Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1995 Jul;61(7):2487–2492. doi: 10.1128/aem.61.7.2487-2492.1995

Role of fadR and atoC(Con) mutations in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in recombinant pha+ Escherichia coli.

H G Rhie 1, D Dennis 1
PMCID: PMC167520  PMID: 7618860

Abstract

Recombinant Escherichia coli fadR atoC(Con) mutants containing the polyhydroxyalkanoate (PHA) biosynthesis genes from Alcaligenes eutrophus are able to incorporate significant levels of 3-hydroxyvalerate (3HV) into the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]. We have used E. coli fadR (FadR is a negative regulator of fatty acid oxidation) and E. coli atoC(Con) (AtoC is a positive regulator of fatty acid uptake) mutants to demonstrate that either one of these mutations alone can facilitate copolymer synthesis but that 3HV levels in single mutant strains are much lower than in the fadR atoC(Con) strain. E. coli atoC(Con) mutants were used alone and in conjunction with atoA and atoD mutants to determine that the function of the atoC(Con) mutation is to increase the uptake of propionate and that this uptake is mediated, at least in part, by atoD+. Similarly, E. coli fadR mutants were used alone and in conjunction with fadA, fadB, and fadL mutants to show that the effect of the fadR mutation is dependent on fadB+ and fadA+ gene products. Strains that were mutant in the fadB or fadA locus were unable to complement a PHA biosynthesis pathway that was mutant at the phaA locus (thiolase), but a strain containing a fadR mutation and which was fadA+ fadB+ was able to complement the phaA mutation and incorporated 3HV into P(3HB-co-3HV) to a level of 29 mol%.

