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. 1990 May;172(5):2491–2497. doi: 10.1128/jb.172.5.2491-2497.1990

Cloning of the Zymomonas mobilis structural gene encoding alcohol dehydrogenase I (adhA): sequence comparison and expression in Escherichia coli.

K F Keshav 1, L P Yomano 1, H J An 1, L O Ingram 1
PMCID: PMC208888  PMID: 2185223

Abstract

Zymomonas mobilis ferments sugars to produce ethanol with two biochemically distinct isoenzymes of alcohol dehydrogenase. The adhA gene encoding alcohol dehydrogenase I has now been sequenced and compared with the adhB gene, which encodes the second isoenzyme. The deduced amino acid sequences for these gene products exhibited no apparent homology. Alcohol dehydrogenase I contained 337 amino acids, with a subunit molecular weight of 36,096. Based on comparisons of primary amino acid sequences, this enzyme belongs to the family of zinc alcohol dehydrogenases which have been described primarily in eucaryotes. Nearly all of the 22 strictly conserved amino acids in this group were also conserved in Z. mobilis alcohol dehydrogenase I. Alcohol dehydrogenase I is an abundant protein, although adhA lacked many of the features previously reported in four other highly expressed genes from Z. mobilis. Codon usage in adhA is not highly biased and includes many codons which were unused by pdc, adhB, gap, and pgk. The ribosomal binding region of adhA lacked the canonical Shine-Dalgarno sequence found in the other highly expressed genes from Z. mobilis. Although these features may facilitate the expression of high enzyme levels, they do not appear to be essential for the expression of Z. mobilis adhA.

