Production of organic acids from aspergillus cis-aconitic acid decarboxylase (cada) deletion strains

Abstract

This application provides recombinant Aspergillus fungi having an endogenous cis-aconitic acid decarboxylase (cadA) gene genetically inactivated, which allows aconitic acid production by the recombinant fungi. Such recombinant fungi can further include an exogenous nucleic acid molecule encoding aspartate decarboxylase (panD), an exogenous nucleic acid molecule encoding β-alanine-pyruvate aminotransferase (BAPAT), and an exogenous nucleic acid molecule encoding 3-hydroxypropironate dehydrogenase (HPDH). Kits including these fungi, and methods of using these fungi to produce aconitic acid and 3-hydroxypropionic acid (3-HP) are also provided.

We claim: 
     
         1 . An isolated recombinant  Aspergillus  fungus comprising a genetic inactivation of an endogenous cis-aconitic acid decarboxylase (cadA) gene. 
     
     
         2 . The isolated recombinant  Aspergillus  fungus of  claim 1 , wherein the  Aspergillus  fungus is  Aspergillus pseudoterreus.    
     
     
         3 . The isolated recombinant  Aspergillus  fungus of  claim 1 , wherein the  Aspergillus  fungus is  Aspergillus terreus.    
     
     
         4 . The isolated recombinant  Aspergillus  fungus of  claim 1 , wherein the endogenous cadA gene is genetically inactivated by complete or partial deletion mutation or by insertional mutation. 
     
     
         5 . The isolated recombinant  Aspergillus  fungus of  claim 1 , wherein the cadA gene prior to its genetic inactivation encodes a protein having at least 80% sequence identity to SEQ ID NO: 50 or 52. 
     
     
         6 . The isolated recombinant  Aspergillus  fungus of  claim 1 , wherein the cadA gene prior to its genetic inactivation comprises a coding sequence comprising at least 80% sequence identity to SEQ ID NO: 49, 51, 59 or 92. 
     
     
         7 . A composition comprising the isolated recombinant  Aspergillus  fungus of  claim 1 . 
     
     
         8 . A kit, comprising:
 the isolated recombinant  Aspergillus  fungus of  claim 1 ; and   a medium for culturing the fungus.   
     
     
         9 . A method of making aconitic acid, comprising:
 culturing the isolated recombinant  Aspergillus  fungus of  claim 1  under conditions that permit the fungus to make aconitic acid; thereby making aconitic acid.   
     
     
         10 . The method of  claim 9 , wherein the fungus is cultured in Riscaldati medium. 
     
     
         11 . The method of  claim 9 , further comprising isolating the aconitic acid from culture media or from the fungus. 
     
     
         12 . The method of  claim 9 , wherein the aconitic acid is cis-aconitic acid.