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PHA synthase activity controls the molecular weight and polydispersity of polyhydroxybutyrate in vivo

Nature Biotechnology volume 15, pages 63–67 (1997)Cite this article

Abstract

A synthetic operon for polyhydroxyalkanoate (PHA) biosynthesis designed to yield high levels of PHA synthase activity in vivo was constructed by positioning a genetic fragment encoding β-ketothiolase and acetoacetyl-CoA reductase behind a modified synthase gene containing an Escherichia coif promoter and ribosome binding site. Plasmids containing the synthetic operon and the native Alcaligenes eutro-phus PHA operon were transformed into E. coli DH5α and analyzed for polyhydroxybutyrate production. The molecular weight of polymer isolated from recombinant E. coli containing the modified synthase construct, determined by multiangle light scattering, was lower than that of the polymer from E. coli containing the natjve A. eutrophus operon. A further decrease in polyester molecular weight was observed with increased induction of the PHA biosynthetic genes in the synthetic operon. Comparison of the enzyme activity levels of PHA biosynthetic enzymes in a strain encoding the native operon with a strain possessing the synthetic operon indicates that the amount of polyhydroxyalkanoate synthase in a host organism plays a key role in controlling the molecular weight and the polydispersity of polymer.

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References

  1. Williams, S.F. and Peoples, O.P. 1996.Biodegradable plastics from plants. Chemtech. 26: 38–44.

    CAS  Google Scholar 

  2. Marchessault, R.H. 1996.Tender morsels for bacteria: recent developments in microbial polyesters. Trends Polym. Sci. 4: 163–168.

    CAS  Google Scholar 

  3. Lee, S.Y. 1996. Bacterial polyhydroxyalkanoates. Biotechnol. Bioeng. 49: 1–14.

    Article  CAS  Google Scholar 

  4. Poirer, Y., Nawrath, C. and Somerville, C. 1995. Production of polyhydroxyalkanoates, a family of biodegradable plastics and elastomers, in bacteria and plants. Bio/Technology 13: 142–150.

    Google Scholar 

  5. Muller, H.M. and Seebach, D. 1993. Poly(hydroxyalkanoates): a fifth class of physiologically important organic biopolymers?. Angew. Chem. Int. Ed. Engl. 32: 477–502.

    Article  Google Scholar 

  6. Anderson, A. and Dawes, E.A. 1990. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol. Rev. 54: 450–472.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Reusch, R., Hiske, T., Sadoff, H., Harris, R. and Beveridge, T. 1987. Cellular incorporation of poly-β-hydroxybutyrate into plasma membranes of Escherichia coli and Azotobacter vinelandii alters native membrane structure. Can. J. Microbiol. 33: 435–444.

    Article  CAS  Google Scholar 

  8. Reusch, R. and Sadoff, H. 1988. Putative structure and functions of a poly-β-hydroxybutyrate/calcium polyphosphate channel in bacterial plasma membranes. Proc. Natl. Acad. Sci. USA 85: 4176–4180.

    Article  CAS  Google Scholar 

  9. Reusch, R.N. 1995. Low molecular weight complexed poly(3-hydroxybutyrate): a dynamic and versatile molecule in vivo.Can. J. Microbiol. 41(suppl.1): 50–54.

    Article  CAS  Google Scholar 

  10. Seebach, D., Brunner, A., Burger, H.M., Reusch, R.N., and Bramble, L.L. 1996.Channel-forming activity of 3-hydroxybutanoic-acid oligomers in planar lipid bilayers.Helv. Chim. Act. 79: 507–517.

    Article  CAS  Google Scholar 

  11. Hahn, S.K., Chang, Y.K. and Lee, S.Y. 1995. Recovery and characterization of poly(3-hydroxybutyric acid) synthesized in Alcaligenes eutrophus and recombi-nant Escherichia coli. Appl. Environ. Microbiol. 61: 34–39.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Seebach, D., Brunner, A., Burger, H.M., Schneider, J. and Reusch, R.N. 1994. Isolation and 1H-NMR spectroscopic identification of poly(3-hydroxybutanoate) from prokaryotic and eukaryotic organisms: determination of the absolute configuration (R) of the monomeric unit 3-hydroxybutanoic acid from Escherichia coli and spinach. Eur. J. Biochem. 224: 317–328.

