Strains, media, and growth conditions
Plasmids used in this study were propagated in Escherichia coli DH5α, which was grown in Luria-Bertani (LB) medium (Bertani 1951) supplemented with 50 μg mL−1 ampicillin (Sigma-Aldrich, St. Louis, MO, USA). The fungal strains used in this study were derived from A. niger CBS 138852 (cspA1, pyrA−, kusA::amdS) (Meyer et al. 2007), which was obtained from the Westerdijk Fungal Biodiversity Institute culture collection (Utrecht, the Netherlands). All strains generated in this study were deposited at the culture collection of Westerdijk Fungal Biodiversity Institute under accession numbers indicated in Supplemental Table S1. All fungal strains were maintained by growing at 30°C on Aspergillus minimal medium (MM) or complete medium (CM) (de Vries et al. 2004) supplemented with 1% D-glucose and 1.22 g L−1 uridine (Sigma-Aldrich, St. Louis, MO, USA).
Growth profiles were performed using Aspergillus MM with the addition of 25 mM D-glucose, D-galacturonic acid, or D-xylose (Sigma-Aldrich, St. Louis, MO, USA) or 1% beechwood xylan, cellulose, xyloglucan, or apple/citrus pectin. All media were supplemented with 1.22 g L−1 uridine. For antibiotic resistance tests, the media were supplemented with 10–25 μg mL−1 hygromycin B (InvivoGen, San Diego, CA, USA). All growth profile plates were inoculated with 1000 freshly harvested spores and performed in duplicates and were incubated at 30°C for up to 14 days. Growth was evaluated by visual inspection, and pictures were taken at multiple time points.
For liquid cultures, freshly harvested conidia were pre-grown in 250 mL CM containing 2% D-fructose (Sigma-Aldrich, St. Louis, MO, USA) and 1.22 g L−1 uridine for 16 h at 30°C in a rotary shaker at 250 rpm. After 16 h incubation, mycelia were harvested by filtration through sterile cheesecloth, rinsed with MM, and approximately 2.5 g (wet weight) mycelium was transferred in triplicates into 50 mL MM containing 2% wheat bran, 1% D-xylose, 1% citrus pectin (CP), or 1% citrus pectin supplemented with 0.075% (5 mM) D-xylose (CPX). Supernatant samples were taken after 24 h incubation at 30°C in a rotary shaker at 250 rpm. The samples were centrifuged (10 min, 3220 × g, 4°C), and cell-free supernatant samples were stored at −20°C until further processing.
Construction of mutant strains
CRISPR/Cas9 genome editing was performed using the ANEp8-Cas9-pyrG plasmid, which contains the pyrG gene as selection marker (Song et al. 2018). The guide RNA (gRNA) sequences were selected by using the Geneious 11.1.4 software (https://www.geneious.com) based on the methodology described by Doench et al. (2014). Repair templates, which include ~ 750–1000 bp of the 5′ and 3′ flanking regions of the target sequences, were amplified and fused together using fusion-PCR and were used to repair the target sequence cleaved by the Cas9 nuclease.
The construction of CRISPR/Cas9 plasmids, generation of A. niger protoplasts, transformation, and colony purification of putative mutant strains was performed as previously described (Kun et al. 2020). The PpgaX-hph reporter strain CBS 147359 was generated by replacing the exopolygalacturonase X (pgaX) ORF (open reading frame) with the hygromycin-B-phosphotransferase (hph) ORF originated from E. coli (Kaster et al. 1983) in the A. niger CBS 138852 background strain. The mutants carrying D-xylose-inducible chimeric GaaR-XlnR constructs were generated by replacing the N-terminal region of XlnR with that of the GaaR in the A. niger CBS 138852, CBS 147359 (CBS 138852 PpgaX-hph), and CBS 146901 (CBS 138852 ΔgaaR) background strains. The constitutively active form of chimeric GaaR-XlnR V756F (corresponding to amino acid mutation V732F in the chimeric sequence) has been generated by simultaneous replacement of XlnR DNA-binding domain and insertion of a point mutation via a single-stranded oligonucleotide in the C-terminal region of XlnR as described before (Kun et al. 2020).
