Poly(3-hydroxyalkanoate) polymerase synthesis and in vitro activity in recombinant Escherichia coli and Pseudomonas putida.
We tested the synthesis and in vitro activity of the poly(3-hydroxyalkanoate) (PHA) polymerase 1 from Pseudomonas putida GPo1 in both P. putida GPp104 and Escherichia coli JMU193. The polymerase encoding gene phaC1 was expressed using the inducible PalkB promoter. It was found that the production of polymerase could be modulated over a wide range of protein levels by varying inducer concentrations. The optimal inducer dicyclopropylketone concentrations for PHA production were at 0.03% (v/v) for P. putida and 0.005% (v/v) for E. coli. Under these concentrations the maximal polymerase level synthesized in the E. coli host (6% of total protein) was about three- to fourfold less than that in P. putida (20%), whereas the maximal level of PHA synthesized in the E. coli host (8% of total cell dry weight) was about fourfold less than that in P. putida (30%). In P. putida, the highest specific activity of polymerase was found in the mid-exponential growth phase with a maximum of 40 U/g polymerase, whereas in E. coli, the maximal specific polymerase activity was found in the early stationary growth phase (2 U/g polymerase). Our results suggest that optimal functioning of the PHA polymerase requires factors or a molecular environment that is available in P. putida but not in E. coli.
Ren
Qun
Q
Biocompatible Materials, Materials Science and Technology (EMPA), 9014 St. Gallen, Switzerland. qun.ren@empa.ch
de Roo
Guy
G
van Beilen
Jan B
JB
Zinn
Manfred
M
Kessler
Birgit
B
Witholt
Bernard
B
D000217:Acyltransferases / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000217:Acyltransferases; C067032:poly(3-hydroxyalkenoate)polymerase