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WO2024168219A2 - Compositions et procédés pour production améliorée d'oligosaccharides de lait humain - Google Patents

Compositions et procédés pour production améliorée d'oligosaccharides de lait humain Download PDF

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WO2024168219A2
WO2024168219A2 PCT/US2024/015115 US2024015115W WO2024168219A2 WO 2024168219 A2 WO2024168219 A2 WO 2024168219A2 US 2024015115 W US2024015115 W US 2024015115W WO 2024168219 A2 WO2024168219 A2 WO 2024168219A2
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amino acid
acid sequence
host cell
seq
hmo
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WO2024168219A3 (fr
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Christopher F. MUGLER
Jacqueline T. HUMPHRIES
Jessica Walter
Joshua A. LERMAN
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Amyris Inc
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Amyris Inc
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    • C12Y501/03014—UDP-N-acetylglucosamine 2-epimerase (non-hydrolysing) (5.1.3.14)

Definitions

  • HMOs Human milk oligosaccharides
  • 6’-sialyllactose 6’- SL
  • exemplary benefits include the promotion of the growth of protective intestinal microbes such as bifidobacteria, an increase in protection from gastrointestinal infections, a strengthening of the immune system, and an improvement in cognitive development.
  • HMOs are not found in other milk sources, such as cow or goat, the only source of HMOs has traditionally been mother's milk. In efforts to improve the nutritional value of infant formula and expand the use of HMOs for child and adult nutrition, there has been an increased interest in the synthetic production of these compounds.
  • Heterologous production of 6’-SL in yeast generally involves four non-native enzymes: sialic acid synthase, UDP-N-acetylglucosamine 2-epimerase, p-galactoside-a-2,6-sialyltransferase, and CMP-Neu5Ac synthetase.
  • sialic acid synthase UDP-N-acetylglucosamine 2-epimerase
  • p-galactoside-a-2,6-sialyltransferase p-galactoside-a-2,6-sialyltransferase
  • CMP-Neu5Ac synthetase CMP-Neu5Ac synthetase
  • the present disclosure provides host cells that are capable of producing a human milk oligosaccharide (HMO) or HMO precursor and that have been genetically modified to express one or more heterologous nucleic acids that encode an enzyme of the biosynthetic pathway for the corresponding HMO or HMO precursor.
  • the disclosure also features particular biosynthetic enzymes useful for producing certain HMOs, such as 6’-sialyllactose (6’-SL), or HMO precursors, such as sialic acid, A/-acetylmannosamine (ManNAc), and cytidine-5'-monophosphate (CMP) sialic acid, as well as nucleic acids encoding such enzymes.
  • the disclosure provides a series of sialic acid synthase, UDP-N-acetylglucosamine 2-epimerase, p-galactoside-a-2,6-sialyltransferase, and CMP- Neu5Ac synthetase polypeptides, nucleic acids encoding the same, and host cells expressing such polypeptides, as well as methods of using these compositions to produce an HMO or HMO precursor in a host cell, such as a yeast cell.
  • a host cell such as a yeast cell.
  • the enzymes described herein exhibit a series of advantageous biochemical properties and, importantly, have been shown to be capable of producing 6’-SL in a host cell (e.g., yeast cell) along with key HMO precursors of interest, including sialic acid, ManNAc, and CMP sialic acid. This, in turn, provides the benefit of allowing for the production of a given HMO in high yield and with high productivity.
  • a host cell e.g., yeast cell
  • HMO precursors of interest including sialic acid, ManNAc, and CMP sialic acid.
  • the disclosure provides a host cell capable of producing an HMO or HMO precursor.
  • the host cell comprises one or more heterologous nucleic acids that each, independently, encode: (a) a sialic acid synthase having an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOS: 1 -5; and/or (b) a UDP-N- acetylglucosamine 2-epimerase having an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 6; and/or (c) a p-galactoside-a-2,6-sialyltransferase having an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 7; and/or (d) a CMP-Neu5Ac synthetase having an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 8.
  • the disclosure provides a host cell that is capable of producing an HMO or HMO precursor and that comprises one or more heterologous nucleic acids that each, independently, encode: (a) a sialic acid synthase; and/or (b) an UDP-N-acetylglucosamine 2-epimerase; and/or (c) a p-galactoside-a-2,6-sialyltransferase; and/or (d) a CMP-Neu5Ac synthetase, wherein the host cell produces the HMO or HMO precursor at a peak yield of at least 0.2% (w/w), and/or wherein the host cell produces the HMO or HMO precursor at a peak productivity of at least 0.01 g/L/h.
  • a sialic acid synthase and/or (b) an UDP-N-acetylglucosamine 2-epimerase; and/or (c) a p-galactoside-a-2,6-s
  • the host cell comprises a heterologous nucleic acid encoding a sialic acid synthase.
  • the sialic acid synthase has an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of 99%) identical to any one of SEQ ID NOs: 1 -5.
  • the sialic acid synthase has an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to any one of SEQ ID NOs: 1 -5. In some embodiments, the sialic acid synthase has an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99%) identical to any one of SEQ ID NOs: 1 -5. In some embodiments, the sialic acid synthase has the amino acid sequence of any one of SEQ ID NOs: 1 -5.
  • the sialic acid synthase has an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of 99%) identical to SEQ ID NO: 1 . In some embodiments, the sialic acid synthase has an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 1 .
  • the sialic acid synthase has an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 1 . In some embodiments, the sialic acid synthase has the amino acid sequence of SEQ ID NO: 1 .
  • the host cell comprises a heterologous nucleic acid encoding a UDP- N-acetylglucosamine 2-epimerase.
  • the UDP-N-acetylglucosamine 2- epimerase has an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of 99%) identical to the amino acid sequence of SEQ ID NO: 6.
  • the UDP-N-acetylglucosamine 2-epimerase has an amino acid sequence that is at least 90% (e.g., at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the UDP-N-acetylglucosamine 2-epimerase has an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the UDP-N-acetylglucosamine 2-epimerase has the amino acid sequence SEQ ID NO: 6.
  • the host cell comprises a heterologous nucleic acid encoding a p- galactoside-a-2,6-sialyltransferase.
  • the p-galactoside-a-2,6-sialyltransferase has an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of 99%) identical to the amino acid sequence of SEQ ID NO: 7.
  • the p-galactoside-a-2,6-sialyltransferase has an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the p- galactoside-a-2,6-sialyltransferase has an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the p-galactoside-a-2,6-sialyltransferase has the amino acid sequence SEQ ID NO: 7.
  • the host cell comprises a heterologous nucleic acid encoding a CMP- Neu5Ac synthetase.
  • the CMP-Neu5Ac synthetase has an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of 99%) identical to the amino acid sequence of SEQ ID NO: 8.
  • the CMP-Neu5Ac synthetase has an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the CMP-Neu5Ac synthetase has an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the CMP-Neu5Ac synthetase has the amino acid sequence SEQ ID NO: 8.
  • the host cell further comprises a heterologous nucleic acid encoding an ABC transporter having an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of 99%) identical to the amino acid sequence of SEQ ID NO: 9.
  • the ABC transporter has an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 9.
  • the ABC transporter has an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the ABC transporter has an amino an acid sequence of SEQ ID NO: 9.
  • the host cell further comprises one or more heterologous nucleic acids encoding a protein that transports lactose into the cell.
  • the protein that transports lactose into the cell is a lactose permease.
  • the lactose permease has an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of 99%) identical to the amino acid sequence of SEQ ID NO: 10.
  • the lactose permease has an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the lactose permease has an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the lactose permease has the amino acid sequence SEQ ID NO: 10.
  • one or more of the heterologous nucleic acids are integrated into the genome of the cell. In some embodiments, one or more of the heterologous nucleic acids are present within one or more plasmids. In some embodiments, expression of one or more of the heterologous nucleic acids is driven by an inducible promoter or is negatively regulated by the activity of a promoter that is responsive to a small molecule.
  • the HMO is 6’-SL, lacto-N-neotetraose (LNnT), lacto-N-tetraose (LNT), lacto-N-fucopentaose (LNFP) I, LNFP II, LNFP III, LNFP V, LNFP VI, lacto-N-difucohexaose (LNDFH) I, LNDFH II, lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), fucosyllacto-N-hexaose (F-LNH) I, F-LNH II, difucosyllacto-N-hexaose (DF-LNH) I, DF-LNH II, difucosyllacto-N-neohexaose (DF-LNnH), difucosyl-para-lacto-N-
  • the host cell is a yeast cell.
  • the yeast cell is a Saccharomyces sp. cell or a Kluveromyces sp. cell.
  • the yeast cell is a Saccharomyces cerevisiae cell.
  • the yeast cell is a Kluveromyces marxianus cell.
  • the host cell produces 6’-SL at a peak yield of at least 0.2% (w/w). In some embodiments, the host cell produces 6’-SL at a peak yield of between 1% (w/w) and 10% (w/w) (e.g., between 1% (w/w) and 8% (w/w), 1% (w/w) and 6 % (w/w), 1 % (w/w) and 4 % (w/w), 1% (w/w) and 2% (w/w), 2% (w/w) and 10% (w/w), 4% (w/w) and 10% (w/w), 6% (w/w) and 10 % (w/w), or 8% (w/w) and 10% (w/w)).
  • w/w e.g., between 1% (w/w) and 8% (w/w), 1% (w/w) and 6 % (w/w), 1 % (w/w) and 4 % (w/w), 1% (w/w
  • the host cell produces 6’-SL at a peak productivity of at least 0.01 g/L/h. In some embodiments, the host cell produces 6’-SL at a peak productivity of between 0.04 g/L/h and 0.1 g/L/h (e.g., between 0.04 g/L/h and 0.09 g/L/h, 0.04 g/L/h and 0.08 g/L/h, 0.04 g/L/h and 0.07 g/L/h, 0.04 g/L/h and 0.06 g/L/h, 0.04 g/L/h and 0.05 g/L/h, 0.05 g/L/h and 0.1 g/L/h, 0.06 g/L/h and 0.1 g/L/h, 0.07 g/L/h and 0.1 g/L/h, 0.08 g/L/h and 0.1 g/L/h, or 0.09 g/L/h and 0.1 g/L/h).
  • the disclosure provides a method of producing an HMO or HMO precursor, the method comprising culturing a population of any one of the host cells described herein in a culture medium under conditions suitable for the host cells to produce the HMO or HMO precursor.
  • the culture medium comprises sucrose and lactose, optionally wherein the mass ratio of the sucrose to the lactose is less than 40.
  • the method comprises growing the population of host cells in a growth medium comprising a small molecule, wherein expression of the one or more heterologous nucleic acids is negatively regulated by the activity of a promoter responsive to the small molecule, and wherein the concentration of the small molecule in the culture medium during the culturing is sufficiently low that the promoter is no longer active.
  • the HMO is 6’-SL, LNnT, LNT, LNFP I, LNFP II, LNFP III, LNFP V, LNFP VI, LNDFH I, LNDFH II, LNH, LNnH, F-LNH I, F-LNH II, DF-LNH I, DF-LNH II, DF-LNnH, DF- para-LNH, DF-para-LNnH, TF-LNH, LST a, LST b, LST c, DS-LNT, F-LST a, F-LST b, FS-LNH, FS- LNnH I, or FDS-LNH II.
  • the HMO is 6’-SL.
  • the HMO precursor is sialic acid.
  • the HMO precursor is ManNAc.
  • the HMO precursor is CMP-sialic acid.
  • the disclosure provides a fermentation composition comprising (i) a population of host cells comprising any one of the host cells described herein and (ii) a culture medium comprising an HMO or HMO precursor produced from the host cells.
  • the HMO is 6’-SL, LNnT, LNT, LNFP I, LNFP II, LNFP III, LNFP V, LNFP VI, LNDFH I, LNDFH II, LNH, LNnH, F-LNH I, F-LNH II, DF-LNH I, DF-LNH II, DF-LNnH, DF-para-LNH, DF-para-LNnH, TF- LNH, LST a, LST b, LST c, DS-LNT, F-LST a, F-LST b, FS-LNH, FS-LNnH I, or FDS-LNH II.
  • the HMO is 6’-SL.
  • the disclosure provides a method of genetically modifying a yeast cell to produce one or more HMOs or HMO precursors, the method comprising introducing a heterologous nucleic acid that each, independently, encode (a) a sialic acid synthase having an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of 99%) identical to the amino acid sequence of any one of SEQ ID NOS: 1 -5; and/or (b) a UDP-N-acetylglucosamine 2-epimerasehaving an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of 99%) identical to the amino acid sequence of SEQ ID NO
  • the disclosure provides a method of genetically modifying a yeast cell to produce one or more HMOs or HMO precursors, the method comprising introducing a heterologous nucleic acid that each, independently, encode (a) a sialic acid synthase; and/or (b) an UDP-N- acetylglucosamine 2-epimerase; and/or (c) a p-galactoside-a-2,6-sialyltransferase; and/or (d) a CMP- Neu5Ac synthetase, wherein the host cell produces the HMO or HMO precursor at a peak yield of at least 0.2% (w/w), and/or wherein the host cell produces 6’-SL at a peak productivity of at least 0.01 g/L/h.
  • a heterologous nucleic acid that each, independently, encode (a) a sialic acid synthase; and/or (b) an UDP-N- acetylglucos
  • the sialic acid synthase has an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of 99%) identical to any one of SEQ ID NOs: 1 -5. In some embodiments, the sialic acid synthase has an amino acid sequence that is at least 90% (e.g., at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to any one of SEQ ID NOs: 1 -5.
  • the sialic acid synthase has an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99%) to any one of SEQ ID NOs: 1 -5. In some embodiments, the sialic acid synthase has the amino acid sequence of any one of SEQ ID NOs: 1 -5.
