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Plasmid-free and inducer-free genetically engineered bacterium for producing D-pantothenic acid and construction method

A technology of genetically engineered bacteria and pantothenic acid, applied in the field of metabolic engineering, can solve the problems of expensive precursors, uneconomical, difficult strain improvement, etc., to achieve the effect of removing negative feedback inhibition, strengthening pyruvate accumulation, and improving sugar uptake ability

Active Publication Date: 2021-08-20
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the biological production of D-pantothenic acid, although the biological enzyme method has made great progress, it still needs expensive precursors, which is uneconomical, and the fermentation method lacks suitable strains, so a high-yielding D-pantothenic acid strain was constructed by metabolic engineering. - pantothenic acid producing strains
[0006] Commonly used production host strains in industry include Escherichia coli and Corynebacterium glutamicum, but the engineering transformation of Corynebacterium glutamicum is complicated and takes a long time, making it difficult to improve strains
At present, the production of D-pantothenic acid by Corynebacterium glutamicum is still less than 2g / L, which is not conducive to the industrial production of D-pantothenic acid

Method used

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  • Plasmid-free and inducer-free genetically engineered bacterium for producing D-pantothenic acid and construction method
  • Plasmid-free and inducer-free genetically engineered bacterium for producing D-pantothenic acid and construction method
  • Plasmid-free and inducer-free genetically engineered bacterium for producing D-pantothenic acid and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1: Charge Trum DPA8 (E.COLI W3110 TRC-PANC / TRC-PANE / TRC-PANB / TRC-ILVC / ILVG * / ΔAVTA / ILVE * / COAA * Construction

[0089] (1) As the Escherichia.coli W3110, it is used as a strain using Crispr-Cas9 gene editing techniques, and the PANC gene promoter in the genome plant ESCHERICHIA.COLI W3110 genome is replaced with a TRC promoter to obtain recombinant strain Escherichia.coli W3110 / TRC- PANC, recorded as DPA1;

[0090] (2) Use the CRISPR-Cas9 gene editing technology to replace the PANE gene promoter in the strain DPA1 genome to TRC promoter to obtain recombinant strain Escherichia.coli W3110 / TRC-PANCPANE, remembered as DPA2;

[0091] (3) Use the CRISPR-Cas9 gene editing technique to replace the PANB gene promoter in the strain DPA2 genome with a TRC promoter to obtain recombinant strain Escherichia.coli W3110 / TRC-PANCPANEPANB, which is recorded as DPA3;

[0092] (4) Use crisPR-Cas9 gene editing techniques to replace the ILVC gene promoter in the stra...

Embodiment 2

[0097] Example 2: Construction and shake flask fermentation of strain DPA8-1 knockout PoxB gene

[0098] Gene Engineering DPA8 (ie E.COLI W3110 TRC-PANC / TRC-PANE / TRC-PANB / TRC-ILVC / ILVG * / ΔAVTA / ILVE * / COAA * ) As starting strain, the use of CRISPR-Cas9 mediated gene editing technology (YuJiang et al.2015Multigene Editing in the Escherichia coli Genome via theCRISPR-Cas9 System.Applied Environmental Microbiology.81: 2506-2514), on acetic acid in the genome Gene Poxb is knocked out to reduce pyruvate synthesis acetic acid:

[0099](1) Constructing a PTarget-PoxB plasmid: Template as a PTARGET F plasmid (AddGene Plasmid # 62226), PT- △ PoxB F / PT- △ POXB R is amplified, and the obtained PCR product passes through DPN I at 37 ° C Insulation is 30 min, then transformed into E.Coli DH5α chemotactic admissions, magnificent maternal (SD) flat screen, sequencing and verifying the correct PTARGET-POXB plasmid for subsequent connection Donor DNA.

[0100] (2) Constructing a PTD...

Embodiment 3

[0108] Example 3: Construction of strain DPA8-2 knockout PTA gene

[0109] (1) Construction of PTarget- △ PTA plasmid: Template as a Ptarget F Plasmid (AddGene Plasmid # 62226), PTARGET-△ PTA F / PTARGET- △ PTA R is amplified, and the PCR product is incubated at 37 ° C through DPN I. Digestion 3 h, then transformed into E.Coli DH5α chemotactic cells, spectacular enzyme flat screen screening, sequencing and verifying the correct PTarget-PTA plasmid for subsequent connection Donordna.

[0110] (2) Constructing a PTD-PTA plasmid: Take the E.Coli W3110 genome as analog, ΔPTA P1, ΔPtAP2, ΔPTAP3 and ΔPTA P4 as primers, and the construction step is the same as in Example 2 (2) to obtain a PTD-ΔPTA plasmid. .

[0111] (3) Introduce PCAS plasmid (AddGene Plasmid # 62225) into the strain DPA8-1 perception obtained in Example 2, the strain DPA8-1 sensing state preparation method as in Example 2 (3).

[0112] (4) Constructing a strain DPA11 positive colonies, the construction method is the sa...

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PUM

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Abstract

The invention relates to a genetically engineered bacterium for producing D-pantothenic acid at high yield, a construction method and application of the genetically engineered bacterium to preparation of the D-pantothenic acid through microbial fermentation. According to the genetically engineered bacterium, related genes of an organic acid synthesis path of escherichia coli are blocked, so that side effects of organic acid are reduced and the accumulation of a pyruvic acid pool is increased; a pyruvic acid synthesis path gene pykA is enhanced and the accumulation of pyruvic acid is enhanced; negative feedback inhibition of a key enzyme is relieved and a bottleneck step of synthesizing the D-pantothenic acid is solved; the sugar uptake capability is improved and the accumulation of a precursor pyruvic acid is increased; over-expression genes lpd and ilvD are used for improving an expression condition of a main path synthesis path gene; finally, the engineered bacterium for producing the D-pantothenic acid at higher yield is obtained; an exogenous enzyme does not need to be introduced into a plasmid to enhance the enzyme activity of the key enzyme and the shake-flask fermentation yield reaches 3.99g / L; a repressor protein coding gene lacI is knocked out to form constitutive expression; after a culture medium is optimized, the shake-flask fermentation yield reaches 4.72g / L; materials are supplemented in batches in a 5L fermentation tank and fermentation is carried out; and the yield reaches 34.28g / L.

Description

[0001] (1) Technical field [0002] The present invention belongs to the field of metabolic engineering, particularly to plasmid-free, high yield without inducing agent D- pantothenic acid using strain genetically engineered construction method and application. [0003] (2) Background [0004] Pantothenic acid belonging to vitamin B family, also known as vitamin B5, is a water soluble vitamin, the presence of D and L forms two configurations, but only the type D- (D-PA) biological activity. Pantothenic acid in vivo can be used as precursors of coenzyme A (CoA), and play a key role in almost all biological processes, promoting energy metabolism and energy exchange in vivo. Pantothenic acid mercaptoethylamine phosphorylated product may Synthesis of 4-phosphopantetheine mercaptoethylamine, coenzyme A (CoA) and acyl carrier protein part (ACP), which constitutes a coenzyme CoA and ACP acyltransferase widely involved in glucose , lipid, protein metabolism and biotransformation effects on ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/53C12N15/54C12N15/31C12N15/113C12P13/02C12R1/19
CPCC12N9/0008C12N9/1029C12N9/0006C12N9/1205C12N9/1022C12N9/0051C07K14/245C12P13/02C12Y102/03003C12Y203/01008C12Y101/01027C12Y207/0104C12Y202/01006C12Y108/01004
Inventor 柳志强张博陈力金洁漪李波王培郑裕国
Owner ZHEJIANG UNIV OF TECH
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