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. 1996 Feb;178(3):774–779. doi: 10.1128/jb.178.3.774-779.1996

Molecular analysis of the poly(3-hydroxyalkanoate) synthase gene from a methylotrophic bacterium, Paracoccus denitrificans.

S Ueda 1, T Yabutani 1, A Maehara 1, T Yamane 1
PMCID: PMC177724  PMID: 8550512

Abstract

A 3.6-kb EcoRI-SalI fragment of Paracoccus denitrificans DNA hybridized with a DNA probe carrying the poly(3-hydroxyalkanoate) (PHA) synthase gene (phaC) of Alcaligenes eutrophus. Nucleotide sequence analysis of this region showed the presence of a 1,872-bp open reading frame (ORF), which corresponded to a polypeptide with a molecular weight of 69,537. Upstream of the ORF, a promoter-like sequence was found. Escherichia coli carrying the fusion gene between lacZ and the ORF accumulated a level of poly(3-hydroxybutyrate) that was as much as 20 wt% of the cell dry weight in the presence of beta-ketothiolase and acetoacetylcoenzyme A reductase genes of A. eutrophus. The ORF was designated phaCPd. A plasmid vector carrying the phaCPd'-'lacZ fusion gene downstream of the promoter-like sequence expressed beta-galactosidase activity in P. denitrificans. When a multicopy and broad-host-range vector carrying the ORF along with the promoter-like sequence was introduced into P. denitrificans, the PHA content in the cells increased by twofold compared with cells carrying only a vector sequence.

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Selected References

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  1. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  2. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  3. Dawes E. A., Senior P. J. The role and regulation of energy reserve polymers in micro-organisms. Adv Microb Physiol. 1973;10:135–266. doi: 10.1016/s0065-2911(08)60088-0. [DOI] [PubMed] [Google Scholar]
  4. Ditta G., Schmidhauser T., Yakobson E., Lu P., Liang X. W., Finlay D. R., Guiney D., Helinski D. R. Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid. 1985 Mar;13(2):149–153. doi: 10.1016/0147-619x(85)90068-x. [DOI] [PubMed] [Google Scholar]
  5. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Huisman G. W., Wonink E., Meima R., Kazemier B., Terpstra P., Witholt B. Metabolism of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas oleovorans. Identification and sequences of genes and function of the encoded proteins in the synthesis and degradation of PHA. J Biol Chem. 1991 Feb 5;266(4):2191–2198. [PubMed] [Google Scholar]
  8. Kovach M. E., Phillips R. W., Elzer P. H., Roop R. M., 2nd, Peterson K. M. pBBR1MCS: a broad-host-range cloning vector. Biotechniques. 1994 May;16(5):800–802. [PubMed] [Google Scholar]
  9. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  10. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  11. Liebergesell M., Steinbüchel A. Cloning and molecular analysis of the poly(3-hydroxybutyric acid) biosynthetic genes of Thiocystis violacea. Appl Microbiol Biotechnol. 1993 Jan;38(4):493–501. doi: 10.1007/BF00242944. [DOI] [PubMed] [Google Scholar]
  12. Liebergesell M., Steinbüchel A. Cloning and nucleotide sequences of genes relevant for biosynthesis of poly(3-hydroxybutyric acid) in Chromatium vinosum strain D. Eur J Biochem. 1992 Oct 1;209(1):135–150. doi: 10.1111/j.1432-1033.1992.tb17270.x. [DOI] [PubMed] [Google Scholar]
  13. Peoples O. P., Masamune S., Walsh C. T., Sinskey A. J. Biosynthetic thiolase from Zoogloea ramigera. III. Isolation and characterization of the structural gene. J Biol Chem. 1987 Jan 5;262(1):97–102. [PubMed] [Google Scholar]
  14. Peoples O. P., Sinskey A. J. Fine structural analysis of the Zoogloea ramigera phbA-phbB locus encoding beta-ketothiolase and acetoacetyl-CoA reductase: nucleotide sequence of phbB. Mol Microbiol. 1989 Mar;3(3):349–357. doi: 10.1111/j.1365-2958.1989.tb00180.x. [DOI] [PubMed] [Google Scholar]
  15. Peoples O. P., Sinskey A. J. Poly-beta-hydroxybutyrate (PHB) biosynthesis in Alcaligenes eutrophus H16. Identification and characterization of the PHB polymerase gene (phbC). J Biol Chem. 1989 Sep 15;264(26):15298–15303. [PubMed] [Google Scholar]
  16. Rose R. E. The nucleotide sequence of pACYC177. Nucleic Acids Res. 1988 Jan 11;16(1):356–356. doi: 10.1093/nar/16.1.356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Schembri M. A., Bayly R. C., Davies J. K. Cloning and analysis of the polyhydroxyalkanoic acid synthase gene from an Acinetobacter sp.: evidence that the gene is both plasmid and chromosomally located. FEMS Microbiol Lett. 1994 May 1;118(1-2):145–152. doi: 10.1111/j.1574-6968.1994.tb06817.x. [DOI] [PubMed] [Google Scholar]
  19. Schubert P., Krüger N., Steinbüchel A. 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. 1991 Jan;173(1):168–175. doi: 10.1128/jb.173.1.168-175.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schubert P., Steinbüchel A., Schlegel H. G. Cloning of the Alcaligenes eutrophus genes for synthesis of poly-beta-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli. J Bacteriol. 1988 Dec;170(12):5837–5847. doi: 10.1128/jb.170.12.5837-5847.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Slater S. C., Voige W. H., Dennis D. E. Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway. J Bacteriol. 1988 Oct;170(10):4431–4436. doi: 10.1128/jb.170.10.4431-4436.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  23. Steinbüchel A., Schlegel H. G. Physiology and molecular genetics of poly(beta-hydroxy-alkanoic acid) synthesis in Alcaligenes eutrophus. Mol Microbiol. 1991 Mar;5(3):535–542. doi: 10.1111/j.1365-2958.1991.tb00725.x. [DOI] [PubMed] [Google Scholar]
  24. Steinrücke P., Ludwig B. Genetics of Paracoccus denitrificans. FEMS Microbiol Rev. 1993 Jan;10(1-2):83–117. doi: 10.1016/0378-1097(93)90505-v. [DOI] [PubMed] [Google Scholar]
  25. Timm A., Steinbüchel A. Cloning and molecular analysis of the poly(3-hydroxyalkanoic acid) gene locus of Pseudomonas aeruginosa PAO1. Eur J Biochem. 1992 Oct 1;209(1):15–30. doi: 10.1111/j.1432-1033.1992.tb17256.x. [DOI] [PubMed] [Google Scholar]
  26. Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
  27. Ueda S., Matsumoto S., Takagi A., Yamane T. Synthesis of Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) from Methanol and n-Amyl Alcohol by the Methylotrophic Bacteria Paracoccus denitrificans and Methylobacterium extorquens. Appl Environ Microbiol. 1992 Nov;58(11):3574–3579. doi: 10.1128/aem.58.11.3574-3579.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Valentin H. E., Steinbüchel A. Cloning and characterization of the Methylobacterium extorquens polyhydroxyalkanoic-acid-synthase structural gene. Appl Microbiol Biotechnol. 1993 Jun;39(3):309–317. doi: 10.1007/BF00192084. [DOI] [PubMed] [Google Scholar]

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