Recombinant host cell with altered membrane lipid composition
Inventors
Grillitsch, Karlheinz • Daum, Guenther • Grutsch, Andreas
Assignees
Austrian Centre Of Industrial Biotechnology • Boehringer Ingelheim RCV GmbH and Co KG • Validogen GmbH • Biomin Holding GmbH • Lonza AG • Sandoz AG
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Publication Number
US-11479798-B2
Publication Date
2022-10-25
Expiration Date
Abstract
The present invention is in the field of recombinant biotechnology, in particular in the field of protein expression. The invention generally relates to a method of expressing a protein of interest (POI) from a host cell. The invention relates particularly to improving a host cell's capacity to express and/or secrete a protein of interest and use of the host cell for protein expression. The invention also relates to cell culture technology, and more specifically to culturing cells to produce desired molecules for medical purposes or food products.
Core Innovation
The invention relates to increasing the yield of a protein of interest in a eukaryotic host cell by overexpressing at least one polynucleotide encoding a protein involved in lipid metabolism, where the lipid-metabolism protein is involved in sphingolipid biosynthesis. The disclosed engineering targets sphingolipid molecular species and biomembrane lipid composition, including shifts toward mature M(IP)2C and/or increases in ceramide/IPC classes.
The lipid-metabolism protein comprises an amino acid sequence as shown in SEQ ID NO: 1 or a homologue thereof having at least 70% sequence identity to the amino acid sequence as shown in SEQ ID NO: 1. The disclosure also includes helper proteins and KO helper proteins, with functional homologue/identity thresholds and sequence-based selection such as SEQ ID NOs 1–17.
In the described embodiments, the engineered host cell is used for manufacturing a protein of interest, and compatible host cell types are limited to specified yeast/fungal species. The disclosure further includes overexpression and underexpression approaches, depletion of triacylglycerol storage, and examples in Pichia pastoris with increased yields for model protein of interest formats.
Claims Coverage
The independent claims cover two complementary aspects: a method that increases the yield of a protein of interest by overexpressing a specific lipid-metabolism protein involved in sphingolipid biosynthesis with sequence/identity constraints, and a recombinant eukaryotic host cell engineered for manufacturing the protein of interest with the same sequence/identity constraints. Across the independent claims, there are two main inventive feature groupings.
Overexpressing a sphingolipid-biosynthesis lipid-metabolism protein to increase protein yield
A method of increasing the yield of a protein of interest in a eukaryotic host cell by overexpressing at least one polynucleotide encoding at least one protein involved in lipid metabolism, wherein the protein is involved in sphingolipid biosynthesis and comprises an amino acid sequence as shown in SEQ ID NO: 1 or a homologue thereof having at least 70% sequence identity to an amino acid sequence as shown in SEQ ID NO: 1.
Recombinant eukaryotic host cell overexpressing a sphingolipid-biosynthesis lipid-metabolism protein
A recombinant eukaryotic host cell for manufacturing a protein of interest, wherein the host cell is engineered to overexpress at least one polynucleotide encoding at least one protein involved in lipid metabolism, wherein the lipid-metabolism protein is involved in sphingolipid biosynthesis and comprises an amino acid sequence as shown in SEQ ID NO: 1 or a homologue thereof, wherein the homologue has at least 70% sequence identity to an amino acid sequence as shown in SEQ ID NO: 1.
Overall, the claim set centers on engineered overexpression of a sphingolipid-biosynthesis lipid-metabolism protein defined by SEQ ID NO: 1 or a homologue with at least 70% identity to increase the yield of a protein of interest, implemented either as a method or as a recombinant eukaryotic host cell for manufacturing.
Stated Advantages
Increase yields of a protein of interest in a eukaryotic host cell.
Increasing the yield of a protein of interest in a eukaryotic host cell compared to a host cell that does not overexpress a polynucleotide encoding a protein involved in lipid metabolism.
Documented Applications
Manufacturing a protein of interest in a recombinant eukaryotic host cell.
Manufacturing a protein of interest using a recombinant eukaryotic host cell engineered to overexpress lipid-metabolism proteins involved in sphingolipid biosynthesis.
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