  • the sialic acid synthase has an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of 99%) identical to SEQ ID NO: 1 . In some embodiments, the sialic acid synthase has an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 1 .
  • the sialic acid synthase has an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 1 . In some embodiments, the sialic acid synthase has the amino acid sequence of SEQ ID NO: 1 . In some embodiments, the host cell comprises a heterologous nucleic acid encoding a UDP-N- acetylglucosamine 2-epimerase.
  • the UDP-N-acetylglucosamine 2-epimerase has an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of 99%) identical to the amino acid sequence of SEQ ID NO: 6.
  • the UDP-N-acetylglucosamine 2-epimerase has an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 6.
  • the UDP-N- acetylglucosamine 2-epimerase has an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the UDP-N-acetylglucosamine 2-epimerase has the amino acid sequence SEQ ID NO: 6.
  • the host cell comprises a heterologous nucleic acid comprising a p- galactoside-a-2,6-sialyltransferase.
  • the p-galactoside-a-2,6-sialyltransferase has an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of 99%) identical to the amino acid sequence of SEQ ID NO: 7.
  • the p-galactoside-a-2,6-sialyltransferase has an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the p- galactoside-a-2,6-sialyltransferase has an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the p-galactoside-a-2,6-sialyltransferase has the amino acid sequence SEQ ID NO: 7.
  • the host cell comprises a heterologous nucleic acid encoding a CMP- Neu5Ac synthetase.
  • the CMP-Neu5Ac synthetase has an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of 99%) identical to the amino acid sequence of SEQ ID NO: 8.
  • the CMP-Neu5Ac synthetase has an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the CMP-Neu5Ac synthetase has an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the CMP-Neu5Ac synthetase has the amino acid sequence SEQ ID NO: 8.
  • the host cell further comprises a heterologous nucleic acid encoding an ABC transporter that is (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of 99%) identical to the amino acid sequence of SEQ ID NO: 9.
  • the ABC transporter has an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 9.
  • the ABC transporter has an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the ABC transporter has an amino an acid sequence of SEQ ID NO: 9.
  • the host cell further comprises a heterologous nucleic acid encoding a protein that transports lactose into the cell.
  • the protein that transports lactose into the cell is a lactose permease.
  • the term “capable of producing” refers to a host cell that is genetically modified to express the enzyme(s) necessary for the production of a given compound in accordance with a biochemical pathway that produces the compound.
  • a host cell e.g., a yeast cell
  • HMO human milk oligosaccharide
  • HMO precursor is one that expresses the enzymes necessary for production of the HMO or HMO precursor according to the biosynthetic pathway for the HMO or HMO precursor of interest.
  • endogenous describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell).
  • a particular organism e.g., a human
  • a particular location within an organism e.g., an organ, a tissue, or a cell, such as a human cell.
  • exogenous describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell).
  • Exogenous materials include those that are provided from an external source to an organism or to cultured matter extracted therefrom.
  • the term "express” refers to any one or more of the following events: (1 ) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.
  • Expression of a gene of interest in a cell, tissue sample, or subject can manifest, for example, as: an increase in the quantity or concentration of mRNA encoding a corresponding protein (as assessed, e.g., using RNA detection procedures described herein or known in the art, such as quantitative polymerase chain reaction (qPCR) and RNA seq techniques), an increase in the quantity or concentration of a corresponding protein (as assessed, e.g., using protein detection methods described herein or known in the art, such as enzyme-linked immunosorbent assays (ELISA), among others), and/or an increase in the activity of a corresponding protein (e.g., in the case of an enzyme, as assessed using an enzymatic activity assay described herein or known in the art).
  • RNA detection procedures described herein or known in the art such as quantitative polymerase chain reaction (qPCR) and RNA seq techniques
  • qPCR quantitative polymerase chain reaction
  • RNA seq techniques an increase in the quantity or concentration of a corresponding protein (
  • expression cassette or “expression construct” refers to a nucleic acid construct that, when introduced into a host cell, results in transcription and/or translation of an RNA or polypeptide, respectively.
  • expression of transgenes one of skill will recognize that the inserted polynucleotide sequence need not be identical but may be only substantially identical to a sequence of the gene from which it was derived. As explained herein, these substantially identical variants are specifically covered by reference to a specific nucleic acid sequence.
  • an expression cassette is a polynucleotide construct that includes a polynucleotide sequence encoding a polypeptide for use in the invention operably linked to a promoter, e.g., its native promoter, where the expression cassette is introduced into a heterologous microorganism.
  • an expression cassette includes a polynucleotide sequence encoding a polypeptide of the invention where the polynucleotide that is targeted to a position in the genome of a microorganism such that expression of the polynucleotide sequence is driven by a promoter that is present in the microorganism.
  • the term “gene” refers to the segment of DNA involved in producing or encoding a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). Alternatively, the term “gene” can refer to the segment of DNA involved in producing or encoding a non-translated RNA, such as an rRNA, tRNA, gRNA, or micro RNA.
  • a “genetic pathway” or “biosynthetic pathway” as used herein refers to a set of at least two different coding sequences, where the coding sequences encode enzymes that catalyze different parts of a synthetic pathway to form a desired product (e.g., an HMO or HMO precursor).
  • a first encoded enzyme uses a substrate to make a first product which in turn is used as a substrate for a second encoded enzyme to make a second product.
  • the genetic pathway includes 3 or more members (e.g., 3, 4, 5, 6, 7, 8, 9, etc.), wherein the product of one encoded enzyme is the substrate for the next enzyme in the synthetic pathway.
  • host cell refers to a microorganism, such as yeast, and includes an individual cell or cell culture including a heterologous vector or heterologous polynucleotide as described herein.
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change.
  • a host cell includes cells into which a recombinant vector or a heterologous polynucleotide of the invention has been introduced, including by transformation, transfection, and the like.
  • human milk oligosaccharide and “HMO” are used interchangeably herein to refer to a group of nearly 200 identified sugar molecules that are found as the third most abundant component in human breast milk.
  • HMOs in human breast milk are a complex mixture of free, indigestible carbohydrates with many different biological roles, including promoting the development of a functional infant immune system.
  • HMOs include, without limitation, 6’-siallylactose (6’-SL), 2’- fucosyllactose (2’-FL), lacto-N-neotetraose (LNnT), 3-fucosyllactose (3-FL), difucosyllactose (DFL), lacto-N-tetraose (LNT), lacto-N-fucopentaose (LNFP) I, LNFP II, LNFP III, LNFP V, LNFP VI, lacto-N- difucohexaose (LNDFH) I, LNDFH II, lacto-N-hexaose (LNH), lacto-N-neohexaose (LNnH), fucosyllacto-N-hexaose (F-LNH) I, F-LNH II, difucosyllacto-N-hexaose (DFLNH
  • HMO precursor refers to an intermediate in an HMO biosynthetic pathway.
  • sialic acid, A/-acetylmannosamine (ManNAc), and cytidine-5'-monophosphate (CMP) sialic acid are HMO precursors as they are intermediates in the 6’SL biosynthetic pathway.
  • Percent (%) sequence identity with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software.
  • percent sequence identity values may be generated using the sequence comparison computer program BLAST.
  • percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows:
  • nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid.
  • polynucleotide and nucleic acid are used interchangeably and refer to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
  • a nucleic acid as used in the present disclosure will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs may be used that may have alternate backbones, including, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O- methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); positive backbones; non-ionic backbones, and non-ribose backbones. Nucleic acids or polynucleotides may also include modified nucleotides that permit correct read-through by a polymerase.
  • Polynucleotide sequence or “nucleic acid sequence” includes both the sense and antisense strands of a nucleic acid as either individual single strands or in a duplex. As will be appreciated by those in the art, the depiction of a single strand also defines the sequence of the complementary strand; thus, the sequences described herein also provide the complement of the sequence. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • the nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribonucleotides, and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc. Nucleic acid sequences are presented in the 5’ to 3’ direction unless otherwise specified.
  • polypeptide As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to a polymer of amino acid residues. The terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
  • Two sequences are "substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e. , 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection as described above.
  • the identity exists over a region that is at least about 50 nucleotides (or 20 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 50, 100, or 200 or more amino acids) in length.
  • Nucleic acid or protein sequences that are substantially identical to a reference sequence include “conservatively modified variants.” With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine
  • each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
  • amino acid sequences one of skill will recognize that individual substitutions in a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • amino acid groups defined in this manner can include: a "charged/polar group” including Glu (Glutamic acid or E), Asp (Aspartic acid or D), Asn (Asparagine or N), Gin (Glutamine or Q), Lys (Lysine or K), Arg (Arginine or R) and His (Histidine or H); an "aromatic or cyclic group” including Pro (Proline or P), Phe (Phenylalanine or F), Tyr (Tyrosine or Y) and Trp (Tryptophan or W); and an "aliphatic group” including Gly (Glycine or G), Ala (Alanine or A), Vai (Valine or V), Leu (Leucine or L), lie (Isoleucine or I), Met (Methionine or M), Ser (Serine or S), Thr (Threonine or T) and Cys (Cysteine or C).
  • a "charged/polar group” including Glu (Glutamic acid
  • subgroups can also be identified.
  • the group of charged/polar amino acids can be sub-divided into sub-groups including: the "positively-charged subgroup” comprising Lys, Arg and His; the "negatively-charged sub-group” comprising Glu and Asp; and the "polar sub-group” comprising Asn and Gin.
  • the aromatic or cyclic group can be sub-divided into sub-groups including: the "nitrogen ring sub-group” comprising Pro, His and Trp; and the "phenyl sub-group” comprising Phe and Tyr.
  • the aliphatic group can be sub-divided into sub-groups including: the "large aliphatic non-polar sub-group” comprising Vai, Leu, and lie; the "aliphatic si ightly-polar sub-group” comprising Met, Ser, Thr and Cys; and the "small-residue sub-group” comprising Gly and Ala.
  • conservative mutations include amino acid substitutions of amino acids within the sub-groups above, such as, but not limited to: Lys for Arg or vice versa, such that a positive charge can be maintained; Glu for Asp or vice versa, such that a negative charge can be maintained; Ser for Thr or vice versa, such that a free -OH can be maintained; and Gin for Asn or vice versa, such that a free -NH2 can be maintained.
  • the following six groups each contain amino acids that further provide illustrative conservative substitutions for one another.
  • the terms “conservative mutation,” “conservative substitution,” and “conservative amino acid substitution” refer to a substitution of one or more amino acids for one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric volume. These properties are summarized for each of the twenty naturally-occurring amino acids in Table 1 , below. Table 1. Representative physicochemical properties of naturally-occurring amino acids
  • production generally refers to an amount of compound produced by a genetically modified host cell provided herein. In some embodiments, production is expressed as a yield of the compound by the host cell. In other embodiments, production is expressed as a productivity of the host cell in producing the compound.
  • the term “overexpression” refers to a process of genetically modifying a host cell to express a polypeptide or RNA molecule in an amount that exceeds the amount of the polypeptide or RNA that would be observed in a host cell of the same species but that has not been subject to the genetic modification.
  • Exemplary methods of overexpressing a polypeptide or RNA molecule of the disclosure include expressing the polypeptide or RNA molecule in a host cell under the control of a highly active transcription regulatory element, such as a promoter or enhancer that fosters expression of the polypeptide or RNA at levels that exceed wild-type expression levels observed in an unmodified host cell of the same species.
  • promoter refers to a synthetic or naturally-derived nucleic acid that is capable of activating, increasing, or enhancing expression of a DNA coding sequence, or inactivating, decreasing, or inhibiting expression of a DNA coding sequence.
  • a promoter may contain one or more specific transcriptional regulatory sequences to further enhance or repress expression and/or to alter the spatial expression and/or temporal expression of the coding sequence.
  • a promoter may be positioned 5' (upstream) of the coding sequence under its control.
  • a promoter may also initiate transcription in the downstream (3’) direction, the upstream (5’) direction, or be designed to initiate transcription in both the downstream (3’) and upstream (5’) directions.
  • the distance between the promoter and a coding sequence to be expressed may be approximately the same as the distance between that promoter and the native nucleic acid sequence it controls. As is known in the art, variation in this distance may be accommodated without loss of promoter function.
  • the term also includes a regulated promoter, which generally allows transcription of the nucleic acid sequence while in a permissive environment (e.g., microaerobic fermentation conditions, or the presence of maltose), but ceases transcription of the nucleic acid sequence while in a non-permissive environment (e.g., aerobic fermentation conditions, or in the absence of maltose). Promoters used herein can be constitutive, inducible, or repressible.
  • heterologous refers to what is not normally found in nature.
  • heterologous nucleic acid refers to a nucleic acid not normally found in a given cell in nature.
  • a heterologous nucleic acid can be: (a) foreign to its host cell, i.e., exogenous to the host cell such that a host cell does not naturally contain the nucleic acid; (b) naturally found in the host cell, i.e., endogenous or native to the host cell, but present at an unnatural quantity in the cell (i.e., greater or lesser quantity than naturally found in the host cell); (c) be naturally found in the host cell but positioned outside of its natural locus.
  • a “heterologous” polypeptide refers to a polypeptide that is encoded by a “heterologous nucleic acid”.
  • a “heterologous” polypeptide may be naturally produced by a host cell but is encoded by a heterologous nucleic acid that has been introduced into the host cell by genetic engineering.
  • a “heterologous” polypeptide can include embodiments in which an endogenous polypeptide is produced by an expression construct and is overexpressed in the host cell compared to native levels of the polypeptide produced by the host cell.
  • the term “introducing” in the context of a nucleic acid or protein in a host cell refers to any process that results in the presence of a heterologous nucleic acid or polypeptide inside the host cell.
  • the term encompasses introducing a nucleic acid molecule (e.g., a plasmid or a linear nucleic acid) that encodes the nucleic acid of interest (e.g., an RNA molecule) or polypeptide of interest and results in the transcription of the RNA molecules and translation of the polypeptides.
