Lonza Inc.

Boehringer Ingelheim Rcv Gmbh & Co. Kg

Universitat Fur Bodenkultur Wien

Validogen Gmbh

Vaitdogen Gmbh

Univeristät Für Bodenkultur Wien

Sandoz Ag

Brigitte Gasser

Diethard Mattanovich

Roland Prielhofer

Michael Maurer

Joachim Klein

Jana Wenger

Silvia Heiss

Markus Buchetics

Michael Sauer

Christoph Kiziak

Corinna Rebnegger

Domen Zavec

Mirelle Citiali Flores Villegas

Martin Dragosits

Mirelle Citlali Flores Villegas

Rebecca Göngrich

A recombinant methanol utilization pathway deficient methylotrophic yeast (Mut−) host cell which is engineered: a) by one or more genetic modifications to reduce expression of a first and a second endogenous gene compared to the host cell prior to said one or more genetic modifications, wherein i. the first endogenous gene encodes alcohol oxidase 1 (AOX1) comprising the amino acid sequence identified as SEQ ID NO:1 or a homologue thereof, and ii. the second endogenous gene encodes alcohol oxidase 2 (AOX2) comprising the amino acid sequence identified as SEQ ID NO:3 or a homologue thereof, and b) by one or more genetic modifications to increase expression of an alcohol dehydrogenase (ADH2) gene compared to the host cell prior to said one or more genetic modifications, wherein the ADH2 gene encodes an alcohol dehydrogenase (ADH2).

Mut- methylotrophic yeast

A method of producing a protein of interest (POI) by culturing a recombinant eukaryotic cell line comprising an expression construct comprising a regulatable promoter and a nucleic acid molecule encoding a POI under the transcriptional control of said promoter, comprising the steps a) cultivating the cell line with a basal carbon source repressing the promoter, b) cultivating the cell line with a limited amount of a supplemental carbon source de-repressing the promoter to induce production of the POI at a transcription rate of at least 15% as compared to the native pGAP promoter, and c) producing and recovering the POI; and further an isolated regulatable promoter and a respective expression system.

Promoter variants for protein production

A recombinant host cell comprising an endogenous gene encoding a FLO8 protein comprising the amino acid sequence identified as SEQ ID NO:1 or a homologue thereof, which host cell is engineered by one or more genetic modifications to reduce expression of said gene compared to the host cell prior to said one or more genetic modifications, and which host cell comprises a heterologous expression cassette comprising a gene of interest (GO!) under the control of an expression cassette promoter (ECP) which ECP is repressible by a non-methanol carbon source, and a method of producing a protein of interest using said recombinant host cell.

Carbon-source regulated protein production in a recombinant host cell

Methods of producing promoter variants

An isolated nucleic acid encoding a leader, which has a specific sequence, an isolated leader peptide encoded by such nucleic acid, an expression cassette comprising such nucleic acid encoding a leader operably linked to a nucleic acid sequence encoding a POI a recombinant yeast host cell or a vector comprising such expression cassette, a method of producing a POI in such yeast host cell, and further the use of the specific nucleic acid for the secretion of a POT from a host cell and/or to increase the secretion of a POT from a host cell.

Expression sequences

The invention relates to an isolated nucleic acid sequence comprising a promoter, which is a native sequence ofcomprising the nucleic acid sequence of pCS1 of SEQ ID 1, or a functionally active variant thereof which is a size variant, a mutant or hybrid of SEQ ID 1, or a combination thereof, expression constructs and recombinant host cells comprising the promoter, and a method of producing a protein of interest under the control of the promoter. It further relates to a method to identify a constitutive promoter from eukaryotic cells, and an isolated nucleic acid sequence comprising a promoter which when operatively linked to a nucleotide sequence encoding a protein of interest directs the expression thereof in a host cell at an expression level that is higher than under control of the native pGAP promoter at high and low growth rates.

Constitutive promoter

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.

Recombinant host cell engineered to overexpress helper proteins

An isolated and/or artificial pG1-x promoter, which is a functional variant of the carbon source regulatable pG1 promoter ofidentified by SEQ ID 1, which pG1-x promoter consists of or comprises at least a part of SEQ ID 1 with a length of at least 293 bp, characterized by the following promoter regions:

Promoter variants

Recombinant host cell for expressing proteins of interest

Constitiutive promoter

A method of producing a protein of interest (POI) by culturing a recombinant eukaryotic cell line comprising an expression construct comprising a regulatable promoter and a nucleic acid molecule encoding a POI under the transcriptional control of said promoter, comprising the steps a) cultivating the cell line with a basal carbon source repressing the promoter, b) cultivating the cell line with a limited amount of a supplemental carbon source de-repressing the promoter to induce production of the POI at a transcription rate or at least 15% as compared to the native pGAP promoter, and c) producing and recovering the POI; and further an isolated regulatable promoter and a respective expression system.

Regulatable promoter

An isolated nucleic acid encoding a leader, which has a specific sequence, an isolated leader peptide encoded by such nucleic acid, an expression cassette comprising such nucleic acid encoding a leader operably linked to a nucleic acid sequence encoding a POI, a recombinant yeast host cell or a vector comprising such expression cassette, a method of producing a POI in such yeast host cell, and further the use of the specific nucleic acid for the secretion of a POI from a host cell and/or to increase the secretion of a POI from a host cell.

The invention relates to an isolated nucleic acid sequence comprising a promoter, which is a native sequence ofcomprising the nucleic acid sequence of pCS1 of SEQ ID NO:1, or a functionally active variant thereof which is a size variant, a mutant or hybrid of SEQ ID NO:1, or a combination thereof, expression constructs and recombinant host cells comprising the promoter, and a method of producing a protein of interest under the control of the promoter. It further relates to a method to identify a constitutive promoter from eukaryotic cells, and an isolated nucleic acid sequence comprising a promoter which when operatively linked to a nucleotide sequence encoding a protein of interest directs the expression thereof in a host cell at an expression level that is higher than under control of the native pGAP promoter at high and low growth rates.

The invention refers to a method of producing a recombinant polypeptide of interest (POI) in a cell culture, comprising genetically engineering a eukaryotic cell line&#x2014;to specifically cause prolongation of the G2+M cell cycle phase in a pre-culture phase, and&#x2014;to produce the POI in a producing phase following the pre-culture phase, a high producer cell line and cell culture as well as a method of increasing the yield of a recombinant POI production in a cell culture.

Growing community of inventors

Vienna, Austria

Brigitte Gasser