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

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.

[1]  Makoto Kato,et al.  Preparation of aliphatic poly(thioester) by the lipase-catalyzed direct polycondensation of 11-mercaptoundecanoic acid. , 2005, Biomacromolecules.

[2]  E. Laskowska,et al.  The Small Heat Shock Protein IbpA of Escherichia coli Cooperates with IbpB in Stabilization of Thermally Aggregated Proteins in a Disaggregation Competent State* , 2005, Journal of Biological Chemistry.

[3]  A. Steinbüchel,et al.  Application of the BPEC Pathway for Large-Scale Biotechnological Production of Poly(3-Mercaptopropionate) by Recombinant Escherichia coli, Including a Novel In Situ Isolation Method , 2005, Applied and Environmental Microbiology.

[4]  A. Steinbüchel,et al.  Poly(3-mercaptopropionate): a nonbiodegradable biopolymer? , 2005, Biomacromolecules.

[5]  A. Steinbüchel,et al.  Poly(3-hydroxybutyrate) granule-associated proteins: impacts on poly(3-hydroxybutyrate) synthesis and degradation. , 2005, Biomacromolecules.

[6]  S. Lee,et al.  Roles and applications of small heat shock proteins in the production of recombinant proteins in Escherichia coli. , 2004, Biotechnology and bioengineering.

[7]  T. Lütke-Eversloh,et al.  Studies on the biodegradability of polythioester copolymers and homopolymers by polyhydroxyalkanoate (PHA)-degrading bacteria and PHA depolymerases , 2004, Archives of Microbiology.

[8]  A. Steinbüchel,et al.  The complex structure of polyhydroxybutyrate (PHB) granules: four orthologous and paralogous phasins occur in Ralstonia eutropha. , 2004, Microbiology.

[9]  S. Taguchi,et al.  Evolution of polyhydroxyalkanoate (PHA) production system by "enzyme evolution": successful case studies of directed evolution. , 2004, Macromolecular bioscience.

[10]  Hyun‐dong Shin,et al.  Functional role of granule-associated genes, phaP and phaR, in poly-β-hydroxybutyrate biosynthesis in recombinant E. coli harboring phbCAB operon , 2003, Biotechnology Letters.

[11]  A. Steinbüchel,et al.  Microbial polythioesters. , 2004, Macromolecular bioscience.

[12]  Masatsugu Shimomura,et al.  Design of novel biointerfaces (II). Fabrication of self-organized porous polymer film with highly uniform pores. , 2004, Bio-medical materials and engineering.

[13]  A. Steinbüchel,et al.  Physical properties of microbial polythioesters: characterization of poly(3-mercaptoalkanoates) synthesized by engineered Escherichia coli. , 2003, Biomacromolecules.

[14]  V. Gerke,et al.  Ca2+-dependent binding and activation of dormant ezrin by dimeric S100P. , 2003, Molecular biology of the cell.

[15]  S. Iwata,et al.  Enzyme-catalyzed preparation of aliphatic polyesters containing thioester linkages , 2003 .

[16]  A. Steinbüchel,et al.  Novel precursor substrates for polythioesters (PTE) and limits of PTE biosynthesis in Ralstonia eutropha. , 2003, FEMS microbiology letters.

[17]  Hellmut Eckert,et al.  Biosynthesis of novel thermoplastic polythioesters by engineered Escherichia coli , 2002, Nature materials.

[18]  L. Willmitzer,et al.  Constitutive Expression of the β-Ketothiolase Gene in Transgenic Plants. A Major Obstacle for Obtaining Polyhydroxybutyrate-Producing Plants1 , 2002, Plant Physiology.

[19]  A. Sinskey,et al.  The Ralstonia eutropha PhaR Protein Couples Synthesis of the PhaP Phasin to the Presence of Polyhydroxybutyrate in Cells and Promotes Polyhydroxybutyrate Production , 2002, Journal of bacteriology.

[20]  A. Steinbüchel,et al.  Characterization of microbial polythioesters: physical properties of novel copolymers synthesized by Ralstonia eutropha. , 2002, Biomacromolecules.

