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Genetically engineered bacteria, construction and application of high-yielding pantothenic acid without the addition of β-alanine

A technology of genetically engineered bacteria and alanine, applied in the direction of genetic engineering, application, plant genetic improvement, etc., can solve the problem of lack of in-depth research on D-pantothenic acid fermentation, and achieve the effect of enhancing the utilization ability

Active Publication Date: 2020-07-31
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the above reasons, the research on the fermentation of D-pantothenic acid has not been in-depth

Method used

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  • Genetically engineered bacteria, construction and application of high-yielding pantothenic acid without the addition of β-alanine
  • Genetically engineered bacteria, construction and application of high-yielding pantothenic acid without the addition of β-alanine
  • Genetically engineered bacteria, construction and application of high-yielding pantothenic acid without the addition of β-alanine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1: the HPLC determination of D-pantothenic acid content

[0063] The detection method is as follows:

[0064] Chromatographic conditions: C 18 Column (250×4.6mm, particle size 5μm, Agilent Technologies Co., Santa Clara, CA, USA), detection wavelength: 200nm, column temperature: 30°C;

[0065] Sample treatment: Dilute the sample with ultrapure water to keep the D-pantothenic acid content between 0.05g / L and 0.40g / L;

[0066] Mobile phase: acetonitrile / water / phosphoric acid: (50 / 949 / 1);

[0067] Data collection time: 18min.

Embodiment 2

[0068] Example 2: Construction and shake flask fermentation of effectively utilizing extracellular β-alanine strain W3110 (Trc-panC)

[0069] Using Escherichia coli W3110 as the starting strain, the gene editing technology mediated by CRISPR-Cas9 was used (Yu Jiang et al.2015Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9System.Applied Environmental Microbiology.81:2506-2514), derived from The trc promoter of pTrc99A (nucleotide sequence shown in SEQ ID No.1) replaces the original promoter of panC (GeneID: 12932172) in the genome to enhance the expression intensity of panC.

[0070] (1) Construction of pTarget-panC plasmid: Use pTarget F plasmid (Addgene Plasmid#62226) as a template, use pTarget-panC-1 / pTarget-panC-2 as primers for PCR amplification, and the PCR product is digested by Dpn I at 37°C 3h, then transformed into E.coli DH5α, screened with spectacle enzyme plate, and sequenced to verify that the correct pTarget-panC plasmid was obtained, which w...

Embodiment 3

[0078] Example 3: Construction of high-expression ketopantoate reductase strain W3110 (Trc-panCpanE) and shake flask fermentation

[0079] (1) Construction of pTarget-panE plasmid: Use pTarget F plasmid (Addgene Plasmid#62226) as a template, use pTarget-panE-1 / pTarget-panE-2 as primers for PCR amplification, and digest the PCR product with Dpn I at 37°C 3h, then transformed into E.coli DH5α, screened with spectacle enzyme plate, and sequenced to verify that the correct pTarget-panE plasmid was obtained, which was used for subsequent connection of DonorDNA.

[0080] (2) Construction of pTD-panE plasmid: with the E.coli W3110 genome as a template, pTD-panE-1, pTD-panE-2, pTD-panE-3 and pTD-panE-4 as primers, the construction steps are the same as in Example 2 (2), obtain pTD-panE plasmid.

[0081] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the competent W3110 (Trc-panC) obtained in Example 2, and the preparation method of the competent W3110 (Trc-panC) wa...

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Abstract

The invention relates to genetic engineering bacteria capable of producing pantothenic acid at high yield without addition of beta-alanine, a construction method of the genetic engineering bacteria, and application of the genetic engineering bacteria in preparation of D-pantothenic acid by microbial fermentation. According to the invention, (1), the final step of an escherichia coli D-pantothenicacid synthesis pathway is enhanced, and the utilizing ability of escherichia coli to extracellular beta-alanine, (2), a pantoic acid synthesis pathway is enhanced, (3), ilvG gene is repaired, and thefeedback inhibition effect of by-products on a pantoic acid synthesis pathway is weakened, (4), according to the change in cell phenotype, flux of a valine synthesis pathway is weakened, (5), a CRISPRi technique is used to screen metabolic modification sites of TCA cycle, a PPP pathway and a by-product metabolic pathway, according to the result, an isoleucine synthesis pathway is blocked, and thecompetition of 2-butanoic acid for reaction of acetolactate synthesized from pyruvic acid under catalysis of acetolactate synthase is relieved, and (6), aspartate decarboxylase from other strains is subjected to heterologous expression to obtain a genetical engineering strain capable of producing the pantothenic acid at high yield without addition of the beta-alanine. By combined expression of panB and panC which are derived from corynebacterium glutamicum and panD derived from bacillus subtilis together on pTrc99A plasmids, 1.2g / L of D- pantothenic acid is obtained without adding the beta-alanine.

Description

[0001] (1) Technical field [0002] The invention relates to a high-yield pantothenic acid-producing genetically engineered bacterium without the addition of beta-alanine and a construction method thereof, as well as the application of the genetically engineered bacterium in the preparation of D-pantothenic acid by microbial fermentation. [0003] (2) Background technology [0004] Pantothenic acid is usually stored, transported and traded in the form of stable calcium salts. Calcium pantothenate is a kind of B vitamins and one of the nutrients necessary for the normal growth of organisms. Calcium D-pantothenate is a vitamin drug and is a One of the components of coenzyme A, which exists in living cells, its main function is to participate in the metabolism of protein, fat, and sugar in the body, and it can treat vitamin B deficiency, peripheral neuritis, and intestinal colic after surgery. It can be used in combination with vitamin C. Treatment of disseminated lupus erythemato...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/90C12N15/70C12N15/54C12N15/53C12N15/52C12N15/60C12P13/02C12R1/19
Inventor 柳志强张博郑裕国张小明
Owner ZHEJIANG UNIV OF TECH
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