Use of homologous amdS genes as selectable markers

Abstract

The present invention discloses novel amdS genes from fungi previously not known to contain and amdS gene, such as Aspergillus niger and Penicillium chrysogenum. The novel amdS genes can be used as homologous selectable marker genes in the transformation of these fungi. Alternatively, the cloned amdS genes can be used to inactivate the endogenous copy of the gene in order to reduce the background in transformation experiments.

1 . A DNA sequence encoding an acentamidase and characterized in that the DNA sequence is derived from an organism other than  Aspergillus nidulans, Aspergillus oryzae  and  Saccharomyces cerevisiae.    
     
     
         2 . A DNA sequence encoding an acetamidase which comprises an a amino acid sequence which is characterized in that: 
 a) its amino acid positional identity with one of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 is more than 30%, and    b) its amino acid positional identity with each of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 is less than 100%.    
     
     
         3 . A DNA sequences according to claims  1  or  2 , and characterized in that the DNA sequence is derived from an organism selected from the group consisting of: 
 fungi from the  Aspergillus niger  group, the  Aspergillus glaucus  group, the  Aspergillus terreus  group, the  Aspergillus restrictus  group, the  Aspergillus fumigatus  group, the  Aspergillus cervinus  group, the  Aspergillus ornatus  group, the  Aspergillus clavatus  group, the  Aspergillus versicolor  group, the  Aspergillus ustus  group, the  Aspergillus wentii  group, the  Aspergillus ochraceus  group, the  Aspergillus candidus  group, the  Aspergillus cremeus  group, the  Aspergillus sparsus  group, Penicillius species, Trichoderma species, Mucor species, Rhizopus species, Phanerochaete species, Neurospora species, Humicola species, Claviceps species, Sordaria species, Ustilago species, Fusarium species, Schizophyllum species, Cephalosporium species, Acremonium species, edible fungi of which preferably Agaricus bisporus, Kluyveromyces species, Yarrowia species, Candia species, Hansenula species, Pichia species and Phaffia species.  
 
     
     
         4 . A DNA sequence according to  claim 3  and characterized in that the DNA sequence is derived from  Aspergillus niger  or  Penicillium chrysogenum.    
     
     
         5 . A recombinant DNA construct which comprises a DNA sequence as defined in any one of claims  1 - 4 .  
     
     
         6 . A recombinant DNA construct according to  claim 5  and characterized in that the DNA sequence is operably linked to a promoter which is native to the DNA sequence.  
     
     
         7 . A recombinant DNA construct according to  claim 5  and characterized in that the DNA sequence is operably linked to a promoter which is foreign to the DNA sequence.  
     
     
         8 . A recombinant DNA construct according to  claim 7 , wherein the promoter foreign to the DNA sequence is a promoter derived from a gene which is selected from the group consisting of the genes encoding glycolytic enzymes and enzymes involved in alcohol metabolism.  
     
     
         9 . A recombinant DNA construct according to any one of claims  7 - 8 , wherein the DNA sequence and the promoter are derived from the same species.  
     
     
         10 . A recombinant DNA construct according to any one of claims  5 - 9 , further comprising a gene of interest.  
     
     
         11 . A cell which comprises a recombinant DNA construct as defined in any one of claims  5 - 10 .  
     
     
         12 . A cell characterized in that the cell does not contain an active endogenous copy of an acetamidase gene comprising a DNA sequence as defined in any one of claims  1 - 4 .  
     
     
         13 . A cell according to  claim 12 , characterized in that the endogenous copy of the acetamidase gene has been inactivated by a homologous recombination event.  
     
     
         14 . A cell according to  claim 13  and characterized in that the homologous recombination event is a gene-replacement.  
     
     
         15 . A cell according to any one of claims  11 - 14 , further comprising a recombinant gene of interest.  
     
     
         16 . A cell according to any one of claims  11 - 15 , further characterized in that an endogenous gene of interest has been inactivated.  
     
     
         17 . A process for the production of a desired product, and characterized in that the process comprises the steps of: 
 a) culturing a recombinant cell as defined in claims  15  or  16 , under conditions conducive to the production of the product, and    b) recovering the product.    
     
     
         18 . A method for obtaining an DNA sequence as defined in any one of claims  1 - 4 , wherein the method comprises the steps of: 
 a) identification of conserved regions in the amino acid sequences of known acetamidase genes,    b) amplification of a DNA fragment in a PCR, using as primers a set of degenerate oligonucleotides corresponding to the conserved regions identified in a), on a template nucleic acid containing the DNA sequence,    c) isolation of the DNA sequence using the DNA fragment amplified in b) as hybridization probe to screen a DNA library containing the DNA sequence.    
     
     
         19 . A DNA sequence as defined in any one of claims  1 - 4 , wherein the DNA sequence is obtainable by the method of  claim 18 .  
     
     
         20 . A method for inactivation of an endogenous copy of an acetamidase gene comprising a DNA sequence as defined in any one of claims  1 - 4 , wherein the method comprises the steps of: 
 a) construction of an inactivation vector comprising a disrupted copy of the acetamidase gene,    b) transformation of a host cell with the inactivation vector,    c) selection of transformants with an inactivated copy of the acetamidase gene.    
     
     
         21 . A method according to  claim 20 , wherein the inactivation vector is capable of replacing the endogenous copy of the acetamidase gene.  
     
     
         22 . A method of culturing cells at least a proportion of which consists of cells according to  claim 15  or  16  in a culture medium, wherein the culture medium comprises acetamide as sole carbon and/or nitrogen source.  
     
     
         23 . The method according to  claim 22 , wherein said culturing results in the enrichment of the proportion of cells according to  claim 15  or  16 .  
     
     
         24 . A living cell selected from the group consisting of fungi from the  Aspergillus niger  group, the  Aspergillus glaucus  group, the  Aspergillus terreus  group, the  Aspergillus restrictus  group, the  Aspergillus fumigatus  group, the  Aspergillus cervinus  group, the  Aspergillus ornatus  group, the  Aspergillus clavatus  group, the  Aspergillus versicolor  group, the  Aspergillus ustus  group, the  Aspergillus wentii  group, the  Aspergillus ochraceus  group, the  Aspergillus candidus  group, the  Aspergillus cremeus  group, the  Aspergillus sparsus  group, Penicillium species, Trichoderma species, Mucor species, Rhizopus species, Phanerochaete species, Neurospora species, Humicola species, Claviceps species, Sordaria species, Ustilago species, Fusarium species, Schizophyllum species, Cephalosporium species, Acremonium species, edible fungi of which preferably  Agaricus bisporus,  Kluyveromyces species, Yarrowia species, Candida species, Hansenula species, Pichia species and Phaffia species, characterized by their ability to grow well on a culture medium containing acetamide as sole carbon and/or nitrogen source and wherein said ability is not caused by the expression of a heterologous acetamidase gene.