1887

Abstract

was investigated with respect to polyhydroxybutyrate (PHB) biosynthesis. Polyhydroxyalkanoate (PHA) accumulation contributing to approximately 18% of the cell dry weight was obtained in the presence of glucose. Gas chromatography–mass spectrometry and gel permeation chromatography of the purified PHA showed that this polyester was solely composed of 3-hydroxybutyrate and had a weight average molar mass of 55×10 g mol and a polydispersity of 16. An ORF encoding a conserved, hypothetical protein which shared approximately 47% identity with the PHB synthase from was identified within the complete genomic sequence. This putative PHB synthase gene, , consisted of a 2019 nt stretch of DNA (encoding 673 aa residues), which encoded a PHB synthase with a molecular mass of approximately 73 kDa. This is currently the largest PHA synthase identified. The coding region was subcloned into vector pBBR1-JO2 under promoter control. The resulting plasmid, pQQ4, mediated PHB accumulation in the mutant PHB4 and recombinant JM109(pBHR69), which produced the β-ketothiolase and acetoacetyl-CoA reductase from , contributing to approximately 62% and 6% of cell dry weight, respectively. Functional expression of the coding region of was confirmed by immunoblotting and PHB synthase activity.

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2001-12-01
2024-12-03
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References

  1. 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 Microbiol 54, 1977-1982. [Google Scholar]
  2. Fiedler, S., Steinbüchel, A. & Rehm, B. H. A. (2000). PhaG-mediated synthesis of poly(3-hydroxyalkanoates) consisting of medium-chain-length constituents from nonrelated carbon sources in recombinant Pseudomonas fragi. Appl Environ Microbiol 66, 2117-2124.[CrossRef] [Google Scholar]
  3. Friedrich, B., Hogrefe, C. & Schlegel, H. G. (1981). Naturally-occurring genetic transfer of hydrogen-oxidizing ability between strains of Alcaligenes eutrophus. J Bacteriol 147, 198-205. [Google Scholar]
  4. Fukui, T., Shiomi, N. & Doi, Y. (1998). Expression and characterization of (R)-specific enoyl coenzyme A hydratase involved in polyhydroxyalkanoate biosynthesis by Aeromonas caviae. J Bacteriol 180, 667-673. [Google Scholar]
  5. Gerngross, T. U., Snell, K. D., Peoples, O. P., Sinskey, A. J., Csuhai, E., Masamune, M. & Stubbe, J. (1994). Overexpression and purification of the soluble polyhydroxyalkanoate synthase from Alcaligenes eutrophus – evidence for a required posttranslational modification for catalytic activity. Biochemistry 33, 9311-9320.[CrossRef] [Google Scholar]
  6. Handrick, R., Reinhardt, S. & Jendrossek, D. (2000). Mobilization of poly(3-hydroxybutyrate) in Ralstonia eutropha.J Bacteriol 182, 5916-5918.[CrossRef] [Google Scholar]
  7. Haywood, G. W., Anderson, A. J., Williams, G. A., Dawes, E. A. & Ewing, D. F. (1991). Accumulation of a poly(hydroxyalkanoate) copolymer containing primarily 3-hydroxyvalerate from simple carbohydrate substrates by Rhodococcus sp. NCIMB 40126. Int J Biol Macromol 13, 83-88.[CrossRef] [Google Scholar]
  8. Hoffmann, N., Steinbüchel, A. & Rehm, B. H. A. (2000a). The Pseudomonas aeruginosa phaG gene product is involved in the synthesis of polyhydroxyalkanoic acid consisting of medium-chain-length constituents from non-related carbon sources. FEMS Microbiol Lett 184, 253-259.[CrossRef] [Google Scholar]