[21]  A. Steinbüchel,et al.  Biosynthesis of poly(3-hydroxybutyrate-co-3-mercaptobutyrate) as a sulfur analogue to poly(3-hydroxybutyrate) (PHB). , 2001, Biomacromolecules.

[22]  E. Laskowska,et al.  Transcription of the ibpB Heat-Shock Gene Is under Control of ς32- and ς54-Promoters, a Third Regulon of Heat-Shock Response , 2001 .

[23]  Mee-Jung Han,et al.  Proteome Analysis of Metabolically EngineeredEscherichia coli Producing Poly(3-Hydroxybutyrate) , 2000, Journal of bacteriology.

[24]  A. Steinbüchel,et al.  Identification of a new class of biopolymer: bacterial synthesis of a sulfur-containing polymer with thioester linkages. , 2001, Microbiology.

[25]  E. Laskowska,et al.  Transcription of the ibpB heat-shock gene is under control of sigma(32)- and sigma(54)-promoters, a third regulon of heat-shock response. , 2001, Biochemical and biophysical research communications.

[26]  A. Steinbüchel,et al.  In vitro synthesis of poly(3-hydroxydecanoate): purification and enzymatic characterization of type II polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa , 2000, Applied Microbiology and Biotechnology.

[27]  A. Steinbüchel,et al.  Exploitation of butyrate kinase and phosphotransbutyrylase from Clostridium acetobutylicum for the in vitro biosynthesis of poly(hydroxyalkanoic acid) , 2000, Applied Microbiology and Biotechnology.

[28]  A. Steinbüchel,et al.  A Novel Genetically Engineered Pathway for Synthesis of Poly(Hydroxyalkanoic Acids) in Escherichia coli , 2000, Applied and Environmental Microbiology.

[29]  A. Steinbüchel,et al.  In vitro biosynthesis of poly(3-hydroxybutyric acid) by using purified poly(hydroxyalkanoic acid) synthase of Chromatium vinosum , 1998, Applied Microbiology and Biotechnology.

[30]  C. Föllner,et al.  Considerations on the structure and biochemistry of bacterial polyhydroxyalkanoic acid inclusions. , 1995, Canadian journal of microbiology.

[31]  A. Steinbüchel,et al.  Analysis of a 24-kilodalton protein associated with the polyhydroxyalkanoic acid granules in Alcaligenes eutrophus , 1995, Journal of bacteriology.

[32]  A. Steinbüchel,et al.  Diversity of bacterial polyhydroxyalkanoic acids , 1995 .

[33]  J. Sanders,et al.  Biomimetic, amorphous granules of polyhydroxyalkanoates: composition, mobility, and stabilization in vitro by proteins , 1995 .

[34]  G. Huisman,et al.  Formation of polyester blends by a recombinant strain of Pseudomonas oleovorans: Different poly(3-hydroxyalkanoates) are stored in separate granules , 1993 .

[35]  A. Steinbüchel,et al.  Isolation and identification of granule-associated proteins relevant for poly(3-hydroxyalkanoic acid) biosynthesis in Chromatium vinosum D. , 1992, FEMS microbiology letters.

[36]  J. Gierse,et al.  Two novel heat shock genes encoding proteins produced in response to heterologous protein expression in Escherichia coli , 1992, Journal of bacteriology.

[37]  A. Steinbüchel,et al.  Formation of polyesters consisting of medium-chain-length 3-hydroxyalkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent pseudomonads , 1990, Applied and environmental microbiology.

[38]  R. Gross,et al.  Pseudomonas oleovorans as a Source of Poly(β-Hydroxyalkanoates) for Potential Applications as Biodegradable Polyesters , 1988, Applied and environmental microbiology.

[39]  D. Hanahan.  Studies on transformation of Escherichia coli with plasmids. , 1983, Journal of molecular biology.

[40]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[41]  K. Weber,et al.  The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. , 1969, The Journal of biological chemistry.

[42]  A. Spurr.  A low-viscosity epoxy resin embedding medium for electron microscopy. , 1969, Journal of ultrastructure research.