Current Genetics

, Volume 28, Issue 5, pp 467–473 | Cite as

Cloning and expression of an Aspergillus kawachii endo-1,4-β-xylanase gene in Saccharomyces cerevisiae

  • Johan M. Crous
  • Isak S. Pretorius
  • Willem H. van Zyl
Original Paper


First-strand cDNA was prepared from mRNA isolated from Aspergillus kawachii IFO4308 and the β-xylanase gene (xynC) amplified by using the polymerase chain reaction (PCR) technique. This gene was inserted between the yeast phosphoglycerate kinase (PGK1) gene promoter (PGK1p) and terminator (PGK1T) sequences. The PGK1P-xynC-PGK1T construct (designated XYN3) was cloned into a multicopy episomal plasmid and the XYN3 gene was expressed in Saccharomyces cerevisiae. Functional β-xylanase (Xyn3) was produced and secreted by the recombinant yeast. Xyn3 was stable between 30 and 50°C, and the optimum temperature and pH were shown to be at 60°C and lower than pH3, respectively. An autoselective fur1::LEU2 XYN3 recombinant strain was developed that allowed β-xylanase production at a level of 300 nkat/ml in a non-selective complex medium.

Key words

Aspergillus kawachii β-xylanase Expression Saccharomyces cerevisiae 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Bailey MJ, Biely P, Poutanen K (1992) Interlaboratory testing of methods of assay of xylanase activity. J Biotechnol 23: 257–270Google Scholar
  2. Biely P (1985) Microbial xylanolytic systems. Trends Biotechnol 3: 286–290Google Scholar
  3. Biely P, Mislovicová D, Toman R (1988) Remazol brilliant blue-xylan: a soluble chromogenic substrate for xylanases. Methods Enzymol 160: 536–541Google Scholar
  4. Coughlan MP, Hazlewood GP (1993) β-1,4-D-Xylan-degrading enzyme systems: biochemistry, molecular biology and applications. Biotechnol Appl Biochem 17: 259–289Google Scholar
  5. Farkas V, Lisková M, Biely P (1985) Novel media for detection of microbial producers of cellulase and xylanase. FEMS Microbiol Lett 28: 137–140Google Scholar
  6. Filho EXF, Puls J, Coughlan MP (1993) Biochemical characteristics of two endo-β-1,4-xylanases produced by Penicillium capsulatum. J Industr Microbiol 11: 171–180Google Scholar
  7. Fuller RS, Sterne RE, Thorner J (1988) Enzymes required for yeast prohormone processing. Annu Rev Physiol 50: 345–362Google Scholar
  8. Graham H, Inborr J (1992) Applications of xylanase-based enzymes in commercial pig and poultry production. In: Visser J, Beldman G, Kusters-van Someren MA, Voragen AGJ (eds) Xylans and xylanases. Elsevier Science Publishers BV, Amsterdam, pp 535–538Google Scholar
  9. Hessing JGM, van Rotterdam CO, Verbakel JMA, Roza M, Maat J, van Gorcom RFM, van den Hondel CAMJJ (1994) Isolation and characterization of a 1,4-β-endoxylanase gene of A. awamori. Curr Genet 26: 228–232Google Scholar
  10. Hill J, Ian KA, Donald G, Griffiths DE (1991) DMSO-enhanced whole-cell yeast transformation. Nucleic Acids Res 19: 5791Google Scholar
  11. Hill JE, Myers AM, Koerner TJ, Tzagoloff A (1986) Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast 2: 163–167Google Scholar
  12. Hoffman CS, Winston F (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57: 267–272Google Scholar
  13. Innis MA, Gelfand DH (1990) Optimizing of PCRs. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols. Academic Press, San Diego, California, pp 3–12Google Scholar
  14. Innis MA, Holland MJ, McCabe PC, Cole GE, Wittman VP, Tal R, Watt KWK, Gelfand DH, Holland JP, Meade JH (1985) Expression, glycosylation, and secretion of an Aspergillus glucoamylase by Saccharomyces cerevisiae. Science 228: 21–26Google Scholar
  15. Ito K, Iwashita K, Iwano K (1992a) Cloning and sequencing of the xynC gene encoding acid xylanase of Aspergillus kawachii. Biosci Biotech Biochem 56: 1338–1340Google Scholar
  16. Ito K, Ogasawara H, Sugimoto T, Ishikawa T (1992b) Purification and properties of acid stable xylanases from Aspergillus kawachii. Biosci Biotech Biochem 56: 547–550Google Scholar
  17. Kern L, de Montigny J, Jund R, Lacroute F (1990) The FUR1 gene of Saccharomyces cerevisiae: cloning, structure and expression of wild-type and mutant alleles. Gene 88: 149–157Google Scholar
  18. Kormelink FJM, Searle-Van Leeuwen MJF, Wood TM, Voragen AGJ (1993) Purification and characterization of three endo-(1,4)-β-xylanases and one β-xylosidase from Aspergillus awamori. J Biotechnol 27: 249–265Google Scholar
  19. Kunkel TA, Roberts JD, Zakour RA (1987) Rapid and efficient sitespecific mutagenesis without phenotypic selection. Methods Enzymol 154: 365–382Google Scholar
