Degradation of xylan to D-xylose by recombinant Saccharomyces cerevisiae coexpressing the Aspergillus niger beta-xylosidase (xlnD) and the Trichoderma reesei xylanase II (xyn2) genes

Abstract

The beta-xylosidase-encoding xlnD gene of Aspergillus niger 90196 was amplified by the PCR technique from first-strand cDNA synthesized on mRNA isolated from the fungus. The nucleotide sequence of the cDNA fragment was verified to contain a 2,412-bp open reading frame that encodes a 804-amino-acid propeptide. The 778-amino-acid mature protein, with a putative molecular mass of 85.1 kDa, was fused in frame with the Saccharomyces cerevisiae mating factor alpha1 signal peptide (MFalpha1(s)) to ensure correct posttranslational processing in yeast. The fusion protein was designated Xlo2. The recombinant beta-xylosidase showed optimum activity at 60 degrees C and pH 3.2 and optimum stability at 50 degrees C. The K(i(app)) value for D-xylose and xylobiose for the recombinant beta-xylosidase was determined to be 8.33 and 6.41 mM, respectively. The XLO2 fusion gene and the XYN2 beta-xylanase gene from Trichoderma reesei, located on URA3-based multicopy shuttle vectors, were successfully expressed and coexpressed in the yeast Saccharomyces cerevisiae under the control of the alcohol dehydrogenase II gene (ADH2) promoter and terminator. These recombinant S. cerevisiae strains produced 1,577 nkat/ml of beta-xylanase activity when expressing only the beta-xylanase and 860 nkat/ml when coexpressing the beta-xylanase with the beta-xylosidase. The maximum beta-xylosidase activity was 5.3 nkat/ml when expressed on its own and 3.5 nkat/ml when coexpressed with the beta-xylanase. Coproduction of the beta-xylanase and beta-xylosidase enabled S. cerevisiae to degrade birchwood xylan to D-xylose.

FIG. 1

Schematic representation of the expression cassettes used, indicating the xylanase (XYN2) and β-xylosidase (XLND) genes as well as the ADH2 promoter (ADH2_P) and terminator (ADH2_T) and the mating factor α secretion signal (MFα1_S). XYN2 is indicated by cross-hatched boxes, xlnD is indicated by hatched boxes, the ADH2 promoter and terminator sequences are indicated by open boxes, and the MFα1_S secretion signal is indicated by solid boxes.

FIG. 2

(A and B) Effect of pH at 50°C (A) and temperature at pH 5.0 (B) on the activity of Xlo2. The buffers used in the enzyme reactions were 50 mM citrate buffer (pH 3 to 6.2) (○) and 50 mM phosphate buffer (pH 6.2 to 9) (□). (C) The temperature stability of Xlo2 at 50°C (●), 55°C (▾), 60°C (■), and 65°C (⧫), was determined by preincubating the enzyme at these temperatures in the absence of the substrate for 0, 2, 5, 10, 20, 40, 90, and 120 min before determining the β-xylosidase activity on PNPX. The β-xylosidase activity prior to the preincubations (time 0 min) was taken as 100%.

FIG. 3

(A) Thin-layer chromatogram of the hydrolysis of xylobiose and xylotriose by S. cerevisiae (XLO2). Reaction mixtures were incubated at 50°C, and samples were taken after 0 and 30 min and 1, 2, 3, 4, 5, and 7 h. d-Xylose, xylobiose, and xylotriose were used as standards (S). (B) A plot of (v_o/v_i) − 1 versus the xylobiose concentration to investigate competitive inhibition of β-xylosidase activity on PNPX in the presence of xylobiose (■), d-xylose (●), cellobiose (▾), or d-glucose (▴).

FIG. 4

Time course of β-xylanase (A), β-xylosidase (B), and cell mass (C) produced by S. cerevisiae Y294 (VECT) (▴, ▵), Y294 (XYN2) (●, ○), Y294 (XLO2) (▾, ▿), and Y294 (XYN2 XLO2) (■, □) in shake flask cultures. The β-xylanase activities were assayed by the method of Bailey et al. (2) using the culture supernatant as the source of enzyme, and the β-xylosidase activities were assayed as described by La Grange et al. (25), using cell cultures as the source of enzyme. Enzyme activities were expressed in katals per milliliter and are indicated by solid symbols. The enzyme activities represent the average of three independent cultures. The maximum deviation for the β-xylanase and β-xylosidase activities did not exceed 11 and 12%, respectively. Yeast cell counts were determined with a haemocytometer and are indicated by open symbols.

FIG. 5

Xylan degradation by Y294 (VECT) (lanes 1), Y294 (XYN2) (lanes 2), and Y294 (XYN2 XLO2) (lanes 3). Samples were taken after 48, 72, and 136 h of growth at 30°C. d-Xylose, xylobiose, and xylotriose were used as standards (S). The right-hand lane (labeled X) contains the standard (d-xylose, xylobiose, and xylotriose), as well as the 136-h sample of Y294 (VECT), to monitor the effects of the medium components on the migration of d-xylose, xylobiose, and xylotriose.