1887

Abstract

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 strain. Overexpressed PhaP1 from H16 affected poly(3-mercaptopropionate) [poly(3MP)] and poly(3-hydroxybutyrate) [poly(3HB)] accumulation in recombinant , which expressed the non-natural BPEC pathway consisting of butyrate kinase and phosphotransbutyrylase from and PHA synthase from . For this, BPEC-carrying with and without 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 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 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 , unlike in PhaP1-negative mutants of . 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.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.29260-0
2007-02-01
2020-08-03
Loading full text...

Full text loading...

/deliver/fulltext/micro/153/2/366.html?itemId=/content/journal/micro/10.1099/mic.0.29260-0&mimeType=html&fmt=ahah

References

  1. Allen S. P., Polyzzi J. O., Gierse J. K., Easton A. M.. 1992; Two novel heat shock genes encoding proteins produced in response to heterologous protein expression in Escherichia coli . J Bacteriol174:6938–6947
    [Google Scholar]
  2. Bohmert K., Balbo I., Tischendorf G., Willmitzer L., Steinbüchel A.. 2002; Constitutive expression of the β -ketothiolase gene in transgenic plants. A major obstacle for obtaining polyhydroxybutyrate-producing plants. Plant Physiol128:1282–1290[CrossRef]
    [Google Scholar]
  3. Brandl H., Gross R. A., Lenz R. W., Fuller R. C.. 1988; Pseudomonas oleovorans as a source of poly( β -hydroxyalkanoates) for potential applications as biodegradable polyesters. Appl Environ Microbiol66:2117–2124
    [Google Scholar]
  4. Elbanna K., Jendrossek D., Luftmann H., Lütke-Eversloh T., Steinbüchel A.. 2004; Studies on the biodegradability of polythioester copolymers and homopolymers by polyhydroxyalkanoate (PHA)-degrading bacteria and PHA depolymerases. Arch Microbiol182:212–225
    [Google Scholar]
  5. Han M.-J., Yoon S. S., Lee S. Y.. 2001; Proteome analysis of metabolically engineered Escherichia coli producing poly(3-hydroxybutyrate. J Bacteriol183:301–308[CrossRef]
    [Google Scholar]
  6. Han M.-J., Park S. J., Park T. J., Lee S. Y.. 2004; Roles and applications of small heat shock proteins in the production of recombinant proteins in Escherichia coli . Biotechnol Bioeng88:426–436[CrossRef]
    [Google Scholar]
  7. Hanahan D.. 1983; Studies on transformation of Escherichia coli with plasmids. J Mol Biol166:557–580[CrossRef]
    [Google Scholar]
  8. Horowitz D. M., Sanders J. K. M.. 1995; Biomimetic, amorphous granules of polyhydroxybutyrate: composition, mobility, and stabilization in vitro by proteins. Can J Microbiol41 (Suppl. 1:115–123
    [Google Scholar]
  9. Iwata S., Thoshima S., Matsumara S.. 2003; Enzyme-catalyzed preparation of aliphatic polyesters containing thioester linkages. Macromol Rapid Commun24:467–471[CrossRef]
    [Google Scholar]
  10. Jossek R., Reichelt R., Steinbüchel A.. 1998; In vitro biosynthesis of poly(3-hydroxybutyric acid) by using purified poly(hydroxyalkanoic acid) synthase of Chromatium vinosum . Appl Microbiol Biotechnol49:258–266[CrossRef]
    [Google Scholar]
  11. Kato M., Toshima K., Matsumura S.. 2005; Preparation of aliphatic poly(thioester) by the lipase-catalyzed direct polycondensation of 11-mercaptoundecanoic acid. Biomacromolecules6:2275–2280[CrossRef]
    [Google Scholar]
