Molecular and General Genetics MGG

, Volume 234, Issue 2, pp 201–210 | Cite as

Cloning and expression of the genes for xylose isomerase and xylulokinase from Klebsiella pneumoniae 1033 in Escherichia coli K12

  • Sigrun D. Feldmann
  • Hermann Sahm
  • Georg A. Sprenger


The genes xy1A and xy1B were cloned together with their promoter region from the chromosome of Klehsiella pneumoniae var. aerogenes 1033 and the DNA sequence (3225 bp) was determined. The gene xy1A encodes the enzyme xylose isomerase (XI or XylA) consisting of 440 amino acids (calculated Mr of 49 793). The gene xy1B encodes the enzyme xylulokinase (XK or Xy1B) with a calculated M, of 51 783 (483 amino acids). The two genes successfully complemented xy1 mutants of Escherichia coli K12, but no gene dosage effect was detected. E. coli wild-type cells which harbored plasmids with the intact xylAKp 5′ upstream region in high copy number (but lacking an active xy1B gene on the plasmids) were phenotypically xylose-negative and xylose isomerase and xylulokinase activities were drastically diminished. Deletion of 5′ upstream regions of xy1A on these plasmids and their substitution by a lac promoter resulted in a xylose-positive phenotype. This also resulted in overproduction of plasmid-encoded xylose isomerase and xylulokinase activities in recombinant E. coli cells.

