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Poly(3-hydroxyalkanoate) polymerase synthesis and in vitro activity in recombinant Escherichia coli and Pseudomonas putida

  • Applied Genetics and Molecular Biotechnology
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Abstract

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.

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Acknowledgements

We thank Prof. D. Dennis for providing E. coli strain JMU193, Dr. M. Nieboer for providing plasmid pAlkVSVG1, Prof. S. Panke for providing plasmid pAlkSCm and Prof. H. Ploegh for providing monoclonal antibodies to the VSV G tag. We thank Dr. M. Passarge for preparing partially purified PhaC1.

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Authors and Affiliations

  1. Biocompatible Materials, Materials Science and Technology (EMPA), 9014, St. Gallen, Switzerland

    Qun Ren & Manfred Zinn

  2. Catchmabs, Nieuwe Kanaal 7, 6709 PA, Wageningen, The Netherlands

    Guy de Roo

  3. Institute of Biotechnology, ETH Hönggerberg, 8093, Zurich, Switzerland

    Jan B. van Beilen & Bernard Witholt

  4. ETH-Präsidialstab, ETH Zentrum, HG F 52.2, 8092, Zurich, Switzerland

    Birgit Kessler

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  1. Qun Ren
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Correspondence to Qun Ren.

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Ren, Q., de Roo, G., van Beilen, J.B. et al. Poly(3-hydroxyalkanoate) polymerase synthesis and in vitro activity in recombinant Escherichia coli and Pseudomonas putida . Appl Microbiol Biotechnol 69, 286–292 (2005). https://doi.org/10.1007/s00253-005-1995-1

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  • DOI: https://doi.org/10.1007/s00253-005-1995-1

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