O-Mannosyltransferase Deficient Filamentous Fungal Cells and Methods of Use Thereof

Abstract

The present disclosure relates to compositions and methods useful for the production of heterologous proteins with reduced O-mannosylation in filamentous fungal cells, such as Trichoderma cells. More specifically, the invention provides a PMT-deficient filamentous fungal cell comprising a) at least a first mutation that reduces an endogenous protease activity compared to a parental filamentous fungal cell which does not have said first mutation, and, b) at least a second mutation in a PMT gene that reduces endogenous O-mannosyltransferase activity compared to a parental filamentous fungal cell which does not have said second mutation, wherein said filamentous fungal cell is selected from the group consisting of Trichoderma, Neurospora, Myceliophthora or Chrysosporium cell.

1 . A PMT-deficient filamentous fungal cell comprising:
 a) a first mutation that reduces or eliminates an endogenous protease activity compared to a parental filamentous fungal cell which does not have said first mutation, and   b) a second mutation in a PMT gene that reduces endogenous O-mannosyltransferase activity compared to a parental filamentous fungal cell which does not have said second mutation, wherein said filamentous fungal cell is selected from the group consisting of  Trichoderma, Neurospora, Myceliophthora  and  Chrysosporium  cell.   
     
     
         2 . The PMT-deficient filamentous fungal cell of  claim 1 , wherein said second mutation that reduces the endogenous O-mannosyltransferase activity is a deletion or a disruption of a PMT gene encoding an endogenous protein O-mannosyltransferase activity. 
     
     
         3 . The PMT-deficient filamentous fungal cell of  claim 1 , wherein said second mutation in a PMT gene is a mutation in either:
 a) PMT1 gene comprising the polynucleotide of SEQ ID NO:1,   b) a functional homologous gene of PMT1 gene, which functional homologous gene is capable of restoring parental O-mannosylation level by functional complementation when introduced into a  T. reesei  strain having a disruption in said PMT1 gene, or   c) a polynucleotide encoding a polypeptide having at least 50% identity with SEQ NO:2, said polypeptide having O-mannosyltransferase activity.   
     
     
         4 . The PMT-deficient filamentous fungal cell of  claim 1 , wherein said cell has a third mutation that reduces or eliminates the level of expression of an ALG3 gene compared to the level of expression in a parental cell which does not have such third mutation. 
     
     
         5 . The PMT-deficient filamentous fungal cell of  claim 4 , further comprising a first polynucleotide encoding a N-acetylglucosaminyltransferase I catalytic domain and a second polynucleotide encoding a N-acetylglucosaminyltransferase IT catalytic domain. 
     
     
         6 . The PMT-deficient filamentous fungal cell of  claim 1 - 5 , further comprising one or more polynucleotides encoding a polypeptide selected from the group consisting of:
 a) α1,2 mannosidase;   b) N-acetylglucosaminyltransferase I catalytic domain;   c) α mannosidase   d) N-acetylglucosaminyltransferase II catalytic domain;   e) β1,4 galactosyltransferase; and,   f) fucosyltransferase.   
     
     
         7 . The PMT-deficient filamentous fungal cell of  claim 1 , wherein said cell is a  Trichoderma  cell comprising a mutation that reduces or eliminates the protein O-mannosyltransferase activity of  Trichoderma  pmt1. 
     
     
         8 . The PMT-deficient filamentous fungal cell of  claim 1 , wherein said cell is a  Trichoderma  cell, for example  Trichoderma reesei , and said cell comprises mutations that reduce or eliminate the activity of:
 a) the three endogenous proteases pep1, tsp1 and slp1;   b) the three endogenous proteases gap1, slp1 and pep1;   c) the three endogenous proteases selected from the group consisting of pep1, pep2, pep3, pep4, pep5, pep8, pep11, pep12, tsp1, slp1, slp2, slp3, slp7, gap1 and gap2;   d) three to six proteases selected from the group consisting of pep1, pep2, pep3, pep4, pep5, tsp1, slp1, slp2, slp3, gap1 and gap2; or   e) seven to ten proteases selected from the group consisting of pep1, pep2, pep3, pep4, pep5, pep7, pep8, tsp1, slp1, slp2, slp3, slp5, slp6, slp7, slp8, tpp1, gap1 and gap2.   
     