  • the term also encompasses integrating the nucleic acid encoding the RNA molecules or polypeptides into the genome of a progenitor cell.
  • nucleic acid is then passed through subsequent generations to the host cell, so that, for example, a nucleic acid encoding an RNA-guided endonuclease is “pre-integrated” into the host cell genome.
  • introducing refers to translocation of a nucleic acid or polypeptide from outside the host cell to inside the host cell.
  • Various methods of introducing nucleic acids, polypeptides and other biomolecules into host cells are contemplated, including but not limited to, electroporation, contact with nanowires or nanotubes, spheroplasting, PEG 1000-mediated transformation, biolistics, lithium acetate transformation, lithium chloride transformation, and the like.
  • transformation refers to a genetic alteration of a host cell resulting from the introduction of exogenous genetic material, e.g., nucleic acids, into the host cell.
  • mutation refers to a change in the nucleotide sequence of a gene. Mutations in a gene may occur naturally as a result of, for example, errors in DNA replication, DNA repair, irradiation, and exposure to carcinogens or mutations may be induced as a result of administration of a transgene expressing a mutant gene. Mutations may result from a single nucleotide substitution or deletion.
  • operably linked refers to a functional linkage between nucleic acid sequences such that the sequences encode a desired function.
  • a coding sequence for a gene of interest is in operable linkage with its promoter and/or regulatory sequences when the linked promoter and/or regulatory region functionally controls expression of the coding sequence. It also refers to the linkage between coding sequences such that they may be controlled by the same linked promoter and/or regulatory region; such linkage between coding sequences may also be referred to as being linked in frame or in the same coding frame.
  • “Operably linked” also refers to a linkage of functional but non-coding sequences, such as an autonomous propagation sequence or origin of replication. Such sequences are in operable linkage when they are able to perform their normal function, e.g., enabling the replication, propagation, and/or segregation of a vector bearing the sequence in a host cell.
  • the term “about” is used herein to mean a value that is ⁇ 10% of the recited value.
  • FIG. 1 is a schematic diagram of the enzymes and key intermediates involved in the 6’- sialyllactose (6’-SL) biosynthetic pathway.
  • FIG. 2 is a graph showing the amount of A/-acetylmannosamine (ManNAc) produced by host cells encoding the UDP-N-acetylglucosamine 2-epimerase Cj. NeuC (SEQ ID NO: 6) in comparison to a control strain where the black vertical bars represent 95% confidence interval, and the black horizontal bar represents median value.
  • FIG. 3 shows a series of graphs of ManNAc productivity (g/l/hr) (top graph) and ManNAc yield on sucrose (%) (bottom graph) for host cells expressing the 6’-SL biosynthetic pathway with a low activity sialic acid synthase Cj.
  • NeuB enzyme SEQ ID NO: 1 ).
  • FIG. 4 is a graph showing the amount of 6’-SL produced by host cells that were genetically modified to produce 6’-SL but lack a sialic acid synthase gene and are transformed with a heterologous sialic acid synthase (SEQ ID NOS: 1 -5), where the black vertical bars represent 95% confidence interval, and the black horizontal bar represents median.
  • SEQ ID NOS: 1 -5 a heterologous sialic acid synthase
  • FIG. 5 shows a series of graphs showing the cumulative 6’-SL yield percentage for strains grown on a sucrose feed (top left), the cumulative 6’-SL productivity (g/l/hr) (top right), the cumulative sialic acid yield for strains grown on a sucrose feed (bottom left), and the fermentation cell density in grams dry cell weight (bottom right) for strains expressing the 6’-SL biosynthetic pathway including sialic acid synthases II.
  • NeuB SEQ ID NO: 2 (circles), Bp. NeuB (SEQ ID NO: 3) (diamonds), in comparison to a control expressing Cj. NeuB (SEQ ID NO: 1 ) squares.
  • FIG. 6 shows a series of graphs showing the cumulative 6’-SL yield percentage for strains grown on a sucrose feed (top left), the cumulative 6’-SL productivity (g/l/hr) (top right), the cumulative sialic acid yield for strains grown on a sucrose feed (bottom left), and the fermentation cell density in grams dry cell weight (bottom right) for strains expressing the ABC transporter Vp.
  • Kpol SEQ ID NO: 9 (circles and triangles ) in comparison to a strain that does not express an exporter (squares).
  • FIG. 7 is a graph showing the percentage of 6’-SL found in the supernatant compared to cell- associated fraction of strain of cells expressing the ABC transporter Vp. Kpol (SEQ ID NO: 9) (circles and triangles) in comparison to a control (squares).
  • the present disclosure features host cells capable of producing one or more human milk oligosaccharides (HMOs) or HMO precursors, as well as methods of using such host cells to produce an HMO or HMO precursor in high overall yield and with high productivity.
  • the host cells described herein may, for example, encode one or more heterologous nucleic acids encoding one or more enzymes of the HMO biosynthetic pathway as described in FIG. 1 .
  • the host cell may encode a sialic acid synthase, a UDP-N-acetylglucosamine 2-epimerase, a p-galactoside-a- 2,6-sialyltransferase, and/or a CMP-Neu5Ac synthetase, and the host cell may be capable of producing the HMO 6’-sialyllactose (6’-SL), among other HMOs or HMO precursors described herein, such as sialic acid, N-acetylmannosamine (ManNAc), and/or cytidine-5'-monophosphate (CMP) sialic acid.
  • a sialic acid synthase a UDP-N-acetylglucosamine 2-epimerase, a p-galactoside-a- 2,6-sialyltransferase, and/or a CMP-Neu5Ac synthetase
  • the host cell may be
  • host cells expressing one or more of the HMO biosynthetic enzymes described herein are capable of producing a desired HMO or HMO precursor with overall yield relative to host cells that do not express one or more of the HMO biosynthetic enzymes of the disclosure.
  • the following sections provide a detailed description of the host cells that may be used to produce an HMO or HMO precursor with elevated overall yield and with an overall productivity, as well as exemplary techniques for preparing such modified host cells.
  • host cells described herein may be modified so as to express one or more enzymes of the biosynthetic pathway of a target HMO.
  • host cells of the disclosure e.g., yeast cells
  • Such host cells may be modified to express the remaining or heterologous enzymes of the biosynthetic pathway.
  • a host cell e.g., a yeast cell
  • a desired HMO e.g., 6’-SL
  • HMO precursor e.g., ManNAc, sialic acid, and CMP-sialic acid
  • the host cells may be modified so as to express the remaining enzymes of the biosynthetic pathway for the desired HMO by providing the cells with one or more heterologous nucleic acid molecules that, together, encode the remaining enzymes of the biosynthetic pathway.
  • host cells of the disclosure are modified so as to express one or more enzymes of the biosynthetic pathway of a 6’-SL.
  • the one or more enzymes may include, for example, a sialic acid synthase, a UDP-N-acetylglucosamine 2-epimerase, a p-galactoside-a-2,6- sialyltransferase, or a CMP-Neu5Ac.
  • a host cell of the disclosure is modified to express: a sialic acid synthase having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of any one of SEQ ID NOS: 1 -5; and/or a UDP-N-acetylglucosamine 2- epimerase having an amino acid sequence that is at least 85% identical (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence of any one of SEQ ID NO: 6; and/or a p-galactoside-a-2,6-sialyltransferase having an amino acid
  • the host cell may be modified to express a sialic acid synthase.
  • the sialic acid synthase may have an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of 99%) identical to any one of SEQ ID NOs: 1 -5.
  • the sialic acid synthase may have an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to any one of SEQ ID NOs: 1 -5.
  • the sialic acid synthase may have an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99%) identical to any one of SEQ ID NOs: 1 -5. In some embodiments, the sialic acid synthase has the amino acid sequence of any one of SEQ ID NOs: 1 -5.
  • the host cell may include a heterologous nucleic acid encoding a UDP-N-acetylglucosamine 2-epimerase.
  • the UDP-N-acetylglucosamine 2-epimerase may have an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of 99%) identical to the amino acid sequence of SEQ ID NO: 6.
  • the UDP-N- acetylglucosamine 2-epimerase may have an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 6.
  • the UDP-N-acetylglucosamine 2-epimerase may have an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 6.
  • the UDP-N-acetylglucosamine 2-epimerase has the amino acid sequence SEQ ID NO: 6.
  • the host cell may include a heterologous nucleic acid encoding a p-galactoside-a-2,6- sialyltransferase.
  • the p-galactoside-a-2,6-sialyltransferase has an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of 99%) identical to the amino acid sequence of SEQ ID NO: 7.
  • the p-galactoside-a-2,6-sialyltransferase may have an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 7.
  • the p-galactoside-a-2,6-sialyltransferase may have an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 7.
  • the p-galactoside-a-2,6-sialyltransferase has the amino acid sequence SEQ ID NO: 7.
  • the host cell may include a heterologous nucleic acid encoding a CMP-Neu5Ac synthetase.
  • the CMP-Neu5Ac synthetase may have an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of 99%) identical to the amino acid sequence of SEQ ID NO: 8.
  • the CMP-Neu5Ac synthetase has an amino acid sequence that is at least 90% (e.g., at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 8.
  • the CMP- Neu5Ac synthetase may have an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 8.
  • the CMP-Neu5Ac synthetase has the amino acid sequence SEQ ID NO: 8.
  • host cells of the disclosure may be modified to express other enzymes of a biosynthetic pathway of a desired HMO.
  • a host cell of the disclosure may be modified to express one or more of an ABC transporter or a protein that transports lactose into the cell, such as a lactose permease.
  • Such enzymes may be expressed, for example, in host cells so as to produce an HMO or HMO precursor.
  • the host cell may include a heterologous nucleic acid encoding an ABC transporter having an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of 99%) identical to the amino acid sequence of SEQ ID NO: 9.
  • the ABC transporter may have an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 9.
  • the ABC transporter may have an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the ABC transporter has an amino an acid sequence of SEQ ID NO: 9.
  • the host cell may include one or more heterologous nucleic acids encoding a protein that transports lactose into the cell.
  • the protein that transports lactose into the cell may be a lactose permease.
  • the lactose permease has an amino acid sequence that is at least 85% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, of 99%) identical to the amino acid sequence of SEQ ID NO: 10.
  • the lactose permease may have an amino acid sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 10.
  • the lactose permease has an amino acid sequence that is at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the lactose permease has the amino acid sequence SEQ ID NO: 10.
  • Enzyme activity can be assessed using any number of assays, including assays that evaluate the overall production of at least one HMO (e.g., 6’-SL, LNnT, LNT, LNFP I, LNFP II, LNFP III, LNFP V, LNFP VI, LNDFH I, LNDFH II, LNH, LNnH, F-LNH I, F-LNH II, DFLNH I, DFLNH II, DFLNnH, DF- para-LNH, DF-para-LNnH, TF-LNH, LST a, LST b, LST c, DS-LNT, F-LST a, F-LST b, FS-LNH, FS- LNnH I, or FDS-LNH II) or HMO precursor (e.g., sialic acid, ManNAc, or CMP-sialic acid) by a host cell (e.g., yeast cell) strain.
  • HMO precursor
  • a host cell including one or more heterologous nucleic acids encoding a sialic acid synthase, a UDP-N-acetylglucosamine 2-epimerase, a p-galactoside-a-2,6- sialyltransferase, and/or a CMP-Neu5Ac enzymes described herein increases HMO or HMO precursor production, for example, by at least 1%, at least 5%, at least10%, at least 20%, at least 30%, at least 40%, at least 50%, or greater, when expressed in a host cell (e.g., a yeast strain described herein) as compared to a counterpart host cell of the same strain that does not express the same sialic acid synthase, a UDP-N-acetylglucosamine 2-epimerase, a p-galactoside-a-2,6- sialyltransferase, or a CMP-Neu5Ac enzyme described herein.
  • a host cell including one or more heterologous nucleic acids encoding a sialic acid synthase, a UDP-N-acetylglucosamine 2-epimerase, a p-galactoside-a-2,6- sialyltransferase, and/or a CMP-Neu5Ac enzymes described herein has an increased productivity (g/L/hr) for producing a target HMO (e.g., 6’-SL) by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or greater, when expressed in a host cell compared to a counterpart host cell of the same strain that does not express the same sialic acid synthase, a UDP- N-acetylglucosamine 2-epimerase, a p-galactoside-a-2,6-sialyltransferase, and/or a CMP-Neu5Ac described
  • a host cell including one or more heterologous nucleic acids encoding a sialic acid synthase, a UDP-N-acetylglucosamine 2-epimerase, a p-galactoside-a-2,6- sialyltransferase, and/or a CMP-Neu5Ac enzymes described herein has an increased productivity (g/L/hr) for producing an HMO precursor (e.g., sialic acid, ManNAc, or CMP-sialic acid) by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or greater, when expressed in a host cell compared to a counterpart host cell of the same strain that does not express the same sialic acid synthase, a UDP-N-acetylglucosamine 2-epimerase, a p-galactoside-a-2,6- sialyltransferase, and
  • the host cells (e.g., yeast cells) of the disclosure are capable of producing the HMO precursor sialic acid. In some embodiments, the host cells (e.g., yeast cells) of the disclosure are capable of producing the HMO precursor ManNAc. In some embodiments, the host cells (e.g., yeast cells) of the disclosure are capable of producing the HMO precursor CMP-sialic acid.
  • the host cells e.g., yeast cells
  • the activated sugar UDP-glucose is composed of a pyrophosphate group, the pentose sugar ribose, glucose, and the nucleobase uracil.
  • UDP-glucose is natively produced by yeast cells, and its production levels can be increased with overexpression of, for example, phosphoglucomutase-2 (PGM2) or UTP glucose-1 -phosphate uridylyltransferase (UGP1 ).
