Compositions and methods for improved protein production

Abstract

The present invention relates to the identification of novel nucleic acid sequences, designated herein as 7p, 8k, 7E, 9G, 8Q and 203, in a host cell which effect protein production. The present invention also provides host cells having a mutation or deletion of part or all of the gene encoding 7p, 8k, 7E, 9G, 8Q and 203, which are presented in FIG. 1 , and are SEQ ID NOS.: 1-6, respectively. The present invention also provides host cells further comprising a nucleic acid encoding a desired heterologous protein such as an enzyme.

1 . A filamentous fungus having a mutation or deletion of part or all of a gene having the sequence selected from at least one sequence set forth in any one of SEQ ID NOs:1-6, and said mutation or deletion results in the enhanced production of a desired polypeptide compared to the parent filamentous fungus. 
     
     
         2 . The filamentous fungus of  claim 1 , wherein said filamentous fungus is capable of expressing a heterologous protein. 
     
     
         3 . The filamentous fungus of  claim 1 , wherein said heterologous protein is selected from the group consisting of hormones, enzymes, growth factors, and cytokines. 
     
     
         4 . The filamentous fungus of  claim 3  wherein said heterologous protein is an enzyme. 
     
     
         5 . The filamentous fungus of  claim 4  wherein said enzyme is selected from the group consisting of proteases, carbohydrases, lipases, isomerases, racemases, epimerases, tautomerases, mutases, transferases, kinases and phosphatases. 
     
     
         6 . A method for the production of a heterologous protein in a transformed filamentous fungus host cell according to  claim 1 , comprising the steps of:
 (a) obtaining a filamentous fungus host cell comprising a nucleic acid encoding said heterologous protein wherein said host cell contains a mutation or deletion in at least one nucleic acid sequence having the sequence set forth in any one of SEQ ID NOs:1-6, wherein said mutation or deletion results in the enhanced production of the heterologous protein compared to a parent filamentous fungus: and   (b) growing said filamentous fungus host cell under conditions suitable for the expression of said heterologous protein.   
     
     
         7 . The method of  claim 6 , wherein said gene comprises the nucleic acid sequence set forth in SEQ ID NO:1. 
     
     
         8 . An isolated nucleotide sequence selected from a group consisting of SEQ ID NOs: 1-6. 
     
     
         9 . The isolated nucleotide sequence according to  claim 8  wherein said sequence has been modified. 
     
     
         10 . The isolated nucleotide sequence according to  claim 9  wherein said modification is selected from truncation, deletion, mutation or other means of inactivation. 
     
     
         11 . A vector comprising at least one of the nucleotide sequences according to  claim 9 . 
     
     
         12 . A host cell transformed with a vector according to  claim 11 . 
     
     
         13 . (canceled) 
     
     
         14 . A method of producing a heterologous desired polypeptide said method comprising
 (a) obtaining a parental host cell strain;   (b) transforming said parental cell strain with a vector encoding a desired polypeptide;   (c) transforming said parental cell strain with a vector according to  claim 11  to produce a modified host cell;   (d) selecting modified host cells that produce said heterologous desired polypeptide; and   (e) culturing said modified host cell in a suitable growth medium for production of said heterologous desired polypeptide wherein steps (b) and (c) may be done in any order or simultaneously.   
     
     
         15 . The method of  claim 14 , wherein said suitable growth medium additionally comprises an inducer of cellulase production. 
     
     
         16 . The method of  claim 15 , wherein said inducer of cellulase production is selected from one or more of cellulose, lactose, sophorose and glucose/sophorose. 
     
     
         17 . The method of  claim 14 , wherein the method additionally comprises the at least partial purification of cellulases produced by said culture. 
     
     
         18 . A method for producing a novel strain of  T. reesei  using insertional mutagenesis wherein said novel strain of  T. reesei  has enhanced cellulase production as compared to the parent strain of  T. reesei , comprising:
 (a) preparing a population of competent  Agrobacterium  sp. cells by electroporating into competent  Agrobacterium  sp. cells an expression vector comprising, in operable condition, the left and right T-DNA boarder regions, pV51 plasmid origins for replication in  Agrobacterium  sp. and bacterial markers to confer resistance to chloramphenicol to create a population comprising transformed  Agrobacterium  sp. cells;   (b) selecting for  Agrobacterium  sp. from said population of step (a);   (c) inoculating a culture of  T. reesei  spores with the  Agrobacterium  sp. transformants of step (b) to create an induction culture;   (d) culturing said induction culture of step (c) at about 18° C. and for about 24 hours to create a population;   (e) transferring samples of said population of transformed  T. reesei  of step (d) to selective medium and isolating colonies of  T. reesei  effective in degrading cellulose; and   (f) comparing the effectiveness of cellulose degradation between the  T. reesei  of the isolated colonies of step (e) and the non-transformed parent strain, wherein said  T. reesei  of the isolated colonies of step (e) are enhanced to in cellulose degradation when compared to the non-transformed parent strain.   
     
     
         19 . The method of  claim 18 , wherein said  Agrobacterium  sp. cells are selected from  Agrobacterium tumefaciens  and  Agrobacterium rhizogenes.