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Selected References

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

  1. Bennetzen J. L., Hall B. D. Codon selection in yeast. J Biol Chem. 1982 Mar 25;257(6):3026–3031. [PubMed] [Google Scholar]
  2. Bridgen J., Kolb E., Harris J. I. Amino acid sequence homology in alcohol dehydrogenase. FEBS Lett. 1973 Jun 15;33(1):1–3. doi: 10.1016/0014-5793(73)80144-9. [DOI] [PubMed] [Google Scholar]
  3. Conway T., Ingram L. O. Phosphoglycerate kinase gene from Zymomonas mobilis: cloning, sequencing, and localization within the gap operon. J Bacteriol. 1988 Apr;170(4):1926–1933. doi: 10.1128/jb.170.4.1926-1933.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Conway T., Ingram L. O. Similarity of Escherichia coli propanediol oxidoreductase (fucO product) and an unusual alcohol dehydrogenase from Zymomonas mobilis and Saccharomyces cerevisiae. J Bacteriol. 1989 Jul;171(7):3754–3759. doi: 10.1128/jb.171.7.3754-3759.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Conway T., Osman Y. A., Konnan J. I., Hoffmann E. M., Ingram L. O. Promoter and nucleotide sequences of the Zymomonas mobilis pyruvate decarboxylase. J Bacteriol. 1987 Mar;169(3):949–954. doi: 10.1128/jb.169.3.949-954.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Conway T., Sewell G. W., Ingram L. O. Glyceraldehyde-3-phosphate dehydrogenase gene from Zymomonas mobilis: cloning, sequencing, and identification of promoter region. J Bacteriol. 1987 Dec;169(12):5653–5662. doi: 10.1128/jb.169.12.5653-5662.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Conway T., Sewell G. W., Osman Y. A., Ingram L. O. Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis. J Bacteriol. 1987 Jun;169(6):2591–2597. doi: 10.1128/jb.169.6.2591-2597.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dennis E. S., Gerlach W. L., Pryor A. J., Bennetzen J. L., Inglis A., Llewellyn D., Sachs M. M., Ferl R. J., Peacock W. J. Molecular analysis of the alcohol dehydrogenase (Adh1) gene of maize. Nucleic Acids Res. 1984 May 11;12(9):3983–4000. doi: 10.1093/nar/12.9.3983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fowler P. W., Ball A. J., Griffiths D. E. The control of alcohol dehydrogenase isozyme synthesis in Saccharomyces cerevisiae. Can J Biochem. 1972 Jan;50(1):35–43. doi: 10.1139/o72-007. [DOI] [PubMed] [Google Scholar]
  10. Hoekema A., Kastelein R. A., Vasser M., de Boer H. A. Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression. Mol Cell Biol. 1987 Aug;7(8):2914–2924. doi: 10.1128/mcb.7.8.2914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ikemura T. Correlation between the abundance of yeast transfer RNAs and the occurrence of the respective codons in protein genes. Differences in synonymous codon choice patterns of yeast and Escherichia coli with reference to the abundance of isoaccepting transfer RNAs. J Mol Biol. 1982 Jul 15;158(4):573–597. doi: 10.1016/0022-2836(82)90250-9. [DOI] [PubMed] [Google Scholar]
  12. Ikuta T., Szeto S., Yoshida A. Three human alcohol dehydrogenase subunits: cDNA structure and molecular and evolutionary divergence. Proc Natl Acad Sci U S A. 1986 Feb;83(3):634–638. doi: 10.1073/pnas.83.3.634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jendrossek D., Steinbüchel A., Schlegel H. G. Alcohol dehydrogenase gene from Alcaligenes eutrophus: subcloning, heterologous expression in Escherichia coli, sequencing, and location of Tn5 insertions. J Bacteriol. 1988 Nov;170(11):5248–5256. doi: 10.1128/jb.170.11.5248-5256.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jörnvall H. Horse liver alcohol dehydrogenase. The primary structure of the protein chain of the ethanol-active isoenzyme. Eur J Biochem. 1970 Sep;16(1):25–40. doi: 10.1111/j.1432-1033.1970.tb01049.x. [DOI] [PubMed] [Google Scholar]
  15. Jörnvall H., Persson B., Jeffery J. Characteristics of alcohol/polyol dehydrogenases. The zinc-containing long-chain alcohol dehydrogenases. Eur J Biochem. 1987 Sep 1;167(2):195–201. doi: 10.1111/j.1432-1033.1987.tb13323.x. [DOI] [PubMed] [Google Scholar]
  16. Kreitman M. Nucleotide polymorphism at the alcohol dehydrogenase locus of Drosophila melanogaster. Nature. 1983 Aug 4;304(5925):412–417. doi: 10.1038/304412a0. [DOI] [PubMed] [Google Scholar]
  17. Luria S. E., Delbrück M. Mutations of Bacteria from Virus Sensitivity to Virus Resistance. Genetics. 1943 Nov;28(6):491–511. doi: 10.1093/genetics/28.6.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Machlin S. M., Hanson R. S. Nucleotide sequence and transcriptional start site of the Methylobacterium organophilum XX methanol dehydrogenase structural gene. J Bacteriol. 1988 Oct;170(10):4739–4747. doi: 10.1128/jb.170.10.4739-4747.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mackenzie K. F., Conway T., Aldrich H. C., Ingram L. O. Expression of Zymomonas mobilis adhB (encoding alcohol dehydrogenase II) and adhB-lacZ operon fusions in recombinant Z. mobilis. J Bacteriol. 1989 Sep;171(9):4577–4582. doi: 10.1128/jb.171.9.4577-4582.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mackenzie K. F., Eddy C. K., Ingram L. O. Modulation of alcohol dehydrogenase isoenzyme levels in Zymomonas mobilis by iron and zinc. J Bacteriol. 1989 Feb;171(2):1063–1067. doi: 10.1128/jb.171.2.1063-1067.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McKnight G. L., Kato H., Upshall A., Parker M. D., Saari G., O'Hara P. J. Identification and molecular analysis of a third Aspergillus nidulans alcohol dehydrogenase gene. EMBO J. 1985 Aug;4(8):2093–2099. doi: 10.1002/j.1460-2075.1985.tb03897.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Neale A. D., Scopes R. K., Kelly J. M., Wettenhall R. E. The two alcohol dehydrogenases of Zymomonas mobilis. Purification by differential dye ligand chromatography, molecular characterisation and physiological roles. Eur J Biochem. 1986 Jan 2;154(1):119–124. doi: 10.1111/j.1432-1033.1986.tb09366.x. [DOI] [PubMed] [Google Scholar]
  23. Neale A. D., Scopes R. K., Wettenhall R. E., Hoogenraad N. J. Pyruvate decarboxylase of Zymomonas mobilis: isolation, properties, and genetic expression in Escherichia coli. J Bacteriol. 1987 Mar;169(3):1024–1028. doi: 10.1128/jb.169.3.1024-1028.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Osman Y. A., Conway T., Bonetti S. J., Ingram L. O. Glycolytic flux in Zymomonas mobilis: enzyme and metabolite levels during batch fermentation. J Bacteriol. 1987 Aug;169(8):3726–3736. doi: 10.1128/jb.169.8.3726-3736.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pawluk A., Scopes R. K., Griffiths-Smith K. Isolation and properties of the glycolytic enzymes from Zymomonas mobilis. The five enzymes from glyceraldehyde-3-phosphate dehydrogenase through to pyruvate kinase. Biochem J. 1986 Aug 15;238(1):275–281. doi: 10.1042/bj2380275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Russell P. R., Hall B. D. The primary structure of the alcohol dehydrogenase gene from the fission yeast Schizosaccharomyces pombe. J Biol Chem. 1983 Jan 10;258(1):143–149. [PubMed] [Google Scholar]
  27. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Stormo G. D., Schneider T. D., Gold L. M. Characterization of translational initiation sites in E. coli. Nucleic Acids Res. 1982 May 11;10(9):2971–2996. doi: 10.1093/nar/10.9.2971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wills C., Kratofil P., Londo D., Martin T. Characterization of the two alcohol dehydrogenases of Zymomonas mobilis. Arch Biochem Biophys. 1981 Sep;210(2):775–785. doi: 10.1016/0003-9861(81)90245-9. [DOI] [PubMed] [Google Scholar]
  30. Young E. T., Pilgrim D. Isolation and DNA sequence of ADH3, a nuclear gene encoding the mitochondrial isozyme of alcohol dehydrogenase in Saccharomyces cerevisiae. Mol Cell Biol. 1985 Nov;5(11):3024–3034. doi: 10.1128/mcb.5.11.3024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Youngleson Jonathan S., Santangelo Joseph D., Jones David T., Woods David R. Cloning and Expression of a Clostridium acetobutylicum Alcohol Dehydrogenase Gene in Escherichia coli. Appl Environ Microbiol. 1988 Mar;54(3):676–682. doi: 10.1128/aem.54.3.676-682.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

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