    Article  CAS  Google Scholar 

  13. Lee, S.Y., Lee, K.M., Chang, H.N. and Steinbuchel, A. 1994. Comparison of recombinant Escherichia coli strains for synthesis and accumulation of poly(3-hydroxybutyric acid) and morphological changes. Biotechnol. Bioeng. 44: 1337–1347.

    Article  CAS  Google Scholar 

  14. Kalousek, S. and Lubitz, W. 1995. High-level poly(β-hydroxybutyrate) production in recombinant Escherichia coli in sugar-free, complex medium. Can. J. Microbiol. 41(suppl.1): 216–221.

    Article  CAS  Google Scholar 

  15. Kidwell, J., Valentin, H.E. and Dennis, D. 1995. Regulated expression of the Alcaligenes eutrophus pha biosynthesis genes in Escherichia Coli. Appl. Environ. Microbiol. 61: 1391–1398.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Lee, S.Y. and Chang, H.N. 1995. Production of poly(3-hydroxybutyric acid) by recombinant Escherichia coli strains: genetic and fermentation studies. Can. J. Microbiol. 41(supp. 1): 207–215.

    Article  CAS  Google Scholar 

  17. Lee, S.Y., Yim, K.S., Chang, H.N. and Chang, Y.K. 1994. Construction of plasmids, estimation of plasmid stability, and use of stable plasmids for the production of poly(3-hydroxybutyric acid) by recombinant Escherichia coli. J. Biotechnol. 32: 203–211

    Article  CAS  Google Scholar 

  18. Lee, S.Y., Chang, H.N. and Chang, Y.K. 1994. Production of poly(β-hydroxybu-tyric Acid) by recombinant Escherichia coli. Ann. NY Acad. Sci. 721: 43–53.

    Article  CAS  Google Scholar 

  19. Zhang, H., Obias, V., Gonyer, K. and Dennis, D. 1994. Production of polyhydroxyalkanoates in sucrose-utilizing recombinant Escherichia coli and Klebsiella strains. Appl. Environ. Microbiol. 60: 1198–1205.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Kim, B.S., Lee, S.Y. and Chang, H.N. 1992. Production of poly-β-hydroxybutyrate by fed-batch culture of recombinant Escherichia coli. Biotechnol. Lett. 14: 811–818.

    Article  CAS  Google Scholar 

  21. Lee, S.Y. and Chang, H.N. 1993. High cell density cultivation of Escherichia coli W using sucrose as a carbon source. Biotechnol. Lett. 15: 971–974.

    Article  CAS  Google Scholar 

  22. Gilbert, R.G. 1995. Molecular weight distributions in free-radical polymerizations: their cause and cure. Trends Polym. Sci. 3: 222–226.

    CAS  Google Scholar 

  23. Peoples, O.P. and Sinskey, A.J. 1989. Poly-β-hydroxybutyrate (PHB) biosynthesis in Alcaligenes eutrophus H16: identification and characterization of the PHB polymerase gene (phbC). J. Biol. Chem. 264: 15298–15303.

    CAS  PubMed  Google Scholar 

  24. Gerngross, T.U., Snell, K.D., Peoples, O.p., Sinskey, A.J., Csuhai, E., Masamune, S. et al. 1994.Overexpression and purification of the soluble polyhydroxyalka-noate synthase from Alcaligenes eutrophus: evidence for a required posttransla-tional modification for catalytic activity. Biochemistry 33: 9311–9320.

    Article  CAS  Google Scholar 

  25. Kawaguchi, Y., Doi, Y. 1992. Kinetics and mechanism of synthesis and degradation of poly(3-hydroxybutyrate) in Alcaligenes eutrophus. Macromolecules. 25: 2324–2329.

    Article  CAS  Google Scholar 

  26. Fukui, T., Yoshimoto, A., Matsumoto, M., Hosokawa, S., Saito, T., Nishikawa, H. et al. 1976. Enzymatic synthesis of poly-β-hydroxybutyrate in Zoogloea ramigera. Arch. Microbiol. 110: 149–156.

