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Applied and Environmental Microbiology, September 2009, p. 5461-5466, Vol. 75, No. 17
0099-2240/09/$08.00+0     doi:10.1128/AEM.01072-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Tolerance of the Ralstonia eutropha Class I Polyhydroxyalkanoate Synthase for Translational Fusions to Its C Terminus Reveals a New Mode of Functional Display{triangledown} ,{dagger}

Anika C. Jahns and Bernd H. A. Rehm*

Institute for Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North, New Zealand

Received 11 May 2009/ Accepted 23 June 2009

Here, the class I polyhydroxyalkanoate synthase (PhaC) from Ralstonia eutropha was investigated regarding the functionality of its conserved C-terminal region and its ability to tolerate translational fusions to its C terminus. MalE, the maltose binding protein, and green fluorescent protein (GFP) were considered reporter proteins to be translationally fused to the C terminus. Interestingly, PhaC remained active only when a linker was inserted between PhaC and MalE, whereas MalE was not functional. However, the extension of the PhaC N terminus by 458 amino acid residues was required to achieve a functionality of MalE. These data suggested a positive interaction of the extended N terminus with the C terminus. To assess whether a linker and/or N-terminal extension is generally required for a functional C-terminal fusion, GFP was fused to the C terminus of PhaC. Both fusion partners were active without the requirement of a linker and/or N-terminal extension. A further reporter protein, the immunoglobulin G binding ZZ domain of protein A, was translationally fused to the N terminus of the fusion protein PhaC-GFP and resulted in a tripartite fusion protein mediating the production of polyester granules displaying two functional protein domains.

* Corresponding author. Mailing address: Institute for Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North, New Zealand. Phone: 64 6 350 5515, ext. 7890. Fax: 64 6 350 2267. E-mail: b.rehm{at}massey.ac.nz

{triangledown} Published ahead of print on 6 July 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, September 2009, p. 5461-5466, Vol. 75, No. 17
0099-2240/09/$08.00+0     doi:10.1128/AEM.01072-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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