Compositions and methods for producing human milk oligosaccharides

Patent metadata

Field	Value
Publication number	US-12559732-B2
Application number	US-202117999017-A
Country	US
Kind code	B2
Filing date	May 18, 2021
Priority date	May 19, 2020
Publication date	Feb 24, 2026
Grant date	Feb 24, 2026

Abstract

The present disclosure provides compositions and methods related to the production of human milk oligosaccharides (HMOs). In particular, the present disclosure provides compositions and methods for converting lactose and N-acetylglucosamine (GlcNAc) into N-acetyllactosamine (LacNAc)-enriched galactooligosaccharide (GOS) compositions using novel β-hexosyl-transferase (BHT) enzymes.

First claim

What is claimed is:

1 . A functional, recombinant β-hexosyl-transferase (rBHT) polypeptide comprising at least 90% sequence identity with SEQ ID NO: 1 and an N-terminal truncation of 56 amino acids to 81 amino acids in length, with reference to SEQ ID NO: 1.

2 . The polypeptide of claim 1 , wherein the polypeptide comprises at least 95% sequence identity with SEQ ID NO: 1.

3 . The polypeptide of claim 1 , wherein the polypeptide further comprises at least one additional amino acid substitution.

4 . The polypeptide of claim 1 , wherein the polypeptide further comprises a signal sequence.

5 . The polypeptide of claim 4 , wherein the signal sequence comprises an amino acid sequence from a protein from any one of Komagataella ( Pichia ) pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, Hansenula ( Ogataea ) polymorpha , or Kluyveromyces lactis.

6 . The polypeptide of claim 4 , wherein the signal sequence comprises a polypeptide with at least 90% sequence identity to at least one of α-mating factor signal sequence from Saccharomyces cerevisiae (MFα) (SEQ ID NO: 29), Invertase (IV) signal sequence (SEQ ID NO: 30), Glucoamylase (GA) signal sequence (SEQ ID NO: 31), or Inulinase (IN) signal sequence (SEQ ID NO: 32).

7 . The polypeptide of claim 4 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 50, 52, 54, 56, 58, 60, 68, or 70.

8 . The polypeptide of claim 1 , wherein the polypeptide comprises at least one asparagine residue at position 289, 297, 431, or 569 with respect to SEQ ID NO: 1.

9 . The polypeptide of claim 1 , wherein the polypeptide catalyzes the hydrolysis of lactose β-(1-4) glycosidic linkages.

10 . A nucleic acid molecule encoding the polypeptide of claim 1 .

11 . A vector comprising the nucleic acid molecule of claim 10 .

12 . A method of generating a galactooligosaccharide (GOD) composition from lactose in a host cell using the polypeptide of claim 1 .

13 . The method of claim 12 , wherein the GOS composition comprises LacNAc-enriched GOS or GOS lacking GlcNAc.

14 . The method of claim 13 , wherein the host cell is one or more of a yeast cell, a fungal cell, a mammalian cell, an insect cell, a plant cell, or an algal cell.

15 . The method of claim 14 , wherein the host cell comprises one or more cells from Komagataella ( Pichia ) pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, Hansenula ( Ogataea ) polymorpha , or Kluyveromyces lactis, Aspergillus spp., and Trichoderma reesei.

16 . The method of claim 13 , wherein the method produces a LacNAc-enriched GOS yield of at least 10% of initial lactose concentration, and a total GOS concentration of at least 50% of initial lactose concentration.

Assignees

• Univ North Carolina State

Inventors

• Bruno-Barcena Jose M
• Dagher Suzanne
• Azcarate-Peril Maria Andrea

Classifications

• C12P19/28

  N-glycosides · CPC title

• C12P19/18

  produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins · CPC title

• C12N15/79

  Vectors or expression systems specially adapted for eukaryotic hosts · CPC title

• A23L33/125

  containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates (indigestible substances A23L33/21) · CPC title

• C12P19/14

  produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase} · CPC title