Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation from gamma-aminobutyrate and glutamate.
To provide 4-hydroxybutyryl-CoA for poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation from glutamate in Escherichia coli, an acetyl-CoA:4-hydroxybutyrate CoA transferase from Clostridium kluyveri, a 4-hydroxybutyrate dehydrogenase from Ralstonia eutropha, a gamma-aminobutyrate:2-ketoglutarate transaminase from Escherichia coli, and glutamate decarboxylases from Arabidopsis thaliana or E. coli were cloned and functionality tested by expression of single genes in E. coli to verify enzymatic activity, and uniquely assembled as operons under the control of the lac promoter. These operons were independently transformed into E. coli CT101 harboring the runaway replication vector pJM9238 for polyhydroxyalkanoate (PHA) production. Plasmid pJM9238 contains the PHA biosynthetic operon of R. eutropha under tac promoter control. Polyhydroxyalkanoate formation was monitored by nuclear magnetic resonance (NMR) spectroscopic analysis of the chloroform extracted and ethanol precipitated polyesters. Functionality of the biosynthetic pathway for copolymer production was demonstrated through feeding experiments using various carbon sources that supplied different precursors within the 4HB-CoA biosynthetic pathway.
Valentin
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HE
Monsanto Co., 800 North Lindbergh Boulevard, St. Louis, Missouri 63167, USA. henry.e.valentin@monsanto.com
Reiser
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Gruys
K J
KJ
D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular; D015256:Coenzyme A-Transferases / Q000378:metabolism; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005968:Glutamate Decarboxylase / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D006884:Hydroxybutyrate Dehydrogenase / Q000378:metabolism; D006885:Hydroxybutyrates / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011091:Polyesters / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*; D000094122:Polyhydroxybutyrates
D015256:Coenzyme A-Transferases; D017931:DNA Primers; D005968:Glutamate Decarboxylase; D018698:Glutamic Acid; D006884:Hydroxybutyrate Dehydrogenase; D006885:Hydroxybutyrates; D011091:Polyesters; D005680:gamma-Aminobutyric Acid; C068056:poly(3-hydroxybutyrate-co-4-hydroxybutyrate); D000094122:Polyhydroxybutyrates; C019416:4-hydroxybutyrate dehydrogenase; C072231:4-hydroxybutyrate CoA-transferase