Heat-shock protein HspA mimics the function of phasins sensu stricto in recombinant strains of Escherichia coli accumulating polythioesters or polyhydroxyalkanoates

• Published in: Microbiology (Society for General Microbiology) 2007-02, Vol.153 (2), p.366-374
• Main Authors: Tessmer, Nicole, Konig, Simone, Malkus, Ursula, Reichelt, Rudolf, Potter, Markus, Steinbuchel, Alexander
• Format: Article
• Language: English
• Subjects: 3-Hydroxybutyric Acid - metabolism; 3-Mercaptopropionic Acid - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Biological and medical sciences; Clostridium acetobutylicum; Cupriavidus necator - genetics; Cupriavidus necator - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli - ultrastructure; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Heat-Shock Proteins - metabolism; Metabolism. Enzymes; Microbiology; Microscopy, Electron, Transmission; Polyesters - metabolism; Polymers - metabolism; Ralstonia eutropha; Recombinant Proteins - metabolism; Recombination, Genetic
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.29260-0

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 prev