The generated mutant strains have been confirmed by diagnostic PCR, through the amplification of the target gene region and/or Sanger sequencing (Macrogen Europe, Amsterdam, the Netherlands) (data not shown). For each individual mutation, one candidate was selected for subsequent phenotypic analysis. All primers used in this study were ordered from Integrated DNA Technologies, Inc. (IDT, Leuven, Belgium) and are presented in Supplemental Table S2.
In silico analyses
The prediction of coiled-coil motifs (Supplemental Fig. S1a, S1b) was performed using the DeepCoil online tool (Ludwiczak et al. 2019) (https://toolkit.tuebingen.mpg.de/tools/deepcoil).
The estimated protein mass was calculated as follows. Signal peptides for secretion were predicted using SignalP 5.0 software tool (Armenteros et al. 2019) (http://www.cbs.dtu.dk/services/SignalP/). Estimation of mature amino acid sequence was subsequently calculated using the ProtParam tool from the ExPASy web server (https://web.expasy.org/protparam/).
SDS-PAGE and enzyme activity assays
Liquid culture filtrates of the control and mutant strains grown in media containing 1% citrus pectin, 1% D-xylose, or the combination of 1% citrus pectin and 0.075% (5 mM) D-xylose for 24 h were used to evaluate the produced extracellular CAZymes.
Twelve microliters of the culture filtrates were added to 4 μL loading buffer (10% of 1 M Tris–HCl, pH 6.8; 42% glycerol, 4% (w/v) SDS; 0.02% (w/v) bromophenol blue; 4% of 14.7 M mercaptoethanol), incubated at 85°C for 15 min, ice-cooled for 2 min and centrifuged at ~ 10,000 × g for 2 min. Finally, 15 μL of sample were loaded onto 12% (w/v) acrylamide SDS-PAGE gels calibrated with PageRuler prestained protein marker (Thermo Fisher Scientific, Waltham, MA, USA). Visualization was performed by silver staining (Chevallet et al. 2006), while documentation was done by using a HP Scanjet G2410 scanner. All samples were evaluated in biological duplicates.
Enzyme activities were performed by using the colorimetric para-nitrophenol (pNP) or azo-dye substrate assays in 96-well flat bottom microtiter plates. For pNP assays, 10 μL of supernatant samples were mixed with 10 μL of 0.1% 4-nitrophenyl β-D-glucopyranoside for β-glucosidase (BGL) activity or 4-nitrophenyl β-D-xylopyranoside for β-xylosidase (BXL) activity substrates, 50 μL of 50 mM NaAc (pH 5), and 30 μL of demineralized water in a final volume of 100 μL. Both pNP assays were measured after 1 h incubation at 30°C. The reactions were stopped by adding 100 μL of 0.25 M Na2CO3, and the absorption values were measured at 405 nm wavelength using a FLUOstar OPTIMA microplate reader (BMG Labtech, Ortenberg, Germany). For azo-dye substrate assays, 20 μL of supernatant samples were mixed with 30 μL of 100 mM NaAc (pH 4.6) and 50 μL of Azo-CM-Cellulose (Megazyme, Bray, Ireland) or Azo-Xylan (birchwood) (Megazyme, Bray, Ireland) substrate for endoglucanase (EGL) and endoxylanase (XLN) activity measurement, respectively. The reaction mixtures were incubated for 4 h at 30°C and were terminated by the addition of 250 μL of precipitation solution (4% NaAc*3H2O, 0.4% ZnAc, 76% EtOH, pH 5). The plates were centrifuged at 4°C, 1000 × g for 10 min. Supernatant samples were transferred to new microtiter plates, and absorption was measured at 600 nm wavelength using a FLUOstar OPTIMA microplate reader. All measurements were performed by using biological duplicates and technical triplicates.
