Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation from gamma-aminobutyrate and glutamate

To provide 4-hydroxybutyryl-CoA for poly(3-hydroxybutyrate-co-4-hydroxybuty rate) formation from glutamate in Escherichia coli, an acetyl-CoA:4-hydroxy butyrate CoA transferase from Clostridium kluyveri, a 4-hydroxybutyrate deh ydrogenase from Ralstonia eutropha, a gamma-aminobutyrate:2-ketoglutarate t ransaminase from Escherichia coli, and glutamate decarboxylases from Arabid opsis thaliana or E. coli were cloned and functionality tested by expressio n of single genes in E. coil to verify enzymatic activity, and uniquely ass embled as operons under the control of the lac promoter. These operons were independently transformed into E. coil CT101 harboring the runaway replica tion vector pJM9238 for polyhydroxyalkanoate (PHA) production. Plasmid pJM9 238 contains the PHA biosynthetic operon of R. eutropha under tac promoter control. Polyhydroxyalkanoate formation was monitored by nuclear magnetic r esonance (NMR) spectroscopic analysis of the chloroform extracted and ethan ol precipitated polyesters. Functionality of the biosynthetic pathway for c opolymer production was demonstrated through feeding experiments using vari ous carbon sources that supplied different precursors within the 4HB-CoA bi osynthetic pathway. (C) 2000 John Wiley & Sons, Inc.