    Article  CAS  Google Scholar 

  27. Haywood, G.W., Anderson, A.J. and Dawes, E.A. 1989. The importance of PHB-synthase substrate specificity in polyhydroxyalkanoate synthesis by Alcaligenes eutrophus. FEMS Microbiol. Lett. 57: 1–6.

    Article  CAS  Google Scholar 

  28. Nawrath, C., Poirier, Y., and Somerville, C. 1994. Targeting of the polyhydroxybu-tyrate biosynthetic pathway to the plastids of Arabidopsis thaliana results in high levels of polymer accumulation. Proc. Natl. Acad. Sci. USA. 91: 12760–12764.

    Article  CAS  Google Scholar 

  29. Gerngross, T.U. and Martin, D.P. 1995, Enzyme-catalyzed synthesis of poly[(R)(-)-3-hydroxybutyrate]: formation of macroscopic granules in vitro. Proc. Natl. Acad. Sci. USA 92: 6279–6283.

    Article  CAS  Google Scholar 

  30. Huisman, G.W., Wonink, E. de Koning, G., Preusting, H., and Witholt, B. 1992. Synthesis of poly(3-hydroxyalkanoates) by mutant and recombinant Pseudomonas strains. Appl. Microbiol. Biotechnol. 38: 1–5.

    Article  CAS  Google Scholar 

  31. Wodzinska, J., Snell, K.D., Rhomberg, A., Sinskey, A.J., Biemann, K. and Stubbe, J. 1996. Polyhydroxybutyrate synthase: evidence for covalent catalysis. J. Am. Chem. Soc. 118: 6319–6320

    Article  CAS  Google Scholar 

  32. Amann, E., Ochs, B. and Abel, K.J. 1988. Tightly regulated tec promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene. 69: 301–315.

    Article  CAS  Google Scholar 

  33. Ploux, O., Masamune, S. and Walsh, C.T. 1988. The NADPH-linked acetoacetyl-CoA reductase from Zoogloea ramigera: characterization and mechanistic studies of the cloned enzyme over-produced in Escherichia coli. Eur. J. Biochem. 174: 177–182.

    Article  CAS  Google Scholar 

  34. Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, Plainview, NY

    Google Scholar 

  35. Peoples, O.P. and Sinskey, A.J. 1989. Poly-β-hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16: characterization of the genes encoding β-ketothio-lase and acetoacetyl-CoA reductase. J. Biol. Chem. 264: 15293–15297.

    CAS  PubMed  Google Scholar 

  36. Nishimura,T., Saito, T., and Tomita, K. 1978. Purification and properties of β-ketothiolase from Zoogloea ramigera. Arch. Microbiol. 116: 21–27.

    Article  Google Scholar 

  37. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of micro-gram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.

    Article  CAS  Google Scholar 

  38. Law, J.H. and Slepecky, R.A. 1961. Assay of poly-β-hydroxybutyric acid. J. Bacteriol. 82: 33–36.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Grubisic, Z., Rempp, P., and Benoit, H. 1967. A universal calibration for gel permeation chromatography. Polymer Letters. 5: 753–759.

    Article  Google Scholar 

  40. Akita, S., Einaga, Y., Miyaki, Y., and Fugita, H. 1976. Solution properties of poly(D-β-hydroxybutyrate). 1. Biosynthesis and characterization. Macromolecules. 9: 774–780.

    Article  CAS  Google Scholar 

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Author notes

  1. Sang Jun Sim

    Present address: Division of Chemical Engineering, Korea Institute of Science and Technology, Seoul, Korea

Authors and Affiliations

  1. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139

    Sang Jun Sim, Kristi D. Snell, JoAnne Stubbe & Anthony J. Sinskey

  2. Biomaterial Science and Engineering Laboratory, Massachusetts Institute of Technology, Cambridge, MA, 02139

    Scott A. Hogan & Chokyun Rha

  3. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, 02139

    JoAnne Stubbe

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  1. Sang Jun Sim
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Correspondence to Kristi D. Snell.

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Sim, S., Snell, K., Hogan, S. et al. PHA synthase activity controls the molecular weight and polydispersity of polyhydroxybutyrate in vivo. Nat Biotechnol 15, 63–67 (1997). https://doi.org/10.1038/nbt0197-63

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