PHA synthase activity controls the molecular weight and polydispersity of polyhydroxybutyrate in vivo.
A synthetic operon for polyhydroxyalkanoate (PHA) biosynthesis designed to yield high levels of PHA synthase activity in vivo was constructed by positioning a genetic fragment encoding beta-ketothiolase and acetoacetyl-CoA reductase behind a modified synthase gene containing an Escherichia coli promoter and ribosome binding site. Plasmids containing the synthetic operon and the native Alcaligenes eutrophus PHA operon were transformed into E. coli DH5 alpha and analyzed for polyhydroxybutyrate production. The molecular weight of polymer isolated from recombinant E. coli containing the modified synthase construct, determined by multiangle light scattering, was lower than that of the polymer from E. coli containing the native A. eutrophus operon. A further decrease in polyester molecular weight was observed with increased induction of the PHA biosynthetic genes in the synthetic operon. Comparison of the enzyme activity levels of PHA biosynthetic enzymes in a strain encoding the native operon with a strain possessing the synthetic operon indicates that the amount of polyhydroxyalkanoate synthase in a host organism plays a key role in controlling the molecular weight and the polydispersity of polymer.
Sim
S J
SJ
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Snell
K D
KD
Hogan
S A
SA
Stubbe
J
J
Rha
C
C
Sinskey
A J
AJ
D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism*; D000421:Alcaligenes / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D005285:Fermentation; D006885:Hydroxybutyrates / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D009876:Operon / Q000235:genetics; D011091:Polyesters / Q000737:chemistry* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014169:Transformation, Bacterial; D000094122:Polyhydroxybutyrates
D000217:Acyltransferases; D006885:Hydroxybutyrates; D011091:Polyesters; D011994:Recombinant Proteins; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C077225:poly(3-hydroxyalkanoic acid) synthase