Genomic and secretomic analyses of Blastobotrys yeasts reveal key xylanases for biomass decomposition

Abstract

Xylanolytic enzyme systems in ascomycetous yeasts remain underexplored, despite the presence of yeasts in various xylan-rich ecological niches. In this study, we investigated the secreted xylanolytic machineries of three Blastobotrys species-B. mokoenaii, B. illinoisensis, and B. malaysiensis-by integrating genome annotation, bioinformatics, and secretome analyses of cultures grown on beechwood glucuronoxylan. Our findings demonstrate that these yeasts effectively hydrolyze xylan through the secretion of xylanases from the glycoside hydrolase (GH) family 11, which play a central role in cleaving the xylan backbone. Additionally, the yeasts produce a diverse array of other CAZymes, including members of GH families 3, 5, and 67, with putative roles in xylan degradation. We also report on the heterologous expression and functional characterization of the GH30_7 xylanase BmXyn30A from B. mokoenaii, which exhibits both glucuronoxylanase and xylobiohydrolase activities. We demonstrate additive effects between GH family 30 BmXyn30A and GH family 11 BmXyn11A during the hydrolysis of beechwood glucuronoxylan, where the enzymes exhibit complementary roles that enhance the deconstruction of this complex hemicellulose substrate. These findings broaden our understanding of the xylanolytic systems in yeasts and underscore the potential of Blastobotrys species as cell factories and natural xylanase producers. The enzymes they produce hold promise for biorefining applications, enabling efficient utilization of renewable xylan-rich plant biomass resources. KEY POINTS: • Extracellular GH11 xylanases dominate glucuronoxylan degradation in Blastobotrys yeasts. • Yeast GH30_7 enzyme shows multifaceted activity, supporting complex xylan breakdown. • Blastobotrys yeasts show promise as cell factories for industrial biotechnology applications.

Keywords: GH11; GH30; Glycoside hydrolase; Wood; Xylan; Xylanolytic yeast.

Fig. 1

Growth on agar plates and in liquid cultures on different carbon sources. Blastobotrys growth on different monosaccharides or polysaccharides in agar plates (A) with 4 g L⁻¹ carbon source after 5 weeks of growth at room temperature from a 20-µL culture drop with a starting OD = 5. Growth on plates was evaluated visually and categorized from − (no growth) to +  +  + (very good growth), depending on the diameter and density of the yeast colonies and hyphae morphologies. MLG = mixed-linkage glucan (barley), CMC = carboxymethyl cellulose. Growth of B. mokoenaii (B), B. illinoisensis (C), or B. malaysiensis (D) on different monosaccharides (20 g L⁻¹). Growth of Blastobotrys yeast on beechwood glucuronoxylan 10 g L⁻¹ (E), wheat arabinoxylan 10 g L⁻¹ (F), konjac glucomannan 1 g L⁻¹ (G), CMC 20 g L⁻¹ (H), MLG 10 g L⁻¹ (I), and tamarind xyloglucan 5 g L⁻¹ (J). Variations in carbon source concentrations are due to high insolubility and viscosity at 10–20 g L⁻¹ of some carbon sources. OD equivalent = optical density normalized from S. cerevisiae growth in Delft + 20 g L⁻¹ glucose medium in a Growth-Profiler 960. All growth curves are means of biological triplicates

Fig. 2

Xylanolytic activity of Blastobotrys yeasts. Halo formation in Delft medium + 4 g L⁻¹ xylan in agar plates by Blastobotrys yeasts after 48 h of growth at room temperature (A). Growth of Blastobotrys yeasts in 30-mL Delft medium + 20 g L⁻¹ beechwood GX in biological triplicates with error bars representing standard deviations (B). Beechwood GX hydrolysis activity, quantified by DNS assays, in Blastobotrys secretomes from yeasts grown in Delft + 20 g L⁻¹ beechwood GX cultures in biological triplicates (C). β-Xylosidase activity, quantified using pNP-xylose, in Blastobotrys secretomes from yeasts grown in Delft medium + 20 g L⁻¹ beechwood GX, in biological triplicates (D). BGX = beechwood glucuronoxylan, DNS = dinitro salicylate, GX = glucuronoxylan, WAX = wheat arabinoxylan

Fig. 3

Secreted CAZymes during yeast growth on glucuronoxylan. The heatmap shows the detected abundances of annotated CAZyme proteins from three biological replicates of the secretomes from B. mokoenaii, B. illinoisensis, and B. malaysiensis during growth on GX. The colors in the heatmap indicate protein abundance, ranging from high (dark blue, log10 = 8–10 of label-free quantification) to low abundance (white, log10 = 4–5 of label-free quantification). Replicates with missing values are marked in gray (A). Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of proteins from 10 × concentrated secretomes from each biological replicate. MW = molecular protein weight ladder, C = control (recombinant BmXyn11A), lanes 1–3 = B. mokoenaii-concentrated secretomes, lanes 4–6 = B. illinoisensis-concentrated xylan secretomes, lanes 7–9 = B. malaysiensis-concentrated GX secretomes. Black arrows indicate the molecular weight predicted for GH11 proteins, and the red arrow indicates GH30_7 protein (B). The predicted molecular weights (without signal peptide sequence) of the GH11 xylanases are 21 kDa, 20 kDa, and 20 kDa for B. mokoenaii, B. illinoisensis, and B. malaysiensis, respectively, and 50 kDa GH30_7 for B. mokoenaii. CAZyme = Carbohydrate-Active EnZyme, GX = glucuronoxylan

Fig. 4

BmXyn30A xylan degradation assay and xylooligosaccharide analysis. Chromatogram comparing xylooligosaccharide profiles from beechwood GX hydrolysis by BmXyn30A showing more intense peaks at later retention time (indicated by black arrow), compared to BmXyn11A and the two enzymes combined (A). Xylobiohydrolase activity of BmXyn30A using 200-µL xylotetraose analyzed by HPAEC-PAD (B). Additive effects to BmXyn11A BGX hydrolysis with BmXyn30A after 16 h incubation in 10 g L⁻¹ beechwood GX in 100 mM sodium acetate buffer pH 5 at 40 °C, 600 rpm, using 0.1 µM enzyme concentrations and DNS reducing sugar assays. Values are means of triplicates with standard deviations as error bars. Asterisks indicate statistical significance hydrolysis levels with p-values of 0.05 (*) considered significant (n = 3) and evaluated using one-way analysis of variance (ANOVA) with Tukey’s test (C). BGX = beechwood glucuronoxylan. GX = glucuronoxylan. HPAEC-PAD = high-performance anion-exchange chromatography coupled with pulsed amperometric detection