Modified beta-1,3-n-acetylglucosaminyltransferase polypeptides
Provided herein are variant β-1,3-N-acetylglucosaminyltransferase polypeptides capable of producing lacto-n-neotetraose, yeast cells capable of producing one or more human milk oligosaccharides, and methods of making such cells. Also, provided are fermentation compositions including the disclosed genetically modified yeast cells, and related methods of producing and recovering HMOs generated by the yeast cells.
1 . A variant β-1,3-N-acetylglucosaminyltransferase (LgtA) polypeptide comprising a G179R or G179K amino acid substitution relative to the amino acid sequence of SEQ ID NO: 1, wherein the polypeptide has an amino acid sequence that is from 90% to 99.7% identical to the amino acid sequence of SEQ ID NO: 1.
2 . The variant polypeptide of claim 1 , wherein the polypeptide further comprises one or more amino acid substitutions relative to SEQ ID NO: 1 selected from A27G, P89T, E170L, N180D, N180A, I182V, I182Y, H183P, H183S, N185G, T186G, T186D, M187P, W206N, A207V, Q211V, Q211C, Q211I, Q211L, W213S, W213N, V216L, L229A, L229P, V230A, V230D, R2331, H235R, S240N, S240Y, K242D, Y243S, Y243A, Y243L, Y243R, S244T, Q247C, 1250F, I254A, Q255D, A258D, A258R, S265H, S284Y, L288S, K290Q, and/or E294N.
3 . The variant polypeptide of claim 1 , wherein the polypeptide further comprises a E170L, I182V, V216L, or K290Q amino acid substitution relative to SEQ ID NO: 1.
4 . The variant polypeptide of claim 1 , wherein the polypeptide has an amino acid sequence that is from 95% to 99.7% identical to the amino acid sequence of SEQ ID NO: 1.
5 . The variant polypeptide of claim 1 , wherein the polypeptide has an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 5, 10, or 14-22.
6 . The variant polypeptide of claim 5 , wherein the polypeptide has an amino acid sequence of any one of SEQ ID NO: 5, 10, or 14-22.
7 . The variant polypeptide of claim 1 , wherein the polypeptide exhibits increased substrate specificity for lactose over a longer chain oligosaccharide as compared to a polypeptide having the amino acid sequence of SEQ ID NO: 1.
8 . A nucleic acid encoding the variant polypeptide of claim 1 .
9 . A isolated recombinant host cell comprising the variant polypeptide of claim 1 .
10 . The isolated recombinant host cell of claim 9 , wherein the cell is capable of producing a human milk oligosaccharide, wherein the cell comprises one or more heterologous nucleic acids that each, independently, encode one or more additional enzymes of the biosynthetic pathway of the human milk oligosaccharide.
11 . The isolated recombinant host cell of claim 10 , wherein the one or more additional enzymes comprise one or both of a β-1,4-galactosyltransferase (LgtB) and a UDP-N-acetylglucosamine diphosphorylase.
12 . The isolated recombinant host cell of cell of claim 10 wherein the one or more heterologous nucleic acids encode a protein that transports lactose into the cell, wherein the protein that transports lactose into the cell is a lactose permease or a lactose transporter.
13 . A method of producing a human milk oligosaccharide, the method comprising culturing a population of host cells of claim 9 in a culture medium under conditions suitable for the host cells to produce the human milk oligosaccharide.
14 . A fermentation composition comprising (i) a population of host cells comprising the host cell of claim 9 and (ii) a culture medium comprising a human milk oligosaccharide produced from the host cells.
15 . A method of recovering a human milk oligosaccharide from the fermentation composition of claim 14 , the method comprising:
a. separating at least a portion of the population of host cells from the culture medium;
b. contacting the separated host cells with a heated aqueous wash liquid; and
C. removing the wash liquid from the separated host cells.
16 . A method of genetically modifying a yeast cell to produce a human milk oligosaccharide, the method comprising:
a. (i) introducing a heterologous nucleic acid encoding the variant polypeptide of claim 1 into the yeast cell and (ii) introducing one or more heterologous nucleic acids that each, independently, encode one or more additional enzymes of the biosynthetic pathway of the human milk oligosaccharide into the yeast cell; or
b. introducing a heterologous nucleic acid encoding the variant polypeptide of claim 1 into the yeast cell, wherein the yeast cell comprises one or more heterologous nucleic acids that each, independently, encode one or more additional enzymes of the biosynthetic pathway of the human milk oligosaccharide.
17 . The method of claim 16 , wherein the one or more additional enzymes comprise one or both of an LgtB and a UDP-N-acetylglucosamine diphosphorylase, optionally wherein the LgtB has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25.
18 . The method of claim 16 , wherein the one or more heterologous nucleic acids encode a protein that transports lactose into the cell, wherein the protein that transports lactose into the cell is a lactose permease or a lactose transporter.
19 . The method of claim 16 , wherein the human milk oligosaccharide is LNnT, and wherein the method results in:
(i) from about a 2-fold increase to about a 6-fold increase in LNnT titer relative to a corresponding method in which a polypeptide having the amino acid sequence of SEQ ID NO: 1 is used in place of the variant polypeptide, or
(ii) an increase in the ratio of LNnT produced to para-lacto-N-neopentaose (p-LNnP) or para-lacto-N-neohexaose (p-LNnH) produced, by mass, relative to a corresponding method in which a polypeptide having the amino acid sequence of SEQ ID NO: 1 is used in place of the variant polypeptide.
20 . The host cell of claim 11 , wherein the LgtB has an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25.