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Publication Number
US-9206454-B2
Publication Date
2015-12-08
Expiration Date
Abstract
The present invention relates to a genetically modified yeast cell comprising: —at least one recombinant promoter operably linked to at least one gene encoding a polypeptide or protein supporting the biosynthesis of polypeptides or proteins within said cell, said at least one gene being located at the native genomic locus of the genetically unmodified wild-type yeast cell, wherein the naturally occurring promoter of the at least one gene encoding the biosynthesis supporting polypeptide or protein is inactivated by at least one mutation within said naturally occurring promoter and, —a secretion cassette comprising a recombinant nucleic acid molecule encoding a protein or polypeptide of interest and a method for producing a recombinant protein or polypeptide of interest using such a cell.
Core Innovation
The invention provides a method for producing a recombinant protein or polypeptide of interest by cultivating a genetically modified yeast cell. The yeast cell comprises an expression secretion cassette including a recombinant nucleic acid molecule encoding the protein or polypeptide of interest, and at least one recombinant promoter operably linked to at least one gene naturally occurring in the yeast genome and encoding a polypeptide or protein supporting biosynthesis of polypeptides or proteins.
The gene is located at the native genomic locus of the genetically unmodified wild-type yeast cell, and the naturally occurring promoter of the gene encoding the biosynthesis-supporting polypeptide or protein is inactivated by at least one mutation within the naturally occurring promoter. The system further includes cultivating the genetically modified yeast cell under conditions allowing for expression of the protein or polypeptide of interest and the biosynthesis-supporting polypeptide or protein, followed by isolating the protein or polypeptide of interest from the culture medium.
The disclosure emphasizes that keeping the naturally occurring promoter active is inefficient for target production, and therefore inactivating that promoter is used to improve recombinant protein secretion. Biosynthesis-supporting genes are preferably chaperones, including protein disulfide isomerase (PDI), Kar2/BiP, or calnexin, and specific examples use methylotrophic yeasts and AOX1-mutated promoter platforms to enable high-level expression of native support transcripts and secretion of disulfide-bonded proteins.
Claims Coverage
The document provides two independent claims. Both independent claims cover cultivation of a genetically modified yeast cell to produce a protein of interest and the corresponding genetically modified yeast cell configuration itself, with the same core theme of a recombinant promoter driving a gene at the native genomic locus while the naturally occurring promoter of the biosynthesis-supporting gene is inactivated by mutation.
Recombinant promoter at native genomic locus with inactivated naturally occurring promoter
A recombinant promoter operably linked to a gene naturally occurring in the yeast genome, where the gene is located at the native genomic locus of the genetically unmodified wild-type yeast cell, and wherein the naturally occurring promoter of the gene encoding a biosynthesis supporting polypeptide or protein is inactivated by at least one mutation within said naturally occurring promoter.
Expression secretion cassette for protein of interest
An expression secretion cassette comprising a recombinant nucleic acid molecule encoding a protein or polypeptide of interest.
Biosynthesis supporting gene encoding biosynthesis-supporting polypeptide or protein
At least one gene naturally occurring in the genome encoding a polypeptide or protein supporting the biosynthesis of polypeptides or proteins within the genetically modified yeast cell.
Cultivating and isolating recombinant protein or polypeptide
Cultivating the genetically modified yeast cell in a culture medium under conditions that allow for expression of the protein or polypeptide of interest and the at least one gene encoding the biosynthesis supporting polypeptide or protein, and isolating the protein or polypeptide of interest from the culture medium.
Across the independent claims, the inventive concept is the combination of a recombinant secretion cassette for a protein of interest with a recombinant promoter that drives expression of an endogenous biosynthesis-supporting gene at its native genomic locus, while the gene’s naturally occurring promoter is inactivated by mutation. The method claim further adds the cultivation and isolation steps for producing the recombinant protein.
Stated Advantages
Enables efficient expression of the biosynthesis supporting polypeptide or protein supporting target production while preventing efficient target production when the native promoter is kept active.
Increases native biosynthesis supporting gene transcript levels and enables secretion of recombinant proteins from methylotrophic yeast systems.
Documented Applications
Production of secreted recombinant proteins and polypeptides from genetically modified yeast cells, including disulfide-bonded proteins. Examples described include secretion of human serum transferrin (transferrin variants, including transferrin-non-glycosylated), human serum albumin (HSA), HSA-interferon(alpha2a) fusion, interleukin-2 (IL-2), and Fab.
Comparative evaluations of improved secretion and titers under microscale conditions and bioreactor cultivations compared with parental strains and strains expressing native support proteins recombinantly in 1 copy.
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