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US 9,222,083 B2Granted
Microorganisms genetically engineered to have modified N-glycosylation activity
AssigneesOxyrane UK Limited, Vib Vzw, Universiteit Gent
InventorsNico Luc Marc Callewaert, Wouter Vervecken, Karen Jacqueline Marcel De Pourcq, Steven Christian Jozef Geysens, Mouna Guerfal
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Abstract
Described herein are methods and genetically engineered cells useful for producing an altered N-glycosylation form of a target molecule. Also described are methods and molecules with altered N-glycosylation useful for treating a variety of disorders such as metabolic disorders.
Claims28
A method of producing an altered N-glycosylation form of a target protein, the method comprising:
- providing a Yarrowia lipolytica or an Arxula adeninivorans cell genetically engineered to express a protein that promotes phosphorylation of mannosyl moieties of N-glycans and to comprise a deficiency in Outer CHain elongation (OCH1) activity;
- and
- introducing into the cell a nucleic acid encoding a target protein, wherein the cell produces the target protein in an altered N-glycosylation form relative to the N-glycosylation form of the target protein produced in a corresponding non-genetically engineered cell, and
- wherein the protein that promotes phosphorylation of mannosyl moieties is:
- (a)(i) a wild-type MNN4 polypeptide;
- (a)(ii) a deletion variant that is the polypeptide of (a)(i) but lacks up to twenty amino acid segments, each segment consisting of two amino acids;
- (a)(iii) a deletion variant that is the polypeptide of (a)(i) or the variant of (a)(ii) but lacks up to twenty single, non-contiguous amino acids of the polypeptide of (a)(i) or the variant of (a)(ii);
- or
- (a)(iv) a substitution variant that is the polypeptide of (a)(i), the variant of (a)(ii), or the variant of (a)(iii), but with no more than 10 conservative substitutions,
- wherein the variants of (a)(ii), (a)(iii) and (a)(iv) have at least 25% of the phosphorylation-promoting activity of the polypeptide of (a)(i),
- (b)(i) a wild-type PNO1 polypeptide;
- (b)(ii) a deletion variant that is the polypeptide of (b)(i) but lacks up to twenty amino acid segments, each segment consisting of two amino acids;
- (b)(iii) a deletion variant that is the polypeptide of (b)(i) or the variant of (b)(ii) but lacks up to twenty single, non-contiguous amino acids of the polypeptide of (b)(i) or the variant of (b)(ii);
- or
- (b)(iv) a substitution variant that is the polypeptide of (b)(i), the deletion variant of (b)(ii), or the deletion variant of (b)(iii), but with no more than 10 conservative substitutions,
- wherein the variants of (b)(ii), (b)(iii), and (b)(iv) have at least 25% of the phosphorylation-promoting activity of the polypeptide of (b)(i), or
- (c) a wild-type MNN6 polypeptide.
14 more elements
An isolated Yarrowia lipolytica or Arxula adeninivorans cell genetically engineered to express a protein that promotes phosphorylation of mannosyl moieties of N-glycans and to comprise a deficiency in Outer CHain elongation (OCH1) activity,.
- wherein the protein that promotes phosphorylation of mannosyl moieties is:
- (a)(i) a wild-type MNN4 polypeptide;
- (a)(ii) a deletion variant that is the polypeptide of (a)(i) but lacks up to twenty amino acid segments, each segment consisting of two amino acids;
- (a)(iii) a deletion variant that is the polypeptide of (a)(i) or the variant of (a)(ii) but lacks up to twenty single, non-contiguous amino acids of the polypeptide of (a)(i) or the variant of (a)(ii);
- or
- (a)(iv) a substitution variant that is the polypeptide of (a)(i), the variant of (a)(ii), or the variant of (a)(iii), but with no more than 10 conservative substitutions,
- wherein the variants of (a)(ii), (a)(iii) and (a)(iv) have at least 25% of the phosphorylation-promoting activity of the polypeptide of (a)(i),
- (b)(i) a wild-type PNO1polypeptide;
- (b)(ii) a deletion variant that is the polypeptide of (b)(i) but lacks up to twenty amino acid segments, each segment consisting of two amino acids;
- (b)(iii) a deletion variant that is the polypeptide of (b)(i) or the variant of (b)(ii) but lacks up to twenty single, non-contiguous amino acids of the polypeptide of (b)(i) or the variant of (b)(ii);
- or
- (b)(iv) a substitution variant that is the polypeptide of (b)(i), the deletion variant of (b)(ii), or the deletion variant of (b)(iii), but with no more than 10 conservative substitutions,
- wherein the variants of (b)(ii), (b)(iii), and (b)(iv) have at least 25% of the phosphorylation-promoting activity of the polypeptide of (b)(i), or
- (c) a wild-type MNN6 polypeptide.
