Expression of PHA polymerase genes of Pseudomonas putida in Escherichia coli and its effect on PHA formation

• Published in: Antonie van Leeuwenhoek Vol. 87; no. 2; pp. 91 - 100
• Main Authors: QUN REN, VAN BEILEN, Jan B, SIERRO, Nicolas, ZINN, Manfred, KESSLER, Birgit, WITHOLT, Bernard
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer 01-02-2005; Springer Nature B.V
• Subjects: Acyltransferases - genetics; Acyltransferases - metabolism; Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Biological and medical sciences; Biosynthesis; Cloning, Molecular; E coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression; Genetic Vectors; Genetics; Microbiology; Plasmids; Polyesters - metabolism; Promoter Regions, Genetic; Pseudomonas putida - enzymology; Pseudomonas putida - genetics; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Pseudomonadales; Gene dosage; Plasmid stability; Escherichia coli; Genetic stability; Gene expression; mcl-PHA; Plasmid; Bacteria; Pseudomonadaceae; PHA monomer composition; Heterologous; Pseudomonas putida; Enterobacteriaceae; Heterologous expression
• ISSN: 0003-6072; 1572-9699
• DOI: 10.1007/s10482-004-1360-x

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.