Improved muconic acid production from genetically engineered microorganisms

Abstract

The subject of this invention is improvements in the yield and titer of biological production of muconic acid by fermentation. Increased activity of one or more enzymes involved in the muconic acid pathway leads to increased production of muconic acid.

1 - 30 . (canceled) 
     
     
         31 . A genetically engineered microorganism that produces muconic acid from a non-aromatic carbon source comprising
 at least one exogenous gene encoding for 3,4-dihydroxybenzoic acid decarboxylase and   at least one gene that codes for a protein that increases the activity of said 3,4-dihydroxybenzoic acid decarboxylase.   
     
     
         32 . The genetically engineered microorganism of  claim 31 , wherein said genetically engineered microorganism produces at least 60 g/L of muconic acid in 72 hours. 
     
     
         33 . The genetically engineered microorganism of  claim 31 , wherein said genetically engineered microorganism is a bacterium. 
     
     
         34 . The genetically engineered microorganism of  claim 31 , wherein said genetically engineered microorganism is  Escherichia coli.    
     
     
         35 . The genetically engineered microorganism of  claim 31 , wherein said protein that increases the activity of said 3,4-dihydroxybenzoic acid decarboxylase is a KpdB protein encoded by a kpdB gene or a homolog thereof, and wherein said KpdB protein or homolog thereof has at least 25% amino acid identity to said KpdB protein. 
     
     
         36 . The genetically engineered microorganism of  claim 31 , wherein said protein that increases the activity of said 3,4-dihydroxybenzoic acid decarboxylase is a UbiX protein encoded by a ubiX gene or a homolog thereof, and wherein said UbiX protein or homolog thereof has at least 25% amino acid identity to said UbiX protein. 
     
     
         37 . The genetically engineered microorganism of  claim 31 , further comprising one or more exogenous genes selected from the group consisting of aroZ, qa-4, asbF, quiC and catAX. 
     
     
         38 . The genetically engineered microorganism of  claim 31 , further comprising one or more exogenous genes selected from the group consisting of aroB, aroD, aroF, aroG, aroH, tktA, talB, rpe, and rpi, wherein said one or more exogenous genes encode one or more proteins that function in a shikimic acid pathway. 
     
     
         39 . The genetically engineered microorganism of  claim 31 , further comprising at least one exogenous gene encoding a pyruvate carboxylase and a mutation of the phosphoenol pyruvate carboxylase gene. 
     
     
         40 . A genetically engineered  Escherichia coli  strain that produces muconic acid from a non-aromatic carbon source comprising at least one exogenous gene encoding for 3,4-dihydroxybenzoic acid decarboxylase and at least one gene that codes for a protein that increases the activity of said 3,4-dihydroxybenzoic acid decarboxylase, wherein said genetically engineered  Escherichia coli  strain produces at least 60 g/L of muconic acid in 72 hours. 
     
     
         41 . A genetically engineered  Escherichia coli  strain that produces muconic acid from a non-aromatic carbon source comprising at least one exogenous gene encoding for 3,4-dihydroxybenzoic acid decarboxylase and at least one gene that codes for a protein that increases the activity of said 3,4-dihydroxybenzoic acid decarboxylase, wherein said genetically engineered  Escherichia coli  strain produces at least 80 g/L of muconic acid in 72 hours. 
     
     
         42 . The genetically engineered  Escherichia coli  strain of  claim 40 , wherein said protein that increases the activity of said 3,4-dihydroxybenzoic acid decarboxylase is a KpdB protein encoded by a kpdB gene or a homolog thereof, and wherein said KpdB protein or homolog thereof has at least 25% amino acid identity to said KpdB protein. 
     
     
         43 . The genetically engineered  Escherichia coli  strain of  claim 40 , wherein said protein that increases the activity of said 3,4-dihydroxybenzoic acid decarboxylase is a UbiX protein encoded by a ubiX gene or a homolog thereof, and wherein said UbiX protein or homolog thereof has at least 25% amino acid identity to said UbiX protein. 
     
     
         44 . The genetically engineered  Escherichia coli  strain of  claim 40 , further comprising one or more exogenous genes selected from the group consisting of aroZ, qa-4, asbF, quiC and catAX. 
     
     
         45 . The genetically engineered  Escherichia coli  strain of  claim 40 , further comprising at least one exogenous gene encoding a pyruvate carboxylase and a mutation of the phosphoenol pyruvate carboxylase gene.