  9. Hoffmann, N., Steinbüchel, A. & Rehm, B. H. A. (2000b). Homologous functional expression of cryptic phaG from Pseudomonas oleovorans establishes the transacylase-mediated polyhydroxyalkanoate biosynthetic pathway. Appl Microbiol Biotechnol 54, 665-670.[CrossRef] [Google Scholar]
  10. Hoppensack, A., Rehm, B. H. A. & Steinbüchel, A. (1999). Analysis of 4-phosphopantetheinylation of polyhydroxybutyrate synthase from Ralstonia eutropha: generation of beta-alanine auxotrophic Tn5 mutants and cloning of the panD gene region. J Bacteriol 181, 1429-1435. [Google Scholar]
  11. Jia, Y., Kappock, T. J., Frick, T., Sinskey, A. J. & Stubbe, J. (2000). Lipases provide a new mechanistic model for polyhydroxybutyrate (PHB) synthases: characterization of the functional residues in Chromatium vinosum PHB synthase. Biochemistry 39, 3927-3936.[CrossRef] [Google Scholar]
  12. Jia, Y., Yuan, W., Wodzinska, J., Park, C., Sinskey, A. J. & Stubbe, J. (2001). Mechanistic studies on class I polyhydroxybutyrate (PHB) synthase from Ralstonia eutropha: class I and III synthases share a similar catalytic mechanism. Biochemistry 40, 1011-1019.[CrossRef] [Google Scholar]
  13. Kovach, M. E., Elzer, P. H., Hill, D. S., Robertson, G. T., Farris, M. A., Roop, R. M. & Peterson, K. M. (1995). 4 new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166, 175-176.[CrossRef] [Google Scholar]
  14. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[CrossRef] [Google Scholar]
  15. Langenbach, S., Rehm, B. H. A. & Steinbüchel, A. (1997). Functional expression of the PHA synthase gene phaC1 from Pseudomonas aeruginosa in Escherichia coli results in poly(3-hydroxyalkanoate) synthesis. FEMS Microbiol Lett 150, 303-309.[CrossRef] [Google Scholar]
  16. Liebergesell, M., Sonomoto, I. L., Madkour, M., Mayer, F. & Steinbüchel, A. (1994). Purification and characterization of the poly(hydroxyalkanoic acid) synthase from Chromatium vinosum and localization of the enzyme at the surface of poly(hydroxyalkanoic acid) granules. Eur J Biochem 226, 71-80.[CrossRef] [Google Scholar]
  17. Mak, Y. M. & Ho, K. K. (1993). An improved procedure for direct phenol extraction of single-stranded M13-DNA for sequencing. Biotechniques 14, 732-734. [Google Scholar]
  18. Nierman, W. C., Feldblyum, T. V., Laub, M. T. & 34 other authors (2001). Complete genome sequence of Caulobacter crescentus. Proc Natl Acad Sci USA 98, 4136–4141.[CrossRef] [Google Scholar]
  19. Peoples, O. P. & Sinskey, A. J. (1989a). Poly-β-hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16 – characterization of the genes encoding β-ketothiolase and acetoacetyl-CoA reductase.J Biol Chem 264, 15293-15297. [Google Scholar]
  20. Peoples, O. P. & Sinskey, A. J. (1989b). Poly-β-hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16 – identification and characterization of the PHB polymerase gene (phbC). J Biol Chem 264, 15298-15303. [Google Scholar]
  21. Poindexter, J. S. (1981). The Caulobacters – ubiquitous unusual bacteria. Microbiol Rev 45, 123-179. [Google Scholar]
  22. Qi, Q. S., Rehm, B. H. A. & Steinbüchel, A. (1997). Synthesis of poly(3-hydroxyalkanoates) in Escherichia coli expressing the PHA synthase gene phaC2 from Pseudomonas aeruginosa: comparison of PhaC1 and PhaC2. FEMS Microbiol Lett 157, 155-162.[CrossRef] [Google Scholar]
  23. Qi, Q. S., Steinbüchel, A. & Rehm, B. H. A. (1998). Metabolic routing towards polyhydroxyalkanoic acid synthesis in recombinant Escherichia coli (fadR): inhibition of fatty acid beta-oxidation by acrylic acid. FEMS Microbiol Lett 167, 89-94. [Google Scholar]
