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Escherichia coli and application thereof to synthesis of fucosylated oligosaccharide

A technology of fucosyl and Escherichia coli, which is applied in the field of metabolic engineering, can solve the problems of unreachable industrial production, low yield of human milk oligosaccharides, and low yield

Active Publication Date: 2020-10-23
武汉中科光谷绿色生物技术有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In summary, the existing technology is limited by the low yield of the intracellular synthesis pathway of GDP-L-fucose, which leads to the low yield of fucosylated human milk oligosaccharides, which cannot meet the needs of industrial production

Method used

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  • Escherichia coli and application thereof to synthesis of fucosylated oligosaccharide
  • Escherichia coli and application thereof to synthesis of fucosylated oligosaccharide
  • Escherichia coli and application thereof to synthesis of fucosylated oligosaccharide

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Embodiment 1

[0062] The research on the efficient synthesis of fucosylated oligosaccharides using engineered Escherichia coli has been carried out for many years. Based on the existing technology, most of them use the metabolic intermediate fructose-6-phosphate of Escherichia coli as the starting point for the de novo synthesis of GDP-L-fucose way.

[0063] For the construction method of metabolic engineering bacteria for efficiently biosynthesizing fucosylated human milk oligosaccharides using sucrose in this example, see figure 1 , including the following steps:

[0064] (1) Construct engineering strains and overexpress the de novo synthesis pathway genes of GDP-L-fucose. Such as image 3 with Figure 4As shown, the 6-phosphomannose isomerase gene manA (Gene ID: 944840) and the phosphomannose mutase manB derived from E. coli str.K-12substr.MG1655 (GenBank: NC_000913.3) were amplified by PCR respectively. (Gene ID: 946574), α-D-mannose 1-phosphate guanyltransferase manC (Gene ID: 946...

Embodiment 2

[0082] Example 2 Using Escherichia coli to ferment and synthesize 2'-fucosyl lactose (5L tank)

[0083] (1) Seed medium LB (g / L): tryptone 10, yeast extract 5, sodium chloride 10, pH7.2-7.4; when preparing solid medium, add 17g / L agar powder;

[0084] Initial fermentation medium (g / L): glucose 10-20, ammonium sulfate 3-7, dipotassium hydrogen phosphate 8-12, potassium dihydrogen phosphate 6-10, citric acid 0.5-1.0, CaCl 2 1.0~1.5, vitamin B10.01~0.1, defoamer 20~80ml / L, trace element mother solution 10~20mL / L; trace element mother solution formula (g / L): nitrilotriacetic acid (add appropriate amount of alkali, separate Preparation) 8~12, ferric ammonium citrate 5~7, zinc sulfate heptahydrate 0.5~1, CoCl 2 ·6H 2 O 0.1~0.5, manganese chloride tetrahydrate 0.6~1.2, CuCl 2 2H 2 O 0.1~0.2, boric acid 0.1~0.5, Na 2 MoO 4 2H 2 O 0.1~0.5; feed solution (g / L): sucrose 500~800, magnesium sulfate 10~20.

[0085] (2) Inoculate the engineered Escherichia coli constructed in Example...

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Abstract

The present invention discloses a construction method of escherichia coli for synthesizing fucosylated oligosaccharide by fermentation. The construction method includes the steps: (1) overexpressing at least one gene for encoding enzymes required for denovo synthesizing GDP-L-fucose in a prokaryotic host cell; (2) expressing an exogenous gene for encoding a fucosyltransferase in the prokaryotic host cell; (3) reducing or eliminating the activity of GDP-mannanohydrolase in the prokaryotic host cell; and (4) reducing or eliminating the activity of beta-galactosidase in the prokaryotic host cell.According to the present invention, the escherichia coli is constructed by using the method, and the escherichia coli may be used for preparing the fucosylated oligosaccharide. Shown by a result generated by performing fermentation production verification on the fucosylated oligosaccharide (with 2'-fucosyllactose as an example) in a 5L tank by adopting an engineering strain constructed by the present invention, the highest production level of the 2'-fucosyllactose (2'-FL) may reach 50 g / L.

Description

technical field [0001] The invention relates to the technical field of metabolic engineering, and also relates to a strain of Escherichia coli, especially a method for using the Escherichia coli to synthesize fucosylated oligosaccharides. Background technique [0002] At present, three elements are necessary for the biosynthesis of fucosylated human milk oligosaccharides (2'-fucosyllactose, 3-fucosyllactose, difucosyllactose, etc.): Nucleotide-activated fucose Sugar GDP-L-fucose (GDP-L-fucose, mainly used as the donor substrate of the synthesis reaction, under normal circumstances, the intracellular level is extremely low, and it is not easy to produce on a large scale, and the price is expensive), acceptor sugar (mainly Substrates that accept fucose in this reaction, such as lactose, N-acetyllactosamine, fucosyllactose, lacto-N-disaccharide, lacto-N-tetraose, sialyllactose, disialyllactose, etc. ), fucosyltransferase (FucT, which requires exogenous introduction and high ex...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/61C12N15/54C12N15/60C12N15/53C12N15/90C12P19/00C12R1/19
CPCC12N9/90C12N9/1241C12N9/88C12N9/0006C12N9/1051C12N9/2471C12N9/2494C12N9/1288C12N9/1029C12N15/902C12P19/00C12Y503/01008C12Y504/02008C12Y207/07013C12Y402/01047C12Y101/01271C12Y302/01023C12Y302/01078C12Y207/08031C12Y203/01018C12Y101/01017
Inventor 倪志坚廖迎雪吴金勇陈祥松李翔宇李忠奎袁丽霞姚建铭
Owner 武汉中科光谷绿色生物技术有限公司
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