Full Text

The Full Text of this article is available as a PDF (245.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson A. J., Dawes E. A. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev. 1990 Dec;54(4):450–472. doi: 10.1128/mr.54.4.450-472.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Binstock J. F., Pramanik A., Schulz H. Isolation of a multi-enzyme complex of fatty acid oxidation from Escherichia coli. Proc Natl Acad Sci U S A. 1977 Feb;74(2):492–495. doi: 10.1073/pnas.74.2.492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brown T. D., Jones-Mortimer M. C., Kornberg H. L. The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli. J Gen Microbiol. 1977 Oct;102(2):327–336. doi: 10.1099/00221287-102-2-327. [DOI] [PubMed] [Google Scholar]
  4. DiRusso C. C., Nunn W. D. Cloning and characterization of a gene (fadR) involved in regulation of fatty acid metabolism in Escherichia coli. J Bacteriol. 1985 Feb;161(2):583–588. doi: 10.1128/jb.161.2.583-588.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DiRusso C. C. Primary sequence of the Escherichia coli fadBA operon, encoding the fatty acid-oxidizing multienzyme complex, indicates a high degree of homology to eucaryotic enzymes. J Bacteriol. 1990 Nov;172(11):6459–6468. doi: 10.1128/jb.172.11.6459-6468.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Feigenbaum J., Schulz H. Thiolases of Escherichia coli: purification and chain length specificities. J Bacteriol. 1975 May;122(2):407–411. doi: 10.1128/jb.122.2.407-411.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Frerman F. E., Bennett W. Studies on the uptake of fatty acids by Escherichia coli. Arch Biochem Biophys. 1973 Nov;159(1):434–443. doi: 10.1016/0003-9861(73)90471-2. [DOI] [PubMed] [Google Scholar]
  8. Frerman F. E. The role of acetyl coenzyme A: butyrate coenzyme A in the transferase uptake of butyrate by isolated membrane vesicles of Escherichia coli. Arch Biochem Biophys. 1973 Nov;159(1):444–452. doi: 10.1016/0003-9861(73)90472-4. [DOI] [PubMed] [Google Scholar]
  9. Henry M. F., Cronan J. E., Jr Escherichia coli transcription factor that both activates fatty acid synthesis and represses fatty acid degradation. J Mol Biol. 1991 Dec 20;222(4):843–849. doi: 10.1016/0022-2836(91)90574-p. [DOI] [PubMed] [Google Scholar]
  10. Jenkins L. S., Nunn W. D. Genetic and molecular characterization of the genes involved in short-chain fatty acid degradation in Escherichia coli: the ato system. J Bacteriol. 1987 Jan;169(1):42–52. doi: 10.1128/jb.169.1.42-52.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Krahenbuhl S., Brass E. P. Inhibition of hepatic propionyl-CoA synthetase activity by organic acids. Reversal of propionate inhibition of pyruvate metabolism. 1991 Mar 15-Apr 1Biochem Pharmacol. 41(6-7):1015–1023. doi: 10.1016/0006-2952(91)90209-n. [DOI] [PubMed] [Google Scholar]
  12. Magnuson K., Jackowski S., Rock C. O., Cronan J. E., Jr Regulation of fatty acid biosynthesis in Escherichia coli. Microbiol Rev. 1993 Sep;57(3):522–542. doi: 10.1128/mr.57.3.522-542.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Maloy S. R., Bohlander M., Nunn W. D. Elevated levels of glyoxylate shunt enzymes in Escherichia coli strains constitutive for fatty acid degradation. J Bacteriol. 1980 Aug;143(2):720–725. doi: 10.1128/jb.143.2.720-725.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Maloy S. R., Nunn W. D. Genetic regulation of the glyoxylate shunt in Escherichia coli K-12. J Bacteriol. 1982 Jan;149(1):173–180. doi: 10.1128/jb.149.1.173-180.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Maloy S. R., Nunn W. D. Role of gene fadR in Escherichia coli acetate metabolism. J Bacteriol. 1981 Oct;148(1):83–90. doi: 10.1128/jb.148.1.83-90.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nunn W. D., Giffin K., Clark D., Cronan J. E., Jr Role for fadR in unsaturated fatty acid biosynthesis in Escherichia coli. J Bacteriol. 1983 May;154(2):554–560. doi: 10.1128/jb.154.2.554-560.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Otaki K., Takahashi K., Hasegawa A., Soga Y., Nakazawa M. [A case of Peutz-Jeghers syndrome]. Shigaku. 1986 Apr;73(7):1693–1697. [PubMed] [Google Scholar]
  18. Overath P., Pauli G., Schairer H. U. Fatty acid degradation in Escherichia coli. An inducible acyl-CoA synthetase, the mapping of old-mutations, and the isolation of regulatory mutants. Eur J Biochem. 1969 Feb;7(4):559–574. [PubMed] [Google Scholar]
  19. Pauli G., Overath P. ato Operon: a highly inducible system for acetoacetate and butyrate degradation in Escherichia coli. Eur J Biochem. 1972 Sep 25;29(3):553–562. doi: 10.1111/j.1432-1033.1972.tb02021.x. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Peoples O. P., Sinskey A. J. Poly-beta-hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16. Characterization of the genes encoding beta-ketothiolase and acetoacetyl-CoA reductase. J Biol Chem. 1989 Sep 15;264(26):15293–15297. [PubMed] [Google Scholar]
  22. Pramanik A., Pawar S., Antonian E., Schulz H. Five different enzymatic activities are associated with the multienzyme complex of fatty acid oxidation from Escherichia coli. J Bacteriol. 1979 Jan;137(1):469–473. doi: 10.1128/jb.137.1.469-473.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Simons R. W., Egan P. A., Chute H. T., Nunn W. D. Regulation of fatty acid degradation in Escherichia coli: isolation and characterization of strains bearing insertion and temperature-sensitive mutations in gene fadR. J Bacteriol. 1980 May;142(2):621–632. doi: 10.1128/jb.142.2.621-632.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Slater S., Gallaher T., Dennis D. Production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) in a recombinant Escherichia coli strain. Appl Environ Microbiol. 1992 Apr;58(4):1089–1094. doi: 10.1128/aem.58.4.1089-1094.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Suzuki F., Zahler W. L., Emerich D. W. Acetoacetyl-CoA thiolase of Bradyrhizobium japonicum bacteroids: purification and properties. Arch Biochem Biophys. 1987 Apr;254(1):272–281. doi: 10.1016/0003-9861(87)90103-2. [DOI] [PubMed] [Google Scholar]
  29. Thompson S., Mayerl F., Peoples O. P., Masamune S., Sinskey A. J., Walsh C. T. Mechanistic studies on beta-ketoacyl thiolase from Zoogloea ramigera: identification of the active-site nucleophile as Cys89, its mutation to Ser89, and kinetic and thermodynamic characterization of wild-type and mutant enzymes. Biochemistry. 1989 Jul 11;28(14):5735–5742. doi: 10.1021/bi00440a006. [DOI] [PubMed] [Google Scholar]
  30. Yang S. Y., Yang X. Y., Healy-Louie G., Schulz H., Elzinga M. Nucleotide sequence of the fadA gene. Primary structure of 3-ketoacyl-coenzyme A thiolase from Escherichia coli and the structural organization of the fadAB operon. J Biol Chem. 1990 Jun 25;265(18):10424–10429. [PubMed] [Google Scholar]
  31. Zhang H., Obias V., Gonyer K., Dennis D. Production of polyhydroxyalkanoates in sucrose-utilizing recombinant Escherichia coli and Klebsiella strains. Appl Environ Microbiol. 1994 Apr;60(4):1198–1205. doi: 10.1128/aem.60.4.1198-1205.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES