Oxaloacetate hydrolase deficient fungal host cells

Abstract

The present invention relates to isolated nucleic acid sequences encoding polypeptides having oxaloacetate hydrolase activity. The invention also relates to nucleic acid constructs vectors, and host cells comprising the nucleic acid sequences as well as recombinant methods for producing the polypeptides.

1 - 49 . (canceled)  
     
     
         50 . A method for producing a mutant of a cell, which comprises disrupting or deleting the nucleic acid sequence encoding an oxaloacetate hydrolase or a control sequence thereof, which results in the mutant producing less of the oxaloacetate hydrolase than the cell, wherein the oxaloacetate hydrolase 
 (a) has an amino acid sequence that has at least 90% identity with amino acids 1-341 of SEQ ID NO:2;    (b) is encoded by a nucleic acid sequence having at least 90% homology with the cDNA sequence of SEQ ID NO: 1; and/or    (c) is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with the nucleic acid sequence of SEQ ID NO: 1, the cDNA sequence of SEQ ID NO: 1, the complete complementary strand of SEQ ID NO: 1, and/or the complete complementary strand of the cDNA sequence of SEQ ID NO: 1, wherein the high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5X SSPE, 0.3%. SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, followed by washing three times for 15 minutes using 2X SSC, 0.2% SDS at 65° C.    
     
     
         51 . The method of  claim 60 , wherein the oxaloacetate hydrolase has an amino acid sequence that has at least 90% identity with amino acids 1-341 of SEQ ID NO: 2.  
     
     
         52 . The method of  claim 50 , wherein the oxaloacetate hydrolase has an amino acid sequence that has at least 95% identity with amino acids 1-341 of SEQ ID NO: 2.  
     
     
         53 . The method of  claim 50 , wherein the oxaloacetate hydrolase has an amino acid sequence that has at least 97% identity with amino acids 1-341 of SEQ ID NO: 2.  
     
     
         54 . The method of  claim 50 , wherein the oxaloacetate hydrolase has an amino acid sequence which comprises SEQ ID NO: 2 or a fragment of SEQ ID NO: 2 that has oxaloacetate hydrolase activity.  
     
     
         55 . The method of  claim 50 , wherein the oxaloacetate hydrolase has the amino acid sequence of SEQ ID NO: 2.  
     
     
         56 . The method of  claim 50 , wherein the parent cell comprises the nucleic acid sequence of SEQ ID NO: 1.  
     
     
         57 . The method of  claim 50 , wherein the parent cell comprises the cDNA sequence of SEQ ID NO: 1.  
     
     
         58 . The method of  claim 50 , wherein the oxaloacetate hydrolase is encoded by a nucleic acid sequence that hybridizes under said high stringency conditions with the nucleic acid sequence of SEQ ID NO: 1, the cDNA sequence of SEQ ID NO: 1, the complete complementary strand of SEQ ID NO: 1, and/or the complete complementary strand of the cDNA sequence of SEQ ID NO: 1.  
     
     
         59 . The method of  claim 50 , comprising: 
 (a) introducing into a parent cell a nucleic acid sequence comprising a modification of at least one of the genes responsible for the production of a oxaloacetate dehydrolase, and    (b) identifying the mutant cell from step (a), wherein the mutant cell produces less of the oxaloacetate dehydrolase than the parent cell when cultured under the same conditions.    
     
     
         60 . A mutant cell produced by the method of  claim 50 .  
     
     
         61 . The mutant cell of  claim 60  which is a mutant cell of a filamentous fungal cell.  
     
     
         62 . The mutant cell of  claim 61  which is a mutant cell of an  Aspergillus, Acrernonium, Aureobasidiumr, Cryptococcus, Filibasidium, Fusarium, Gibberelia, Humicola, Magnaporthe, Mucor, Myceliophthora, Myrothecium, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma cell.    
     
     
         63 . The mutant cell of  claim 62  which is a mutant cell of an  Aspergillus niger  cell.  
     
     
         64 . The mutant cell of  claim 60 , wherein the mutant cell produces at least about 25% less of the oxaloacetate hydrolase or oxalic acid than the parent cell when cultured under identical conditions.  
     
     
         65 . The mutant cell of  claim 64 , wherein the mutant cell produces no oxaloacetate hydrolase or oxalic acid.  
     
     
         66 . The mutant cell of  claim 60 , wherein the mutant cell further comprises one or more modifications of one or more third nucleic acid sequences, wherein the modification reduces or eliminates expression of the one or more third nucleic acid sequences.  
     
     
         67 . The mutant cell of  claim 66 , wherein the third nucleic acid sequence encodes an enzyme selected from the group consisting of an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonucdease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, a pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, and xylanase.  
     
     
         68 . The mutant cell of  claim 60 , wherein the mutant cell further comprises a modification of one or more genes which encode a protease.