Recombinant escherichia coli for synthesizing lactoyl N-neotetraose and construction method and application of recombinant escherichia coli

A technology for recombining Escherichia coli and Escherichia coli, applied in the field of metabolic engineering, can solve the problems of multi-flow of carbon sources, insufficient stability of plasmid expression, regulation of metabolic pathways, etc.

Active Publication Date: 2020-08-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Previously, there have been studies on the transformation of microorganisms to synthesize lactyl N-neotetrasaccharides, including the construction of metabolic pathways in Escherichia coli and Bacillus subtilis, but most of the previous studies used plasmids to introduce key genes, and the expression of plasmids was not stable enough , there are problems such as being easily lost in the fermentation process, or the fermentation yield is unstable
Some studies tried to express key genes on the genome, but did not further regulate the metabolic pathways, resulting in more carbon sources flowing to other branch pathways, low substrate utilization in the metabolic process, and difficulty in increasing product yields.

Method used

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  • Recombinant escherichia coli for synthesizing lactoyl N-neotetraose and construction method and application of recombinant escherichia coli
  • Recombinant escherichia coli for synthesizing lactoyl N-neotetraose and construction method and application of recombinant escherichia coli
  • Recombinant escherichia coli for synthesizing lactoyl N-neotetraose and construction method and application of recombinant escherichia coli

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Experimental program
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Effect test

Embodiment 1

[0028] Example 1: Construction of Gene Knockout Homology Arm Fragments

[0029] According to the sequence information of E.coli MG1655, the primers shown in Table 1 were designed. The integration site of the lacY gene was fliK. Using the above primers, the E.coli MG1655 genome was used as a template to amplify lacZ, nagB, wecB, ugd, The upstream and downstream homology arm fragments of each gene of fliK were respectively fused by Overlapping PCR to obtain fragments lacZ12, nagB12, wecB12, ugd12, and lacY12.

[0030] Table 1

[0031]

Embodiment 2

[0032] Embodiment 2: Construction of pTarget plasmid

[0033] Select the N20 region (20bp base sequence used to target the gene to be knocked out in the CRISPR / Cas9 system) on each gene to be knocked out, design primers, use pTarget as a template, and replace the N20 region of the template by PCR. The PCR product was transferred into E.coli JM109, and the plasmid was extracted to obtain 4 plasmids, pTarget-lacZ, pTarget-nagB, pTarget-wecB, pTarget-ugd, pTarget-lacY.

Embodiment 3

[0034] Example 3: Gene knockout and integration

[0035] The starting strain E.coli MG1655 was recorded as M0, and the pCas9 plasmid was transformed into M0 to obtain the host M0 (pCas9) expressing the Cas9 protein, and then the pTarget-lacZ plasmid and fragment lacZ12 were electrotransformed into M0 (pCas9), and the lacZ gene was knocked out. M01(pCas9), eliminate the pTarget-lacZ plasmid, electrotransfer the pTarget-nagB plasmid and the fragment nagB12 into M01(Cas9), knock out the nagB gene, knock out each gene to be knocked out in this way, and finally eliminate the pTarget and pCas9 plasmids , and M04 with lacZ, nagB, wecB, and ugd gene knockouts was obtained.

[0036] On the basis of the M04 strain that knocked out the lacZ, nagB, wecB, and ugd genes, the pCas9 plasmid was transformed into M04 to obtain the host M04 (pCas9) expressing the Cas9 protein, and then the pTarget-lacY plasmid and the lacY gene, tac promoter , the fusion fragment lacY12 of the upper and lower h...

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Abstract

The invention discloses recombinant escherichia coli for synthesizing lactoyl N-neotetraose and a construction method and application of the recombinant escherichia coli. The recombinant escherichia coli are obtained by knocking out a beta-galactosidase lacZ gene, a glucosamine-6-phosphodeaminase nagB gene, a UDP-acetylglucosamine epitope isomerase wecB gene and a UDP-glucose dehydrogenase ugd gene in the host genome of escherichia coli and overexpressing a lactose transportase lacY gene, beta-1, 3-N-glucosaminidase lgtA gene and a beta-1, 4 galactosyltransferase lgtB gene on the genome. The yield of lactoyl-N-neotetraose synthesized by recombinant escherichia coli reaches 985 mg / L, and a foundation is laid for further metabolic engineering transformation of escherichia coli to produce lactoyl-N-neotetraose.

Description

technical field [0001] The invention relates to a recombinant Escherichia coli for synthesizing lactyl N-neotetrasaccharide and its construction method and application, belonging to the technical field of metabolic engineering. Background technique [0002] Breastmilk has always been regarded as the best food source for infants and young children. In addition to providing infants with the nutrients needed for normal growth, it also has many health-promoting effects that cow's milk does not have. Oligosaccharides are the third largest solid component in breast milk after lactose and fat, and the total content of oligosaccharides in breast milk is 5-15 g / L. [0003] Lacto-N-neotetraose is one of the breast milk oligosaccharides with high content in breast milk, which plays an important role in regulating intestinal flora, enhancing immunity and promoting cell synthesis. As an important nutrient, lactoyl N-neotetrasaccharide has been approved by the US FDA and EU as an additiv...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P19/00C12R1/19
CPCC12N9/1048C12N9/2402C12Y302/01108C12N9/00C12N15/70C12P19/00C12N1/20Y02A50/30
Inventor 刘龙陈坚堵国成李江华吕雪芹张蔚
Owner JIANGNAN UNIV
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