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Alpha (1,2) fucosyltransferase syngenes for use in the production of fucosylated oligosaccharides

A technology of fucosyltransferase and fucosylation, which is applied in the directions of glycosyltransferase, transferase, esterification and saccharide, etc., can solve the problems of impure product, high cost, production limitation, etc.

Active Publication Date: 2017-05-31
GLYCOSYN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, their production by chemical synthesis has been limited due to stereo-specificity issues, precursor availability issues, product impurity issues, and high cost issues

Method used

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  • Alpha (1,2) fucosyltransferase syngenes for use in the production of fucosylated oligosaccharides
  • Alpha (1,2) fucosyltransferase syngenes for use in the production of fucosylated oligosaccharides
  • Alpha (1,2) fucosyltransferase syngenes for use in the production of fucosylated oligosaccharides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] Example 1: Identification of a novel α(1,2)-fucosyltransferase

[0155] In order to identify other novel α(1,2) fucosyltransferases, calibration operations were performed using CLCbio Main Workbench package version 6.9 to generate multiple sequence calibration queries (CLCbio, 10 Rogers Street #101, Cambridge, MA 02142, USA) using Four previously determined lactose-utilizing α(1,2) fucosyltransferase protein sequences: Helicobacter pylori futC (SEQ ID NO: 1), H. mustelae FutL (SEQ ID NO: 2), Bacteroides vulgaris futN (SEQ ID NO: 3) and E. coli 0126wbgL (SEQ ID NO: 4). Figure 3 shows the sequence alignment and percent sequence identity between four predetermined lactose-utilizing alpha(1,2) fucosyltransferase protein sequences. Iterative PSI-BLAST was performed, interrogated using FASTA-type multiple sequence alignments, and the NCBI PSI-BLAST program was run on a local copy of NCBI BLAST+ version 2.2.29. Specific streak matrix files (.pssm) were generated using PSI-...

Embodiment 2

[0162] Example 2: Verification of the new α(1,2)FT

[0163] To detect lactose-utilizing fucosyltransferase activity, fucosylated oligosaccharides (i.e., 2'-fucosyllactose ( 2'-FL)), and it contains cytoplasmic guanosine diphosphate-fucose and lactose pools. The production of fucosylated oligosaccharides indicated that the candidate enzyme-coding sequence functions as a lactose-utilizing alpha(1,2) fucosyltransferase. Of all the samples identified, 12 novel α(1,2) fucosyltransferases were further analyzed for their ability to produce 2′-fucosyltransferases: Prevotella melanogenes FutO, Clostridium Clostridium boltae+13FutP, Lachnospiraceae FutQFutP, Methanosphaerula palustris FutR, Tannerella sp. FutS, Bacteroides faecalis FutU, Butyvibrio FutV, Revobacteria FutW, parabacteroides johnsonii FutX, Akkermansia muciniphilia FutY, Salmonella enterica FutZ, Bacteroides FutZA.

[0164] The construct includes 12 variants of the novel α(1,2)FT with the following structure: EcoRI-T7...

Embodiment 3

[0173] Example 3: Characterization of cultures expressing novel α(1,2)FTs

[0174] Bacteria expressing the novel α(1,2)FTs FutO, FutQ and FutX were further characterized. Specifically, proliferation ratio and exogenous α(1,2) FT experiments were examined.

[0175] Expression plasmids containing the fucosyltransferases WbgL (plasmid pG204), FutN (plasmid pG217), and the novel α(1,2)FT FutO (plasmid pG393), FutQ (plasmid pG395), and FutX (pG401) were introduced into host bacteria strain. For example, the host strain utilized here has the following genotype: ΔampC::P trp B cI,Δ(1acI-lacZ)::FRT,P lacIq lac Y + , ΔwcaJ::FRT, thyA::Tn10, Δlon:(npt3, lacZ + ), ΔlacA

[0176] Bacterial cultures expressing each exogenous fucosyltransferase were induced by the addition of tryptophan in the presence of lactose (in order to induce expression of the exogenous fucosyltransferase). Growth of the cultures was monitored by recording spectrophotometer readings at A600 at the following...

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Abstract

The invention provides compositions and methods for engineering E. coli or other host production bacterial strains to produce fucosylated oligosaccharides, and the use thereof in the prevention or treatment of infection.

Description

technical field [0001] The present invention provides compositions and methods for producing purified oligosaccharides, particularly certain fucosylated oligosaccharides commonly found in human milk. [0002] technical background [0003] Human milk contains a wide variety of rich neutral and acidic oligosaccharides. More than 130 different complex oligosaccharides have been identified in humans, and their structural diversity and distribution are unique to humans. Although these molecules may not be directly available to infants as nutrients, they can play a critical role in establishing a healthy lipid microenvironment, in preventing disease, and in establishing immune function. Prior to the invention described here, the ability to inexpensively produce human milk oligosaccharides (HMOS) was problematic. For example, their production by chemical synthesis has been limited due to stereo-specificity issues, precursor availability issues, product impurity issues, and high co...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/63C12P19/18
CPCC12P19/18C12N9/1051C12P19/00C12Y204/01069C07H13/04Y02A50/30C12P19/04C12N15/70C12Y204/01149C12Y204/01086
Inventor J·M·麦科伊M·I·海特曼M·梅里吉
Owner GLYCOSYN
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