Skip to main content
SpringerLink
Log in

Expression of PHA polymerase genes of Pseudomonas putida in Escherichia coli and its effect on PHA formation

  • Published:
Antonie van Leeuwenhoek Aims and scope Submit manuscript

Abstract

Poly-3-hydroxyalkanoates (PHAs) are synthesized by many bacteria as intracellular storage material. The final step in PHA biosynthesis is catalyzed by two PHA polymerases (phaC) in Pseudomonas putida. The expression of these two phaC genes (phaC1 and phaC2)was studied in Escherichia coli, either under control of the native promoter or under control of an external promoter. It was found that the two phaC genes are not expressed in E. coli without an external promoter. During heterologous expression of phaC from Plac on a high copy number plasmid, a rapid reduction of the number of colony forming units was observed, especially for phaC2. It appears that the plasmid instability was partially caused by high-level production of PHA polymerase. Subsequently, tightly regulated phaC2 expression systems on a low copy number vector were applied in E. coli. This resulted in PHA yields of over 20 of total cell dry weight, which was 2 fold higher than that obtained from the system where phaC2 is present on a high copy number vector. In addition, the PHA monomer composition differed when different gene expression systems or different phaC genes were applied.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • M.M. Bagdasarian E. Amman R. Lurz B. Rückert M. Bagdasarian (1983) ArticleTitleActivity of the hybrid trp-lac(tac) promoter of Escherichia coli in Pseudomonas putida. Construction of broadhost-range, controlled-expression vectors. Gene. 931 273–282

    Google Scholar 

  • P.N. Black C.C. DiRusso (1994) ArticleTitleMolecular and biochemical analyses of fatty acid transport, metabolism, and gene regulation in Escherichia coli Biochimica et Biophysica Acta. 1210 123–145 Occurrence Handle1:CAS:528:DyaK2cXhsFyhsL0%3D Occurrence Handle8280762

    CAS  PubMed  Google Scholar 

  • J. Davison M. Heusterspreute N. Chevalier V. Ha-Thi F. Brunel (1987) ArticleTitleVectors with restriction site banks, V. pJRD215, a wide host range cosmid vector with multiple cloning sites Gene. 51 275–280 Occurrence Handle1:CAS:528:DyaL2sXktVWqsbw%3D Occurrence Handle3036654

    CAS  PubMed  Google Scholar 

  • G.J.M. Koning Particlede B. Witholt (1996) ArticleTitleA biodegradable rubber from bacteria, poly(hydroxyalkanoate) from Pseudomonas Mat. Sci. Eng. C. 4 121–124

    Google Scholar 

  • V. Lorenzo Particlede L. Eltis B. Kessler K.N. Timmis (1993) ArticleTitleAnalysis of Pseudomonas gene products using lacIq/Ptrp-lac plasmids and transposons that confer conditional phenotypes Gene. 123 17–24 Occurrence Handle8380783

    PubMed  Google Scholar 

  • M.J. Smet Particlede G. Eggink B. Witholt J. Kingma H. Wynberg (1983) ArticleTitleCharacterization of intracellular inclusions formed by Pseudomonas oleovorans during growth on octane J. Bacteriol. 154 870–878 Occurrence Handle6841319

    PubMed  Google Scholar 

  • V. Deretic W.M. Konyecsni C.D. Mohr D.W. Martin N.S. Hibler (1989) ArticleTitleCommon denominators of promoter control in Pseudomonas and other bacteria Bio/Technology. 7 1249–1254

    Google Scholar 

  • C.C. DiRusso W.D. Nunn (1985) ArticleTitleCloning and characterization of a gene (fadR) involved in regulation of fatty acid metabolism in Escherichia coli J. Bacteriol. 161 583–588 Occurrence Handle1:CAS:528:DyaL2MXhs1Wnt74%3D Occurrence Handle3881402

    CAS  PubMed  Google Scholar 

  • U.J. Hänggi (1995) ArticleTitleRequirements on bacterial polyesters as future substitute for conventional plastics for consumer goods FEMS Microbiol. Rev. 16 213–220

    Google Scholar 

  • O. Hrabak (1992) Carbohydrates in industrial synthesis M.A. Clarke (Eds) Proceedings of the symposium of the division of carbohydrate chemistry of the American Chemical Society. Verlag Dr. Albert Bartens Berlin 18–26

    Google Scholar 

  • G.W. Huisman O. Leeuw Particlede G. Eggink B. Witholt (1989) ArticleTitleSynthesis of poly-hydroxyalkanoates is a common feature of fluorescent Pseudomonads Appl. Environ. Microbiol. 55 1949–1954 Occurrence Handle1:CAS:528:DyaL1MXlsFSgsL0%3D Occurrence Handle2506811

