# US 5,705,367 A · Glycosyltransferases for biosynthesis of oligosaccharides, and genes encoding them

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- Number: US 5,705,367 A
- Document ID: US5705367A
- Status: Granted
- Kind: A
- Application number: 08/683,426
- Filed: 1996-07-18
- Granted: 1998-01-06
- Earliest claimed: 1994-09-26
- Assignees: The Rockefeller University
- Inventors: Emil C. Gotschlich
- Web page: https://patentgrep.com/patent/US5705367A

## CPC

- C07K 14/22: from Neisseriaceae (F)
- A61P 31/04: Antibacterial agents
- C12N 9/1048: {Glycosyltransferases (2.4)}
- C12N 9/1051: {Hexosyltransferases (2.4.1)}
- C12P 19/18: produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

The present invention is directed to nucleic acids encoding glycosyltransferases, the proteins encoded thereby, and to methods for synthesizing oligosaccharides using the glycosyltransferases of the invention. In particular, the present application is directed to identification a glycosyltransferase locus of Neisseria gonorrhoeae containing five open reading frames for five different glycosyltransferases. The functionally active glycosyltransferases of the invention are characterized by catalyzing reactions such as adding Gal .beta..fwdarw.4 to GlcNAc or Glc; adding GalNAc or GlcNAc .beta.1.fwdarw.3 to Gal; and adding Gal .alpha.1.fwdarw.4 to Gal. The glycosyltransferases of the invention are particularly suited to the synthesis of the oligosaccharides Gal.beta.1.fwdarw.4GlcNAc.beta.1.fwdarw.3Gal.beta.1.fwdarw.4Glc (a mimic of lacto-N-neotetraose), GalNAc.beta.1.fwdarw.3Gal.beta.1.fwdarw.4GlcNAc.beta.1.fwdarw.3Gal.beta.1. fwdarw.4Glc.beta.1.fwdarw.4 (a mimic ganglioside), and Gal.alpha.1.fwdarw.4Gal.beta.1.fwdarw.4Glc.beta.1.fwdarw.4Hep.fwdarw.R (a mimic of the saccharide portion of globo-glycolipids).

## Claims

### Claim 1

A method for preparing an oligosaccharide having the structure Gal.alpha.1.fwdarw.4Gal.beta.1.fwdarw.4Glc, which comprises sequentially performing the steps of:
a) contacting a reaction mixture comprising an activated Gal to an acceptor moiety comprising a Glc residue in the presence of a glycosyltransferase having an amino acid sequence of SEQ ID NO:6; and
b) contacting a reaction mixture comprising an activated Gal to the acceptor moiety comprising Gal.beta..fwdarw.4Glc in the presence of a glycosyltransferase having an amino acid sequence of SEQ ID NO:4.

### Claim 2

A method for preparing an oligosaccharide having the structure GalNA.beta.1.fwdarw.3Gal.beta.1.fwdarw.4GlcNAc.beta.1.fwdarw.3Gal.beta.1.f wdarw.4Glc, which comprises sequentially performing the steps of:
a) contacting a reaction mixture comprising an activated Gal to an acceptor moiety comprising a Glc residue in the presence of a glycosyltransferase having an amino acid sequence of SEQ ID NO:6;
b) contacting a reaction mixture comprising an activated GlcNAc to the acceptor moiety comprising a Gal.beta.1.fwdarw.4Glc residue in the presence of a glycosyltransferase having an amino acid sequence of SEQ ID NO:3 or SEQ ID NO:11;
c) contacting a reaction mixture comprising an activated Gal to the acceptor moiety comprising a GlcNAc.beta.1.fwdarw.3Gal.beta.1.fwdarw.4Glc residue in the presence of a glycosyltransferase having an amino acid of SEQ ID NO:8; and
d) contacting a reaction mixture comprising an activated GalNAc to the acceptor moiety comprising a Gal.beta.1.fwdarw.4GlcNAc.beta.1.fwdarw.3Gal.beta.1.fwdarw.4Glc residue in the presence of a glycosyltransferase having an amino acid sequence of SEQ ID NO:5 or SEQ ID NO:12.

### Claim 3

A method for preparing an oligosaccharide having the structure Gal.beta.1.fwdarw.4GlcNAc.beta.1.fwdarw.3Gal.beta.1.fwdarw.4Glc, which comprises sequentially performing the steps of:
a) contacting a reaction mixture comprising an activated Gal to an acceptor moiety comprising a Glc residue in the presence of a glycosyltransferase having an amino acid sequence of SEQ ID NO:6;
b) contacting a reaction mixture comprising an activated GlcNAc to the acceptor moiety comprising a Gal.beta.1.fwdarw.4Glc residue in the presence of a glycosyltransferase having an amino acid sequence of SEQ ID NO:3 or SEQ ID NO:11; and
c) contacting a reaction mixture comprising an activated Gal to the acceptor moiety comprising a GlcNAc.beta.1.fwdarw.3Gal.beta.1.fwdarw.4Glc residue in the presence of a glycosyltransferase having an amino acid of SEQ ID NO:8.