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1 Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstraße 3, D-48149 Münster, Germany
2 Integrierte Funktionelle Genomik, Westfälische Wilhelms-Universität Münster, Röntgenstraße 21, D-48149 Münster, Germany
3 Institut für Medizinische Physik und Biophysik, Universitätsklinikum, Westfälische Wilhelms-Universität Münster, Robert-Koch-Straße 31, D-48149 Münster, Germany
Correspondence
Alexander Steinbüchel
steinbu{at}uni-muenster.de
Polyhydroxyalkanoic acids (PHAs) are synthesized by unspecific PHA synthases and deposited as energy and carbon storage granules in the cytoplasm of many prokaryotes. The number and size of the granules depend on the presence of phasins which are amphiphilic structural proteins occurring at the granule surface. Recently, it was shown that polythioesters (PTEs) are also synthesized by PHA synthases. To increase the yield of these polymers, the role of recombinant phasins was analysed in an artificial PHA-producing Escherichia coli strain. Overexpressed PhaP1 from Ralstonia eutropha H16 affected poly(3-mercaptopropionate) [poly(3MP)] and poly(3-hydroxybutyrate) [poly(3HB)] accumulation in recombinant E. coli, which expressed the non-natural BPEC pathway consisting of butyrate kinase and phosphotransbutyrylase from Clostridium acetobutylicum and PHA synthase from Thiococcus pfennigii. For this, BPEC-carrying E. coli with and without phaP1 was cultivated in presence of glucose as carbon source for growth plus 3-mercaptopropionate or 3-hydroxybutyrate as precursor substrates for poly(3MP) or poly(3HB) biosynthesis, respectively. In the presence of PhaP1, the recombinant E. coli produced about 50 or 68 % more poly(3MP) or poly(3HB), respectively. Therefore, coexpression of PhaP1 alongside the BPEC pathway is important for optimizing strains towards enhanced PHA or PTE production. Furthermore, in the absence of PhaP1, large amounts of the 16 kDa heat-shock protein HspA were synthesized and bound to the granule surface. Unusual small granules occurred in the cells of the recombinant E. coli strains. The diameter of the poly(3MP) granules was only 55±12 nm or 105±12 nm, and of the poly(3HB) granules only 56±10 or 110±22 nm in the presence or absence of PhaP1, respectively. This explains why no single granules capable of accumulating PHAs or PTEs occurred in the recombinant E. coli, unlike in PhaP1-negative mutants of R. eutropha. Obviously, HspA mimics the phasin, thereby preventing coalescence of granules into one single granule. However, the effect of PhaP1 on granule size and on amounts of accumulated polymers was more severe than that of HspA.
This article has been cited by other articles:
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  D. Jendrossek Polyhydroxyalkanoate Granules Are Complex Subcellular Organelles (Carbonosomes) J. Bacteriol., May 15, 2009; 191(10): 3195 - 3202. [Full Text] [PDF]
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L. Neumann, F. Spinozzi, R. Sinibaldi, F. Rustichelli, M. Potter, and A. Steinbuchel Binding of the Major Phasin, PhaP1, from Ralstonia eutropha H16 to Poly(3-Hydroxybutyrate) Granules J. Bacteriol., April 15, 2008; 190(8): 2911 - 2919. [Abstract] [Full Text] [PDF]
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 A. de Almeida, P. I. Nikel, A. M. Giordano, and M. J. Pettinari Effects of Granule-Associated Protein PhaP on Glycerol-Dependent Growth and Polymer Production in Poly(3-Hydroxybutyrate)-Producing Escherichia coli Appl. Envir. Microbiol., December 15, 2007; 73(24): 7912 - 7916. [Abstract] [Full Text] [PDF]
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