Key words

Klebsiella pneumoniae Xylose isomerase Xylulokinase xy1 gene expression 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Altenbuchner J, Schmid K, Schmitt R (1983) Tn1721-encoded tetracycline resistance: mapping of structural and regulatory genes mediating resistance. J Bacteriol 153:116–123Google Scholar
  2. Batt CA, Bodis MS, Picataggio SK, Claps MC, Jamas S, Sinskey AJ (1985) Analysis of xylose operon regulation by Mud (Apr, lac) fusion: trans effect of plasmid coded xylose operon. Can J Microbiol 31:930–933Google Scholar
  3. Batt CA, O'Neill E, Novak SR, Ko J, Sinskey A (1986) Hyper-expression of Escherichia coli Xylose Isomerase. Biotechnol Prog 2:140–144Google Scholar
  4. Batt CA, Jamieson AC, Vandeyar MA (1990) Identification of essential histidine residues in the active site of Escherichia coli xylose (glucose) isomerase. Proc Natl Acad Sci USA 87:618–622Google Scholar
  5. Biely P (1985) Microbial xylanolytic systems. Trends Biotechnol 3:286–290Google Scholar
  6. Börmann ER, Eikmanns BJ, Sahm H (1992) Molecular analysis of the Corynebacterium glutamicum gdh gene encoding glutamate dehydrogenase. Mol Microbiol 6:317–326Google Scholar
  7. Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–472Google Scholar
  8. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  9. Byun M O-K, Kaper JB, Ingram LO (1986) Construction of a vector for the expression of foreign genes in Zymomonas mobilis. J Industr Microbiol 1:9–15Google Scholar
  10. Carrell HL, Glusker JP, Burger V, Manfre F, Tritsch D, Biellmann J-F (1989) X-ray analysis of D-xylose isomerase at 1.9 Å: Native enzyme in complex with substrate and with a mechanism-designed ianctivator. Proc Natl Acad Sci USA 86:4440–4444Google Scholar
  11. Chen W-P (1980) Glucose isomerase (a review). Part 1. Process Biochem Aug-Sept: 30–35Google Scholar
  12. Chin DT, Goff SA, Webster T, Smith T, Goldberg AL (1988) Sequence of the Ion gene in Escherichia coli. A heat-shock gene which encodes the ATP-dependent protease LA. J Biol Chem 263:11718–11728Google Scholar
  13. Collyer CA, Blow DM (1990) Observations of reaction intermediates and the mechanism of aldose-ketose intervonversion by D-xylose isomerase. Proc Natl Acad Sci USA 87:1362–1366Google Scholar
  14. de Crombrugghe B, Busby S, Buc H (1984) Cyclic AMP Receptor Protein: Role in Transcription Activation. Science 224: 831–838Google Scholar
  15. David JD, Wiesmeyer H (1970) Control of xylose metabolism in Escherichia coli. Biochim Biophys Acta 201:497–499Google Scholar
  16. Ebright RH, Cossart P, Gicquel-Sanzey B, Beckwith J (1984) Mutations that alter the DNA sequence specificity of the catabolite gene activator protein of E. coli. Nature 311:232–235Google Scholar
  17. Gunasekera A, Ebright YW, Ebright RH (1990) DNA-sequence recognition by CAP: role of the adenine N6 atom of base pair 6 of the DNA site. Nucleic Acids Res 18:6853–6856Google Scholar
  18. Hanahan D (1985) Techniques for transformation of E. coli. In: Glover DM (ed) DNA Cloning, vol 1. IRL Press, Oxford Washington, DCGoogle Scholar
  19. Henikoff S (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351–359Google Scholar
  20. Henrick K, Collyer CA, Blow DM (1989) Structures of D-xylose isomerase from Arthrobacter strain B3728 containing the inhibitors xylitol and D-sorbitol at 2.5 Å and 2.3 Å resolution, respectively. J Mol Biol 208:129–157Google Scholar
  21. Hohn B, Collins J (1980) A small cosmid for efficient cloning of large DNA fragments. Gene 11:291–298Google Scholar
  22. Jeffries TW (1983) Utilization of xylose by bacteria, yeasts and fungi. Adv Biochem Eng Biotechnol 27:1–32Google Scholar
  23. Kumar S (1976) Properties of cya and crp mutants of Escherichia coli. J Bacteriol 125:545–555Google Scholar
  24. Lawlis VB, Dennis MS, Chen EY, Smith DH, Henner DJ (1984) Cloning and sequencing of the Xylose Isomerase and Xylulose Kinase Genes of Escherichia coli. Appl Environ Microbiol 47:15–21Google Scholar
  25. Lee C, Bagdasarian M, Meng M, Zeikus JG (1990) Catalytic Mechanism of Xylose (Glucose) Isomerase from Clostridium thermosulfurogenes. J Biol Chem 265:19082–19090Google Scholar
  26. Lengeler J (1980) Characterization of mutants of Escherichia coli K12, selected by resistance to streptozotozine. Mol Gen Genet 179:49–54Google Scholar
  27. Loviny-Anderton T, Shaw P-C, Shin M-K, Hartley BS (1991) D-xylose (D-glucose) isomerase from Arthrobacter strain N.R.R.L. B3728. Biochem J 277:263–271Google Scholar
  28. Maleszka R, Wang PY, Schneider H (1982) A ColEl hybrid plasmid containing Escherichia coli genes complementing D-xylose negative mutants of Escherichia coli and Salmonella typhimurium. Can J Biochem 60:144–151Google Scholar
