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Genetically engineered bacterium capable of increasing yield of lacto-N-fucopentaose and production method of genetically engineered bacterium

A technology of genetically engineered bacteria and fucopentasaccharide, which is applied in the fields of biotechnology and food fermentation engineering, can solve the problems of low yield of lactoyl-N-fucopentaose and the like, and achieve the effect of increasing yield

Pending Publication Date: 2021-12-24
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to solve the problem of low yield of lactoyl-N-fucopentaose synthesized by existing microbial methods, the present invention provides a genetically engineered bacterium producing lactoyl-N-fucopentaose and its construction method

Method used

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  • Genetically engineered bacterium capable of increasing yield of lacto-N-fucopentaose and production method of genetically engineered bacterium
  • Genetically engineered bacterium capable of increasing yield of lacto-N-fucopentaose and production method of genetically engineered bacterium
  • Genetically engineered bacterium capable of increasing yield of lacto-N-fucopentaose and production method of genetically engineered bacterium

Examples

Experimental program
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preparation example Construction

[0040] Preparation of Escherichia coli Competent: Kit of Shanghai Sangon Bioengineering Company.

[0041] LB liquid medium: 10g / L peptone, 5g / L yeast extract, 10g / L sodium chloride.

[0042] LB solid medium: 10g / L peptone, 5g / L yeast extract, 10g / L sodium chloride, 17g / L agar powder.

[0043] The determination method of lactoyl-N-fucopentaose described in the embodiment of the present invention uses HPLC, specifically:

[0044] 1mL of fermentation broth was boiled at 100°C for 10min, centrifuged at 13400rpm for 10min, the supernatant was filtered through a 0.22μm membrane, and the amount of lactoyl-N-fucopentaose was detected by HPLC. HPLC detection conditions: differential refractive index detector; chromatographic column is RezexROA-organic acid (Phenomenex, USA), column temperature is 50°C; mobile phase is 5mM H 2 SO 4 Aqueous solution, the flow rate is 0.6mL / min; the injection volume is 10μL.

[0045] Shake flask fermentation culture method is as follows:

[0046] Ino...

Embodiment 1

[0052] Example 1: Knockout of Escherichia coli BL21 (DE3) genome genes lacZ and wcaJ

[0053] The lacZ and wcaJ genes in the Escherichia coli BL21 (DE3) genome were knocked out using the CRISPR-Cas9 gene knockout system, and the specific steps were as follows (see Table 1 for the primer sequences involved):

[0054] (1) Using the Escherichia coli BL21 (DE3) genome as a template, using the primer pair lacZ-up-F / R and lacZ-down-F / R, wcaJ-up-F / R and wcaJ-down-F / R by PCR The upstream and downstream fragments of lacZ and wcaJ were respectively amplified, and the gel was recovered. Then use the upstream and downstream fragments of lacZ and wcaJ as templates respectively, and use lacZ-up-F / lacZ-down-R and wcaJ-up-F / wcaJ-down-R primers to obtain complete lacZ and wcaJ templates by overlapping PCR, and gel recovery DNA fragments.

[0055] (2) Using the original pTargetF plasmid (Addgene: #62226) as a template, lacZ-sg-F / R and wcaJ-sg-F / R as primers, use PCR amplification to replace t...

Embodiment 2

[0064] Example 2: Construction of recombinant bacteria for de novo synthesis of lacto-N-fucopentaose

[0065] The specific steps for the construction of the recombinant bacteria are as follows (see Table 2 for the primer sequences involved):

[0066] (1) Obtaining of galE, galT, galK, manB, manC, gmd and wcaG gene fragments: Since galE-galT-galK, manB-manC and gmd-wcaG are continuous gene fragments on the Escherichia coli genome, the Escherichia coli The genome of K-12 was used as a template to amplify galE-galT-galK, manB-manC and gmd-wcaG genes using ETK-F / ETK-R, BC-F / BC-R and GW-F / GW-R, respectively Fragments, DNA fragments were recovered by gel, and the recovered galE-galT-galK and manB-manC gene fragments were connected to the BamHI vectors of pETDuet-1 and pACYCDuet-1 respectively through a seamless cloning kit (Nanjing Novizan Life Science and Technology Co., Ltd.) Between the / SaiI restriction sites, the plasmids pET-ETK and pACY-BC were obtained. Using the same liga...

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Abstract

The invention discloses a genetically engineered bacterium for increasing the yield of lacto-N-fucopentaose and a production method of the genetically engineered bacterium, and belongs to the field of biotechnology and food fermentation engineering. Through expression of exogenous genes lgtA, wbgO and futC and overexpression of endogenous genes galE, galT, galK, manB, manC, gmd and wcaG of escherichia coli, a de novo synthesis path of the lacto-N-fucopentaose is constructed, and lacZ and wcaJ expression of a bypass path in a synthesis path of the escherichia coli host lacto-N-fucopentaose is knocked out, so that the yield of the lacto-N-fucopentaose is increased. In the shake flask fermentation, the yield of the lacto-N-fucopentaose reaches 1.99 g / L, and in a 3L fermentation tank, the yield of the lacto-N-fucopentaose reaches 16.8 g / L, so that a more efficient production strain is successfully created, and a foundation is laid for the industrial production of the lacto-N-fucopentaose.

Description

technical field [0001] The invention relates to a genetically engineered bacterium for increasing the yield of lactoyl-N-fucopentaose and a production method thereof, belonging to the fields of biotechnology and food fermentation engineering. Background technique [0002] Human milk oligosaccharides (HMOs) are the third largest solid component in human milk after lactose and lipids. A large number of in vitro studies have shown that HMOs have good health effects, including antagonizing microbial activity, resisting viruses Invasion and prevention of necrotizing enterocolitis and other effects. It plays an irreplaceable role in the early development of the baby's digestive system, the improvement of the immune system after birth and the establishment of ecological balance in the body. Lactoyl-N-fucopentaose (LNFPI) is an important oligosaccharide in human milk oligosaccharides, which is converted to lactoyl-N-tetraose (LNT) Generated by fucosylation, it plays an important r...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/56C12N15/54C12N15/61C12N15/53C12N15/70C12P19/02C12R1/19
CPCC12N9/2471C12N9/1288C12Y207/08031C12N9/90C12Y504/02008C12N9/1205C12N9/0006C12Y101/01132C12Y101/01271C12N9/1003C12Y204/01069C12Y501/03002C12N9/1051C12Y207/01006C12Y204/01056C12Y204/01179C12N15/70C12P19/02
Inventor 张涛胡苗苗江波李梦丽
Owner JIANGNAN UNIV