Bioabsorbable polymer containing 2-hydroxyacid monomers
View Patent ↗Genetically engineered organisms for production of PHA copolymers containing 2-hydroxyacid monomers and the methods of making and using thereof have been developed. The copolymers containing 2-hydroxyacid monomers can be synthesized via biosynthesis by the action of a PHA polymerase in a living cell. By changing the genetic background of the cells, one can control specific metabolic pathways allowing control of the level of glycolic acid co-monomer in the PHA polymer.
1. A method of producing a glycolic acid-containing polyhydroxyalkanoate polymer comprising
expressing in an organism selected from the group consisting of yeast, bacteria, fungi and plants,
(a) heterologous polynucleotides encoding enzymes required for production of polyhydroxyalkanoate selected from the group consisting of a polyhydroxyalkanoate (PHA) synthase, a beta-ketothiolase, and an aceto-acetyl CoA reductase,
(b) an acyl-CoA transferase, and
(c) at least one enzyme for the production of glycolyl Co-A selected from the group consisting of aldehyde dehydrogenases, diol oxidoreductases, enoyl-CoA dehydratase, beta acyl-CoA dehydrogenase, thiolases and acyl-CoA transferases,
wherein at least one of the polynucleotides encoding an enzyme is heterologous, and
providing 1,4-butanediol which is converted into glycolyl-CoA and 4-hydroxybutyryl-CoA monomers by enzymes expressed by the organism and polymerized by a PHA synthase to produce a poly(4-hydroxybutyrate-co-glycolate) polymer.
2. The method of claim 1 wherein the PHA synthase is a synthase of poly(3-hydroxyalkanoate) or poly(4-hydroxyalkanaote).
3. The method of claim 1 wherein the polynucleotides encode enzymes selected from the group consisting of acetoacetyl-CoA reductases and beta-ketoacyl-CoA thiolases.
4. The method of claim 1 wherein the PHA synthase is poly(3-hydroxyalkanoate) synthase.
5. The method of claim 1 wherein the PHA synthase is poly(4-hydroxyalkanoate) synthase.
6. The method of claim 5 wherein the PHA synthase is poly(4-hydroxybutyrate) synthase.
7. The method of claim 1 wherein the organism is a bacterium.
8. The method of claim 7 wherein the organism is E. coli.
9. The method of claim 1 wherein the organism is E. coli having mutations selected from fadR601 and atoC constitutive mutations.
10. The method of claim 1 wherein the aldehyde dehydrogenase is from E. coli , wherein the diol oxidoreductase is from Klebsiella pneumoniae , and wherein the acyl-CoA transferase is from Clostridium kluyveri.
11. The method of claim 1 wherein the polyhydroxyalkanoate is poly(4-hydroxybutyrate-co-glycolate).
12. The method of claim 1 wherein the polyhydroxyalkanoate is poly(3-hydroxybutyrate-co-glycolate).