  • PGM2 phosphoglucomutase-2
  • the host cells e.g., yeast cells
  • the activated sugar UDP-galactose is composed of a pyrophosphate group, the pentose sugar ribose, galactose, and the nucleobase uracil.
  • UDP- galactose is natively produced by yeast cells, and its production levels can be increased with overexpression of, for example, UDP-glucose-4-epimerase (GAL10).
  • GAL10 UDP-glucose-4-epimerase
  • the host cells e.g., yeast cells
  • the activated sugar UDP-N- acetylglucosamine consists of a pyrophosphate group, the pentose sugar ribose, N- acetylglucosamine, and the nucleobase uracil.
  • UDP-N-acetylglucosamine is natively produced by yeast cells, and its production levels can be increased with expression of, for example, UDP-N- acetylglucosamine-diphosphorylase, or overexpression of, for example, glucosamine 6-phosphate N- acetyltransferase (GNA1 ) or phosphoacetylglucosamine mutase (PCM1 ).
  • GUA1 glucosamine 6-phosphate N- acetyltransferase
  • PCM1 phosphoacetylglucosamine mutase
  • the host cells e.g., yeast cells
  • the activated sugar GDP-fucose consists of a pyrophosphate group, the pentose sugar ribose, fucose, and the nucleobase guanine.
  • GDP-fucose is not natively produced by yeast cells, and its production can be enabled with the introduction of, for example, GDP-mannose 4,6-dehydratase, e.g., from Escherichia coli, and GDP-L-fucose synthase, e.g., from Arabidopsis thaliana.
  • the host cells e.g., yeast cells
  • the activated sugar CMP-sialic acid consists of a pyrophosphate group, the pentose sugar ribose, sialic acid, and the nucleobase cytosine.
  • CMP-sialic acid is not natively produced by yeast cells, and its production can be enabled with the introduction of, for example, CMP-Neu5Ac synthetase, e.g., from Campylobacter jejuni, sialic acid synthase, e.g., from C. jejuni, and UDP-N-acetylglucosamine 2-epimerase, e.g., from C. jejuni.
  • the host cells e.g., yeast cells
  • the host cell may further include one or more heterologous nucleic acids encoding one or more of GDP-mannose 4,6-dehydratase, e.g., from Escherichia coli, GDP-L-fucose synthase, e.g., from Arabidopsis thaliana, a-1 ,2-fucosyltransferase, e.g., from Helicobacter pylori, and a fucosidase, e.g., an a-1 ,3-fucosidase.
  • the fucosyltransferase is from Candidata moranbacterium or Pseudoalteromonas haloplanktis.
  • the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of GDP-mannose to GDP-4-dehydro-6-deoxy-D-mannose, e.g., a GDP-mannose 4,6-dehydratase.
  • the GDP-mannose 4,6-dehydratase is from Escherichia coli.
  • GDP-mannose 4,6- dehydratase sources include, for example and without limitation, Caenorhabditis elegans, Homo sapiens, Arabidopsis thaliana, Dictyostelium discoideum, Mus musculus, Drosophila melanogaster, Sinorhizobium fred/ HH103, Sinorhizobium fred/ NGR234, Planctomycetes bacterium RBG_13_63_9, Silicibacter sp. TrichCH4B, Pandoraea vervacti, Bradyrhizobium sp. YR681 , Epulopiscium sp.
  • the GDP-mannose dehydratase is from Caenorhabditis briggsae or Escherichia coli.
  • the host cells e.g., yeast cells
  • the host cells may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of GDP-4-dehydro-6- deoxy-D-mannose to GDP-L-fucose, e.g., a GDP-L-fucose synthase.
  • the GDP-L-fucose synthase is from Arabidopsis thaliana.
  • GDP-L-fucose synthase sources include, for example and without limitation, Mus musculus, Escherichia coli K- ⁇ , Homo sapiens, Marinobacter salaries, Sinorhizobium fred/ NGR234, Oryza sat/'va Japonica Group, Micavibrio aeruginosavorus ARL-13, Citrobacter sp.
  • the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of GDP-L-fucose and lactose to 2’-FL, e.g., an a-1 ,2-fucosyltransferase.
  • the a-1 ,2- fucosyltransferase is from Helicobacter pylori.
  • the fucosyltransferase is from Candidata moranbacterium or Pseudoalteromonas haloplanktis ANT/505.
  • Suitable a-1 ,2- fucosyltransferase sources include, for example and without limitation, Escherichia coli, Sus scrota, Homo sapiens, Chlorocebus sabaeus, Pan troglodytes, Macaca mulatta, Oryctolagus cuniculus, Pongo pygmaeus, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Hylobates lar, Bos taurus, Hylobates agilis, Eulemur fulvus, and Helicobacter hepaticus ATCC 51449.
  • the source of the a-1 ,2-fucosyltransferase is Pseudoalteromonas haloplanktis ANT/505, Candidates moranbacteria bacterium, Acetobacter sp. CAG:267, Bacteroides vulgatus, Sulfurovum lithotrophicum, Thermosynechococcus elongatus BP-1 , Geobacter uraniireducens Rf4, Bacteroides fragilis str. S23L17, Chromobacterium vaccinii, Herbaspirillum sp. YR522, or Helicobacter bills ATCC 43879.
  • the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of difucosyllactose to 2’-FL and fucose, e.g., an a1 -3,4-fucosidase.
  • Suitable a1 -3,4-fucosidase sources include, for example and without limitation, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium longum subsp.
  • the host cells e.g., yeast cells
  • the host cell may further include one or more heterologous nucleic acids encoding one or more of GDP-mannose 4,6-dehydratase, e.g., from Escherichia coli, GDP-L-fucose synthase, e.g., from Arabidopsis thaliana, a-1 ,3-fucosyltransferase, e.g., from Helicobacter pylori, and a fucosidase, e.g., an a-1 ,2-fucosidase.
  • GDP-mannose 4,6-dehydratase e.g., from Escherichia coli
  • GDP-L-fucose synthase e.g., from Arabidopsis thaliana
  • a-1 ,3-fucosyltransferase e.g., from Helicobacter pylori
  • the host cells e.g., yeast cells
  • the host cells may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of GDP-L-fucose and lactose to 3-fucosyllactose, e.g., an a-1 ,3-fucosyltransferase.
  • the a-1 ,3- fucosyltransferase is from Helicobacter pylori.
  • Suitable a-1 ,3-fucosyltransferase sources include, for example and without limitation, Homo sapiens, Escherichia coli, Sus scrofa, Chlorocebus sabaeus, Pan troglodytes, Macaca mulatta, Oryctolagus cuniculus, Pongo pygmaeus, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Hylobates lar, Bos taurus, Hylobates agilis, Eulemur fulvus, Helicobacter hepaticus ATCC 51449, Akkermansia muciniphila, Bacteroides fragilis, and Zea mays.
  • the host cells e.g., yeast cells
  • the host cell may further include one or more heterologous nucleic acids encoding one or more of p-1 ,3-A/-acetylglucosaminyltransferase, e.g., from Neisseria meningitidis, p-1 ,3-galactosyltransferase, e.g., from Escherichia coli, and UDP-N-acetylglucosamine- diphosphorylase, e.g., from E. coli.
  • p-1 ,3-A/-acetylglucosaminyltransferase e.g., from Neisseria meningitidis
  • p-1 ,3-galactosyltransferase e.g., from Escherichia coli
  • UDP-N-acetylglucosamine- diphosphorylase e.g., from E
  • the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of UDP-N-acetyl- alpha-D-glucosamine and lactose to lacto-N-triose II and UDP, e.g., a p-1 ,3-N- acetylglucosaminyltransferase.
  • the p-1 ,3-A/-acetylglucosaminyltransferase is from Neisseria meningitidis.
  • p-1 ,3-A/-acetylglucosaminyltransferase sources include, for example and without limitation, Arabidopsis thaliana, Streptococcus dysgalactiae subsp. equisimilis, Escherichia coli, e.g., Escherichia coli K-12, Pseudomonas aeruginosa PAO1 , Homo sapiens, Mus musculus, Mycobacterium smegmatis str.
  • silvaticum Oenococcus oeni, Neisseria gonorrhoeae, Propionibacterium freudenreichii subsp. shermanii, Escherichia coli 0157:H7, Aggregatibacter actinomycetemcomitans, Bradyrhizobium diazoefficiens USDA 1 10, Francisella tularensis subsp. novicida U1 12, Komagataeibacter xylinus, Haemophilus influenzae Rd KW20, Fusobacterium nucleatum subsp.
  • nucleatum ATCC 25586 Bacillus phage SPbeta, Coccidioides posadasii, Populus tremula x Populus alba, Rhizopus microsporus var. oligosporus, Streptococcus parasanguinis, Shigella flexneri, Caenorhabditis elegans, Hordeum vulgare, Synechocystis sp. PCC 6803 substr.
  • Kazusa Streptococcus agalactiae, Plasmopara viticola, Staphylococcus epidermidis RP62A, Shigella phage Sfll, Plasmid pWQ799, Fusarium graminearum, Sinorhizobium meliloti 1021 , Physcomitrella patens, Sphingomonas sp. S88, Streptomyces hygroscopicus subsp. jinggangensis 5008, Drosophila melanogaster, Phytophthora infestans, Staphylococcus aureus subsp. aureus Mu50, Penicillium chrysogenum, and Tribolium castaneum.
  • the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of UDP-galactose and lacto-N-triose II to lacto-N-tetraose and UDP, e.g., a p-1 ,3-galactosyltransferase.
  • the p-1 ,3-galactosyltransferase is from Escherichia coli.
  • p-1 ,3- galactosyltransferase sources include, for example and without limitation, Arabidopsis thaliana, Streptococcus dysgalactiae subsp. equisimilis, Pseudomonas aeruginosa PAO1 , Homo sapiens, Mus musculus, Mycobacterium smegmatis str.
  • MC2 155 Dictyostelium discoideum, Komagataeibacter hansenii, Aspergillus nidulans FGSC A4, Schizosaccharomyces pombe 972h-, Neurospora crassa OR74A, Aspergillus fumigatus Af293, Ustilago maydis 521 , Bacillus subtilis subsp. subtilis str. 168, Rattus norvegicus, Neisseria meningitidis, Listeria monocytogenes EGD-e, Bradyrhizobium japonicum, Nostoc sp.
  • PCC 7120 Haloferax volcanii DS2, Caulobacter crescentus CB15, Mycobacterium avium subsp. silvaticum, Oenococcus oeni, Neisseria gonorrhoeae, Propionibacterium freudenreichii subsp. shermanii, Aggregatibacter actinomycetemcomitans, Bradyrhizobium diazoefficiens USDA 1 10, Francisella tularensis subsp. novicida U1 12, Komagataeibacter xylinus, Haemophilus influenzae Rd KW20, Fusobacterium nucleatum subsp.
  • nucleatum ATCC 25586 Bacillus phage SPbeta, Coccidioides posadasii, Populus tremula x Populus alba, Rhizopus microsporus var. oligosporus, Streptococcus parasanguinis, Shigella flexneri, Caenorhabditis elegans, Hordeum vulgare, Synechocystis sp. PCC 6803 substr.
  • Kazusa Streptococcus agalactiae, Plasmopara viticola, Staphylococcus epidermidis RP62A, Shigella phage Sfll, Plasmid pWQ799, Fusarium graminearum, Sinorhizobium meliloti 1021 , Physcomitrella patens, Sphingomonas sp. S88, Streptomyces hygroscopicus subsp. jinggangensis 5008, Drosophila melanogaster, Phytophthora infestans, Staphylococcus aureus subsp. aureus Mu50, Penicillium chrysogenum, and Tribolium castaneum.
  • the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of N-acetyl-a-D- glucosamine 1 -phosphate to UDP-N-acetyl-a-D-glucosamine, e.g., a UDP-N-acetylglucosamine- diphosphorylase.
  • the UDP-N-acetylglucosamine-diphosphorylase is from Escherichia coli.
  • the host cells e.g., yeast cells
  • the host cell may further include one or more heterologous nucleic acids encoding one or more of p-1 ,3-A/-acetylglucosaminyltransferase, e.g., from Neisseria meningitidis, p-1 ,4-galactosyltransferase, e.g., from N. meningitidis, and UDP-N-acetylglucosamine- diphosphorylase, e.g., from E. coli.
  • the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of UDP-galactose and lacto-N-triose II to lacto N-neotetraose and UDP, e.g., a p-1 ,4-galactosyltransferase.
  • the p-1 ,4-galactosyltransferase is from Neisseria meningitidis.
  • p-1 ,4- galactosyltransferase sources include, for example and without limitation, Homo sapiens, Neisseria gonorrhoeae, Haemophilus influenzae, Acanthamoeba polyphaga mimivirus, Haemophilus influenzae Rd KW20, Haemophilus ducreyi 35000HP, Moraxella catarrhalis, [Haemophilus] ducreyi, Aeromonas salmonicida subsp. salmonicida A449, and Helicobacter pylori 26695.
  • the host cells e.g., yeast cells
  • the host cells are capable of producing 3’-sialyllactose.
  • the host cells may further include heterologous nucleic acids encoding CMP-Neu5Ac synthetase, e.g., from Campylobacter jejuni, sialic acid synthase, e.g., from C. jejuni, UDP-N-acetylglucosamine 2-epimerase, e.g., from C. jejuni, UDP-N-acetylglucosamine- diphosphorylase, e.g., from E. coli, and CMP-N-acetylneuraminate-p-galactosamide-a-2,3- sialyltransferase, e.g., from N. meningitides MC58.
  • CMP-Neu5Ac synthetase e.g., from Campylobacter jejuni
  • the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of UDP-N-acetyl-a- D-glucosamine to N-acetyl-mannosamine and UDP, e.g., a UDP-N-acetylglucosamine 2-epimerase.
