Recombinant host cell with altered membrane lipid composition
Inventors
Grillitsch, Karlheinz • Daum, Guenther • Grutsch, Andreas
Assignees
Boehringer Ingelheim RCV GmbH and Co KG • Validogen GmbH • Lonza AG
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Publication Number
US-12123044-B2
Publication Date
2024-10-22
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 recombinant fungal host cells engineered to modulate lipid metabolism to increase yield of a protein of interest. In particular, the approach overexpresses proteins involved in sphingolipid biosynthesis, lipid transport, and optionally a chaperone, and the yield of the protein of interest is increased compared to a host cell that does not overexpress the lipid-metabolism polynucleotide.
The core concept is that altering lipid metabolism and lipid handling in the host cell supports production of the protein of interest. The lipid-metabolism protein comprises an amino acid sequence as shown in SEQ ID NO: 1 or a homologue having at least 70% sequence identity, the lipid transport protein comprises an amino acid sequence as shown in SEQ ID NO: 11 or a homologue having at least 70% sequence identity, and the chaperone comprises an amino acid sequence as shown in SEQ ID NO: 18 or a homologue having at least 70% sequence identity.
The document also describes a method of manufacturing a protein of interest using an engineered host cell. The host cell comprises a heterologous polynucleotide encoding the protein of interest, is cultured under suitable conditions to overexpress the lipid-metabolism protein and to express the protein of interest, and optionally the protein of interest is isolated from the cell culture.
Claims Coverage
The independent claim set contains two closely related claims centered on increasing yield and manufacturing a protein of interest in a fungal host cell by overexpressing a polynucleotide encoding a protein involved in lipid metabolism involved in sphingolipid biosynthesis. Across the claims, three inventive features are recited: SEQ ID NO: 1 or a homologue with at least 70% sequence identity, SEQ ID NO: 11 or a homologue with at least 70% sequence identity, and SEQ ID NO: 18 or a homologue with at least 70% sequence identity.
Overexpressing sphingolipid biosynthesis lipid-metabolism protein to increase yield
Overexpressing in a fungal host cell at least one polynucleotide encoding at least one protein involved in lipid metabolism, wherein the yield of the protein of interest is increased compared to a host cell that does not overexpress the at least one polynucleotide encoding the at least one lipid-metabolism protein; 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 having at least 70% sequence identity to the amino acid sequence as shown in SEQ ID NO: 1.
Overexpressing lipid transport protein alongside sphingolipid biosynthesis protein
Overexpressing at least one polynucleotide encoding a protein involved in lipid transport, wherein the lipid transport protein comprises an amino acid sequence as shown in SEQ ID NO: 11 or a homologue having at least 70% sequence identity to the amino acid sequence as shown in SEQ ID NO: 11.
Overexpressing chaperone alongside sphingolipid biosynthesis protein
Overexpressing at least one polynucleotide encoding a chaperone, wherein the chaperone comprises an amino acid sequence as shown in SEQ ID NO: 18 or a homologue thereof having at least 70% sequence identity to the amino acid sequence as shown in SEQ ID NO: 18.
Manufacturing by culturing engineered host cells expressing lipid-related proteins and the heterologous protein of interest
Providing a host cell engineered to overexpress at least one polynucleotide encoding at least one protein involved in lipid metabolism involved in sphingolipid biosynthesis and comprising a heterologous polynucleotide encoding a protein of interest; culturing the host cell to overexpress the lipid-metabolism protein and to express the protein of interest; optionally isolating the protein of interest from the cell culture; wherein at least one polynucleotide encoding a lipid transport protein is also overexpressed and/or wherein a chaperone is overexpressed.
Claim coverage is organized around increasing yield and manufacturing of a protein of interest in a fungal host by overexpressing a defined sphingolipid-biosynthesis lipid-metabolism protein, optionally together with a defined lipid-transport protein and/or a defined chaperone.
Stated Advantages
Increases the yield of the protein of interest in the host cell compared to a host cell that does not overexpress the polynucleotides encoding the lipid-metabolism protein.
Documented Applications
Manufacturing a protein of interest in a fungal host cell by overexpressing a sphingolipid-biosynthesis protein, optionally overexpressing a lipid transport protein and/or a chaperone, with optional isolation of the protein of interest from cell culture.
Producing non-membrane protein of interest, including an enzyme, a therapeutic protein, a food additive, or a feed additive.
Producing therapeutic proteins that are antibodies or antibody fragments that retain antigen-binding activity.
Producing model proteins such as HyHEL with specified yield increase thresholds in connection with sphingolipid biosynthesis helper proteins.
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