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. 1996 Feb;62(2):372–379. doi: 10.1128/aem.62.2.372-379.1996

Metabolic pathway for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) formation in Nocardia corallina: inactivation of mutB by chromosomal integration of a kanamycin resistance gene.

H F Valentin 1, D Dennis 1
PMCID: PMC167808  PMID: 8593043

Abstract

The gene encoding the large subunit of the methylmalonyl-coenzyme A (CoA) mutase in Nocardia corallina (mutBNc) was cloned. A 4.3-kbp BamHI fragment containing almost the entire mutBNc was identified by Southern hybridization experiments employing a digoxigenin-labeled probe deduced from mutB of Streptomyces cinnamonensis, mutBNc was interrupted by insertion of a kanamycin resistance gene block (mutB::kan or mutB::neo) and introduced into N. corallina to obtain mutB-negative strains by homologous recombination. Four of sixteen kanamycin-resistant clones occurred via double-crossover events and harbored only the interrupted mutBNc. These exhibited no growth on odd-chain fatty acids in the presence of kanamycin but exhibited wild-type growth on even-chain fatty acids, glucose, and succinate. Whereas the wild type of N. corallina accumulates a copolyester of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) containing more than 60 mol% 3HV from most carbon sources, mutB-negative strains accumulated poly(3HB-co-3HV) containing only 2 to 6 mol% 3HV. Methylmalonyl-CoA mutase activity was not found in these clones. Therefore, this study provides strong evidence that the majority of 3HV units in poly(3HB-co-3HV) accumulated by N. corallina are synthesized via the methylmalonyl-CoA pathway.

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Selected References

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  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. Brandl H., Gross R. A., Lenz R. W., Fuller R. C. Pseudomonas oleovorans as a Source of Poly(beta-Hydroxyalkanoates) for Potential Applications as Biodegradable Polyesters. Appl Environ Microbiol. 1988 Aug;54(8):1977–1982. doi: 10.1128/aem.54.8.1977-1982.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Haywood G. W., Anderson A. J., Ewing D. F., Dawes E. A. Accumulation of a Polyhydroxyalkanoate Containing Primarily 3-Hydroxydecanoate from Simple Carbohydrate Substrates by Pseudomonas sp. Strain NCIMB 40135. Appl Environ Microbiol. 1990 Nov;56(11):3354–3359. doi: 10.1128/aem.56.11.3354-3359.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Jansen R., Kalousek F., Fenton W. A., Rosenberg L. E., Ledley F. D. Cloning of full-length methylmalonyl-CoA mutase from a cDNA library using the polymerase chain reaction. Genomics. 1989 Feb;4(2):198–205. doi: 10.1016/0888-7543(89)90300-5. [DOI] [PubMed] [Google Scholar]
  6. Loeffelholz M. J., Scholl D. R. Method for improved extraction of DNA from Nocardia asteroides. J Clin Microbiol. 1989 Aug;27(8):1880–1881. doi: 10.1128/jcm.27.8.1880-1881.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Madoń J., Hütter R. Transformation system for Amycolatopsis (Nocardia) mediterranei: direct transformation of mycelium with plasmid DNA. J Bacteriol. 1991 Oct;173(20):6325–6331. doi: 10.1128/jb.173.20.6325-6331.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Marcus J. P., Dekker E. E. Threonine formation via the coupled activity of 2-amino-3-ketobutyrate coenzyme A lyase and threonine dehydrogenase. J Bacteriol. 1993 Oct;175(20):6505–6511. doi: 10.1128/jb.175.20.6505-6511.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Marsh E. N., McKie N., Davis N. K., Leadlay P. F. Cloning and structural characterization of the genes coding for adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii. Biochem J. 1989 Jun 1;260(2):345–352. doi: 10.1042/bj2600345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. SCHLEGEL H. G., KALTWASSER H., GOTTSCHALK G. [A submersion method for culture of hydrogen-oxidizing bacteria: growth physiological studies]. Arch Mikrobiol. 1961;38:209–222. [PubMed] [Google Scholar]
  11. 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]
  12. Timm A., Steinbüchel A. Formation of polyesters consisting of medium-chain-length 3-hydroxyalkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent pseudomonads. Appl Environ Microbiol. 1990 Nov;56(11):3360–3367. doi: 10.1128/aem.56.11.3360-3367.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. WALDE E. [Studies on growth and synthesis of stored substance by Hydrogenomonas]. Arch Mikrobiol. 1962;43:109–137. [PubMed] [Google Scholar]
  14. Wilkemeyer M. F., Crane A. M., Ledley F. D. Primary structure and activity of mouse methylmalonyl-CoA mutase. Biochem J. 1990 Oct 15;271(2):449–455. doi: 10.1042/bj2710449. [DOI] [PMC free article] [PubMed] [Google Scholar]

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