  • the UDP-N-acetylglucosamine 2-epimerase is from Campylobacter jejuni.
  • UDP-N-acetylglucosamine 2-epimerase sources include, for example and without limitation, Homo sapiens, Rattus norvegicus, Mus musculus, Dictyostelium discoideum, Plesiomonas shigelloides, Bacillus subtilis subsp. subtilis str. 168, Bacteroides fragilis, Geobacillus kaustophilus HTA426, Synechococcus sp. CC9311 , Sphingopyxis alaskensis RB2256, Synechococcus sp. RS9916, Moorella thermoacetica ATCC 39073, Psychrobacter sp.
  • MIT 9211 Subdoligranulum variabile DSM 15176, Kordia algicida OT-1 , Bizionia argentinensis JUB59, Tannerella forsythia 92A2, Thiomonas arsenitoxydans, Synechococcus sp. BL107, Escherichia coli, Vibrio campbellii ATCC BAA-1116, Rhodopseudomonas palustris HaA2, Roseobacter litoralis Och 149, Synechococcus sp. CC9311 , Subdoligranulum variabile DSM 15176, Bizionia argentinensis JUB59, Selenomonas sp.
  • the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of N-acetyl- mannosamine and phosphoenolpyruvate to N-acetylneuraminate, e.g., a sialic acid synthase.
  • a sialic acid synthase is from Campylobacter jejuni.
  • Other suitable sialic acid synthase sources include, for example and without limitation, Homo sapiens, groundwater metagenome, Prochlorococcus marinus str.
  • MIT 9211 Rhodospirillum centenum SW, Rhodobacter capsulatus SB 1003, Aminomonas paucivorans DSM 12260, Ictalurus punctatus, Octadecabacter antarcticus 307 , Octadecabacter arcticus 238, Butyrivibrio proteoclasticus B316, Neisseria meningitidis serogroup B., Idiomarina loihiensis L2TR, Butyrivibrio proteoclasticus B316, and Campylobacter jejuni.
  • the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of N- acetylneuraminate and CTP to CMP-N-acetylneuraminate, e.g., a CMP-Neu5Ac synthetase.
  • the CMP-Neu5Ac synthetase is from Campylobacter jejuni.
  • CMP- Neu5Ac synthetase sources include, for example and without limitation, Neisseria meningitidis, Streptococcus agalactiae NEM316, Homo sapiens, Mus musculus, Bacteroides thetaiotaomicron, Pongo abelii, Danio rerio, Oncorhynchus mykiss, Bos taurus, Drosophila melanogaster, and Streptococcus suis BM407.
  • the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of CMP-N- acetylneuraminate and lactose to 3’-siallyllactose and CMP, e.g., a CMP-N-acetylneuraminate-p- galactosamide-a-2,3-sialyltransferase.
  • the CMP-N-acetylneuraminate-p- galactosamide-a-2,3-sialyltransferase is from N. meningitides MC58.
  • CMP-N- acetylneuraminate-p-galactosamide-a-2,3-sialyltransferase sources include, for example and without limitation, Homo sapiens, Neisseria meningitidis alpha14, Pasteurella multocida subsp. multocida str. Pm70, Pasteurella multocida, and Rattus norvegicus.
  • the host cells e.g., yeast cells
  • the host cell may further include one or more heterologous nucleic acids encoding one or more of CMP-Neu5Ac synthetase, e.g., from Campylobacter jejuni, sialic acid synthase, e.g., from C. jejuni, UDP-N-acetylglucosamine 2-epimerase, e.g., from C.
  • UDP-N- acetylglucosamine-diphosphorylase e.g., from E. coli
  • p-galactoside a-2,6-sialyltransferase e.g., from Photobacterium sp. JT-ISH-224.
  • the host cells (e.g., yeast cells) of the disclosure may include a heterologous nucleic acid encoding an enzyme that can catalyze the conversion of CMP-N- acetylneuraminate and lactose to 3’-sialyllactose and CMP, e.g., a p-galactoside-a-2,6- sialyltransferase.
  • the p-galactoside-a-2,6-sialyltransferase is from Photobacterium sp. JT-ISH-224.
  • p-galactoside-a-2,6-sialyltransferase sources include, for example and without limitation, Homo sapiens, Photobacterium damselae, Photobacterium leiognathi, and Photobacterium phosphoreum ANT-2200.
  • the host cell is a yeast cell, such as Saccharomyces cerevisiae.
  • Saccharomyces cerevisiae strains suitable for genetic modification and cultivation to produce HMOs or HMO precursors as disclosed herein include, but are not limited to, Baker's yeast, CBS 7959, CBS 7960, CBS 7961 , CBS 7962, CBS 7963, CBS 7964, IZ-1904, TA, BG-1 , CR-1 , SA-1 , M-26, Y-904, PE-2, PE-5, VR-1 , BR-1 , BR-2, ME-2, VR-2, MA-3, MA-4, CAT-1 , CB-1 , NR-1 , BT-1 , CEN.PK, CEN.PK2, and AL-1 .
  • the host cell is a strain of Saccharomyces cerevisiae selected from the group consisting of PE-2, CAT-1 , VR-1 , BG-1 , CR-1 , and SA-1 .
  • the strain of Saccharomyces cerevisiae is PE-2.
  • the strain of Saccharomyces cerevisiae is CAT-1 .
  • the strain of Saccharomyces cerevisiae is BG-1.
  • the host cell is Kluyveromyces marxianus.
  • Kluyveromyces marxianus can provide several advantages for industrial production, including high temperature tolerance, acid tolerance, native uptake of lactose, and rapid growth rate. Beneficially, this yeast has sufficient genetic similarity to Saccharomyces cerevisiae such that similar or identical promoters and codon optimized genes can be used among the two yeast species. Furthermore, because Kluyveromyces marxianus has a native lactose permease, it is not necessary to introduce a heterologous nucleic acid to introduce this functionality.
  • the modified Kluyveromyces marxianus strain is capable of importing lactose without consuming it.
  • the expression of the p-galactosidase gene in the genetically modified yeast is decreased relative to the expression in wild-type Kluyveromyces marxianus.
  • the modified Kluyveromyces marxianus strain has reduced consumption of imported lactose.
  • the host cells (e.g., yeast cells) of the disclosure may include a promoter that regulates the expression and/or stability of at least one of the heterologous nucleic acids described herein.
  • the promoter negatively regulates the expression and/or stability of the at least one heterologous nucleic acid.
  • the promoter can be responsive to a small molecule that may be present in a culture medium containing the host cell.
  • the small molecule is maltose or an analog or derivative thereof.
  • the small molecule is lysine or an analog or derivative thereof. Maltose and lysine can be attractive selections for the small molecule as they are relatively inexpensive, non-toxic, and stable.
  • the promoter that regulates expression of a heterologous nucleic acid described herein is a relatively weak promoter, or an inducible promoter.
  • Illustrative promoters include, for example, lower-strength GAL pathway promoters, such as GAL10, GAL2, and GAL3 promoters. Additional illustrative promoters for use in conjunction with the heterologous nucleic acids of the disclosure include constitutive promoters from S. cerevisiae, such as the promoter from the native TDH3 gene.
  • a lower strength promoter provides a decrease in expression of at least 25%, or at least 30%, 40%, or 50%, or more, when compared to a GAL1 promoter.
  • heterologous nucleic acid molecule described herein may be accomplished by introducing the heterologous nucleic acid into the host cells under the control of regulatory elements that permit expression in the host cell.
  • the heterologous nucleic acid is an extrachromosomal plasmid.
  • the heterologous nucleic acid is a chromosomal integration vector that can integrate the nucleotide sequence of interest into the chromosome of the host cell.
  • a heterologous nucleic acid of the disclosure is introduced into a host cell (e.g., yeast cell) by way of a gap repair molecular biology technique.
  • a host cell e.g., yeast cell
  • a gap repair molecular biology technique e.g., yeast cell
  • NHEJ non-homologous end joining
  • the NHEJ activity in the host cell can be first disrupted in any of a number of ways.
  • Further details related to genetic modification of host cells (e.g., yeast cells) through gap repair can be found in U.S. Patent No. 9,476,065, the disclosure of which is incorporated herein by reference in its entirety.
  • a heterologous nucleic acid of the disclosure is introduced into the host cell by way of one or more site-specific nucleases capable of causing breaks at designated regions within selected nucleic acid target sites.
  • site-specific nucleases capable of causing breaks at designated regions within selected nucleic acid target sites.
  • nucleases include, but are not limited to, endonucleases, site-specific recombinases, transposases, topoisomerases, zinc finger nucleases, TAL-effector DNA binding domain-nuclease fusion proteins (TALENs), CRISPR/Cas- associated RNA-guided endonucleases, and meganucleases. Further details related to genetic modification of host cells through site specific nuclease activity can be found in U.S. Patent No. 9,476,065, the disclosure of which is incorporated herein by reference in its entirety.
  • changes in a particular gene or polynucleotide including a sequence encoding a polypeptide or enzyme can be performed and screened for activity. Typically, such changes include conservative mutations and silent mutations.
  • modified or mutated polynucleotides and polypeptides can be screened for expression of a functional enzyme using methods known in the art. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or functionally equivalent polypeptides can also be used to clone and express the polynucleotides encoding such enzymes.
  • a coding sequence can be modified to enhance its expression in a particular host.
  • the genetic code is redundant with 64 possible codons, but most organisms typically use a subset of these codons.
  • the codons that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low-usage codons. Codons can be substituted to reflect the preferred codon usage of the host, in a process sometimes called "codon optimization" or "controlling for species codon bias.”
  • Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence.
  • Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocotyledonous plants is UGA, whereas insects and E. coli commonly use UAA as the stop codon (Dalphin et al., 1996, Nucl Acids Res. 24: 216-8).
  • DNA molecules differing in their nucleotide sequences can be used to encode a given heterologous polypeptide of the disclosure.
  • a native DNA sequence encoding the biosynthetic enzymes described above is referenced herein merely to illustrate an embodiment of the disclosure, and the disclosure includes DNA molecules of any sequence that encode the amino acid sequences of the polypeptides and proteins of the enzymes utilized in the methods of the disclosure.
  • a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity.
  • the disclosure includes such polypeptides with different amino acid sequences than the specific proteins described herein so long as the modified or variant polypeptides have the enzymatic anabolic or catabolic activity of the reference polypeptide.
  • the amino acid sequences encoded by the DNA sequences shown herein merely illustrate embodiments of the disclosure.
  • a conservative amino acid substitution is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties, e.g., charge or hydrophobicity.
  • R group side chain
  • a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art (See, e.g., Pearson W. R., 1994, Methods in Mol. Biol. 25: 365-89).
  • any of the genes encoding an enzyme described herein can be optimized by genetic/protein engineering techniques, such as directed evolution or rational mutagenesis, which are known to those of ordinary skill in the art. Such action allows those of ordinary skill in the art to optimize the enzymes for expression and activity in yeast.
  • genes encoding these enzymes can be identified from other fungal and bacterial species and can be expressed for the modulation of this pathway.
  • a variety of organisms could serve as sources for these enzymes, including, but not limited to, Saccharomyces spp., including S. cerevisiae and S. uvarum, Kluyveromyces spp., including K. thermotolerans, K. lactis, and K. marxianus, Pichia spp., Hansenula spp., including H. polymorpha, Candida spp., Trichosporon spp., Yamadazyma spp., including Y. spp.
  • Sources of genes from anaerobic fungi include, but are not limited to, Piromyces spp., Orpinomyces spp., or Neocallimastix spp.
  • Sources of prokaryotic enzymes that are useful include, but are not limited to, Escherichia, coll, Zymomonas mobilis, Staphylococcus aureus, Bacillus spp., Clostridium spp., Corynebacterium spp., Pseudomonas spp., Lactococcus spp., Enterobacter spp., Salmonella spp., or X. dendrorhous.
  • Techniques known to those skilled in the art may be suitable to identify additional homologous genes and homologous enzymes.
  • analogous genes and/or analogous enzymes can be identified by functional analysis and will have functional similarities.
  • Techniques known to those skilled in the art can be suitable to identify analogous genes and analogous enzymes. Techniques include, but are not limited to, cloning a gene by PCR using primers based on a published sequence of a gene/enzyme of interest, or by degenerate PCR using degenerate primers designed to amplify a conserved region among a gene of interest. Further, one skilled in the art can use techniques to identify homologous or analogous genes, proteins, or enzymes with functional homology or similarity.
  • Techniques include examining a cell or cell culture for the catalytic activity of an enzyme through in vitro enzyme assays for said activity, e.g., as described herein or in Kiritani, K., Branched-Chain Amino Acids Methods Enzymology, 1970; then isolating the enzyme with said activity through purification; determining the protein sequence of the enzyme through techniques such as Edman degradation; design of PCR primers to the likely nucleic acid sequence; amplification of said DNA sequence through PCR; and cloning of said nucleic acid sequence.
  • suitable techniques also include comparison of data concerning a candidate gene or enzyme with databases such as BRENDA, KEGG, or MetaCYC.
  • the candidate gene or enzyme can be identified within the above-mentioned databases in accordance with the teachings herein.
  • host cells capable of producing one or more HMOs or HMO precursors and methods of producing one or more HMOs (e.g., one or more of 6’-SL, 2’-FL, LNnT, 3-FL, DFL, LNT, LNFP I, LNFP II, LNFP III, LNFP V, LNFP VI, LNDFH I, LNDFH II, LNH, LNnH, F-LNH I, F-LNH II, DFLNH I, DFLNH II, DFLNnH, DF-para-LNH, DF-para-LNnH, TF-LNH, 3’-SL, LST a, LST b, LST c, DS-LNT, F-LST a, F-LST b, FS-LNH, FS-LNnH I, or FDS-LNH II) or HMO precursors (e.g., sialic acid, ManNAc, or CMP- sialic acid).