  20. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157: 105–132Google Scholar
  21. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685Google Scholar
  22. La Grange DC (1995) MSc thesis. University of Stellenbosch, Stellenbosch, South AfricaGoogle Scholar
  23. Laing E, Pretorius IS (1992) Synthesis and secretion of an Erwinia chrysanthemi pectate lyase in Saccharomyces cerevisiae regulated by different combination of bacterial and yeast promoter and signal sequences. Gene 121: 35–45Google Scholar
  24. Loison G, Nguyen-Juilleret M, Alouani S, Marquet M (1986) Plasmid-transformed URA3 FUR1 double-mutants of S. cerevisiae: an autoselection system applicable to the production of foreign proteins. Bio/Technol 4: 433–437Google Scholar
  25. Maat J, Roza M, Verbakel J, Stam H, Santos da Silva MJ, Bosse M, Egmond MR, Hagemans MLD, van Gorcom RFM, Hessing JGM, van den Hondel CAMJJ, van Rotterdam C (1992) Xylanases and their application in bakery. In: Visser J, Beldman G, Kusters-van Someren MA, Voragen AGJ (eds) Xylans and xylanases. Elsevier Science Publishers BV, Amsterdam, pp 349–360Google Scholar
  26. Mikami S, Iwano K, Shiionki S, Shimada T (1987) Purification and some properties of acid-stable α-amylase from Shochu koji (Aspergíllus kawachii). Agric Biol Chem 51: 2495–2501Google Scholar
  27. Miller GL, Blum R, Glennon WE, Burton AL (1960) Measurement of caboxymethylcellulase activity. Anal Biochem 2: 127–132Google Scholar
  28. Morosoli R, Zalce E, Moreau A, Durand S (1992) Secretion of a xylanase from Cryptococcus albidus by Saccharomyces cerevisiae and Pichia stipitis. In: Visser J, Beldman G, Kusters-van Someren MA, Voragen AGJ (eds) Xylans and xylanases. Elsevier Science Publishers BV, Amsterdam, pp 247–258Google Scholar
  29. Nissen AM, Anker L, Munk N, Lange NK (1992) Xylanases for the pulp and paper industry. In: Visser J, Kusters-van Someren MA, Beldman G, Voragen AGJ (eds) Xylans and xylanases. Elsevier Science Publishers BV, Amsterdam, pp 325–337Google Scholar
  30. Okada H (1990) Expression of the xylanase gene of Bacillus pumilus in Escherichia coli, B. subtilis and Saccharomyces cerevisiae. In: Nga BH, Lee YK (eds) Microbiology applications in food biotechnology. Elsevier Science Publishers, New York, pp 1–12Google Scholar
  31. Olson MV, Dutchik JE, Graham MY, Brodeur GM, Helms C, Frank M, MacCollin M, Scheinman R, Frank T (1986) Random-clone strategy for genomic restriction mapping in yeast. Proc Natl Acad Sci USA 83: 7826–7830Google Scholar
  32. Poutanen K, Rättö M, Puls J, Viikari L (1987) Evaluation of different microbial xylanolytic systems. J Biotechnology 6: 49–60Google Scholar
  33. Puls J, Schuseil J (1993) Chemistry of hemicelluloses: relationship between hemicellulose structure and enzymes required for hydrolysis. In: Coughlan MP, Hazlewood GP (eds) Hemicellulose and hemicellulases. Portland Press, London, pp 1–27Google Scholar
  34. Romanos MA, Scorer CA, Clare JJ (1992) Foreign gene expression in yeast: a review. Yeast 8: 423–488Google Scholar
  35. Rothstein RJ (1983) One-step gene disruption in yeast. Methods Enzymol 101: 202–211Google Scholar
  36. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487–491Google Scholar
  37. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  38. Thomson JA (1993) Molecular biology of xylan degradation. FEMS Microbiol Rev 104: 65–82Google Scholar
  39. Törönnen A, Mach RL, Messner R, Gonzalez R, Kalkkinen N, Harkki A, Kubicek CP (1992) The two major xylanases from Trichoderma reesei: characterization of both enzymes and genes. Bio/Technol 10: 1461–1465Google Scholar
  40. 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
  41. Wong KKY, Saddler JN (1992) Trichoderma xylanases, their properties and application. CRC Crit Rev Biotechnol 12: 413–435Google Scholar
  42. Wong KKY, Saddler JN (1993) Applications of hemicellulases in the food, feed, and pulp and paper industries. In: Coughlan MP, Hazlewood GP (eds) Hemicellulose and hemicellulases. Portland Press, London, pp 127–143Google Scholar
  43. Wong KKY, Tan LUL, Saddler JN (1988) Multiplicity of β-1,4-xylanase in microorganisms: functions and applications. Microbiol Rev 52: 305–317Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • Johan M. Crous
    • 1
  • Isak S. Pretorius
    • 1
  • Willem H. van Zyl
    • 1
  1. 1.Department of MicrobiologyUniversity of StellenboschStellenboschSouth Africa

Personalised recommendations