  12. Kawada J., Marchessault R. H., Lütke-Eversloh T., Steinbüchel A.. 2003; Physical properties of microbial polythioesters: characterization of poly(3-mercaptoalkanoates) synthesized by engineered Escherichia coli . Biomacromolecules4:1698–1702[CrossRef]
    [Google Scholar]
  13. Kim D. Y., Elbanna K., Thakor N., Lütke-Eversloh T., Steinbüchel A.. 2005; Poly(3-mercaptopropionate): a nonbiodegradable biopolymer?. Biomacromolecules6:897–901[CrossRef]
    [Google Scholar]
  14. Koltzscher M., Neumann C., Gerke V., König S.. 2003; A novel approach in search for dimer-specific S100 protein ligands identifies ezrin as a binding partner for S100P: Ca2+ activation by dormant ezrin by S100P. Mol Biol Cell14:2372–2384[CrossRef]
    [Google Scholar]
  15. Kuczyńska-Wiśnik D., Kedzierska S., Matuszewska E., Lund P., Taylor A., Lipinska B., Laskowska E.. 2002; Transcription of the ibpB heat shock gene is under control of sigma(32)- and sigma(54)-promoters, a third regulon of heat shock response. Biochem Biophys Res Commun284:57–64
    [Google Scholar]
  16. Laemmli U. K.. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227:680–685[CrossRef]
    [Google Scholar]
  17. Liebergesell M., Steinbüchel A.. 1992; Isolation and identification of granule-associated proteins relevant for poly(3-hydroxyalkanoic acid) biosynthesis in Chromatium vinosum D. FEMS Microbiol Lett99:227–232[CrossRef]
    [Google Scholar]
  18. Liu S. J., Steinbüchel A.. 2000a; Exploitation of butyrate kinase and phosphotrans-butyrylase from Clostridium acetobutylicum for the in vitro biosynthesis of poly(hydroxyalkanoic acid. Appl Microbiol Biotechnol53:545–552[CrossRef]
    [Google Scholar]
  19. Liu S. J., Steinbüchel A.. 2000b; A novel genetically engineered pathway for synthesis of poly(hydroxyalkanoic acids) in Escherichia coli . Appl Environ Microbiol66:739–743[CrossRef]
    [Google Scholar]
  20. Lütke-Eversloh T., Steinbüchel A.. 2003; Novel precursor substrates for polythioesters (PTE) and limits of PTE synthesis in Ralstonia eutropha . FEMS Microbiol Lett221:191–196[CrossRef]
    [Google Scholar]
  21. Lütke-Eversloh T., Steinbüchel A.. 2004; Microbial polythioesters. Macromol Biosci4:165–174[CrossRef]
    [Google Scholar]
  22. Lütke-Eversloh T., Bergander K., Luftmann H., Steinbüchel A.. 2001a; Identification of a new class of biopolymer: bacterial synthesis of a sulfur-containing polymer with thioester linkages. Microbiology147:11–19
    [Google Scholar]
  23. Lütke-Eversloh T., Bergander K., Luftmann H., Steinbüchel A.. 2001b; Biosynthesis of poly(3-hydroxybutyrate- co -3-mercaptobutyrate) as a sulfur analogue to poly(3-hydroxybutyrate) (PHB. Biomacromolecules2:1061–1065[CrossRef]
    [Google Scholar]
  24. Lütke-Eversloh T., Fischer A., Remminghorst U., Kawada J., Marchessault R. H., Kalwei M., Eckert H., other authors Bögershausen A.. 2002a; Biosynthesis of novel thermoplastic polythioesters by engineered Escherichia coli . Nat Mater1:236–240[CrossRef]
    [Google Scholar]
  25. Lütke-Eversloh T., Kawada J., Marchessault H., Steinbüchel A.. 2002b; Characterization of microbial polythioesters: physical properties of novel copolymers synthesized by Ralstonia eutropha . Biomacromolecules3:159–166[CrossRef]
    [Google Scholar]
  26. Matuszewska M., Laskowska E., Liberek K., Kuczyńska-Wiśnik D.. 2005; The small heat shock protein IbpA of Escherichia coli cooperates with IbpB in stabilization of thermally aggregated proteins in a disaggregation competent state. J Biol Chem280:12292–12298[CrossRef]
    [Google Scholar]
  27. Pötter M., Steinbüchel A.. 2005; Poly(3-hydroxybutyrate) granule-associated proteins: impacts on poly(3-hydroxybutyrate) synthesis and degradation. Biomacromolecules6:552–560[CrossRef]