  • the methods may include, for example, providing a population of host cells (e.g., yeast cells) capable of producing one or more HMOs or HMO precursors and subsequently introducing one or more heterologous nucleic acids encoding one or more enzymes of the HMO biosynthetic pathway.
  • a population of host cells e.g., yeast cells
  • heterologous nucleic acids encoding one or more enzymes of the HMO biosynthetic pathway.
  • the host cells of the disclosure are cultured under conditions suitable for the production of a desired HMO or HMO precursor.
  • the culturing can be performed in a suitable culture medium in a suitable container, such as a cell culture plate, a flask, or a fermentor.
  • Any suitable fermentor may be used, including, but not limited to, a stirred tank fermentor, an airlift fermentor, a bubble fermentor, or any combination thereof.
  • Saccharomyces cerevisiae as the host cell, strains can be grown in a fermentor as described in detail by Kosaric et al., in Ullmann's Encyclopedia of Industrial Chemistry, Sixth Edition, Volume 12, pages 398-473, Wiley-VCH Verlag GmbH & Co.
  • the methods can be performed at any scale of fermentation known in the art to support industrial production of microbial products.
  • Materials and methods for the maintenance and growth of cell cultures are well known to those skilled in the art of microbiology or fermentation science (see, for example, Bailey et al., Biochemical Engineering Fundamentals, second edition, McGraw Hill, New York, 1986). Consideration should be given to appropriate culture medium, pH, temperature, and requirements for aerobic, microaerobic, or anaerobic conditions, depending on the specific requirements of the host cell, the fermentation, and the process.
  • the culturing is carried out for a period of time sufficient for the transformed population to undergo a plurality of doublings until a desired cell density is reached. In some embodiments, the culturing is carried out for a period of time sufficient for the host cell population to reach a cell density (GD600) of between 0.01 and 400 in the fermentation vessel or container in which the culturing is being carried out.
  • the culturing can be carried out until the cell density is, for example, between 0.1 and 14, between 0.22 and 33, between 0.53 and 76, between 1 .2 and 170, or between 2.8 and 400.
  • the culturing can be carried until the cell density is no more than 400, e.g., no more than 170, no more than 76, no more than 33, no more than 14, no more than 6.3, no more than 2.8, no more than 1 .2, no more than 0.53, or no more than 0.23.
  • the culturing can be carried out until the cell density is greater than 0.1 , e.g., greater than 0.23, greater than 0.53, greater than 1 .2, greater than 2.8, greater than 6.3, greater than 14, greater than 33, greater than 76, or greater than 170.
  • Higher cell densities, e.g., greater than 400, and lower cell densities, e.g., less than 0.1 are also contemplated.
  • the culturing is carried for a period of time, for example, between 12 hours and 92 hours, e.g., between 12 hours and 60 hours, between 20 hours and 68 hours, between 28 hours and 76 hours, between 36 hours and 84 hours, or between 44 hours and 92 hours. In some embodiments, the culturing is carried out for a period of time, for example, between 5 days and 20 days, e.g., between 5 days and 14 days, between 6.5 days and 15.5 days, between 8 days and 17 days, between 9.5 days and 18.5 days, or between 11 days and 20 days.
  • the culturing can be carried out for less than 20 days, e.g., less than 18.5 days, less than 17 days, less than 15.5 days, less than 14 days, less than 12.5 day, less than 11 days, less than 9.5 days, less than 8 days, less than 6.5 days, less than 5 day, less than 92 hours, less than 84 hours, less than 76 hours, less than 68 hours, less than 60 hours, less than 52 hours, less than 44 hours, less than 36 hours, less than 28 hours, or less than 20 hours.
  • 20 days e.g., less than 18.5 days, less than 17 days, less than 15.5 days, less than 14 days, less than 12.5 day, less than 11 days, less than 9.5 days, less than 8 days, less than 6.5 days, less than 5 day, less than 92 hours, less than 84 hours, less than 76 hours, less than 68 hours, less than 60 hours, less than 52 hours, less than 44 hours, less than 36 hours, less than 28 hours, or less than 20 hours.
  • the culturing can be carries out for greater than 12 hours, e.g., greater than 20 hours, greater than 28 hours, greater than 36 hours, greater than 44 hours, greater than 52 hours, greater than 60 hours, greater than 68 hours, greater than 76 hours, greater than 84 hours, greater than 92 hours, greater than 5 days, greater than 6.5 days, greater than 8 days, greater than 9.5 days, greater than 11 days, greater than 12.5 days, greater than 14 days, greater than 15.5 days, greater than 17 days, or greater than 18.5 days.
  • the production of the one or more HMOs or HMO precursors by the population of host cells is inducible by an inducing compound.
  • Such host cells can be manipulated with ease in the absence of the inducing compound.
  • the inducing compound is then added to induce the production of one or more HMOs or HMO precursors by the host cells.
  • production of the one or more HMOs or HMO precursors by the host cells is inducible by changing culture conditions, such as, for example, the growth temperature, media constituents, and the like.
  • an inducing agent is added during a production stage to activate a promoter or to relieve repression of a transcriptional regulator associated with a biosynthetic pathway to promote production of one or more HMOs or HMO precursors.
  • an inducing agent is added during a build stage to repress a promoter or to activate a transcriptional regulator associated with a biosynthetic pathway to repress the production of one or more HMOs or HMO precursors, and an inducing agent is removed during the production stage to activate a promoter or to relieve repression of a transcriptional regulator to promote the production of one or more HMOs or HMO precursors.
  • the host cells may include a promoter that regulates the expression and/or stability of a heterologous nucleic acid described herein.
  • the promoter can be used to control the timing of gene expression and/or stability of proteins.
  • HMO or HMO precursor production is substantially reduced or eliminated.
  • HMO or HMO precursor production is stimulated.
  • Such a system enables the use of the presence or concentration of a selected small molecule in a fermentation medium as a switch for the production of an HMO or HMO precursor. Controlling the timing of non-catabolic compound production so as to occur only when production is desired redirects the carbon flux during the non-production phase into cell maintenance and biomass. This more efficient use of carbon can greatly reduce the metabolic burden on the host cells, improve cell growth, increase the stability of the heterologous genes, reduce strain degeneration, and/or contribute to better overall health and viability of the cells.
  • the fermentation method includes a two-step process that utilizes a small molecule as a switch to affect the “off” and “on” stages.
  • the first step i.e. , the “build” stage
  • the host cells are grown in a growth or “build” medium including the small molecule in an amount sufficient to induce the expression of genes under the control of a responsive promoter, and the induced gene products act to negatively regulate production of the non-catabolic compound.
  • the fermentation is carried out in a culture medium including a carbon source wherein the small molecule is absent or present in sufficiently low amounts such that the activity of a responsive promoter is reduced or inactive. As a result, the production of the desired non-catabolic compound by the host cells is stimulated.
  • the culture medium is any culture medium in which a host cell (e.g., yeast cell) can subsist, i.e., maintain growth and viability.
  • the culture medium is an aqueous medium including assimilable carbon, nitrogen, and phosphate sources.
  • Such a medium can also include appropriate salts, minerals, metals, and other nutrients.
  • the carbon source and each of the essential cell nutrients are added incrementally or continuously to the fermentation media, and each required nutrient is maintained at essentially the minimum level needed for efficient assimilation by growing cells, for example, in accordance with a predetermined cell growth curve based on the metabolic or respiratory function of the cells, which convert the carbon source to a biomass.
  • the method of producing one or more HMOs or HMO precursors includes culturing host cells in separate build and production culture media.
  • the method can include culturing the host cells in a build stage, wherein the cells are cultured under nonproducing conditions, e.g., non-inducing conditions, thereby producing an inoculum.
  • the inoculum may then be transferred into a second fermentation medium under conditions suitable to induce production of one or more HMOs or HMO precursors, e.g., inducing conditions.
  • Steady state conditions may then be maintained in the second fermentation stage so as to produce a cell culture containing one or more desired HMOs or HMO precursors.
  • the culture medium includes sucrose and lactose.
  • the carbon sources in the culture medium consist essentially of sucrose and lactose.
  • the carbon sources in the culture medium consist of sucrose and lactose.
  • the mass ratio of the sucrose to the lactose is selected to influence, adjust, or control the relative production rates of HMO(s) or HMO precursor(s) produced by the yeast cells. Controlling the composition of the produced HMO(s) or HMO precursor(s) in this way can advantageously permit the increasing of desired products, the decreasing of undesired products, the targeting of a desired product ratio, and the simplification of downstream product separation processes.
  • the mass ratio of the sucrose to the lactose in the culture medium can be, for example, between 3 and 40, e.g., between 3 and 25.6, between 7.6 and 29.2, between 11 .2 and 32.8, between 14.8 and 36.4, between 18.4 and 40, between 3 and 10, between 3 and 5, or between 3 and 4.
  • the mass ratio of the sucrose to the lactose can be less than 40, e.g., less than
  • the mass ratio of the sucrose to the lactose can be greater than 3, e.g., greater than 7.6, greater than 11 .2, greater than 14.8, greater than
  • Sources of assimilable nitrogen that can be used in a suitable culture medium include, but are not limited to, simple nitrogen sources, organic nitrogen sources and complex nitrogen sources. Such nitrogen sources include anhydrous ammonia, ammonium salts and substances of animal, vegetable and/or microbial origin. Suitable nitrogen sources include, but are not limited to, protein hydrolysates, microbial biomass hydrolysates, peptone, yeast extract, ammonium sulfate, urea, and amino acids. Typically, the concentration of the nitrogen sources in the culture medium is greater than about 0.1 g/L, preferably greater than about 0.25 g/L, and more preferably greater than about 1 .0 g/L.
  • the addition of a nitrogen source to the culture medium beyond a certain concentration is not advantageous for the growth of the yeast.
  • the concentration of the nitrogen sources in the culture medium can be less than about 20 g/L, e.g., less than about 10 g/L or less than about 5 g/L. Further, in some instances it may be desirable to allow the culture medium to become depleted of the nitrogen sources during culturing.
  • the effective culture medium can contain other compounds, such as inorganic salts, vitamins, trace metals, or growth promoters. Such other compounds can also be present in carbon, nitrogen or mineral sources in the effective medium or can be added specifically to the medium.
  • the culture medium can also contain a suitable phosphate source.
  • phosphate sources include both inorganic and organic phosphate sources.
  • Preferred phosphate sources include, but are not limited to, phosphate salts such as mono or dibasic sodium and potassium phosphates, ammonium phosphate and mixtures thereof.
  • the concentration of phosphate in the culture medium is greater than about 1 .0 g/L, e.g., greater than about 2.0 g/L or greater than about 5.0 g/L.
  • the addition of phosphate to the culture medium beyond certain concentrations is not advantageous for the growth of the yeast. Accordingly, the concentration of phosphate in the culture medium can be less than about 20 g/L, e.g., less than about 15 g/L or less than about 10 g/L.
  • a suitable culture medium can also include a source of magnesium, preferably in the form of a physiologically acceptable salt, such as magnesium sulfate heptahydrate, although other magnesium sources in concentrations that contribute similar amounts of magnesium can be used.
  • a source of magnesium preferably in the form of a physiologically acceptable salt, such as magnesium sulfate heptahydrate, although other magnesium sources in concentrations that contribute similar amounts of magnesium can be used.
  • the concentration of magnesium in the culture medium is greater than about 0.5 g/L, e.g., greater than about 1 .0 g/L or greater than about 2.0 g/L.
  • the addition of magnesium to the culture medium beyond certain concetrations is not advantageous for the growth of the yeast.
  • the concentration of magnesium in the culture medium can be less than about 10 g/L, e.g, less than about 5 g/L or less than about 3 g/L. Further, in some instances it may be desirable to allow the culture medium to become depleted of a magnesium source during cul
  • the culture medium can also include a biologically acceptable chelating agent, such as the dihydrate of trisodium citrate.
  • a biologically acceptable chelating agent such as the dihydrate of trisodium citrate.
  • the concentration of a chelating agent in the culture medium can be greater than about 0.2 g/L, e.g., greater than about 0.5 g/L or greater than about 1 g/L.
  • the addition of a chelating agent to the culture medium beyond certain concentrations is not advantageous for the growth of the yeast. Accordingly, the concentration of a chelating agent in the culture medium can be less than about 10 g/L, e.g., less than about 5 g/L or less than about 2 g/L.
  • the culture medium can also initially include a biologically acceptable acid or base to maintain the desired pH of the culture medium.
  • Biologically acceptable acids include, but are not limited to, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and mixtures thereof.
  • Biologically acceptable bases include, but are not limited to, ammonium hydroxide, sodium hydroxide, potassium hydroxide, and mixtures thereof. In some embodiments, the base used is ammonium hydroxide.
  • the culture medium can also include a biologically acceptable calcium source, including, but not limited to, calcium chloride.
  • a biologically acceptable calcium source including, but not limited to, calcium chloride.
  • concentration of the calcium source, such as calcium chloride, dihydrate, in the culture medium is within the range of from about 5 mg/L to about 2000 mg/L, e.g., within the range of from about 20 mg/L to about 1000 mg/L or in the range of from about 50 mg/L to about 500 mg/L.
  • the culture medium can also include sodium chloride.
  • concentration of sodium chloride in the culture medium is within the range of from about 0.1 g/L to about 5 g/L, e.g., within the range of from about 1 g/L to about 4 g/L or in the range of from about 2 g/L to about 4 g/L.
  • the culture medium can also include trace metals.
  • trace metals can be added to the culture medium as a stock solution that, for convenience, can be prepared separately from the rest of the culture medium.
  • the volume of such a trace metal solution added to the culture medium is greater than about 1 mL/L, e.g., greater than about 5 mL/L, and more preferably greater than about 10 mL/L.
