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. 1995 Feb;61(2):413–420. doi: 10.1128/aem.61.2.413-420.1995

Cloning and nucleotide sequencing of the membrane-bound L-sorbosone dehydrogenase gene of Acetobacter liquefaciens IFO 12258 and its expression in Gluconobacter oxydans.

M Shinjoh 1, N Tomiyama 1, A Asakura 1, T Hoshino 1
PMCID: PMC167301  PMID: 7574579

Abstract

Cloning and expression of the gene encoding Acetobacter liquefaciens IFO 12258 membrane-bound L-sorbosone dehydrogenase (SNDH) were studied. A genomic library of A. liquefaciens IFO 12258 was constructed with the mobilizable cosmid vector pVK102 (mob+) in Escherichia coli S17-1 (Tra+). The library was transferred by conjugal mating into Gluconobacter oxydans OX4, a mutant of G. oxydans IFO 3293 that accumulates L-sorbosone in the presence of L-sorbose. The transconjugants were screened for SNDH activity by performing a direct expression assay. One clone harboring plasmid p7A6 converted L-sorbosone to 2-keto-L-gulonic acid (2KGA) more rapidly than its host did and also converted L-sorbose to 2KGA with no accumulation of L-sorbosone. The insert (25 kb) of p7A6 was shortened to a 3.1-kb fragment, in which one open reading frame (1,347 bp) was found and was shown to encode a polypeptide with a molecular weight of 48,222. The SNDH gene was introduced into the 2KGA-producing strain G. oxydans IFO 3293 and its derivatives, which contained membrane-bound L-sorbose dehydrogenase. The cloned SNDH was correctly located in the membrane of the host. The membrane fraction of the clone exhibited almost stoichiometric formation of 2KGA from L-sorbosone and L-sorbose. Resting cells of the clones produced 2KGA very efficiently from L-sorbosone and L-sorbose, but not from D-sorbitol; the conversion yield from L-sorbosone was improved from approximately 25 to 83%, whereas the yield from L-sorbose was increased from 68 to 81%. Under fermentation conditions, cloning did not obviously improve the yield of 2KGA from L-sorbose.

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Selected References

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  1. Anderson S., Marks C. B., Lazarus R., Miller J., Stafford K., Seymour J., Light D., Rastetter W., Estell D. Production of 2-Keto-L-Gulonate, an Intermediate in L-Ascorbate Synthesis, by a Genetically Modified Erwinia herbicola. Science. 1985 Oct 11;230(4722):144–149. doi: 10.1126/science.230.4722.144. [DOI] [PubMed] [Google Scholar]
  2. Anthony C. The structure of bacterial quinoprotein dehydrogenases. Int J Biochem. 1992;24(1):29–39. doi: 10.1016/0020-711x(92)90226-q. [DOI] [PubMed] [Google Scholar]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cleton-Jansen A. M., Dekker S., van de Putte P., Goosen N. A single amino acid substitution changes the substrate specificity of quinoprotein glucose dehydrogenase in Gluconobacter oxydans. Mol Gen Genet. 1991 Oct;229(2):206–212. doi: 10.1007/BF00272157. [DOI] [PubMed] [Google Scholar]
  5. Grindley J. F., Payton M. A., van de Pol H., Hardy K. G. Conversion of Glucose to 2-Keto-l-Gulonate, an Intermediate in l-Ascorbate Synthesis, by a Recombinant Strain of Erwinia citreus. Appl Environ Microbiol. 1988 Jul;54(7):1770–1775. doi: 10.1128/aem.54.7.1770-1775.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Harms N., de Vries G. E., Maurer K., Hoogendijk J., Stouthamer A. H. Isolation and nucleotide sequence of the methanol dehydrogenase structural gene from Paracoccus denitrificans. J Bacteriol. 1987 Sep;169(9):3969–3975. doi: 10.1128/jb.169.9.3969-3975.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Inoue T., Sunagawa M., Mori A., Imai C., Fukuda M., Takagi M., Yano K. Cloning and sequencing of the gene encoding the 72-kilodalton dehydrogenase subunit of alcohol dehydrogenase from Acetobacter aceti. J Bacteriol. 1989 Jun;171(6):3115–3122. doi: 10.1128/jb.171.6.3115-3122.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Knauf V. C., Nester E. W. Wide host range cloning vectors: a cosmid clone bank of an Agrobacterium Ti plasmid. Plasmid. 1982 Jul;8(1):45–54. doi: 10.1016/0147-619x(82)90040-3. [DOI] [PubMed] [Google Scholar]
  9. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Murray N. E., Brammar W. J., Murray K. Lambdoid phages that simplify the recovery of in vitro recombinants. Mol Gen Genet. 1977 Jan 7;150(1):53–61. doi: 10.1007/BF02425325. [DOI] [PubMed] [Google Scholar]
  13. Rothmel R. K., Chakrabarty A. M., Berry A., Darzins A. Genetic systems in Pseudomonas. Methods Enzymol. 1991;204:485–514. doi: 10.1016/0076-6879(91)04025-j. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Sonoyama T., Tani H., Matsuda K., Kageyama B., Tanimoto M., Kobayashi K., Yagi S., Kyotani H., Mitsushima K. Production of 2-Keto-l-Gulonic Acid from d-Glucose by Two-Stage Fermentation. Appl Environ Microbiol. 1982 May;43(5):1064–1069. doi: 10.1128/aem.43.5.1064-1069.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Tamaki T., Fukaya M., Takemura H., Tayama K., Okumura H., Kawamura Y., Nishiyama M., Horinouchi S., Beppu T. Cloning and sequencing of the gene cluster encoding two subunits of membrane-bound alcohol dehydrogenase from Acetobacter polyoxogenes. Biochim Biophys Acta. 1991 Feb 16;1088(2):292–300. doi: 10.1016/0167-4781(91)90066-u. [DOI] [PubMed] [Google Scholar]
  17. Tamaki T., Horinouchi S., Fukaya M., Okumura H., Kawamura Y., Beppu T. Nucleotide sequence of the membrane-bound aldehyde dehydrogenase gene from Acetobacter polyoxogenes. J Biochem. 1989 Oct;106(4):541–544. doi: 10.1093/oxfordjournals.jbchem.a122889. [DOI] [PubMed] [Google Scholar]
  18. Tsukada Y., Perlman D. The fermentation of L-sorbose by Gluconobacter melanogenus. I. General characteristics of the fermentation. Biotechnol Bioeng. 1972 Sep;14(5):799–810. doi: 10.1002/bit.260140509. [DOI] [PubMed] [Google Scholar]

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