  24. Qi, Q., Steinbüchel, A. & Rehm, B. H. A. (2000).In vitro synthesis of poly(3-hydroxydecanoate): purification and enzymatic characterization of type II polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa.Appl Microbiol Biotechnol 54, 37-43.[CrossRef] [Google Scholar]
  25. Rehm, B. H. A. & Steinbüchel, A. (1999). Biochemical and genetic analysis of PHA synthases and other proteins required for PHA synthesis. Int J Biol Macromol 25, 3-19.[CrossRef] [Google Scholar]
  26. Rehm, B. H. A. & Steinbüchel, A. (2001a). Heterologous expression of the acyl–acyl carrier protein thioesterase gene from the plant Umbellularia californica mediates polyhydroxyalkanoate biosynthesis in recombinant Escherichia coli. Appl Microbiol Biotechnol 55, 205-209.[CrossRef] [Google Scholar]
  27. Rehm, B. H. A. & Steinbüchel, A. (2001b). PHA synthases – the key enzymes of PHA synthesis. In Handbook ‘Biopolymers’. Edited by A. Steinbüchel & Y. Doi. Weinheim: Wiley–VCH (in press).
  28. Rehm, B. H. A., Krüger, N. & Steinbüchel, A. (1998). A new metabolic link between fatty acid de novo synthesis and polyhydroxyalkanoic acid synthesis – the phaG gene from Pseudomonas putida KT2440 encodes a 3-hydroxyacyl–acyl carrier protein coenzyme A transferase. J Biol Chem 273, 24044-24051.[CrossRef] [Google Scholar]
  29. Rehm, B. H. A., Qi, Q. S., Beermann, B. B., Hinz, H.-J. & Steinbüchel, A. (2001a). Matrix-assisted in vitro refolding of Pseudomonas aeruginosa class II polyhydroxyalkanoate synthase from inclusion bodies produced in recombinant Escherichia coli. Biochem J 358, 263-268.[CrossRef] [Google Scholar]
  30. Rehm, B. H. A., Hoffmann, N., Qi, Q., Fiedler, S. & Steinbüchel, A. (2001b). Biosynthesis of latex-like polyhydroxyalkanoates. In Proceedings of the International Symposium on Bioconversion of Renewable Raw Materials. Braunschweig, Germany: GBF (in press).
  31. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989).Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  32. Schlegel, H. G., Lafferty, R. & Krauss, I. (1970). The isolation of mutants not accumulating poly-beta-hydroxybutyric acid. Arch Microbiol 71, 283-294. [Google Scholar]
  33. Schubert, P., Steinbüchel, A. & Schlegel, H. G. (1988). Cloning of the Alcaligenes eutrophus genes for synthesis of poly-β-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli. J Bacteriol 170, 5837-5847. [Google Scholar]
  34. Schubert, P., Krüger, N. & Steinbüchel, A. (1991). Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxybutyrate) biosynthetic operon – identification of the N-terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter. J Bacteriol 173, 168-175. [Google Scholar]
  35. Simon, R., Priefer, U. & Pühler, A. (1983). A broad host range mobilization system for in vivo gentetic engineering – transposon mutagenesis in Gram-negative bacteria. Bio/Technology 1, 784-791.[CrossRef] [Google Scholar]
  36. Spiekermann, P., Rehm, B. H. A., Kalscheuer, R., Baumeister, D. & Steinbüchel, A. (1999). A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Arch Microbiol 171, 73-80.[CrossRef] [Google Scholar]
  37. Valentin, H. E. & Steinbüchel, A. (1993). Cloning and characterization of the Methylobacterium extorquens polyhydroxyalkanoic-acid-synthase structural gene. Appl Microbiol Biotechnol 39, 309-317. [Google Scholar]
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