    CAS  PubMed  Google Scholar 

  • G.W. Huisman E. Wonink G.J.M. De Koning H. Preusting B. Witholt (1992) ArticleTitleSynthesis of poly (3-hydroxyalkanoates) by mutant and recombinant Pseudomonas strains Appl. Microbiol. Biotechnol. 38 1–5 Occurrence Handle1:CAS:528:DyaK3sXitFWgu7Y%3D

    CAS  Google Scholar 

  • G.W. Huisman E. Wonink R. Meima B. Kazemier P. Terpstra B. Witholt (1991) ArticleTitleMetabolism of poly (3-hydroxyalkanoates) (PHAs) by Pseudomonas oleovorans J. Biol. Che. 266 2191–2198 Occurrence Handle1:CAS:528:DyaK3MXmt1Cqs7k%3D

    CAS  Google Scholar 

  • L.J. Jenkins W.D. Nunn (1987) ArticleTitleGenetic and molecular characterization of the genes involved in short-chain fatty acid degradation in Escherichia coli: the ato system J. Bacteriol. 169 42–52 Occurrence Handle1:CAS:528:DyaL2sXhtlamsb0%3D Occurrence Handle3025185

    CAS  PubMed  Google Scholar 

  • J. Kidwell H.E. Valentin D. Dennis (1995) ArticleTitleRegulated expression of the Alcaligenes eutrophus pha biosynthesis genes in Escherichia coli Appl. Environ. Microbiol. 61 1391–1398 Occurrence Handle1:CAS:528:DyaK2MXksl2ls7Y%3D Occurrence Handle7747959

    CAS  PubMed  Google Scholar 

  • R.G. Lageveen G.W. Huisman H. Preusting P. Ketelaar G. Eggink B. Witholt (1988) ArticleTitleFormation of polyesters by Pseudomonas oleovorans: effect of substrates on formation and composition of poly-(R)-3-hydroxyalkanoates and poly-(R)-3-hydroxyalkenoates Appl. Environ. Microbiol. 54 2924–2932 Occurrence Handle1:CAS:528:DyaL1MXptFOqtQ%3D%3D Occurrence Handle16347790

    CAS  PubMed  Google Scholar 

  • S. Langenbach B.H.A. Rehm A. Steinbüchel (1997) ArticleTitleFunctional expression of the PHA synthase gene phaC1 from Pseudomonas aeruginosa inEscherichia coliresults in poly(3-hydroxyalkanoate) synthesis FEMS Microbiol. Lett. 150 303–309 Occurrence Handle1:CAS:528:DyaK2sXjsFyisbk%3D Occurrence Handle9170275

    CAS  PubMed  Google Scholar 

  • S.Y. Lee H.N. Chang (1995) ArticleTitleProduction of acid by recombinant Escherichia coli strains: genetic and fermentation studies Can. J. Microbiol. 41 207–215 Occurrence Handle1:CAS:528:DyaK2MXmsFyru70%3D Occurrence Handle7606664

    CAS  PubMed  Google Scholar 

  • S.Y. Lee K.S. Yim H.N. Chang Y.K. Chang (1994) ArticleTitleConstruction 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 Occurrence Handle1:CAS:528:DyaK2cXktVaisr0%3D Occurrence Handle7764567

    CAS  PubMed  Google Scholar 

  • W.D. Nunn (1986) Two-carbon compounds and fatty acids as carbon sources F.C. Neidhardt (Eds) Escherichia coli and Salmonella typhimurium. American Society for Microbiology Washington 285–301

    Google Scholar 

  • W.J. Page (1995) ArticleTitleBacterial polyhydroxyalkanoates, natural biodegradable plastics with a great future Can. J. Microbiol. 41 1–3 Occurrence Handle10.1139/m95-161 Occurrence Handle1:CAS:528:DyaK2MXmsFyrtLs%3D

    Article  CAS  Google Scholar 

  • M.A. Prieto B. Bühler K. Jung B. Witholt B. Kessler (1999) ArticleTitlePhaF, a polyhdroxyalkanoate-granule-associated protein of Pseudomonas oleovorans GPo1 involved in the regulatory expression system for pha genes J. Bacteriol. 181 858–868 Occurrence Handle1:CAS:528:DyaK1MXhtVGju70%3D Occurrence Handle9922249

    CAS  PubMed  Google Scholar 

  • Q. Qi B.H.A. Rehm A. Steinbüchel (1997) ArticleTitleSynthesis of poly(3-hydroxyalkanoates) in Escherichia coli expressing the PHA synthase gene phaC2 from Pseudomonas aeruginosa: comparison of PhaC1 and PhaC2 FEMS Microbiol. Lett. 157 156–162

    Google Scholar 

  • Ren Q. 1997. Biosynthesis of medium chain length poly-3- hydroxyalkanoates: from Pseudomonas to Escherichia coli. PhD. thesis, ETH Zürich Switzerland.