Saccharification tests were performed in 96-well flat bottom microtiter plates. Each reaction had 50 mM sodium citrate (pH 5) containing 3% soybean hulls (SBH) or 3% CP mixed with 20 μL culture filtrate in a final volume of 250 μL. The reaction plates were incubated for 6 h at 30°C and 400 rpm. Reactions were stopped by heat inactivation for 15 min at 95°C. The reaction plates were centrifuged for 20 min at 3220 × g, and the supernatants were subsequently 10-fold diluted in MilliQ water prior to analysis. The experiment was performed using biological duplicates and technical triplicates.
Monosaccharides were analyzed from peak areas in HPAEC-PAD (Dionex ICS-5000 + system; Thermo Fisher Scientific, Waltham, MA, USA) equipped with CarboPac PA1 column (2×250 mm with 2×50 mm guard column; Thermo Fisher Scientific, Waltham, MA, USA). The column was pre-equilibrated with 18 mM NaOH followed by a multi-step gradient: 0–20 min, 18 mM NaOH; 20–30 min: 0–40 mM NaOH and 0–400 mM sodium acetate; 30–35 min, 40–100 mM NaOH and 400 mM to 1 M sodium acetate; and 35–40 min, 100 mM NaOH and 1 M to 0 M sodium acetate followed by re-equilibration of 18 mM NaOH for 10 min (20°C; flow rate: 0.30 mL min−1). Concentrations of 5–250 mM of D-xylose, D-galacturonic acid, and L-arabinose (Sigma-Aldrich, St. Louis, MO, USA) were used as standards for quantification. Blank samples containing 3% SBH or CP, with the addition of sterile MilliQ water instead of culture filtrates were measured as well. These values were subtracted from each corresponding saccharification sample result in order to exclude the amount of free sugar already present in the experimental condition.
Proteomics sample preparation and analysis
Proteins from 600 μL cell-free liquid culture filtrates were precipitated by mixing them with two volumes of −20°C methanol, followed by overnight incubation at −20°C. The precipitated protein solution was centrifuged at 20800 × g, 4°C for 20 min. The supernatant was aspirated, and the pellet was washed once with 60% cold methanol in water and was resuspended in 6M urea, 100 mM ammonium bicarbonate pH 8 solution. An aliquot was subsequently taken for protein quantification performed colorimetrically using the RCDC kit assay (BioRad, Mississauga, Ontario). In total 7.5 μg of protein samples of biological duplicates were immobilized in acrylamide and processed for in-gel digestion with trypsin as previously described (Balliau et al. 2018). Dried digest peptide extracts were solubilized in a solution of 5% acetonitrile, 0.1% formic acid, and 4 fmol μL−1 of trypsin-digested bovine serum albumin (BSA) (Michrom, Auburn, CA) used as internal standard. Five microliters were analyzed by LC-MS/MS using an Easy-LC II Nano-HPLC system connected in-line with a Velos LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA). LC-MS/MS data peptide and protein identification were done using the A. niger NRRL3 protein sequence databases. Protein identification and quantification were performed using the Proteome Discoverer 2.4 (Thermo Fisher Scientific, Waltham, MA, USA) precursor ion quantitation workflow. Normalized individual protein area values were expressed as a fold value of the protein area value determined for the BSA internal standard. Data analysis was performed based on the percentage values of the total exoproteome.
Statistical analyses were performed on all enzyme assays and saccharification experiments, which were carried out in biological duplicates and technical triplicates. Statistically significant differences (p value < 0.05) were determined using the one-way analysis of variance (ANOVA) and Tukey’s honestly significant difference (HSD) test (Supplemental Table S3). Analyses were done using STATGRAPHICS Centurion XVI Version 16.1.17 (www.statgraphics.com/centurion-xvi).