title: Efficient production of active polyhydroxyalkanoate synthase in Escherichia coli by coexpression of molecular chaperones.
abstract: The type I polyhydroxyalkanoate synthase from Cupriavidus necator was heterologously expressed in Escherichia coli with simultaneous overexpression of chaperone proteins. Compared to expression of synthase alone (14.55 mg liter(-1)), coexpression with chaperones resulted in the production of larger total quantities of enzyme, including a larger proportion in the soluble fraction. The largest increase was seen when the GroEL/GroES system was coexpressed, resulting in approximately 6-fold-greater enzyme yields (82.37 mg liter(-1)) than in the absence of coexpressed chaperones. The specific activity of the purified enzyme was unaffected by coexpression with chaperones. Therefore, the increase in yield was attributed to an enhanced soluble fraction of synthase. Chaperones were also coexpressed with a polyhydroxyalkanoate production operon, resulting in the production of polymers with generally reduced molecular weights. This suggests a potential use for chaperones to control the physical properties of the polymer.
authors: Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom.
authors: Nicholas M
authors: NM
authors: Thomson
authors: Azusa
authors: A
authors: Saika
authors: Kazunori
authors: K
authors: Ushimaru
authors: Smith
authors: S
authors: Sangiambut
authors: Takeharu
authors: T
authors: Tsuge
authors: David K
authors: DK
authors: Summers
authors: Easan
authors: E
authors: Sivaniah
mesh_terms: D000217:Acyltransferases / Q000096:biosynthesis* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D018833:Chaperonins / Q000096:biosynthesis* / Q000235:genetics; D020118:Cupriavidus necator / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C097970:GroESL protein, Bacteria; D011994:Recombinant Proteins; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; D018833:Chaperonins