Genetically engineered bacterium with high pantothenic acid yield as well as construction method and application

A technology of genetically engineered bacteria and pantothenic acid, applied in the direction of genetic engineering, microbe-based methods, applications, etc., to achieve the effect of enhancing utilization ability and weakening feedback inhibition

Active Publication Date: 2019-06-11
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While BASF and DSM companies use Bacillus subtilis for transformation, but there is no high-yield strain

Method used

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  • Genetically engineered bacterium with high pantothenic acid yield as well as construction method and application
  • Genetically engineered bacterium with high pantothenic acid yield as well as construction method and application
  • Genetically engineered bacterium with high pantothenic acid yield as well as construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1: the HPLC determination of D-pantothenic acid content

[0054] The detection method is as follows:

[0055] 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;

[0056] Sample treatment: Dilute the sample with ultrapure water to keep the D-pantothenic acid content between 0.05g / L and 0.40g / L;

[0057] Mobile phase: acetonitrile / water / phosphoric acid: (50 / 949 / 1);

[0058] Data collection time: 18min.

Embodiment 2

[0059] Example 2: Construction and shake flask fermentation of effectively utilizing extracellular β-alanine strain W3110 (Trc-panC)

[0060] Using Escherichia coli W3110 as the starting strain, using CRISPR-Cas9-mediated gene editing technology such as figure 1 (Yu Jiang et al.2015Multigene Editing in the Escherichia coli Genome viathe CRISPR-Cas9System.Applied Environmental Microbiology.81:2506-2514), with the trc promoter derived from pTrc99A (nucleotide sequence shown in SEQ ID No.1 ), to replace the original promoter of panC (GeneID: 12932172) in the genome to enhance the expression intensity of panC.

[0061] (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 obta...

Embodiment 3

[0070] Example 3: Construction of high-expression ketopantoate reductase strain W3110 (Trc-panCpanE) and shake flask fermentation

[0071] (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.

[0072] (2) Construction of pTD-panE plasmid: E.coli W3110 genome is used as a template, pTD-panE-1, pTD-panE-2, pTD-panE-3 and pTD-panE-4 are used as primers, and the construction steps are the same as in Example 2 (2), obtain pTD-panE plasmid.

[0073] (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 (...

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Abstract

The invention relates to a genetically engineered bacterium with a high pantothenic acid yield, a construction method of the bacterium, and application of the genetically engineered bacterium to preparation of D-pantothenic acid through microbial fermentation. The construction method comprises the following steps: (1) reinforcing a last step of an escherichia coli D-pantothenic acid synthesis wayto reinforce the utilization capacity of escherichia coli to extracellular beta-alanine; (2) reinforcing a pantoic acid synthetic way; (3) repairing an ilvG gene and weakening the feedback inhibitioneffect of byproducts on the pantoic acid synthetic way; and (4) weakening the flux of a valine synthetic way according to the phenotypic change of cells to obtain an escherichia coli genetically engineered strain with high yield of D-pantothenic acid. According to the invention, the expression of key enzymes PanB, PanC, PanE and IlvC in the D-pantothenic acid biogenic way is enhanced; the ilvG gene is repaired to weaken feedback regulation and control and reinforce the pantoic acid synthesis path, so that the extracellular accumulation of D-pantothenic acid and valine reaches 0.48 g/L and 0.51g/L respectively, a competitive branch is weakened by knocking out avtA and knocking down ilvE to obtain a plasmid-free high-yield bacterium, and the titer of D-pantothenic acid is increased from 0.48 g/L to 1.54 g/L.

Description

(1) Technical field [0001] The invention relates to a genetically engineered bacterium with high pantothenic acid production and a construction method thereof, as well as the application of the genetically engineered bacterium in preparing D-pantothenic acid by microbial fermentation. (2) Background technology [0002] D-pantothenic acid, as a component of coenzyme A, participates in the regulation of protein, sugar and fat metabolism, improves hair color and prevents diseases. In particular, the growth and development of poultry, livestock and fish, as well as the synthesis and decomposition of fat, are all indispensable to the demand for calcium pantothenate. Lack of pantothenic acid can lead to growth retardation, reproductive dysfunction and reduced adaptability of poultry and livestock. Calcium D-pantothenate, as a B vitamin, is widely used in the pharmaceutical industry at home and abroad. The main indication of its unilateral preparation is pantothenic acid deficienc...

Claims

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Application Information

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