Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1996 Jan;62(1):209–213. doi: 10.1128/aem.62.1.209-213.1996

Expression of Aureobasidium pullulans xynA in, and secretion of the xylanase from, Saccharomyces cerevisiae.

X L Li 1, L G Ljungdahl 1
PMCID: PMC167788  PMID: 8572698

Abstract

A previous report dealt with the cloning in Escherichia coli and sequencing of both the cDNA and genomic DNA encoding a highly active xylanase (XynA) of Aureobasidium pullulans (X.-L. Li and L. G. Ljungdahl, Appl. Environ. Microbiol. 60:3160-3166, 1994). Now we show that the gene was expressed in Saccharomyces cerevisiae under the GAL1 promoter in pYES2 and that its product was secreted into the culture medium. S. cerevisiae clone pCE4 with the whole open reading frame of xynA, including the part coding for the signal peptide, had xylanase activity levels of 6.7 U ml-1 in the cell-associated fraction and 26.2 U ml-1 in the culture medium 4 h after galactose induction. Two protein bands with sizes of 25 and 27 kDa and N-terminal amino acid sequences identical to that of APX-II accounted for 82% of the total proteins in the culture medium of pCE4. These proteins were recognized by anti-APX-II antibody. The results suggest that the XynA signal peptide supported the posttranslational processing of xynA product and the efficient secretion of the active xylanase from S. cerevisiae. Clones pCE3 and pGE3 with inserts of cDNA and genomic DNA, respectively, containing only the mature enzyme region attached by a Met codon had low levels of xylanase activity in the cell-associated fractions (1.6 U ml-1) but no activity in the culture media. No xylanase activity was detected in clone pGE4, which was the same as pCE4, except that pGE4 had a 59-bp intron in the signal peptide region. A comparison of the A. pullulans and S. cerevisiae signal peptides demonstrated that the XynA signal peptide was at least three times more efficient than those of S. cerevisiae invertase or mating alpha-factor pheromone in secreting the heterologous xylanase from S. cerevisiae cells.

Full Text

The Full Text of this article is available as a PDF (253.8 KB).

Selected References

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

  1. Achstetter T., Wolf D. H. Hormone processing and membrane-bound proteinases in yeast. EMBO J. 1985 Jan;4(1):173–177. doi: 10.1002/j.1460-2075.1985.tb02333.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Biely P., Mislovicová D., Toman R. Soluble chromogenic substrates for the assay of endo-1,4-beta-xylanases and endo-1,4-beta-glucanases. Anal Biochem. 1985 Jan;144(1):142–146. doi: 10.1016/0003-2697(85)90095-8. [DOI] [PubMed] [Google Scholar]
  3. Carlson M., Taussig R., Kustu S., Botstein D. The secreted form of invertase in Saccharomyces cerevisiae is synthesized from mRNA encoding a signal sequence. Mol Cell Biol. 1983 Mar;3(3):439–447. doi: 10.1128/mcb.3.3.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chaudhuri B., Steube K., Stephan C. The pro-region of the yeast prepro-alpha-factor is essential for membrane translocation of human insulin-like growth factor 1 in vivo. Eur J Biochem. 1992 Jun 15;206(3):793–800. doi: 10.1111/j.1432-1033.1992.tb16986.x. [DOI] [PubMed] [Google Scholar]
  5. Demolder J., Fiers W., Contreras R. Efficient synthesis of secreted murine interleukin-2 by Saccharomyces cerevisiae: influence of 3'-untranslated regions and codon usage. Gene. 1992 Feb 15;111(2):207–213. doi: 10.1016/0378-1119(92)90688-l. [DOI] [PubMed] [Google Scholar]
  6. Emr S. D., Schekman R., Flessel M. C., Thorner J. An MF alpha 1-SUC2 (alpha-factor-invertase) gene fusion for study of protein localization and gene expression in yeast. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7080–7084. doi: 10.1073/pnas.80.23.7080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gilkes N. R., Henrissat B., Kilburn D. G., Miller R. C., Jr, Warren R. A. Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbiol Rev. 1991 Jun;55(2):303–315. doi: 10.1128/mr.55.2.303-315.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hitzeman R. A., Hagie F. E., Levine H. L., Goeddel D. V., Ammerer G., Hall B. D. Expression of a human gene for interferon in yeast. Nature. 1981 Oct 29;293(5835):717–722. doi: 10.1038/293717a0. [DOI] [PubMed] [Google Scholar]
  9. Innis M. A., Holland M. J., McCabe P. C., Cole G. E., Wittman V. P., Tal R., Watt K. W., Gelfand D. H., Holland J. P., Meade J. H. Expression, Glycosylation, and Secretion of an Aspergillus Glucoamylase by Saccharomyces cerevisiae. Science. 1985 Apr 5;228(4695):21–26. doi: 10.1126/science.228.4695.21. [DOI] [PubMed] [Google Scholar]
  10. Kurjan J., Herskowitz I. Structure of a yeast pheromone gene (MF alpha): a putative alpha-factor precursor contains four tandem copies of mature alpha-factor. Cell. 1982 Oct;30(3):933–943. doi: 10.1016/0092-8674(82)90298-7. [DOI] [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. Leathers T. D. Color Variants of Aureobasidium pullulans Overproduce Xylanase with Extremely High Specific Activity. Appl Environ Microbiol. 1986 Nov;52(5):1026–1030. doi: 10.1128/aem.52.5.1026-1030.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Li X. L., Ljungdahl L. G. Cloning, sequencing, and regulation of a xylanase gene from the fungus Aureobasidium pullulans Y-2311-1. Appl Environ Microbiol. 1994 Sep;60(9):3160–3166. doi: 10.1128/aem.60.9.3160-3166.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Li X. L., Zhang Z. Q., Dean J. F., Eriksson K. E., Ljungdahl L. G. Purification and characterization of a new xylanase (APX-II) from the fungus Aureobasidium pullulans Y-2311-1. Appl Environ Microbiol. 1993 Oct;59(10):3212–3218. doi: 10.1128/aem.59.10.3212-3218.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Moreau A., Durand S., Morosoli R. Secretion of a Cryptococcus albidus xylanase in Saccharomyces cerevisiae. Gene. 1992 Jul 1;116(1):109–113. doi: 10.1016/0378-1119(92)90637-5. [DOI] [PubMed] [Google Scholar]
  16. Moreau A., Durand S., Morosoli R. Secretion of a Cryptococcus albidus xylanase in Saccharomyces cerevisiae. Gene. 1992 Jul 1;116(1):109–113. doi: 10.1016/0378-1119(92)90637-5. [DOI] [PubMed] [Google Scholar]
  17. Penttilä M. E., André L., Saloheimo M., Lehtovaara P., Knowles J. K. Expression of two Trichoderma reesei endoglucanases in the yeast Saccharomyces cerevisiae. Yeast. 1987 Sep;3(3):175–185. doi: 10.1002/yea.320030305. [DOI] [PubMed] [Google Scholar]
  18. Vainio A. E., Torkkeli H. T., Tuusa T., Aho S. A., Fagerström B. R., Korhola M. P. Cloning and expression of Hormoconis resinae glucoamylase P cDNA in Saccharomyces cerevisiae. Curr Genet. 1993 Jul-Aug;24(1-2):38–44. doi: 10.1007/BF00324663. [DOI] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES