Biological Systems for Manufacture of Polyhydroxyalkanoate Polymers Containing 4-Hydroxyacids

Abstract

The gene encoding a 4-hydroxybutyryl-Co A transferase has been isolated from bacteria and integrated into the genome of bacteria also expressing a polyhydroxyalkanoate synthase, to yield an improved production process for 4HB-containing polyhydroxyalkanoates using transgenic organisms, including both bacteria and plants. The new pathways provide means for producing 4HB containing PHAs from cheap carbon sources such as sugars and fatty acids, in high yields, which are stable. Useful strains are obtaining by screening strains having integrated into their genomes a gene encoding a 4HB-CoA transferase and/or PHA synthase, for polymer production. Processes for polymer production use recombinant systems that can utilize cheap substrates. Systems are provided which can utilize amino acid degradation pathways, α-ketoglutarate, or succinate as substrate.

1 - 13 . (canceled) 
     
     
         14 . A recombinant bacterial host expressing enzymes to convert succinate or α-ketoglutarate to 4-hydroxybutyryl-CoA, a polyhydroxyalkanoate synthase, and a 4-hydroxybutyrate-CoA transfer, wherein at least one of the enzymes is encoded by a heterologous gene encoding an enzyme selected from the group consisting of a succinyl CoA-CoAtransferase, succinate semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, α-ketoglutamate decarboxylase, glutamate decarboxylase, glutamate dehydrogenase, polyhydroxyalkanoate synthase and a 4-hydroxybutyrate-CoA (4HB-CoA) transferase, wherein the host produces a 4-hydroxybutyrate polymer or copolymer. 
     
     
         15 . The host of  claim 14 , where the 4-hydroxybutyrate copolymer is a poly-3-hydroxybutyrate-4-hydroxybutyrate copolymer. 
     
     
         16 . The host of  claim 14  wherein the host expresses a polyhydroxyalkanoate synthase, a 4HB-CoA transferase, a succinyl CoA-CoA transferase, succinate semialdehyde dehydrogenase, and a 4-hydroxybutyrate dehydrogenase. 
     
     
         17 . The host of  claim 14  wherein the host is  E. coli.    
     
     
         18 . The host of  claim 14  wherein the host expresses a polyhydroxyalkanoate synthase, a 4HB-CoA transferase, 4-hydroxybutyrate dehydrogenase, α-ketoglutamate decarboxylase, glutamate decarboxylase, and a glutamate dehydrogenase. 
     
     
         19 . The host of  claim 14  further comprising genes expressing enzymes selected from the group consisting of β-ketothiolase and acetoacetyl CoA reductase. 
     
     
         20 . A method for enhancing production of polymers containing 4-hydroxybutyrate in a host comprising
 transforming a host expressing enzymes to convert succinate or α-ketoglutarate to 4-hydroxybutyryl-CoA, a polyhydroxyalkanoate synthase, and a 4-hydroxybutyrate-CoA transfer, wherein at least one of the enzymes is encoded by a heterologous gene encoding an enzyme selected from the group consisting of a succinyl CoA-CoAtransferase, succinate semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, α-ketoglutamate decarboxylase, glutamate decarboxylase, glutamate dehydrogenase, polyhydroxyalkanoate synthase and a 4-hydroxybutyrate-CoA (4HB-CoA) transferase, wherein the host produces a 4-hydroxybutyrate polymer or copolymer.   
     
     
         21 . The method of  claim 20  wherein the 4-hydroxybutyrate copolymer is a poly-3-hydroxybutyrate-4-hydroxybutyrate copolymer 
     
     
         22 . The method of  claim 21  wherein the host is transformed with genes encoding both a polyhydroxyalkanoate synthase and a 4HB-CoA transferase. 
     
     
         23 . The method of  claim 20  further comprising enhancing expression of the heterologous enzyme. 
     
     
         24 . The method of  claim 23  wherein production is enhanced by providing the host with 4-hydroxybutyrate as a substrate. 
     
     
         25 . The method of  claim 20  comprising providing a host expressing enzymes selected from the group consisting of α-ketoglutarate transaminase, glutamate-succinic semialdehyde transaminase, glutamate dehydrogenase, glutamate decarboxylase, 4-hydroxybutyrate dehydrogenase and 4-hydroxybutyryl CoA transferase. 
     
     
         26 . The method of  claim 20  comprising providing a host expressing a polyhydroxyalkanoate synthase, a 4HB-CoA transferase, 4-hydroxybutyrate dehydrogenase, α-keto glutamate decarboxylase, glutamate decarboxylase, and a glutamate dehydrogenase. 
     
     
         27 . (canceled)