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. 1996 Oct;62(10):3840–3846. doi: 10.1128/aem.62.10.3840-3846.1996

Cloning of genes encoding alpha-L-arabinofuranosidase and beta-xylosidase from Trichoderma reesei by expression in Saccharomyces cerevisiae.

E Margolles-Clark 1, M Tenkanen 1, T Nakari-Setälä 1, M Penttilä 1
PMCID: PMC168192  PMID: 8837440

Abstract

A cDNA expression library of Trichoderma reesei RutC-30 was constructed in the yeast Saccharomyces cerevisiae. Two genes, abf1 and bxl1, were isolated by screening the yeast library for extracellular alpha-L-arabinofuranosidase activity with the substrate p-nitrophenyl-alpha-L-arabinofuranoside. The genes abf1 and bxl1 encode 500 and 758 amino acids, respectively, including the signal sequences. The deduced amino acid sequence of ABFI displays high-level similarity to the alpha-L-arabinofuranosidase B of Aspergillus niger, and the two can form a new family of glycosyl hydrolases. The deduced amino acid sequence of BXLI shows similarities to the beta-glucosidases grouped in family 3. The yeast-produced enzymes were tested for enzymatic activities against different substrates. ABFI released L-arabinose from p-nitrophenyl-alpha-L-arabinofuranoside and arabinoxylans and showed some beta-xylosidase activity toward p-nitrophenyl-beta-D-xylopyranoside. BXLI did not release L-arabinose from arabinoxylan. It showed alpha-L-arabinofuranosidase, alpha-L-arabinopyranosidase, and beta-xylosidase activities against p-nitrophenyl-alpha-L-arabinofuranosidase, p-nitrophenyl-alpha-L-arabinopyranoside, and p-nitrophenyl-beta-D- xylopyranoside, respectively, with the last activity being the highest. It was also able to hydrolyze xylobiose and slowly release xylose from polymeric xylan. ABFI and BXLI correspond to a previously purified alpha-L-arabinofuranosidase and a beta-xylosidase from T. reesei, respectively, as confirmed by partial amino acid sequencing of the Trichoderma-produced enzymes. Both enzymes produced in yeasts displayed hydrolytic properties similar to those of the corresponding enzymes purified from T. reesei.