  • the addition of a trace metals to the culture medium beyond certain concentrations is not advantageous for the growth of the host cells (e.g., yeast cells).
  • the amount of such a trace metals solution added to the culture medium may desirably be less than about 100 mL/L, e.g., less than about 50 mL/L or less than about 30 mL/L. It should be noted that, in addition to adding trace metals in a stock solution, the individual components can be added separately, each within ranges corresponding independently to the amounts of the components dictated by the above ranges of the trace metals solution.
  • the culture media can include other vitamins, such as pantothenate, biotin, calcium, inositol, pyridoxine-HCI, thiamine-HCI, and combinations thereof.
  • vitamins can be added to the culture medium as a stock solution that, for convenience, can be prepared separately from the rest of the culture medium.
  • the addition of vitamins to the culture medium beyond certain concentrations is not advantageous for the growth of the host cells (e.g., yeast cells).
  • the fermentation methods described herein can be performed in conventional culture modes, which include, but are not limited to, batch, fed-batch, cell recycle, continuous, and semi-continuous.
  • the fermentation is carried out in fed-batch mode.
  • some of the components of the medium are depleted during culture, e.g., during the production stage of the fermentation.
  • the culture may be supplemented with relatively high concentrations of such components at the outset, for example, of the production stage, so that growth and/or HMO production (e.g., HMO production) or HMO precuror production is supported for a period of time before additions are required.
  • HMO production e.g., HMO production
  • HMO precuror production is supported for a period of time before additions are required.
  • the preferred ranges of these components can be maintained throughout the culture by making additions as levels are depleted by culture.
  • Levels of components in the culture medium can be monitored by, for example, sampling the culture medium periodically and assaying for concentrations.
  • additions can be made at timed intervals corresponding to known levels at particular times throughout the culture.
  • the rate of consumption of nutrient increases during culture as the cell density of the medium increases.
  • addition can be performed using aseptic addition methods, as are known in the art.
  • a small amount of anti-foaming agent may be added during the culture.
  • the temperature of the culture medium can be any temperature suitable for growth of the host cells (e.g., yeast cells).
  • the culture medium prior to inoculation of the culture medium with an inoculum, can be brought to and maintained at a temperature in the range of from about 20 °C to about 45 °C, e.g., to a temperature in the range of from about 25 °C to about 40 °C, such as from about 28 °C to about 32 °C.
  • the culture medium can be brought to and maintained at a temperature of 25 °C, 25.5 °C, 26 °C, 26.5 °C, 27 °C, 27.5 °C, 28 °C, 28.5 °C, 29 °C, 29.5 °C, 30 °C, 30.5 °C, 31 °C, 31 .5 °C, 32 °C, 32.5 °C, 33 °C, 33.5 °C, 34 °C, 34.5 °C, 35 °C, 35.5 °C, 36 °C, 36.5 °C, 37 °C, 37.5 °C, 38 °C, 38.5 °C, 39 °C, 39.5 °C, or 40 °C.
  • the pH of the culture medium can be controlled by the addition of acid or base to the culture medium. In such cases, when ammonia is used to control pH, it also conveniently serves as a nitrogen source in the culture medium. In some embodiments, the pH is maintained at from about 3.0 to about 8.0, e.g., at from about 3.5 to about 7.0 or from about 4.0 to about 6.5.
  • the host cells e.g., yeast cells
  • the concentration of produced 6’-SL in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 115 g/l, between 10 g/l and 110 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced 6’-SL in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the 6’-SL concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced 6’-SL can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced 6’-SL in the culture medium can be 100 g/l or greater.
  • the yield of produced 6’-SL on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of 6’-SL on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced 2’-FL in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 115 g/l, between 10 g/l and 110 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced 2’-FL in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the 2’-FL concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced 2’-FL can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced 2’-FL in the culture medium can be 100 g/l or greater.
  • the yield of produced 2’-FL on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of 2’-FL on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced LNnT in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 115 g/l, between 10 g/l and 110 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced LNnT in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the LNnT concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced LNnT can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced LNnT in the culture medium can be 100 g/l or greater.
  • the yield of produced LNnT on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of LNnT on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced LNT in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 1 15 g/l, between 10 g/l and 1 10 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced LNT in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the LNT concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced LNT can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced LNT in the culture medium can be 100 g/l or greater.
  • the yield of produced LNT on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of LNT on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced 3-FL in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 1 15 g/l, between 10 g/l and 1 10 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced 3-FL in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the 3-FL concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced 3-FL can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced 3-FL in the culture medium can be 100 g/l or greater.
  • the yield of produced 3-FL on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of 3-FL on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced LNFP I in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 1 15 g/l, between 10 g/l and 1 10 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced LNFP I in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the LNFP I concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced LNFP I can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced LNFP I in the culture medium can be 100 g/l or greater.
  • the yield of produced LNFP I on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of LNFP I on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced LNFP II in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 1 15 g/l, between 10 g/l and 1 10 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced LNFP II in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the LNFP II concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced LNFP II can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced LNFP II in the culture medium can be 100 g/l or greater.
  • the yield of produced LNFP II on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of LNFP II on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced LNFP III in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 1 15 g/l, between 10 g/l and 1 10 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced LNFP III in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the LNFP III concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced LNFP III can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced LNFP III in the culture medium can be 100 g/l or greater.
  • the yield of produced LNFP III on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of LNFP III on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced LNFP V in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 1 15 g/l, between 10 g/l and 1 10 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced LNFP V in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the LNFP V concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced LNFP V can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced LNFP V in the culture medium can be 100 g/l or greater.
  • the yield of produced LNFP V on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of LNFP V on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced LNFP VI in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 1 15 g/l, between 10 g/l and 1 10 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced LNFP VI in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the LNFP VI concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced LNFP VI can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced LNFP VI in the culture medium can be 100 g/l or greater.
  • the yield of produced LNFP VI on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of LNFP VI on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced 3’-SL in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 115 g/l, between 10 g/l and 110 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced 3’-SL in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the 3’-SL concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced 3’-SL can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced 3’-SL in the culture medium can be 100 g/l or greater.
  • the yield of produced 3’-SL on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of 3’-SL on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced LNDFH I in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 115 g/l, between 10 g/l and 110 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced LNDFH I in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the LNDFH I concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced LNDFH I can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced LNDFH I in the culture medium can be 100 g/l or greater.
  • the yield of produced LNDFH I on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of LNDFH I on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced LNDFH II in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 115 g/l, between 10 g/l and 110 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced LNDFH II in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the LNDFH II concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced LNDFH II can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced LNDFH II in the culture medium can be 100 g/l or greater.
  • the yield of produced LNDFH II on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of LNDFH II on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced LNH in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 115 g/l, between 10 g/l and 110 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced LNH in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the LNH concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced LNH can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced LNH in the culture medium can be 100 g/l or greater.
  • the yield of produced LNH on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of LNH on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced LNnH in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 1 15 g/l, between 10 g/l and 1 10 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced LNnH in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the LNnH concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced LNnH can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced LNnH in the culture medium can be 100 g/l or greater.
  • the yield of produced LNnH on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of LNnH on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced F-LNH I in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 1 15 g/l, between 10 g/l and 1 10 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced F-LNH I in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the F-LNH I concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced F-LNH I can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced F-LNH I in the culture medium can be 100 g/l or greater.
  • the yield of produced F-LNH I on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of F-LNH I on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced F-LNH II in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 1 15 g/l, between 10 g/l and 1 10 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced F-LNH II in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the F-LNH II concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced F-LNH II can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced F-LNH II in the culture medium can be 100 g/l or greater.
  • the yield of produced F-LNH II on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of F-LNH II on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • DFL DFL
  • the concentration of produced DFL in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 1 15 g/l, between 10 g/l and 1 10 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced DFL in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the DFL concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced DFL can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced DFL in the culture medium can be 100 g/l or greater.
  • the yield of produced DFL on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of DFL on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • DFLNH I DFLNH I.
  • the concentration of produced DFLNH I in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 1 15 g/l, between 10 g/l and 1 10 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced DFLNH I in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the DFLNH I concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced DFLNH I can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced DFLNH I in the culture medium can be 100 g/l or greater.
  • the yield of produced DFLNH I on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of DFLNH I on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • DFLNH II DFLNH II.
  • the concentration of produced DFLNH II in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 1 15 g/l, between 10 g/l and 1 10 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced DFLNH II in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the DFLNH II concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced DFLNH II can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced DFLNH II in the culture medium can be 100 g/l or greater.
  • the yield of produced DFLNH II on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of DFLNH II on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • DFLNnH The concentration of produced DFLNnH in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 115 g/l, between 10 g/l and 110 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced DFLNnH in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the DFLNnH concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced DFLNnH can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced DFLNnH in the culture medium can be 100 g/l or greater.
  • the yield of produced DFLNnH on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of DFLNnH on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced DF-para-LNH in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 115 g/l, between 10 g/l and 110 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced DF-para-LNH in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the DF-para-LNH concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced DF-para-LNH can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced DF-para-LNH in the culture medium can be 100 g/l or greater.
  • the yield of produced DF-para-LNH on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of DF-para-LNH on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced DF-para-LNnH in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 1 15 g/l, between 10 g/l and 1 10 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced DF-para-LNnH in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the DF-para-LNnH concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced DF-para-LNnH can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced DF-para-LNnH in the culture medium can be 100 g/l or greater.
  • the yield of produced DF-para-LNnH on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of DF-para-LNnH on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced TF-LNH in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 1 15 g/l, between 10 g/l and 1 10 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced TF-LNH in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the TF-LNH concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced TF-LNH can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced TF-LNH in the culture medium can be 100 g/l or greater.
  • the yield of produced TF-LNH on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of TF-LNH on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced LST a in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 1 15 g/l, between 10 g/l and 1 10 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced LST a in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the LST a concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced LST a can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced LST a in the culture medium can be 100 g/l or greater.
  • the yield of produced LST a on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of LST a on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced LST b in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 1 15 g/l, between 10 g/l and 1 10 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced LST b in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the LST b concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced LST b can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced LST b in the culture medium can be 100 g/l or greater.
  • the yield of produced LST b on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of LST b on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced LST c in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 1 15 g/l, between 10 g/l and 1 10 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced LST c in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the LST c concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced LST c can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced LST c in the culture medium can be 100 g/l or greater.
  • the yield of produced LST c on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of LST c on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced DS-LNT in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 1 15 g/l, between 10 g/l and 1 10 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced DS-LNT in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the DS-LNT concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced DS-LNT can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced DS-LNT in the culture medium can be 100 g/l or greater.
  • the yield of produced DS-LNT on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of DS-LNT on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced F-LST a in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 1 15 g/l, between 10 g/l and 1 10 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced F-LST a in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the F-LST a concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced F-LST a can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced F-LST a in the culture medium can be 100 g/l or greater.
  • the yield of produced F-LST a on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of F-LST a on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced F-LST b in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 115 g/l, between 10 g/l and 110 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced F-LST b in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the F-LST b concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced F-LST b can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced F-LST b in the culture medium can be 100 g/l or greater.
  • the yield of produced F-LST b on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of F-LST b on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced FS-LNH in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 115 g/l, between 10 g/l and 110 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced FS-LNH in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the FS-LNH concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced FS-LNH can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced FS-LNH in the culture medium can be 100 g/l or greater.
  • the yield of produced FS-LNH on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of FS-LNH on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced FS-LNnH in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 115 g/l, between 10 g/l and 110 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced FS- LNnH in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the FS-LNnH concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced FS-LNnH can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced FS- LNnH in the culture medium can be 100 g/l or greater.
  • the yield of produced FS-LNnH on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of FS-LNnH on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the host cells e.g., yeast cells
  • the concentration of produced FDS-LNH II in the culture medium can be, for example, between 1 g/l and 125 g/l, e.g., between 5 g/l and 115 g/l, between 10 g/l and 110 g/l, between 15 g/l and 100 g/l, between 20 g/l and 100 g/l, or between 25 g/l and 100 g/l.
  • the concentration of produced FDS-LNH II in the culture medium can be, for example, between 5 g/l and 100 g/l, e.g., between 5 g/l and 90 g/l, between 10 g/l and 80 g/l, between 10 g/l and 75 g/l, between 20 g/l and 80 g/l, or between 20 g/l and 80 g/l.
  • the FDS-LNH II concentration can be greater than 5 g/l, e.g., greater than 8.5 g/l, greater than 12 g/l, greater than 15.5 g/l, greater than 19 g/l, greater than 22.5 g/l, greater than 26 g/l, greater than 29.5 g/l, greater than 33 g/l, or greater than 36.5 g/l.
  • concentrations of produced FDS-LNH II can be 40 g/l or greater, e.g., 50 g/l, 60 g/l, 70 g/l, 80 g/l, 90 g/l, or greater.
  • concentrations of produced FDS-LNH II in the culture medium can be 100 g/l or greater.
  • the yield of produced FDS-LNH II on the sucrose in the culture medium can be, for example, between 0.01 g/g and 0.4 g/g, e.g., between 0.01 g/g and 0.3 g/g, between 0.01 g/g and 0.2 g/g, between 0.02 g/g and 0.2 g/g, between 0.03 g/g and 0.2 g/g, between 0.04 g/g and 0.2 g/g, or between 0.04 g/g and 0.2 g/g.
