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US 2023/0407273 A1Published application
Glycosyltransferase variants for improved protein production
AssigneeNovozymes A/S
InventorsMichiko Ihara, Takashi Nakanishi
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Abstract
The present invention relates to a polynucleotide variant encoding a glycosyltransferase variant, and to nucleic acid constructs, vectors and host cells comprising said polynucleotide variant. The invention also relates to methods of producing a polypeptide of interest in host cells comprising said polynucleotide and/or glycosyltransferase variant.
Claims21
1-13. (canceled)
14:
- A fungal host cell comprising in its genome:
- a) a first polynucleotide encoding a polypeptide of interest;
- and
- b) a second polynucleotide encoding a polypeptide having a sequence identity of at least 60% but less than 100% to SEQ ID NO:
- 7 and comprising an alteration at a position corresponding to position 17 and/or 137 of SEQ ID NO:
- 7, or encoding a polypeptide having a sequence identity of at least 60% but less than 100% to SEQ ID NO:
- 21 and comprising an alteration at a position corresponding to position 19 and/or 139 of SEQ ID NO:
- 21.
5 more elements
15:
- The fungal host cell according to claim 14, wherein the alteration of said polypeptide at the position corresponding to position 17 or 137 of SEQ ID NO:
- 7, or corresponding to position 19 or 139 of SEQ ID NO:
- 21 is independently chosen from one another, an amino acid substitution, an amino acid insertion, an amino acid deletion, or a premature polypeptide termination.
16:
- The fungal host cell according to claim 14, wherein the alteration of said polypeptide at the position corresponding to position 17 or 137 of SEQ ID NO:
- 7 or corresponding to position 19 or 139 of SEQ ID NO:
- 21 is an amino acid substitution.
17:
- The fungal host cell according to claim 16, wherein the substitution is a substitution of leucine by phenylalanine at a position corresponding to position 137 of SEQ ID NO:
- 7 (L137F), or a substitution of leucine by phenylalanine at a position corresponding to position 139 of SEQ ID NO:
- 21 (L139F), or an amino acid substitution of threonine by isoleucine at a position corresponding to position 17 of SEQ ID NO:
- 7 (T17I), or an amino acid substitution of serine by isoleucine at a position corresponding to position 19 of SEQ ID NO:
- 21 (S19I).
2 more elements
18:
- The fungal host cell according to claim 14, wherein said fungal host cell is a yeast host cell.
19:
- The fungal host cell according to claim 14, wherein said fungal host cell is a filamentous fungal host cell.
20:
- The fungal host cell according to claim 19, wherein said filamentous fungal host cell is selected from an Aspergillus cell and a Trichoderma cell.
21:
- The fungal host cell according to claim 14, wherein the polypeptide of interest comprises an enzyme.
22:
- The fungal host cell according to claim 14, wherein the polypeptide of interest is a glycoprotein.
23:
- The fungal host cell according to claim 22, wherein the polypeptide of interest is a 1,4-alpha-glucosidase.
24:
- The fungal host cell according to claim 14, wherein the polypeptide of interest is a hydrolase.
25:
- The fungal host cell according to claim 24, wherein the polypeptide of interest is a lysozyme.
26:
- A method for producing a polypeptide of interest, the method comprising:
- i) providing a fungal host cell according claim 14, and
- ii) cultivating said host cell under conditions conducive for expression of the polypeptide of interest.
27:
- The method of claim 26, further comprising:
- iii) recovering the polypeptide of interest.
28:
- A nucleic acid construct comprising a heterologous promoter operably linked to a polynucleotide encoding a polypeptide having a sequence identity of at least 60% but less than 100% to SEQ ID NO:
- 7, and comprising an alteration at a position corresponding to position 17 and/or 137 of SEQ ID NO:
- 7, or
- encoding a polypeptide having a sequence identity of at least 60% but less than 100% to SEQ ID NO:
- 21, and comprising an alteration at a position corresponding to position 19 and/or 139 of SEQ ID NO:
- 21.
3 more elements
29:
- The nucleic acid construct according to claim 28, wherein the alteration at the position corresponding to position 17 and/or 137 of SEQ ID NO:
- 7 is a substitution.
30:
- The nucleic acid construct according to claim 28, wherein the alteration at the position corresponding to position 17 and/or 137 of SEQ ID NO:
- 7 is a substitution of leucine for phenylalanine at a position corresponding to position 137 of SEQ ID NO:
- 7 (L137F), and/or a substitution of threonine for isoleucine at a position corresponding to position 17 of SEQ ID NO:
- 7 (T17I).
1 more element
31:
- The nucleic acid construct according to claim 28, wherein the alteration at the position corresponding to position 19 and/or 139 of SEQ ID NO:
- 21 is a substitution.
32:
- The nucleic acid construct according to claim 28, wherein the alteration at the position corresponding to position 19 and/or 139 of SEQ ID NO:
- 21 is a substitution of leucine for phenylalanine at a position corresponding to position 139 of SEQ ID NO:
- 21 (L139F), and/or a substitution of serine for isoleucine at a position corresponding to position 19 of SEQ ID NO:
- 21 (S19I).
1 more element
33:
- An expression vector comprising a nucleic acid construct according to claim 28.