Expression of PHA polymerase genes of Pseudomonas putida in Escherichia coli and its effect on PHA formation.
Poly-3-hydroxyalkanoates (PHAs) are synthesized by many bacteria as intracellular storage material. The final step in PHA biosynthesis is catalyzed by two PHA polymerases (phaC) in Pseudomonas putida. The expression of these two phaC genes (phaC1 and phaC2)was studied in Escherichia coli, either under control of the native promoter or under control of an external promoter. It was found that the two phaC genes are not expressed in E. coli without an external promoter. During heterologous expression of phaC from Plac on a high copy number plasmid, a rapid reduction of the number of colony forming units was observed, especially for phaC2. It appears that the plasmid instability was partially caused by high-level production of PHA polymerase. Subsequently, tightly regulated phaC2 expression systems on a low copy number vector were applied in E. coli. This resulted in PHA yields of over 20 of total cell dry weight, which was 2 fold higher than that obtained from the system where phaC2 is present on a high copy number vector. In addition, the PHA monomer composition differed when different gene expression systems or different phaC genes were applied.
Ren
Qun
Q
Biocompatible Materials, Swiss Federal Laboratories for Materials Testing and Research (EMPA), Lerchenfeldstrasse 5, 9014 St., Gallen, Switzerland. qun.ren-zulian@empa.ch
van Beilen
Jan B
JB
Sierro
Nicolas
N
Zinn
Manfred
M
Kessler
Birgit
B
Witholt
Bernard
B
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D010957:Plasmids; D011091:Polyesters / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D011091:Polyesters; D011994:Recombinant Proteins; D000217:Acyltransferases; C067032:poly(3-hydroxyalkenoate)polymerase