  • the yield of FDS-LNH II on sucrose can be greater than 0.01 g/g, e.g., greater than 0.02 g/g, greater than 0.03 g/g, greater than 0.04 g/g, greater than 0.05 g/g, greater than 0.06 g/g, greater than 0.07 g/g, greater than 0.08 g/g, or greater than 0.09 g/g.
  • Higher yields e.g., greater than 0.1 g/g, or greater than 0.15, or greater than 0.2 g/g, are also contemplated.
  • yields are at least 0.25 g/g, e.g., 0.25 g/g, 0.26 g/g, or greater.
  • the fermentation composition further includes at least one HMO (e.g., 6’-SL, LNnT, 2’-FL, 3-FL, DFL, LNT, LNFP I, LNFP II, LNFP III, LNFP V, LNFP VI, LNDFH I, LNDFH II, LNH, LNnH, F-LNH I, F-LNH II, DFLNH I, DFLNH II, DFLNnH, DF-para-LNH, DF-para-LNnH, TF-LNH, 3’- SL, LST a, LST b, LST c, DS-LNT, F-LST a, F-LST b, FS-LNH, FS-LNnH I, or FDS-LNH II) or HMO precursor produced from the yeast cells.
  • HMO e.g., 6’-SL, LNnT, 2’-FL, 3-FL, DFL, LNT, LNFP I, LNFP II
  • the at least one HMO in the fermentation composition can include, for example, 6’-sialyllactose, 2’-fucosyllactose, difucosyllactose, 3-fucosyllactose, LNT, LNnT, para-LNnH , LNFP I, LNFP II, LNFP III, LNFP V, LNFP VI, LNDFH I, LNDFH II, LNH, LNnH, F-LNH I, F-LNH II, DF-LNH I, DF-LNH II, DF-LNnH, DF-para-LNH, DF-para-LNnH, TF-LNH, LST a, LST b, LST c, DS-LNT, F-LST a, F-LST b, FS-LNH, FS-LNnH I, FDS-LNH II, or 3’-sialyllactose.
  • the fermentation composition includes at least two HMOs.
  • the at least two HMOs in the fermentation composition can include, for example, LNnT and para-LNnH, 2’-fucosyllactose and difucosyllactose, 2’-fucosyllactose and 3-fucosyllactose, 2’-fucosyllactose and lacto-N-tetraose, 2’- fucosyllactose and lacto-N-neotetraose, 2’-fucosyllactose and 3’-sialyllactose, 2’-fucosyllactose and 6’-sialyllactose, difucosyllactose and 3-fucosyllactose, difucosyllactose and lacto-N-tetraose, difucosyllactose and lacto-N-neote,
  • the fermentation composition includes at least three HMOs produced from the yeast cells. In some embodiments, the fermentation composition includes at least four HMOs produced from the yeast cells. In some embodiments, the fermentation composition includes at least five HMOs produced from the yeast cells. In some embodiments, the fermentation composition includes at least six HMOs produced from the yeast cells. In some embodiments, the fermentation composition includes at least seven HMOs produced from the yeast cells.
  • the mass fraction of 6’-SL within the one or more produced HMOs can be, for example, between 0 and 50%, e.g., between 0 and 30%, between 5% and 35%, between 10% and 40%, between 15% and 45%, or between 20% and 40%.
  • the mass fraction of difucosyl lactose in the HMOs can be less than 50%, e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5%.
  • HMOs e.g., one or more of 6’-SL, LNnT, 2’-FL, 3-FL, DFL, LNT, LNFP I, LNFP II, LNFP III, LNFP V, LNFP VI, LNDFH I, LNDFH II, LNH, LNnH, F-LNH I, F-LNH II, DFLNH I, DFLNH II, DFLNnH, DF-para-LNH, DF-para-LNnH, TF-LNH, 3’- SL, LST a, LST b, LST c, DS-LNT, F-LST a, F-LST b, FS-LNH, FS-LNnH I, or FDS-LNH II)) or HMO precursors (e.g., sialic acid, ManNAc, or CMP-sialic acid) from a fermentation composition.
  • HMO precursors e.g., sialic acid, ManNA
  • the fermentation composition is any of the fermentation composition disclosed herein and described above.
  • the method may include separating at least a portion of a population of yeast cells from a culture medium.
  • the separating includes centrifugation.
  • the separating includes filtration.
  • HMOs e.g., one or more of 6’-SL, LNnT, 2’-FL, 3-FL, DFL, LNT, LNFP I, LNFP II, LNFP III, LNFP V, LNFP VI, LNDFH I, LNDFH II, LNH, LNnH, F-LNH I, F- LNH II, DFLNH I, DFLNH II, DFLNnH, DF-para-LNH, DF-para-LNnH, TF-LNH, 3’-SL, LST a, LST b, LST c, DS-LNT, F-LST a, F-LST b, FS-LNH, FS-LNnH I, or FDS-LNH II) or HMO precursors (e.g., sialic acid, ManNAc, or CMP-sialic acid produced by the cells during fermentation can be expected to partition with the culture medium during the separation of the yeast
  • the provided recovery methods further include contacting the separated yeast cells with a heated wash liquid.
  • the heated wash liquid is a heated aqueous wash liquid.
  • the heated wash liquid consists of water.
  • the heated wash liquid includes one or more other liquid or dissolved solid components.
  • the temperature of the heated aqueous wash liquid can be, for example, between 30 °C and 90 °C, e.g., between 30 °C and 66 °C, between 36 °C and 72 °C, between 42 °C and 78 °C, between 48 °C and 84 °C, or between 54 °C and 90 °C.
  • the wash temperature can be less than 90 °C, e.g., less than 84 °C, less than 78 °C, less than 72 °C, less than 66 °C, less than 60 °C, less than 54 °C, less than 48 °C, less than 42 °C, or less than 36°C.
  • the wash temperature can be greater than 30 °C, e.g., greater than 36 °C, greater than 42 °C, greater than 48 °C, greater than 54 °C, greater than 60 °C, greater than 66 °C, greater than 72 °C, greater than 78 °C, or greater than 84 °C.
  • Higher temperatures e.g., greater than 90 °C, and lower temperatures, e.g., less than 30 °C, are also contemplated.
  • the method may further include, subsequent to the contacting of the separated yeast cells with the heated wash liquid, removing the wash liquid from the yeast cells.
  • the removed wash liquid is combined with the separated culture medium and further processesed to isolate the produced one or more HMOs (e.g., one or more of 6’-SL, LNnT, 2’-FL, 3-FL, DFL, LNT, LNFP I, LNFP II, LNFP III, LNFP V, LNFP VI, LNDFH I, LNDFH II, LNH, LNnH, F-LNH I, F-LNH II, DFLNH I, DFLNH II, DFLNnH, DF-para-LNH, DF-para-LNnH, TF-LNH, 3’-SL, LST a, LST b, LST c, DS-LNT, F-LST a, F-LST b, FS-LNH, FS-LNnH I, or FDS-LNH
  • the recovery yield can be such that, for at least one of the one or HMOs (e.g., one or more of 6’-SL, LNnT, 2’-FL, 3-FL, DFL, LNT, LNFP I, LNFP II, LNFP III, LNFP V, LNFP VI, LNDFH I, LNDFH II, LNH, LNnH, F-LNH I, F-LNH II, DFLNH I, DFLNH II, DFLNnH, DF-para-LNH, DF-para-LNnH, TF- LNH, 3’-SL, LST a, LST b, LST c, DS-LNT, F-LST a, F-LST b, FS-LNH, FS-LNnH I, or FDS-LNH II) or HMO precursors (e.g., sialic acid, ManNAc, or CMP-sialic acid) produced from the yeast cells, the mass fraction of the
  • the recovery yield of at least one of the one or more HMOs or HMO precursors can be greater than 70%, e.g., greater than 73%, greater than 76%, greater than 79%, greater than 82%, greater than 85%, greater than 88%, greater than 91%, greater than 94%, or greater than 97%.
  • the recovery yield can be such that, for each of the one or more HMOs or HMO precursors produced from the yeast cells, the mass fraction recovered in the combined culture medium and wash liquid is, for example, between 70% and 100%, e.g., between 70% and 88%, between 73% and 91%, between 76% and 94%, between 79% and 97%, or between 82% and 100%.
  • the recovery yield of each of the one or more HMOs or HMO precursors can be greater than 70%, e.g., greater than 73%, greater than 76%, greater than 79%, greater than 82%, greater than 85%, greater than 88%, greater than 91%, greater than 94%, or greater than 97%.
  • compositions and methods provided herein have been described with respect to a limited number of embodiments, one or more features from any of the embodiments described herein or in the figures can be combined with one or more features of any other embodiment described herein in the figures without departing from the scope of the disclosure. No single embodiment is representative of all aspects of the methods or compositions. In certain embodiments, the methods can include numerous steps not mentioned herein. In certain embodiments, the methods do not include any steps not enumerated herein. Variations and modifications from the described embodiments exist.
  • an infant formula particularly an infant formula produced by: (i) culturing any one of the host cells of the disclosure in a culture medium, thereby producing a desired HMO or HMO precursor, (ii) extracting the HMO or HMO precursor, and (iii) formulating the HMO or HMO precursor for administration to an infant human subject.
  • the infant formula may be in a liquid form as a concentrate or a ready-to-drink liquid.
  • the infant formula may be in the form of a dry powder that may be reconstituted by the addition of water.
  • the infant formula may be used as a human milk replacement or supplement.
  • the infant formula is formulated such that it is suitable for consumption by an infant of less than 2 years of age, such as an infant of 23 months or less, 22 months or less, 21 months or less, 20 months or less, 19 months or less, 18 months or less, 17 months or less, 16 months or less, 15 months or less, 14 months or less, 13 months or less, 12 months or less, 11 months or less, 10 months or less, 9 months or less, 8 months or less, 7 months or less, 6 months or less, 5 months or less, 4 months or less, 3 months or less, 2 months or less, or 1 month or less.
  • the methods may include, for example, (i) culturing any one of the host cells of the disclosure in a culture medium, thereby producing a desired HMO or HMO precursor, (ii) extracting the HMO or HMO precursor, and (Hi) formulating the HMO or HMO precursor for administration to an infant human subject.
  • the infant formula of the disclosure includes one or more HMOs selected from 6’-SL, LNnT, 2’-FL, 3-FL, DFL, LNT, LNFP I, LNFP II, LNFP III, LNFP V, LNFP VI, LNDFH I, LNDFH II, LNH, LNnH, F-LNH I, F-LNH II, DFLNH I, DFLNH II, DFLNnH, DF-para-LNH, DF- para-LNnH, TF-LNH, 3’-SL, LST a, LST b, LST c, DS-LNT, F-LST a, F-LST b, FS-LNH, FS-LNnH I, and FDS-LNH II.
  • HMOs selected from 6’-SL, LNnT, 2’-FL, 3-FL, DFL, LNT, LNFP I, LNFP II, LNFP III, LNFP V, LNFP VI
  • Example 1 Generation of a yeast strain encoding enzymes of the 6’-sialyllactose (6’SL) biosynthetic pathway for the production of 6’-SL and pathway intermediates
  • a DNA construct encoding the UDP-N-acetylglucosamine 2-epimerase Cj. neuC (SEQ ID NO: 6) was introduced into a yeast strain and assayed for ManNAc production.
  • NeuB enzyme SEQ ID NO: 1
  • the product of Cj. NeuC, ManNAc was measured by ion chromatography across time in fermentation for both productivity (g/l/hr) and yield (FIG. 3).
  • DNA constructs encoding candidate sialic acid synthase proteins from various organisms were amplified and introduced into a yeast strain genetically modified to produce 6’-SL that lack the sialic acid synthase gene and transformants were assayed for increased 6’-SL production.
  • yeast strains were cultured for 2 days in 96-well plates in rich media then diluted into growth media containing a 4% sucrose/0.1% lactose minimal nutrient medium. Cultures were incubated for 3 days to sucrose exhaustion, the wells were extracted, and 6’-SL production was determined by mass spectrometry.
  • Strains expressing the 6’-SL biosynthetic pathway were run in an aerobic constant feed fermentation process continuously at 30 °C with 6 g/l/hr TRS feed rate and a mixed lactose/sucrose feed at a ratio of 1 :16 lactose:sucrose. Measurements were taken across time in 0.25 L AMBR fermentation scale and key measurements for a strain expressing the sialic acid synthases II. NeuB (SEQ ID NO: 2), Bp. NeuB (SEQ ID NO: 3), and a control strain expressing the previous-best Cj. NeuB (SEQ ID NO: 1 ) are shown.
  • strains were assessed for cumulative 6’-SL yield percentage on sucrose feed, cumulative 6’-SL productivity in grams of 6’-SL per liter of culture per hour, cumulative sialic acid yield percentage on sucrose feed, and fermentation cell density in grams dry cell weight (FIG. 5).
  • SEQ ID NO: 2 Idiomarina loihiensis sialic acid synthase
  • SEQ ID NO: 11 Wildtype Neisseria meningitidis LgtA p-1 ,3-N-acetylglucosaminyltransferase

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Abstract

L'invention concerne des cellules hôtes capables de produire un oligosaccharide de lait humain (HMO) ou un précurseur de HMO, telles que des cellules de levure qui comprennent un ou plusieurs acides nucléiques hétérologues codant pour une ou plusieurs enzymes d'une voie de biosynthèse de HMO, telle qu'une synthase d'acide sialique, une UDP-N-acétylglucosamine 2-épimérase, une β-galactoside-α-2,6-sialyltransférase et/ou une CMP-Neu5Ac synthétase. L'invention concerne également des compositions de fermentation comprenant les cellules hôtes décrites, ainsi que des procédés associés de production et de récupération de HMO ou de précurseurs de HMO générés par les cellules hôtes.
PCT/US2024/015115 2023-02-09 2024-02-09 Compositions et procédés pour production améliorée d'oligosaccharides de lait humain Ceased WO2024168219A2 (fr)

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