Abstract The endo-β-xylanase-encoding gene (xynA) of Bacillus pumilus PLS was isolated from a genomic DNA library and the open reading frame (ORF) was inserted in expression vectors for the yeast Saccharomyces cere- visiae. Plasmid pFN3 harboured the xynA ORF fused to the yeast mating pheromone alpha-factor signal sequence (MFα1 S ) under the control of the alcohol dehydrogenase II gene promotor (ADH2 P ) and terminator (ADH2 T ) se- quences. In plasmid pFN4, the MFα1 S -xynA ORF was brought under the control of the phosphoglycerate kinase I gene promotor (PGK1 P ) and terminator (PGK1 T ) se- quences. Autoselective, recombinant S. cerevisiae [fur1::LEU2] strains bearing pFN3 or pFN4 secreted functional endo-β-xylanase when grown in complex me- dium. Enzymatic activities in the culture supernatants reached maximum levels of 8.5 nkat/ml and 4.5 nkat/ml, respectively. The temperature and pH optimum for both the bacterial and the recombinant xylanase were 58 °C and pH 6.2. Introduction Xylans are heterogeneous polysaccharides found in the cell walls of all land plants and in almost all plant parts (Coughlan and Hazlewood 1993). Arabinoxylan is a ma- jor xylan in grains, representing up to 4% of the dry weight of wheat endosperm. It consists of a β-1,4-linked D-xylopyranoside backbone substituted with acetyl and/or arabinofuranosyl groups (Henry 1988). Endo- β-1,4-xylanases (E.C. 3.2.1.8) hydrolyse the internal xylosidic linkages of the xylan backbone and thus have a major function in xylan degradation. In the bakery, the application of xylanases has a positive effect on the specific bread volume obtained after dough rising and baking (Maat et al. 1992; Linko et al. 1997). However, recent research supports the hypothesis that only xylanases with a preferential activity towards water-unex- tractable arabinoxylan contribute to an increased bread vol- ume. This property seems to be mainly associated with Ba- cillus xylanases and not with fungal xylanases (Popper 1997; Courtin et al. 1999). The baker's yeast Saccharomy- ces cerevisiae does not produce xylanases; and it has been proposed that the use of suitable xylanase-producing, re- combinant S. cerevisiae strains could contribute to an im- proved production process. Endoxylanases from several micro-organisms have been expressed in S. cerevisiae, mainly with the perspective of enzymatic bleaching of pa- per pulps or biomass regeneration (Donald et al. 1994; Crous et al. 1995; La Grange et al. 1996, 2000; Li and Ljungdahl 1996; Pérèz-Gonzalez et al. 1996; Luttig et al. 1997). Monfort et al. (1996) reported improved properties of an industrial baker's yeast by coexpression of the Asper- gillus nidulans endoxylanase and the A. oryzae α-amylase. The endo-β-xylanase encoding gene xynA from B. pu- milus IPO was isolated and sequenced by Fukusaki et al. (1984). In this paper, we present the expression of the ho- mologous xynA gene of B. pumilus PLS in S. cerevisiae. Materials and methods Strains, plasmids and media Escherichia coli XL1-Blue MRF′ endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F′ proAB lacl q Z∆M15 Tn10 (tet)] (Stratagene, The Netherlands) was used as the host for the different plasmid constructs. B. pumilus PLS (wild-type strain) and the construction of a genomic library in E. coli has been described by La Grange et al. (1997). Plasmids pBluescript SK II (+) (Stratagene), pRLR1 and pJC1 (Crous et al. 1995) were used for cloning, characterisa- tion and expression of the xynA gene. S. cerevisiae Y294 MATα, leu2-3,12, ura3-52, his3, trpl-289 (La Grange et al. 1996) was used for expression of xynA in yeast. E. coli XL1-Blue was cultivated at 37 °C in Luria-Bertani me- dium supplemented with 100 μg ampicillin/ml, for plasmid selec- tion (Sambrook et al. 1989). S. cerevisiae Y294 recombinants were F. Nuyens ( ✉ ) · W.H. van Zyl · D. Iserentant · H. Verachter C. Michiels Laboratory for Food Microbiology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, 3001 Leuven (Heverlee), Belgium e-mail: filip.nuyens@agr.kuleuven.ac.be Fax: +32-16-321960 Appl Microbiol Biotechnol (2001) 56:431–434 DOI 10.1007/s002530100670 ORIGINAL PAPER Filip Nuyens · Willem H. van Zyl · Dirk Iserentant Hubert Verachtert · Chris Michiels Heterologous expression of the Bacillus pumilus endo-β-xylanase (xynA) gene in the yeast Saccharomyces cerevisiae Received: 12 February 2001 / Accepted: 18 March 2001 / Published online: 2 June 2001 © Springer-Verlag 2001