US 12,286,658 B2
Enhancing formation of milk oligosaccharides (HMOs) by modifying lactose import in the cell
Manos Papadakis, Hørsholm (DK)
Assigned to DSM IP Assets B.V., Heerlen (DK)
Appl. No. 18/561,258
Filed by DSM IP Assets B.V., Heerlen (DK)
PCT Filed May 17, 2022, PCT No. PCT/EP2022/063311
§ 371(c)(1), (2) Date Nov. 15, 2023,
PCT Pub. No. WO2022/243308, PCT Pub. Date Nov. 24, 2022.
Claims priority of application No. PA202170249 (DK), filed on May 17, 2021.
Prior Publication US 2024/0279697 A1, Aug. 22, 2024
Int. Cl. C12P 19/18 (2006.01); C12N 9/10 (2006.01); C12N 15/52 (2006.01); C12P 19/00 (2006.01)
CPC C12P 19/00 (2013.01) [C12N 9/1051 (2013.01); C12N 15/52 (2013.01); C12Y 204/01038 (2013.01); C12Y 204/01122 (2013.01); C12Y 204/01147 (2013.01)] 20 Claims
 
1. A genetically engineered cell capable of producing lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT), or LNT and LNnT and wherein the cell
a) overexpresses one or more lactose permease genes,
b) expresses a heterologous MFS transporter protein selected from the group consisting of:
i. Vag with the amino acid sequence according to SEQ ID NO: 62, or a functional homologue thereof having an amino acid sequence which is at least 95% identical to the amino acid sequence of SEQ ID NO: 62,
ii. Nec with the amino acid sequence according to SEQ ID NO: 66, or a functional homologue thereof having an amino acid sequence which is at least 95% identical to the amino acid sequence of SEQ ID NO 66:
iii. Fred, with the amino acid sequence according to SEQ ID NO: 63, or a functional homologue thereof having an amino acid sequence which is at least 95% identical to the amino acid sequence of SEQ ID NO: 63,
iv. Marc with the amino acid sequence according to SEQ ID NO: 64, or a functional homologue thereof having an amino acid sequence which is at least 95% identical to the amino acid sequence of SEQ ID NO: 64 and
v. Bad with the amino acid sequence according to SEQ ID NO: 65, or a functional homologue thereof having an amino acid sequence which is at least 95% identical to the amino acid sequence of SEQ ID NO: 65, and
c) expresses two or more glycosyltransferases selected from the group consisting of β-1,3-GlcNAc-transferases, β-1,3-Gal-transferases and β-1,4-gal-transferases, and
d) expresses one or more polypeptides involved in the biosynthesis of activated sugars.