     
         9 . A method for producing a protein having reduced O-mannosylation, comprising:
 a) providing a PMT-deficient filamentous fungal cell having a mutation in a PMT gene that reduces endogenous O-mannosyltransferase activity in comparison to a parental strain which does not have such mutation, and further comprising a polynucleotide encoding a protein with serine or threonine residue,   b) culturing said PMT-deficient filamentous fungal cell to produce said protein having reduced O-mannosylation,   wherein said filamentous fungal cell is selected from the group consisting of  Trichoderma, Neurospora, Myceliophthora  or  Chrysosporium  cell.   
     
     
         10 . The method according to  claim 9 , wherein said mutation in a PMT gene is a mutation in either:
 a) PMT1 gene comprising the polynucleotide of SEQ ID NO:1,   b) a functional homologous gene of PMT1 gene, which gene is capable of restoring parental O-mannosylation level by functional complementation when introduced into a  T. reesei  strain having a disruption in said PMT1 gene, or   c) a polynucleotide encoding a polypeptide having at least 50% identity with SEQ ID NO:2, said polypeptide having protein O-mannosyltransferase activity.   
     
     
         11 . The method according to  claim 9 , wherein said filamentous fungal cell expresses functional endogenous chaperone protein, such as Protein Disulphide Isomerase (PDI). 
     
     
         12 . The method according to any one of  claim 9 , wherein said PMT-deficient filamentous fungal cell is selected from the cells as defined in any one of  claims 1 - 8 . 
     
     
         13 . The method of  claim 9 , wherein said produced protein is a heterologous mammalian protein selected from the group consisting of
 a) an immunoglubulin, such as IgG,   b) a light chain or heavy chain of an immunoglobulin,   c) a heavy chain or a light chain of an antibody,   d) a single chain antibody,   e) a camelid antibody,   f) a monomeric or multimeric single domain antibody,   g) a FAb-fragment, a FAb2-fragment, and,   h) their antigen-binding fragments.   
     
     
         14 . A method for producing an antibody having reduced O-mannosylation, comprising:
 a) providing a PMT-deficient filamentous fungal cell having:
 i. a mutation that reduces endogenous protein O-mannosyltransferase activity as compared to parental strain which does not have such mutation, and, 
 ii. a polynucleotide encoding a light chain antibody and a polynucleotide encoding a heavy chain antibody, 
   b) culturing the cell to produce said antibody, consisting of heavy and light chains, having reduced O-mannosylation,   wherein said filamentous fungal cell is selected from the group consisting of  Trichoderma, Neurospora, Myceliophthora  and  Chrysosporium  cell.   
     
     
         15 . A protein or antibody composition obtainable by the method of  claim 9 . 
     
     
         16 . The protein or antibody composition according to  claim 15 , wherein said protein or antibody comprises, as a major glycoform, either,
 a) Manα3[Manα6(Manα3)Manα6]Manβ4GlcNAβ4GlcNAc (Man5 glycoform);   b) Manα6(Manα3)Manβ4GlcNAβ4GlcNAc (Man3 glycoform);   c) hybrid or complex type N-glycans, such as the glycoforms selected from the subgroup consisting of GlcNAcMan3, G0, hybrid glycan GlcNAcMan5, and galactosylated derivatives, such as GalGlcNAcMan3, G1, G2; and, GalGlcNAcMan5 glycoforms.   
     
     
         17 . The PMT-deficient filamentous fungal cell of  claim 2 , wherein said second mutation in a PMT gene is a mutation in either:
 a) PMT1 gene comprising the polynucleotide of SEQ ID NO:1,   b) a functional homologous gene of PMT1 gene, which functional homologous gene is capable of restoring parental O-mannosylation level by functional complementation when introduced into a  T. reesei  strain having a disruption in said PMT1 gene, or   c) a polynucleotide encoding a polypeptide having at least 50% identity with SEQ ID NO:2, said polypeptide having O-mannosyltransferase activity.   
     
     
         18 . The PMT-deficient filamentous fungal cell of  claim 2 , wherein said cell has a third mutation that reduces or eliminates the level of expression of an ALG3 gene compared to the level of expression in a parental cell which does not have such third mutation. 
     
     
         19 . The PMT-deficient filamentous fungal cell of  claim 3 , wherein said cell has a third mutation that reduces or eliminates the level of expression of an ALG3 gene compared to the level of expression in a parental cell which does not have such third mutation. 
     
     
         20 . A protein or antibody composition obtainable by the method of  claim 14 .