Construction method of high-yield pantothenic-acid genetically engineered bacterium and bacterium strain

A technology of genetically engineered strains and genetically engineered bacteria, which is applied in the field of improving the production of pantothenic acid in genetically engineered bacteria and in the field of strains, which can solve problems such as insufficient final yield

Active Publication Date: 2020-05-05
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Rogers R. Yocum and others are committed to constructing high-yielding D-pantothenic acid Bacillus subtilis (Bacillus subtilis), by releasing the regulation of panBCD, ilvBNC, panE and ilvD to enhance the synthesis of D-pantothenic acid, and constructing a plasmid that overexpresses panBD. The optimization method of fermentation medium conditions found that the replacement of glucose with maltose and the addition of serine can effectively promote the accumulation of D-pantothenic acid. At the same time, it was found that the one-carbon unit synthesis pathway starting from serine is closely related to D-pantothenic acid. A series of studies were conducted to find that serA The overexpression of glyA and glyA will be beneficial to the synthesis of D-pantothenic acid, but no amplification report has been seen
Christophe Chassagnole et al. co-expressed pECM3-ilvBNCD and pEKEx2-panBC plasmids on ilvA-knockout Corynebacterium glutamicum ATCC13032, and added β-alanine exogenously in fermentation engineering for the production of D-pantothenic acid, but The final output is less than 2g / L. In addition, there are many patent reports on the production of α-ketoisovaleric acid, which are beneficial to improve the fermentation titer of D-pantothenic acid.

Method used

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  • Construction method of high-yield pantothenic-acid genetically engineered bacterium and bacterium strain
  • Construction method of high-yield pantothenic-acid genetically engineered bacterium and bacterium strain
  • Construction method of high-yield pantothenic-acid genetically engineered bacterium and bacterium strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1: Determination of D-pantothenic acid content in fermented liquid by HPLC method

[0083] The detection method is as follows:

[0084] Sample treatment: Take 1ml of fermentation broth and centrifuge to get the supernatant, dilute the supernatant with ultrapure water to an appropriate multiple, and keep the D-pantothenic acid content between 0.05g / L and 0.40g / L;

[0085] 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, flow rate: 0.9ml / min;

[0086] Mobile phase: acetonitrile / water / phosphoric acid: (50 / 949 / 1);

[0087] Data collection time: 23min.

Embodiment 2

[0088] Embodiment 2: Construction and shake flask fermentation of strain DPA10 overexpressing lpd gene

[0089] Using genetically engineered bacteria DPA9 (CCTCC NO: M 2018914) as the starting strain, using CRISPR-Cas9-mediated gene editing technology such as figure 1 (Yu Jiang et al.2015Multigene Editing in the Escherichiacoli Genome via the 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 natural promoter of the lpd gene in the genome to enhance the expression intensity of the lpd gene.

[0090] (1) Construction of pTarget-panC plasmid: use pTarget F plasmid (Addgene Plasmid#62226) as a template, use pT-lpd F / pT-lpd R as primers for PCR amplification, and the obtained PCR product is digested by Dpn I at 37°C 3h, and then transformed into E.coli DH5αtransformation-competent medium, screened with spectinomycin (SD) plate, and sequenced to verify that the c...

Embodiment 3

[0099] Embodiment 3: Construction and shake flask fermentation of the strain DPA11 of knockout glk gene

[0100] (1) Construction of pTarget-glk plasmid: use pTarget F plasmid (Addgene Plasmid#62226) as a template, use pTarget-glk F / pTarget-glk R as primers for PCR amplification, and digest the PCR product with Dpn I at 37°C for 3 hours. Then transformed into E.coli DH5α-transformation competent cells, screened with spectizyme plate, and sequenced to verify that the correct pTarget-glk plasmid was obtained for subsequent connection with DonorDNA.

[0101] (2) Construction of the pTD-glk plasmid: using the E.coli W3110 genome as a template, pTD-glk P1, pTD-glk P2, pTD-glk P3 and pTD-glk P4 as primers, the construction steps are the same as in Example 2 (2), The pTD-glk plasmid was obtained.

[0102] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the competent strain DPA10 obtained in Example 2, and the preparation method of the competent strain DPA10 was the...

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Abstract

The present invention relates to a construction method of high-yield pantothenic-acid genetically engineered bacterium and the bacterium strain, and an application of the genetically engineered bacterium in preparation of D-pantothenic acid by microbial fermentation. The construction method is as follows: (1) enhancing expression of lpd gene; (2) knocking out glk and galP, destroying a glucose non-PTS transport system, enhancing expression of ptsG gene and enhancing a glucose PTS transport system; (3) knocking out yfbQ and ppsA genes; (4) knocking out poxB, pflB and ldhA genes; (4) knocking out ilvE gene; (5) introducing heterologous acetolactate synthase gene alsS; and (6) introducing heterologous pantothenic-acid transporter panT into plasmids and finally obtaining the optimal D-pantothenic acid high-yield escherichia coli genetically engineered bacterium strain. D-pantothenic acid potency increases from 2.76 g/L to 6.33 g/L.

Description

[0001] (1) Technical field [0002] The invention relates to a construction method and bacterial strain for increasing the yield of genetic engineering bacteria pantothenic acid, and the application of the genetic engineering bacteria in the preparation of D-pantothenic acid by microbial fermentation. [0003] (2) Background technology [0004] Pantothenic acid, also known as vitamin B5, is an acidic substance that exists widely in organisms. It is an important premise substance for the biosynthesis of coenzyme A, and it can significantly promote the growth of organisms. However, it has been found that pantothenic acid can only be produced by plants and microbial synthesis. Pantothenic acid can be widely used in food, feed, cosmetics and pharmaceutical industries, and has a good market value. It has been reported that feed grade D-pantothenic acid can be obtained by heating the mixture of D-pantoic acid and β-alanine in a methanol or ethanol environment, and has been put into ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P13/02C12R1/19
CPCC12N9/1205C12N9/1096C12N9/1294C12N9/0008C12N9/1029C12N9/0006C12N9/1022C07K14/47C07K14/315C12P13/02C12Y207/01002C12Y206/01C12Y207/09002C12Y102/03003C12Y203/01054C12Y101/01027C12Y206/01042C12Y202/01006
Inventor 柳志强王微张博郑裕国
Owner ZHEJIANG UNIV OF TECH
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