Recombinant host cell for expressing proteins of interest
Inventors
Gasser, Brigitte • Mattanovich, Diethard • Buchetics, Markus
Assignees
Sandoz GmbH • Boehringer Ingelheim RCV GmbH and Co KG • Universitaet fuer Bodenkultur Wien BOKU • Validogen GmbH • Biomin Holding GmbH • Lonza AG
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Publication Number
US-10590427-B2
Publication Date
2020-03-17
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 yeast host cells engineered to underexpress one or more polynucleotides encoding KO proteins having amino acid sequences as shown in SEQ ID NO: 10, 11, or 12, or functional homologues thereof. The underexpression is achieved by knocking-out the polynucleotide from the genome, disrupting the polynucleotide, disrupting a promoter operably linked to the polynucleotide, replacing that promoter with another promoter having lower promoter activity, or disrupting expression control sequences.
The engineered yeast host cell is further configured to express a heterologous protein of interest. The disclosed system is directed to increasing yield or producing heterologous protein of interest by using the underexpressed KO strategy in a yeast host cell.
In certain embodiments, the heterologous protein of interest includes model antibody Fab fragments, including SDZ-Fab and/or HyHEL-Fab, and the recombinant cell and methods are further refined by combining underexpression of specific KO protein(s) with overexpression of helper proteins having amino acid sequences as shown in SEQ ID NO: 1–9 and/or 162, or functional homologues.
Claims Coverage
The independent claims cover engineered underexpression of specified KO protein genes or functional homologues and expression of a heterologous protein of interest. Across the claims, the main inventive features are KO-protein underexpression in yeast, sequence-identity-based definition of functional homologues, and use of the engineered host cell in yield increase and production workflows.
Yeast host cell underexpressing KO-protein encoding polynucleotides
A recombinant host cell for manufacturing a heterologous protein of interest, wherein the host cell is a yeast and is engineered to underexpress at least one polynucleotide encoding a KO protein having an amino acid sequence as shown in SEQ ID NO: 10, 11, 12 or a functional homologue thereof, achieved by knocking-out the polynucleotide, disrupting the polynucleotide, disrupting a promoter operably linked with the polynucleotide, replacing the promoter with another promoter having lower promoter activity, or disrupting expression control sequences, and wherein the host cell is engineered to express a heterologous protein of interest.
Yeast host cell engineered from a cell expressing KO proteins to underexpress KO1, KO2 or KO3
A yeast host cell obtained by providing a cell which expresses KO protein 1, KO protein 2, and/or KO protein 3, or functional homologue thereof, and engineering the cell to underexpress the gene which encodes KO1, KO2 and/or KO3, or functional homologue thereof, by the same underexpression mechanisms, while expressing a heterologous protein of interest.
Method of increasing yield by underexpressing KO-protein encoding polynucleotides in yeast
A method of increasing the yield of a heterologous protein of interest in a yeast host cell engineered to underexpress at least one polynucleotide encoding a KO protein having an amino acid sequence as shown in SEQ ID NO: 10, 11, 12 or a functional homologue thereof, comprising underexpressing the at least one polynucleotide.
Method of producing heterologous protein by culturing a yeast host underexpressing KO-protein encoding polynucleotides
A method of producing a heterologous protein of interest in a yeast host cell comprising providing a yeast host cell engineered to underexpress at least one polynucleotide encoding a KO protein or functional homologue thereof, wherein the yeast host cell comprises a heterologous polynucleotide encoding a protein of interest, culturing the yeast host cell under suitable conditions to express the heterologous protein of interest, and optionally isolating the protein of interest from the cell culture.
The claims center on engineering a yeast host cell to underexpress KO protein-encoding polynucleotides defined by SEQ ID NO: 10, 11, or 12, or functional homologues with at least 70% sequence identity, using knockout, disruption, promoter replacement, or expression-control sequence disruption. The claims further require expression of a heterologous protein of interest, with method claims directed to increasing yield and producing the heterologous protein by culturing the engineered yeast host cell.
Stated Advantages
Increase the yield of a heterologous protein of interest in a yeast host cell compared to the yeast host cell prior to genetic manipulation.
Produce a heterologous protein of interest in a yeast host cell engineered to underexpress KO proteins corresponding to SEQ ID NO: 10, 11, 12 or functional homologues.
Increase yield of model protein Fab proteins including SDZ-Fab and HyHEL-Fab compared to the host cell prior to engineering.
Documented Applications
Manufacturing or producing heterologous protein of interest in a yeast host cell engineered to underexpress KO proteins defined by SEQ ID NO: 10, 11, 12 or functional homologues.
Increasing the yield of heterologous proteins by using yeast hosts with underexpressed KO-protein polynucleotides.
Production or yield improvement for model antibody Fab proteins SDZ-Fab and HyHEL-Fab in an engineered yeast system.
Producing a heterologous protein of interest by culturing an engineered yeast host cell and optionally isolating the protein from the cell culture.
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