11 more elements
A method of producing an altered N-glycosylation form of a target protein, the method comprising contacting a target protein with a cell lysate prepared from a Yarrowia lipolytica or an Arxula adeninivorans cell genetically engineered to express a protein that promotes phosphorylation of mannosyl moieties of N-glycans and to comprise a deficiency in Outer CHain elongation (OCH1) activity,
- wherein the contacting of the target protein with the cell lysate results in an altered N-glycosylation form of the target protein relative to the N-glycosylation form of the target protein produced from the cell lysate of a corresponding non-genetically engineered cell, and
- wherein the protein that promotes phosphorylation of mannosyl moieties is:
- (a)(i) a wild-type MNN4 polypeptide;
- (a)(ii) a deletion variant that is the polypeptide of (a)(i) but lacks up to twenty amino acid segments, each segment consisting of two amino acids;
- (a)(iii) a deletion variant that is the polypeptide of (a)(i) or the variant of (a)(ii) but lacks up to twenty single, non-contiguous amino acids of the polypeptide of (a)(i) or the variant of (a)(ii);
- or
- (a)(iv) a substitution variant that is the polypeptide of (a)(i), the variant of (a)(ii), or the variant of (a)(iii), but with no more than 10 conservative substitutions,
- wherein the variants of (a)(ii), (a)(iii) and (a)(iv) have at least 25% of the phosphorylation-promoting activity of the polypeptide of (a)(i),
- (b)(i) a wild-type PNO1 polypeptide;
- (b)(ii) a deletion variant that is the polypeptide of (b)(i) but lacks up to twenty amino acid segments, each segment consisting of two amino acids;
- (b)(iii) a deletion variant that is the polypeptide of (b)(i) or the variant of (b)(ii) but lacks up to twenty single, non-contiguous amino acids of the polypeptide of (b)(i) or the variant of (b)(ii);
- or
- (b)(iv) a substitution variant that is the polypeptide of (b)(i), the deletion variant of (b)(ii), or the deletion variant of (b)(iii), but with no more than 10 conservative substitutions,
- wherein the variants of (b)(ii), (b)(iii), and (b)(iv) have at least 25% of the phosphorylation-promoting activity of the polypeptide of (b)(i), or
- (c) a wild-type MNN6 polypeptide.
12 more elements
A substantially pure culture of Yarrowia lipolytica or Arxula adeninivorans cells, a substantial number of which are genetically engineered to express a protein that promotes phosphorylation of mannosyl moieties of N-glycans and to comprise a deficiency in Outer CHain elongation (OCH1) activity, and
- wherein the protein that promotes phosphorylation of mannosyl moieties is:
- (a)(i) a wild-type MNN4 polypeptide;
- (a)(ii) a deletion variant that is the polypeptide of (a)(i) but lacks up to twenty amino acid segments, each segment consisting of two amino acids;
- (a)(iii) a deletion variant that is the polypeptide of (a)(i) or the variant of (a)(ii) but lacks up to twenty single, non-contiguous amino acids of the polypeptide of (a)(i) or the variant of (a)(ii);
- or
- (a)(iv) a substitution variant that is the polypeptide of (a)(i), the variant of (a)(ii), or the variant of (a)(iii), but with no more than 10 conservative substitutions,
- wherein the variants of (a)(ii), (a)(iii) and (a)(iv) have at least 25% of the phosphorylation-promoting activity of the polypeptide of (a)(i),
- (b)(i) a wild-type PNO1 polypeptide;
- (b)(ii) a deletion variant that is the polypeptide of (b)(i) but lacks up to twenty amino acid segments, each segment consisting of two amino acids;
- (b)(iii) a deletion variant that is the polypeptide of (b)(i) or the variant of (b)(ii) but lacks up to twenty single, non-contiguous amino acids of the polypeptide of (b)(i) or the variant of (b)(ii);
- or
- (b)(iv) a substitution variant that is the polypeptide of (b)(i), the deletion variant of (b)(ii), or the deletion variant of (b)(iii), but with no more than 10 conservative substitutions,
- wherein the variants of (b)(ii), (b)(iii), and (b)(iv) have at least 25% of the phosphorylation-promoting activity of the polypeptide of (b)(i), or
- (c) a wild-type MNN6 polypeptide.
11 more elements
Dependencies are inferred from the claim text.