Carbon-source regulated protein production in a recombinant host cell

Abstract

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 (GOI) 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.

1 . 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 (GOI) under the control of an expression cassette promoter (ECP) which ECP is repressible by a non-methanol carbon source. 
     
     
         2 . The host cell of wherein said endogenous polynucleotide is a gene encoding said FLO8 protein, wherein said one or more genetic modifications comprises a disruption, substitution, deletion or knockout of (i) one or more endogenous polynucleotides, or a part thereof; or (ii) an expression control sequence, preferably wherein said expression control sequence is selected from the group consisting of a promoter, a ribosomal binding site, transcriptional or translational start and stop sequences, an enhancer and activator sequence. 
     
     
         3 . The host cell of  claim 1  or  2 , wherein said endogenous gene encoding said FLO8 protein or said homologue is knocked out by said one or more genetic modifications. 
     
     
         4 . The host cell of any one of  claims 1  to  3 , wherein the ECP is inducible in the presence of a growth-limiting amount of a non-methanol carbon source, preferably in the absence of methanol; and repressible in the presence of an excess amount of a non-methanol carbon source that is higher than the growth-limiting amount. 
     
     
         5 . The host cell of any one of  claims 1  to  4 , wherein the ECP comprises at least 60% sequence identity to at least 300 nt of any one of the sequences SEQ ID NO:10-16, or any one of SEQ ID NO:41-45. 
     
     
         6 . The host cell of  claim 5 , wherein the ECP comprises or consists of SEQ ID NO:10 or SEQ ID NO:11. 
     
     
         7 . The host cell of any one of  claims 1  to  6 , wherein the expression cassette further comprises a nucleotide sequence encoding a signal peptide enabling the secretion of a protein of interest (POI) which is encoded by the GOI, preferably wherein the nucleotide sequence encoding the signal peptide is fused adjacent to the 5′-end of the GOI. 
     
     
         8 . The host cell of any one of  claims 1  to  7 , wherein the GOI encodes a protein of interest (POI) which is a peptide or protein selected from the group consisting of an antigen-binding protein, a therapeutic protein, an enzyme, a peptide, a protein antibiotic, a toxin fusion protein, a carbohydrate-protein conjugate, a structural protein, a regulatory protein, a vaccine antigen, a growth factor, a hormone, a cytokine, a process enzyme, and a metabolic enzyme. 
     
     
         9 . The host cell of any one of  claims 1  to  8 , which is
 a) a yeast cell of a genus selected from the group consisting of  Pichia, Hansenula, Komagataella, Saccharomyces, Kluyveromyces, Candida, Ogataea, Yarrowia , and  Geotrichum , such as  Pichia pastoris, Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris, Saccharomyces cerevisiae, Ogataea minuta, Kluyveromyces lactis, Kluyveromes marxianus, Yarrowia lipolytica  or  Hansenula polymorpha ; or 
 b) a cell of filamentous fungi, such as  Aspergillus awamori  or  Trichoderma reesei.    
 
     
     
         10 . A method of increasing the yield of a protein of interest (POI) produced by a host cell expressing a gene of interest (GOI) encoding said POI under the control of a promoter which is repressible by a non-methanol carbon source, by reducing in said host cell expression of a gene encoding a FLO8 protein comprising the amino acid sequence identified as SEQ ID NO:1 or a homologue thereof. 
     
     
         11 . A method for producing a protein of interest (POI) encoded by a gene of interest (GOI) by culturing the host cell of any one of  claims 1  to  9  under conditions to produce said POI. 
     
     
         12 . The method of  claim 10  or  11 , comprising the steps:
 a) culturing the host cell under growing conditions; and 
 b) culturing the host cell under growth-limiting conditions in the presence of up to 1 g/L of a second non-methanol carbon source, resulting in expression of said GOI to produce said POI, preferably wherein said step a) culturing is performed in a batch phase; and said step b) culturing is performed in fed-batch or a continuous cultivation phase. 
 
     
     
         13 . The method of  claim 12 , wherein said first or second carbon source is selected from saccharides, polyols, alcohols, or mixtures of any one or more of the foregoing. 
     
     
         14 . A method for producing a protein of interest (POI) in a host cell, comprising the steps:
 a) genetically engineering the host cell to reduce expression of an endogenous gene encoding a FLO8 protein comprising the amino acid sequence identified as SEQ ID N0:1 or a homologue thereof;   b) introducing into the host cell a heterologous expression cassette comprising a gene of interest (GOI) expressing said POI under the control of an expression cassette promoter (ECP) that is operably linked to the GOI, which ECP is repressible by a non-methanol carbon source;   c) culturing said host cell under conditions to produce said POI;   d) optionally isolating said POI from the cell culture; and   e) optionally purifying said POI.   
     
     
         15 . The method of  claim 14 , wherein the ECP comprises at least 60% sequence identity to at least 300 nt of any one of the sequences SEQ ID NO:10-16, or any one of SEQ ID NO:41-45.