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Selected References

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  1. Barnett C. C., Berka R. M., Fowler T. Cloning and amplification of the gene encoding an extracellular beta-glucosidase from Trichoderma reesei: evidence for improved rates of saccharification of cellulosic substrates. Biotechnology (N Y) 1991 Jun;9(6):562–567. doi: 10.1038/nbt0691-562. [DOI] [PubMed] [Google Scholar]
  2. Bause E., Legler G. Isolation and structure of a tryptic glycopeptide from the active site of beta-glucosidase A3 from Aspergillus wentii. Biochim Biophys Acta. 1980 Dec 16;626(2):459–465. doi: 10.1016/0005-2795(80)90142-7. [DOI] [PubMed] [Google Scholar]
  3. Becker D. M., Guarente L. High-efficiency transformation of yeast by electroporation. Methods Enzymol. 1991;194:182–187. doi: 10.1016/0076-6879(91)94015-5. [DOI] [PubMed] [Google Scholar]
  4. Castle L. A., Smith K. D., Morris R. O. Cloning and sequencing of an Agrobacterium tumefaciens beta-glucosidase gene involved in modifying a vir-inducing plant signal molecule. J Bacteriol. 1992 Mar;174(5):1478–1486. doi: 10.1128/jb.174.5.1478-1486.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dalbøge H., Heldt-Hansen H. P. A novel method for efficient expression cloning of fungal enzyme genes. Mol Gen Genet. 1994 May 10;243(3):253–260. doi: 10.1007/BF00301060. [DOI] [PubMed] [Google Scholar]
  6. Dear S., Staden R. A sequence assembly and editing program for efficient management of large projects. Nucleic Acids Res. 1991 Jul 25;19(14):3907–3911. doi: 10.1093/nar/19.14.3907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Flipphi M. J., Panneman H., van der Veen P., Visser J., de Graaff L. H. Molecular cloning, expression and structure of the endo-1,5-alpha-L-arabinase gene of Aspergillus niger. Appl Microbiol Biotechnol. 1993 Nov;40(2-3):318–326. doi: 10.1007/BF00170387. [DOI] [PubMed] [Google Scholar]
  8. Flipphi M. J., Visser J., van der Veen P., de Graaff L. H. Arabinase gene expression in Aspergillus niger: indications for coordinated regulation. Microbiology. 1994 Oct;140(Pt 10):2673–2682. doi: 10.1099/00221287-140-10-2673. [DOI] [PubMed] [Google Scholar]
  9. Flipphi M. J., van Heuvel M., van der Veen P., Visser J., de Graaff L. H. Cloning and characterization of the abfB gene coding for the major alpha-L-arabinofuranosidase (ABF B) of Aspergillus niger. Curr Genet. 1993 Dec;24(6):525–532. doi: 10.1007/BF00351717. [DOI] [PubMed] [Google Scholar]
  10. Gasparic A., Marinsek-Logar R., Martin J., Wallace R. J., Nekrep F. V., Flint H. J. Isolation of genes encoding beta-D-xylanase, beta-D-xylosidase and alpha-L-arabinofuranosidase activities from the rumen bacterium Prevotella ruminicola B1(4). FEMS Microbiol Lett. 1995 Jan 15;125(2-3):135–141. doi: 10.1111/j.1574-6968.1995.tb07349.x. [DOI] [PubMed] [Google Scholar]
  11. Greve L. C., Labavitch J. M., Hungate R. E. alpha-L-arabinofuranosidase from Ruminococcus albus 8: purification and possible role in hydrolysis of alfalfa cell wall. Appl Environ Microbiol. 1984 May;47(5):1135–1140. doi: 10.1128/aem.47.5.1135-1140.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gräbnitz F., Rücknagel K. P., Seiss M., Staudenbauer W. L. Nucleotide sequence of the Clostridium thermocellum bgIB gene encoding thermostable beta-glucosidase B: homology to fungal beta-glucosidases. Mol Gen Genet. 1989 May;217(1):70–76. doi: 10.1007/BF00330944. [DOI] [PubMed] [Google Scholar]
  13. Henrissat B., Bairoch A. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J. 1993 Aug 1;293(Pt 3):781–788. doi: 10.1042/bj2930781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kellett L. E., Poole D. M., Ferreira L. M., Durrant A. J., Hazlewood G. P., Gilbert H. J. Xylanase B and an arabinofuranosidase from Pseudomonas fluorescens subsp. cellulosa contain identical cellulose-binding domains and are encoded by adjacent genes. Biochem J. 1990 Dec 1;272(2):369–376. doi: 10.1042/bj2720369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kohchi C., Toh-e A. Nucleotide sequence of Candida pelliculosa beta-glucosidase gene. Nucleic Acids Res. 1985 Sep 11;13(17):6273–6282. doi: 10.1093/nar/13.17.6273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Machida M., Ohtsuki I., Fukui S., Yamashita I. Nucleotide sequences of Saccharomycopsis fibuligera genes for extracellular beta-glucosidases as expressed in Saccharomyces cerevisiae. Appl Environ Microbiol. 1988 Dec;54(12):3147–3155. doi: 10.1128/aem.54.12.3147-3155.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Manin C., Shareek F., Morosoli R., Kluepfel D. Purification and characterization of an alpha-L-arabinofuranosidase from Streptomyces lividans 66 and DNA sequence of the gene (abfA). Biochem J. 1994 Sep 1;302(Pt 2):443–449. doi: 10.1042/bj3020443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Penttilä M. E., André L., Lehtovaara P., Bailey M., Teeri T. T., Knowles J. K. Efficient secretion of two fungal cellobiohydrolases by Saccharomyces cerevisiae. Gene. 1988;63(1):103–112. doi: 10.1016/0378-1119(88)90549-5. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Reinikainen T., Ruohonen L., Nevanen T., Laaksonen L., Kraulis P., Jones T. A., Knowles J. K., Teeri T. T. Investigation of the function of mutated cellulose-binding domains of Trichoderma reesei cellobiohydrolase I. Proteins. 1992 Dec;14(4):475–482. doi: 10.1002/prot.340140408. [DOI] [PubMed] [Google Scholar]
  21. Robinson A. B., Scotchler J. W., McKerrow J. H. Rates of nonenzymatic deamidation of glutaminyl and asparaginyl residues in pentapeptides. J Am Chem Soc. 1973 Nov 28;95(24):8156–8159. doi: 10.1021/ja00805a032. [DOI] [PubMed] [Google Scholar]
  22. Saloheimo A., Henrissat B., Hoffrén A. M., Teleman O., Penttilä M. A novel, small endoglucanase gene, egl5, from Trichoderma reesei isolated by expression in yeast. Mol Microbiol. 1994 Jul;13(2):219–228. doi: 10.1111/j.1365-2958.1994.tb00417.x. [DOI] [PubMed] [Google Scholar]
  23. Schwarz W. H., Adelsberger H., Jauris S., Hertel C., Funk B., Staudenbauer W. L. Xylan degradation by the thermophile Clostridium stercorarium: cloning and expression of xylanase, beta-D-xylosidase, and alpha-L-arabinofuranosidase genes in Escherichia coli. Biochem Biophys Res Commun. 1990 Jul 16;170(1):368–374. doi: 10.1016/0006-291x(90)91283-x. [DOI] [PubMed] [Google Scholar]
  24. Sherman F. Getting started with yeast. Methods Enzymol. 1991;194:3–21. doi: 10.1016/0076-6879(91)94004-v. [DOI] [PubMed] [Google Scholar]
  25. Strathmann M., Hamilton B. A., Mayeda C. A., Simon M. I., Meyerowitz E. M., Palazzolo M. J. Transposon-facilitated DNA sequencing. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1247–1250. doi: 10.1073/pnas.88.4.1247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stålbrand H., Saloheimo A., Vehmaanperä J., Henrissat B., Penttilä M. Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei beta-mannanase gene containing a cellulose binding domain. Appl Environ Microbiol. 1995 Mar;61(3):1090–1097. doi: 10.1128/aem.61.3.1090-1097.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Utt E. A., Eddy C. K., Keshav K. F., Ingram L. O. Sequencing and expression of the Butyrivibrio fibrisolvens xylB gene encoding a novel bifunctional protein with beta-D-xylosidase and alpha-L-arabinofuranosidase activities. Appl Environ Microbiol. 1991 Apr;57(4):1227–1234. doi: 10.1128/aem.57.4.1227-1234.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]

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