Crz1 mutant fungal cells

Abstract

The present invention provides CRZ1 mutant fungal host cells, such as Pichia pastoris . The mutant fungal host cells exhibit temperature-resistance, enhanced fermentation robustness and increased expression of heterologous polypeptides such as immunoglobulins. Methods for producing heterologous polypeptides, such as immunoglobulins, using such mutant fungal host cells are within the scope of the present invention.

1 . An isolated fungal host cell lacking functional CRZ1 polypeptide; optionally with the proviso that the cell comprises functional ATT1 polypeptide. 
     
     
         2 . The isolated fungal host cell of  claim 1 :
 wherein endogenous CRZ1 is mutated, disrupted, partially deleted or fully deleted;   or,   wherein CRZ1 polypeptide expression is reduced by interfering with CRZ1 transcription or CRZ1 translation or wherein CRZ1 polypeptide degradation is increased;   or,   wherein CRZ1 polypeptide activity is inhibited by chemical inhibitor.   
     
     
         3 . The isolated fungal host cell of  claim 1  wherein:
 (i) endogenous CRZ1 encodes a polypeptide that differs from wild-type CRZ1 in that comprises one or more mutations selected from the group consisting of: 
 L33→STOP; 
 Q214→STOP; 
 L294H→STOP; 
 S298→STOP; 
 E403→G; 
 F406H→S; 
 F406→L; 
 C411→F; and 
 K469→N; or 
 (ii) endogenous CRZ1 comprises a nucleotide sequence that differs from wild-type CRZ1 in that it comprises one or more mutations selected from the group consisting of: 
 a1407c; 
 g1232t; 
 t1216c; 
 t1217c; 
 a1208g; 
 c893a; 
 t881g; 
 c640t; and 
 t98a; or 
 (iii) endogenous CRZ1 differs from wild-type CRZ1 in that it does not encode a functional C-terminal zinc-finger domain. 
 
     
     
         4 . The isolated fungal host cell of  claim 1 :
 (i) wherein one or more endogenous beta-mannosyltransferase genes are mutated, disrupted, truncated or partially or fully deleted;   (ii) comprising a polynucleotide encoding an alpha-1,2 mannosidase enzyme;   (iii) wherein one or more endogenous phosphomannosyl transferases are mutated, disrupted, truncated or partially or fully deleted;   (iv) comprising a single-subunit oligosaccharyltransferase;   (v) wherein endogenous ALG3 is mutated, disrupted, truncated or partially or fully deleted;   (vi) comprising a polynucleotide encoding an endomannosidase;   (vii) comprising one or more polynucleotides encoding a bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase, an N-acetylneuraminate-9-phosphate synthase, or a CMP-sialic acid synthase;   (viii) wherein endogenous ATT1 gene is mutated, disrupted, truncated or partially or fully deleted;   (ix) wherein endogenous OCH1 is mutated, disrupted, truncated or partially or fully deleted;   (x) comprising a polynucleotide encoding galactosyltransferase;   (xi) comprising a polynucleotide encoding nucleotide sugar transporter;   (xii) comprising a polynucleotide encoding sialyltransferase;   (xiii) comprising a polynucleotide encoding acetylglucosaminyl transferase; and/or   (xiv) wherein one or more endogenous proteases are mutated, disrupted, truncated or partially or fully deleted.   
     
     
         5 . The isolated fungal host cell of  claim 1  comprising a heterologous polynucleotide that encodes a heterologous polypeptide. 
     
     
         6 . The isolated fungal host cell of  claim 5  wherein the heterologous polypeptide is an immunoglobulin. 
     
     
         7 . The isolated fungal host cell of  claim 1  which is yeast, methylotrophic yeast,  Pichia  or  Pichia pastoris.    
     
     
         8 . An isolated polynucleotide which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3 which comprises a mutation selected from the group consisting of:
 L33→STOP;   Q214→STOP;   L294→STOP;   S298→STOP;   E403→G;   F406→S;   F406→L;   C411→F; and   K469→N.   
     
     
         9 . The polynucleotide of  claim 8  comprising a nucleotide sequence of SEQ ID NO: 2 comprising a mutation selected from the group consisting of:
 a1407c; 
 g1232t; 
 t1216c; 
 t1217c; 
 a1208g; 
 c893a; 
 t881g; 
 c640t; and 
 t98a. 
 
     
     
         10 . An isolated vector comprising a polynucleotide of  claim 8 . 
     
     
         11 . A method for producing an isolated fungal cell having improved viability under bioprocess fermentation conditions comprising introducing a mutation selected from the group consisting of:
 L33→STOP;   Q214→STOP;   L294→STOP;   S298→STOP;   E403→G;   F406→S;   F406→L;   C411→F; and   K469→N;   to endogenous CRZ1 in the fungal cell.   
     
     
         12 . An isolated fungal host cell produced by the method of  claim 11 . 
     
     
         13 . A method for making the isolated fungal host cell of  claim 1  comprising introducing a heterologous polynucleotide into the cell which homologously recombines with the endogenous CRZ1 and partially or fully deletes the endogenous CRZ1 or disrupts the endogenous CRZ1. 
     
     
         14 . An isolated fungal host cell produced by the method of  claim 13 . 
     
     
         15 . A method for producing one or more heterologous polypeptides comprising:
 (i) introducing a heterologous polynucleotide encoding the heterologous polypeptide(s) into an isolated fungal host cell of  claim 1 ; and   (ii) culturing the fungal host cell under conditions favorable to expression of the heterologous polypeptide(s) in the fungal host cell and; optionally,   (iii) isolating the heterologous polypeptide(s) from the fungal host cell.   
     
     
         16 . The method of  claim 15  wherein the heterologous polynucleotide that encodes the heterologous polypeptide is operably linked to a methanol inducible promoter and wherein the isolated fungal host cell is cultured under conditions favorable to expression of the heterologous polypeptide in the presence of methanol.