    [Google Scholar]
  28. Pötter M., Müller H., Reinecke F., Wieczorek R., Fricke F., Bowien B., Friedrich B., Steinbüchel A.. 2004; The complex structure of polyhydroxybutyrate (PHB) granules: four orthologous and paralogous phasins occur in Ralstonia eutropha . Microbiology150:2301–2311[CrossRef]
    [Google Scholar]
  29. Preusting H., Kingma J., Huisman G., Witholt B., Steinbüchel A.. 1993; Formation of polyester blends by a recombinant strain of Pseudomonas oleovorans : different poly(3-hydroxyalkanoates) are stored in separate granules. J Environ Polym Degrad1:11–21[CrossRef]
    [Google Scholar]
  30. Qi Q., Rehm B. H. A., Steinbüchel A.. 2000; In vitro synthesis of poly(3-hydroxydecanoate): purification of type II polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa and development of an enzyme assay. Appl Microbiol Biotechnol54:37–43[CrossRef]
    [Google Scholar]
  31. Sambrook J., Fritsch E. F., Maniatis T.. 1989; Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  32. Seo M.-C., Shin H.-D., Lee Y.-H.. 2003; Functional role of granule-associated genes, phaP and phaR , in poly- β -hydroxybutyrate biosynthesis in recombinant E. coli harbouring phbCAB operon. Biotechnol Lett25:1243–1249[CrossRef]
    [Google Scholar]
  33. Spurr A. R.. 1969; A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res26:31–43[CrossRef]
    [Google Scholar]
  34. Steinbüchel A., Valentin H. E.. 1995; Diversity of bacterial polyhydroxyalkanoic acids. FEMS Microbiol Lett128:219–228[CrossRef]
    [Google Scholar]
  35. Steinbüchel A., Aerts K., Babel W., Follner C., Liebergesell M., Madkour M. H., Mayer F.,other authors Pieper-Fürst U.. 1995; Considerations on the structure and biochemistry of bacterial polyhydroxyalkanoic acid inclusions. Can J Microbiol41:Suppl. 194–105
    [Google Scholar]
  36. Taguchi S., Doi Y.. 2004; Evolution of polyhydroxyalkanoate (PHA) production system by ‘enzyme evolution’: successful case studies of directed evolution. Macromol Biosci4:146–156
    [Google Scholar]
  37. Tanaka M., Takebayashi M., Miyama M., Nishada J., Shimomura M.. 2004; Design of novel biointerfaces (II). Fabrication of self-organized porous polymer film with highly uniform pores. Biomed Mater Eng14:439–446
    [Google Scholar]
  38. Thakor N., Lütke-Eversloh T., Steinbüchel A.. 2005; 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. Appl Environ Microbiol71:835–841[CrossRef]
    [Google Scholar]
  39. Timm A., Steinbüchel A.. 1990; Formation of polyesters consisting of medium-chain-length 3-hydroxyalkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent pseudomonads. Appl Environ Microbiol56:3360–3367
    [Google Scholar]
  40. Weber K., Osborn M.. 1969; The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem244:4406–4412
    [Google Scholar]
  41. Wieczorek R., Pries A., Mayer F., Steinbüchel A.. 1995; Analysis of a 24-kilodalton protein associated with the polyhydroxyalkanoic acid granules in Alcaligenes eutrophus . J Bacteriol177:2425–2435
    [Google Scholar]
  42. York G. M., Stubbe J., Sinskey A. J.. 2002; The Ralstonia eutropha PhaR protein couples synthesis of the PhaP phasin to the presence of polyhydroxybutyrate in cells and promotes polyhydroxybutyrate production. J Bacteriol184:59–66[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.29260-0
Loading
/content/journal/micro/10.1099/mic.0.29260-0
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error