Enhancing formation of human milk oligosaccharides (hmos) by modifying lactose import in the cell

A method for producing one or more human milk oligosaccharides (HMOs) comprising:

• a) providing a genetically engineered cell which
• i. overexpresses one or more lactose permeases,
• ii. expresses a heterologous MFS transporter protein selected from the group consisting of
• I. Vag with the amino acid sequence according to SEQ ID NO:
• 9, or a functional homologue thereof having an amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO:
• 9,
• II. Nec with the amino acid sequence according to SEQ ID NO:
• 6, or a functional homologue thereof having an amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO:
• 6
• III. Fred with the amino acid sequence according to SEQ ID NO:
• 8, or a functional homologue thereof having an amino acid sequence which is at least 99% identical to the amino acid sequence of SEQ ID NO:
• 8,
• IV. Marc with the amino acid sequence according to SEQ ID NO:
• 10, or a functional homologue thereof having an amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO:
• 10, and
• V. Bad with the amino acid sequence according to SEQ ID NO:
• 5, or a functional homologue thereof having an amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO:
• 5, and
• iii. 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,
• b) culturing said cell in a suitable media with added lactose, and
• c) harvesting the one or more HMOs,
• wherein the one or more HMOs are LNT, LNnT, or both.

A genetically engineered cell capable of producing LNT, LNnT, or LNT and LNnT and wherein the cell

• f) overexpresses one or more lactose permease genes,
• g) expresses a heterologous MFS transporter protein selected from the group consisting of:
• i. Vag with the amino acid sequence according to SEQ ID NO:
• 9, or a functional homologue thereof having an amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO:
• 9,
• ii. Nec with the amino acid sequence according to SEQ ID NO:
• 6, or a functional homologue thereof having an amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO 6:
• iii. Fred, with the amino acid sequence according to SEQ ID NO:
• 8, or a functional homologue thereof having an amino acid sequence which is at least 99% identical to the amino acid sequence of SEQ ID NO:
• 8,
• iv. Marc with the amino acid sequence according to SEQ ID NO:
• 10, or a functional homologue thereof having an amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO:
• 10 and
• v. Bad with the amino acid sequence according to SEQ ID NO:
• 5, or a functional homologue thereof having an amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO:
• 5, and
• h) 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 optionally,
• i) expresses a sucrose utilisation system and/or
• j) expresses one or more polypeptides involved in the biosynthesis of activated sugars.