  29. Murray NE, Brammar WJ, Murray K (1977) Lambdoid phages that simplify the recovery of in vitro recombinants. Mol Gen Genet 150:53–61Google Scholar
  30. Neuberger MS, Hartley BS, Walker JE (1981) Purification and properties of D-ribulokinase and D-xylulokinase from Klebsiella aerogenes. Biochem J 193:513–524Google Scholar
  31. Postma PW, Lengeler JW (1985) Phosphoenolpyruvate: carbohydrate phosphotransferase systems in bacteria. Microbiol Rev 49:232–269Google Scholar
  32. Rosenberg M, Court D (1979) Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Microbiol 13:319–353Google Scholar
  33. Rosenfeld SA, Stevis PE, Ho NWY (1984) Cloning and characterization of the xy1 genes from Escherichia coli. Mol Gen Genet 194:410–415Google Scholar
  34. Rygus T, Scheler A, Allmansberger R, Hillen W (1991) Molecular cloning, structure, promoters and regulatory elements for transcription of the Bacillus megaterium encoded regulon for xylose utilization. Arch Microbiol 155:535–542Google Scholar
  35. Saari GC, Kumar AA, Kawasaki GH, Insley MY, O'Hara PJ (1987) Sequence of the Ampulariella sp. strain 3876 gene encoding xylose isomerase. J Bacteriol 169:612–618Google Scholar
  36. Sambrook J, Maniatis T, Fritsch EF (1989) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  37. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  38. Scheler A, Rygus T, Allmansberger R, Hillen W (1991) Molecular cloning, structure, promoters and regulatory elements for transcription of the Bacillus licheniformis encoded regulon for xylose utilization. Arch Microbiol 155:526–534Google Scholar
  39. Schellenberg GD, Sarthy A, Larson AE, Backer MP, Crabb JW, Lidstrom M, Hall BD, Furlong CE (1984) Xylose Isomerase from Escherichia coli. Characterization of the Protein and the Structural Gene. J Biol Chem 259:6826–6832Google Scholar
  40. Shine J, Dalgarno L (1974) The 3′ terminal sequence of Escherichia coli 16 S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 71:1342–1346Google Scholar
  41. Sizemore C, Buchner E, Rygus T, Witke C, Götz F, Hillen W (1991) Organization, promoter analysis and transcriptional regulation of the Staphylococcus xylosus xylose utilization operon. Mol Gen Genet 227:377–384Google Scholar
  42. Sprenger GA, Lengeler JW (1984) L-sorbose metabolism in Klebsiella pneumoniae and Sor+ derivatives of Escherichia coli K-12 and chemotaxis toward sorbose. J Bacteriol 157:39–45Google Scholar
  43. Stevis PE, Ho NWY (1985) Overproduction of D-xylose isomerase in Escherichia coli by cloning the D-xylose isomerase gene. Enzyme Microbiol Technol 7:592–596Google Scholar
  44. Tabor S, Richardson CC (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci USA 84:4767–4771Google Scholar
  45. Tanaka S, Lerner SA, Lin ECC (1967) Replacement of a phosphoenol-pyruvate-dependent phosphotransferase by a nicotinamide adenine dinucleotide-linked dehydrogenase for the utilization of mannitol. J Bacteriol 93:642–648Google Scholar
  46. Tiraby G, Bejar S, Drocourt D, Reynes JP, Sicard PJ, Farber GK, Glasfeld A, Ringe D, Petsko GA (1990) Genetic, Enzymatic, and Crystallographic Studies of the Glucose Isomerases of Two Streptomyces Species. In: Hershberger CL et al (eds) Genetics of Industrial Microorganisms, American Society for Microbiology, Washington, DCGoogle Scholar
  47. Ubben D, Schmitt R (1987) A transposable promotor and transposable promotor probes derived from Tn1721. Gene 53:127–134Google Scholar
  48. Vangrysperre W, van Damme J, Vandekerckhove J, De Bruyne CK, Cornelis R, Kersters-Hilderson H (1990) Localization of the essential histidine and carboxylate group in D-xylose isomerases. Biochem J 265:699–705Google Scholar
  49. Vieira J, Messing J (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268Google Scholar
  50. Wilhelm M, Hollenberg CP (1985) Nucleotide sequence of the Bacillus subtilis xylose isomerase gene: extensive homology between the Bacillus and Escherichia coli enzyme. Nucleic Acids Res 13:5713–5722Google Scholar
  51. Wilson BL, Mortlock RP (1973) Regulation of D-xylose and D-arabitol catabolism by Aerobacter aerogenes. J Bacteriol 113:1404–1411Google Scholar
  52. Wong HC, Ting Y, Lin H-C, Reichert F, Myambo K, Watt KWK, Toy PL, Drummond RJ (1991) Genetic organization and regulation of the xylose degradation genes in Streptomyces rubiginosus. J Bacteriol 173:6849–6858Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • Sigrun D. Feldmann
    • 1
  • Hermann Sahm
    • 1
  • Georg A. Sprenger
    • 1
  1. 1.Institut für Biotechnologie 1 des Forschungszentrums Jülich GmbHJülichGermany

Personalised recommendations