Improved process for the production of fucosylated oligosaccharides

A technology of fucosyl and oligosaccharides, applied in biochemical equipment and methods, glycosyltransferases, oxidoreductases, etc., can solve problems such as inability to achieve HMO titer

Pending Publication Date: 2019-05-21
CHR HANSEN HMO GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, HMO titers greater than 20 g / L in the fermented broth cannot or rarely be achieved even with the most efficient methods currently available based on bacterial fermentation

Method used

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  • Improved process for the production of fucosylated oligosaccharides
  • Improved process for the production of fucosylated oligosaccharides
  • Improved process for the production of fucosylated oligosaccharides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Transformation of Escherichia coli BL21(DE3) strain for producing 2'-fucosyllactose

[0113] Using Escherichia coli BL21(DE3) as a parental host, a strain for producing 2'-fucosyllactose in a whole-cell biosynthesis method was constructed. Genome engineering of the strains includes gene disruption and deletion events as well as integration of heterologous genes.

[0114] Since 2'-fucosyllactose is synthesized from lactose (suitable for bacterial cultures), and from GDP-L-fucose produced in living cells, the encoding Inactivation of the wild-type copy of the lacZ gene of the endogenous β-galactosidase (see Ellis et al., "High efficiency mutagenesis, repair, and engineering of chromosome DNA using single-stranded oligonucleotides", Proc. Natl. Acad. Sci. USA 98:6742-6746 (2001)). Using the same method, the arabinose isomerase gene araA was disrupted.

[0115] The lacZΩ gene fragment was introduced under the control of the temperature-sensitive transcriptional repress...

Embodiment 2

[0121] Confirmation of increased 2'-fucosyllactose export by the Yersinia burgdorferi ATCC 43970 sugar efflux transporter

[0122] Knockout of yberc0001_9420

[0123] To demonstrate the functionality of the heterologous sugar transporter from Yersinia burgdorferi ATCC 43970, the gentamicin resistance cassette aacC1 from plasmid pBBR-MCS5 was used (Kovach, Elzer et al. 1995, "Four new derivatives of the broad-host-range cloning vector pBBRI MCS, carrying different antibiotic-resistance cassettes", Gene 166, 175-176) - which was inserted into the gene yberc0001_9420 from strain E. coli BL21(DE3) lacZ - 、araA - 、fucl - 、fucK - , wcaJ - (which contained manB, manC, gmd, wcaG, lacY, wbgL, and yberc0001_9420 chromosomal integration by homologous recombination of Datsenko and Wanner (2000; see above)) the gene yberc0001_9420 was deleted, resulting in strain Δyberc0001_9420.

[0124] Culture conditions for 2’-fucosyllactose production

[0125] The Escherichia coli BL21(DE3...

Embodiment 3

[0132] Production of 2’-fucosyllactose during fermentation

[0133] Fermentations were carried out in 3 L fermenters at 30°C and pH 7.0; the pH was adjusted by titration with 25% ammonia. The strain described in Example 2 was cultivated in the mineral salt medium described in Example 2 using glycerol as a carbon source and energy source. A fermenter with an initial volume of 1 L was inoculated with a preculture grown in the same medium. Glycerol was added continuously (60% v / v) after the 2% glycerol contained in the batch was consumed. When OD 600nm When 6 was reached, lactose was added at a concentration of 0.66 M in three portions (10 ml each) (1 hour apart). Thereafter, lactose was administered in a continuous flow to maintain a lactose concentration in the fermentor of at least 10 mM. After culturing for 86 h, the final titer of 91.3 mM (44.6 g / L) 2'-fucosyllactose was reached. By changing the temperature to 42°C and expressing the β-galactosidase gene, lactose and ...

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Abstract

The present invention relates to a method for producing fucosylated oligosaccharides by using a recombinant prokaryotic host cell that is cultivated on a gluconeogenic substrate, as well as to the host cell and its use. The host cell is genetically modified in that the activity of a fructose-6-phosphate converting enzyme is abolished or lowered, and the transport of the produced fucosylated oligosaccharide through the cell membrane is facilitated by an exogenous transport protein.

Description

technical field [0001] The present invention relates to a method for the production of fucosylated oligosaccharides by using a genetically modified prokaryotic host cell, and to the host cell used in the method and its use for the production of high titers of fucosylated oligosaccharides . Background technique [0002] Human milk is a complex mixture of carbohydrates, fats, proteins, vitamins, minerals and trace elements. By far the most dominant fraction is carbohydrates, which can be further divided into lactose and more complex oligosaccharides (human milk oligosaccharides, HMOs). Whereas lactose is used as an energy source, complex oligosaccharides are not metabolized by the baby. The fraction of complex oligosaccharides accounts for up to 20% of the total carbohydrate fraction and consists of more than 200 different oligosaccharides. The presence and concentration of these complex oligosaccharides are unique to humans and thus cannot be found in significant amounts i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/18C12N1/21C12N9/10C12N9/12
CPCC12P19/18C12N9/12C12Y204/01065C12N9/1051C12N15/52C07K14/24C12N9/1205C12Y207/01011C12Y301/03011C12Y401/02013C12N9/88C12N9/16C07K14/245C12Y204/01C12Y204/01152C12N9/0006C12N9/1241C12N9/90C12N15/70C12Y101/01271C12Y207/07013C12Y402/01047C12Y504/02008
Inventor S·詹尼温D·瓦滕伯格K·帕沙特
Owner CHR HANSEN HMO GMBH
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