Applied Microbiology and Biotechnology

, Volume 89, Issue 5, pp 1527–1536 | Cite as

Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16

  • Toshiaki Fukui
  • Kei Ohsawa
  • Jun Mifune
  • Izumi Orita
  • Satoshi Nakamura
Applied Genetics and Molecular Biotechnology


Five kinds of promoters were evaluated as tools for regulated gene expression in the PHA-producing bacterium Cupriavidus necator. Several broad-host-range expression vectors were constructed by which expression of a reporter gene gfp was controlled by Plac, Ptac, or PBAD derived from Escherichia coli, or promoter regions of phaC1 (PphaC) or phaP1 (PphaP) derived from C. necator. Then, the gfp-expression profiles were determined in C. necator strains harboring the constructed vectors when the cells were grown on fructose or soybean oil. Plac, Ptac, PphaC, and PphaP mediated constitutive gene expression, among which Ptac was the strongest promoter. lacI-Ptac was not thoroughly functional even after addition of isopropyl-β-d-thiogalactopyranoside (IPTG), probably due to inability of C. necator to uptake IPTG. Gene expression by araC-PBAD could be regulated by varying l-arabinose concentration in the medium, although P(3HB) production rate was slightly decreased in the recombinant. phaR-PphaP exhibited an expression profile tightly coupled with P(3HB) accumulation, suggesting application of the vector harboring phaR-PphaP for gene expression specific at the PHA-biosynthesis phase. The properties of these promoters were expected to be useful for effective engineering of PHA biosynthesis in C. necator.