  • Q. Ren N. Sierro M. Kellerhals B. Kessler B. Witholt (2000) ArticleTitleProperties of engineered poly-3-hydroxyalkanoates (PHAs) produced in Escherichia coli recombinants Appl. Environ. Microbiol. 66 1311–1320 Occurrence Handle1:CAS:528:DC%2BD3cXisVWntLs%3D Occurrence Handle10742205

    CAS  PubMed  Google Scholar 

  • Q. Ren N. Sierro B. Witholt B. Kessler (2000) ArticleTitleFabG, an NADPH-dependent 3-ketoacyl reductase of Pseudomonas aeruginosa, provides precursors for medium-chain-length poly- 3-hydroxyalkanoate biosynthesis in Escherichia coli J. Bacteriol. 182 2978–2981 Occurrence Handle1:CAS:528:DC%2BD3cXivFKqtro%3D Occurrence Handle10781572

    CAS  PubMed  Google Scholar 

  • H.G. Rhie D. Dennis (1995) ArticleTitleRole of fadR and atoC(Con) mutations in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in recombinant pha) Escherichia coli Appl. Environ. Microbiol. 61 2487–2492 Occurrence Handle1:CAS:528:DyaK2MXms1Gktbs%3D Occurrence Handle7618860

    CAS  PubMed  Google Scholar 

  • J. Sambrook E.F. Fritsch T. Maniatis (1989) Molecular cloning: a laboratory manual. 2nd edn Cold Spring Harbor Laboratory Press New York, USA

    Google Scholar 

  • P. Schubert N. Krueger A. Steinbuechel (1991) ArticleTitleMolecular 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 Occurrence Handle1:CAS:528:DyaK3MXls1Ghtw%3D%3D Occurrence Handle1987116

    CAS  PubMed  Google Scholar 

  • A. Timm A. Steinbüchel (1992) ArticleTitleCloning and molecular analysis of the poly(3-hydroxyalkanoic acid) gene locus of Pseudomonas aeruginosa PAO1 Eur. J. Biochem. 209 15–30 Occurrence Handle1:CAS:528:DyaK3sXks1Cgsrc%3D Occurrence Handle1396693

    CAS  PubMed  Google Scholar 

  • J.B. Beilen Particlevan S. Panke S. Lucchinib A.G. Franchini M. Röthlisberger B. Witholt (2001) ArticleTitleAnalysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes Microbiology. 147 1621–1630 Occurrence Handle11390693

    PubMed  Google Scholar 

  • F.R. Leij Particlevan der B. Witholt (1995) ArticleTitleStrategies for the sustainable production of new biodegradable polyesters in plants: a review. Can J. Microbiol. 41 222–238

    Google Scholar 

  • S.F. Williams D.P. Martin D.M. Horowitz O.P. Peoples (1999) ArticleTitlePHA applications: Addressing the price performance issue I. Tissue engineering Int. J. Biol. Macromol. 25 111–121 Occurrence Handle1:CAS:528:DyaK1MXksV2qtrw%3D Occurrence Handle10416657

    CAS  PubMed  Google Scholar 

  • B. Witholt (1972) ArticleTitleMethod for isolating mutants overproducing nicotinamide adenine dinucleotide and its precursors J. Bacteriol. 109 350–364 Occurrence Handle1:CAS:528:DyaE38Xktlyktw%3D%3D Occurrence Handle4333379

    CAS  PubMed  Google Scholar 

  • C. Yanisch-Perron J. Vieira J. Messing (1985) ArticleTitleImproved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors Genes. 33 103–119 Occurrence Handle1:CAS:528:DyaL2MXktVWgtL0%3D

    CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Biocompatible Materials, Swiss Federal Laboratories for Materials Testing and Research (EMPA), Lerchenfeldstrasse 5, 9014 St., Gallen, Switzerland

    Qun Ren & Manfred Zinn

  2. Institute of Biotechnology, Swiss Federal Institute of Technology, CH-8093, Zurich, Switzerland

    Jan B. van Beilen, Nicolas Sierro & Bernard Witholt

  3. Prädialstab, Swiss Federal Institute of Technology, HG F 52.2, CH-8092, Zurich, Switzerland

    Birgit Kessler

Authors
  1. Qun Ren
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jan B. van Beilen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nicolas Sierro
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Manfred Zinn
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Birgit Kessler
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bernard Witholt
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Qun Ren.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ren, Q., Beilen, J.B.v., Sierro, N. et al. Expression of PHA polymerase genes of Pseudomonas putida in Escherichia coli and its effect on PHA formation. Antonie Van Leeuwenhoek 87, 91–100 (2005). https://doi.org/10.1007/s10482-004-1360-x

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10482-004-1360-x

Keywords

Search

Navigation