C, 250rpm / min culture.

[0050] Result analysis: During the fermentation process, samples were taken every 4 hours for the first 12 hours, and samples were taken every 12 hours...

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Abstract

The invention discloses a full-biosynthesis method of malonic acid, and belongs to the technical field of bioengineering. According to the full-biosynthesis method of malonic acid, escherichia coli BL21 (DE3) is taken as a host, a phosphoenolpyruvate carboxylase gene (ppc), a succinate dehydrogenase gene (sdhC), a succinate semialdehyde dehydrogenase gene (yne1) and an asparaginase gene (apa) from the escherichia coli, and a heterologous gene aspartic acid-alpha-dehydrogenase gene (panD) from Corynebacterium glutamicum are subjected to modular overexpression, and a heterologous gene beta-alanine pyruvate transaminase gene (pa0123) from pseudomonas aeruginosa constructs a full biosynthetic pathway of the malonic acid, so that the engineering bacteria successfully accumulate the malonic acid. Biological manufacturing of the malonic acid and a precursor thereof has the advantages of low pollution, high product quality and the like, and has a great development prospect.

Description

technical field [0001] The invention relates to a method for fully biosynthesizing malonic acid, which belongs to the field of bioengineering. Background technique [0002] Malonic acid (malonate; Propanedioic acid; Propane diacid) is also known as carrot acid, malic acid or beet acid. There are two functional groups of active methylene and carboxyl in the molecular structure, so it can participate in various chemical reactions and is a very important organic synthesis intermediate. It is mainly used in spices, adhesives, resin additives, pharmaceutical intermediates, electroplating polishing agents, explosion control agents, heat welding flux additives, etc. [0003] At present, the method of hydrolysis of cyanoacetic acid and malonate hydrolysis is commonly used in industry to prepare malonic acid. However, NaCN is used in the hydrolysis of cyanoacetic acid, and the cyanide ion is highly toxic and harmful to the environment, and the reaction process is complicated. Malo...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/44C12R1/19
CPCC12N9/88C12N9/001C12N9/0008C12N9/1096C12N15/70C12P7/44C12Y401/01031C12Y103/99001C12Y102/01C12Y403/01001C12Y206/01C12N9/0016C12P13/20C12P13/06C12P13/08C12P13/12C12Y104/01021Y02A50/30
Inventor 邓禹赵运英李诗韵李国辉毛银周胜虎
Owner JIANGNAN UNIV
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