Polyhydroxyalkanoate Poly(3-hydroxybutyrate) Promoter Cupriavidus necator 


  1. de Lorenzo V, Herrero M, Jakubzik U, Timmis KN (1990) Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol 172:6568–6572Google Scholar
  2. Delamarre SC, Batt CA (2006) Comparative study of promoters for the production of polyhydroxyalkanoates in recombinant strains of Wautersia eutropha. Appl Microbiol Biotechnol 71:668–679CrossRefGoogle Scholar
  3. Fukui T, Doi Y (1997) Cloning and analysis of the poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate) biosynthesis genes of Aeromonas caviae. J Bacteriol 179:4821–4830Google Scholar
  4. Fukui T, Doi Y (1998) Efficient production of polyhydroxyalkanoates from plant oils by Alcaligenes eutrophus and its recombinant strain. Appl Microbiol Biotechnol 49:333–336CrossRefGoogle Scholar
  5. 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 180:667–673Google Scholar
  6. Fukui T, Abe H, Doi Y (2002) Engineering of Ralstonia eutropha for production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from fructose and solid-state properties of the copolymer. Biomacromolecules 3:618–624CrossRefGoogle Scholar
  7. Fukui T, Suzuki M, Tsuge T, Nakamura S (2009) Microbial synthesis of poly((R)-3-hydroxybutyrate-co-3-hydroxypropionate) from unrelated carbon sources by engineered Cupriavidus necator. Biomacromolecules 10:700–706CrossRefGoogle Scholar
  8. Guzman LM, Belin D, Carson MJ, Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130Google Scholar
  9. Hustede E, Steinbüchel A, Schlegel HG (1992) Cloning of poly(3-hydroxybutyric acid) synthase genes of Rhodobacter sphaeroides and Rhodospirillum rubrum and heterologous expression in Alcaligenes eutrophus. FEMS Microbiol Lett 72:285–290CrossRefGoogle Scholar
  10. Kahar P, Tsuge T, Taguchi K, Doi Y (2004) High yield production of polyhydroxyalkanoates from soybean oil by Ralstonia eutropha and its recombinant strain. Polym Deg Stab 83:79–86CrossRefGoogle Scholar
  11. Kato M, Bao HJ, Kang CK, Fukui T, Doi Y (1996) Production of a novel copolyester of 3-hydroxybutyric acid and medium chain length 3-hydroxyalkanaic acids by Pseudomonas sp. 61-3 from sugars. Appl Microbiol Biotechnol 45:363–370CrossRefGoogle Scholar
  12. Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM 2nd, Peterson KM (1995) Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176CrossRefGoogle Scholar
  13. Lee JN, Shin HD, Lee YH (2003) Metabolic engineering of pentose phosphate pathway in Ralstonia eutropha for enhanced biosynthesis of poly-β-hydroxybutyrate. Biotechnol Prog 19:1444–1449CrossRefGoogle Scholar
  14. Liebergesell M, Steinbüchel A (1992) Cloning and nucleotide sequences of genes relevant for biosynthesis of poly(3-hydroxybutyric acid) in Chromatium vinosum strain D. Eur J Biochem 209:135–150CrossRefGoogle Scholar
  15. Liebergesell M, Steinbüchel A (1993) Cloning and molecular analysis of the poly(3-hydroxybutyric acid) biosynthetic genes of Thiocystis violacea. Appl Microbiol Biotechnol 38:493–501CrossRefGoogle Scholar
  16. Liebergesell M, Rahalkar S, Steinbüchel A (2000) Analysis of the Thiocapsa pfennigii polyhydroxyalkanoate synthase: subcloning, molecular characterization and generation of hybrid synthases with the corresponding Chromatium vinosum enzyme. Appl Microbiol Biotechnol 54:186–194CrossRefGoogle Scholar
  17. Madison LL, Huisman GW (1999) Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol Mol Biol Rev 63:21–53Google Scholar
  18. Matsusaki H, Manji S, Taguchi K, Kato M, Fukui T, Doi Y (1998) Cloning and molecular analysis of the poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxy alkanoate) biosynthesis genes in Pseudomonas sp. strain 61-3. J Bacteriol 180:6459–6467Google Scholar
  19. Mifune J, Nakamura S, Fukui T (2008) Targeted engineering of Cupriavidus necator chromosome for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from vegetable oil. Can J Chem 86:621–627CrossRefGoogle Scholar
  20. Mifune J, Nakamura S, Fukui T (2010) Engineering of pha operon on Cupriavidus necator chromosome for efficient biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from vegetable oil. Polym Degrad Stab 95:1305–1312CrossRefGoogle Scholar
  21. Philip S, Keshavarz T, Roy I (2007) Polyhydroxyalkanoates: biodegradable polymers with a range of applications. J Chem Technol Biotechnol 82:233–247CrossRefGoogle Scholar
  22. Pieper U, Steinbüchel A (1992) Identification, cloning and sequence analysis of the poly(3-hydroxyalkanoic acid) synthase gene of the gram-positive bacterium Rhodococcus ruber. FEMS Microbiol Lett 75:73–79CrossRefGoogle Scholar
  23. Pohlmann A, Fricke WF, Reinecke F, Kusian B, Liesegang H, Cramm R, Eitinger T, Ewering C, Pötter M, Schwartz E, Strittmatter A, Voss I, Gottschalk G, Steinbüchel A, Friedrich B, Bowien B (2006) Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16. Nat Biotechnol 24:1257–1262CrossRefGoogle Scholar
  24. Pötter M, Steinbüchel A (2005) Poly(3-hydroxybutyrate) granule-associated proteins: impacts on poly(3-hydroxybutyrate) synthesis and degradation. Biomacromolecules 6:552–560CrossRefGoogle Scholar
  25. Pötter M, Madkour MH, Mayer F, Steinbüchel A (2002) Regulation of phasin expression and polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha H16. Microbiology 148:2413–2426Google Scholar
  26. Schlegel HG, Lafferty R, Krauss I (1970) The isolation of mutants not accumulating poly-β-hydroxybutyric acid. Arch Mikrobiol 71:283–294CrossRefGoogle Scholar
  27. Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering. Transposon mutagenesis in gram negative bacteria. Bio/Technology 1:784–791CrossRefGoogle Scholar
  28. Steinbüchel A, Valentin HE (1995) Diversity of bacterial polyhydroxyalkanoic acids. FEMS Microbiol Lett 128:219–228CrossRefGoogle Scholar
  29. Sudesh K, Fukui T, Doi Y (1998) Genetic analysis of Comamonas acidovorans polyhydroxyalkanoate synthase and factors affecting the incorporation of 4-hydroxybutyrate monomer. Appl Environ Microbiol 64:3437–3443Google Scholar
  30. Sudesh K, Abe H, Doi Y (2000) Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Prog Polym Sci 25:1503–1555CrossRefGoogle Scholar
  31. Tanadchangsaeng N, Kitagawa A, Yamamoto T, Abe H, Tsuge T (2009) Identification, biosynthesis, and characterization of polyhydroxyalkanoate copolymer consisting of 3-hydroxybutyrate and 3-hydroxy-4-methylvalerate. Biomacromolecules 10:2866–2874CrossRefGoogle Scholar
  32. Timm A, Steinbüchel A (1992) Cloning and molecular analysis of the poly(3-hydroxyalkanoic acid) gene locus of Pseudomonas aeruginosa PAO1. Eur J Biochem 209:15–30CrossRefGoogle Scholar
  33. Tsuge T, Watanabe S, Shimada D, Abe H, Doi Y, Taguchi S (2007) Combination of N149S and D171G mutations in Aeromonas caviae polyhydroxyalkanoate synthase and impact on polyhydroxyalkanoate biosynthesis. FEMS Microbiol Lett 277:217–222CrossRefGoogle Scholar
  34. York GM, Stubbe J, Sinskey AJ (2002) The Ralstonia eutropha PhaR protein couples synthesis of the PhaP phasin to the presence of polyhydroxybutyrate in cells and promotes polyhydroxybutyrate production. J Bacteriol 184:59–66CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Toshiaki Fukui
    • 1
  • Kei Ohsawa
    • 1
  • Jun Mifune
    • 1
  • Izumi Orita
    • 1
  • Satoshi Nakamura
    • 1
  1. 1.Department of Bioengineering, Graduate School of Bioscience and BiotechnologyTokyo Institute of TechnologyYokohamaJapan

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