Lignocellulose

Together with L.edodes, 26 fungal species assigned to Basidiomycota or Ascomycota were used in the phylogenetic analysis. The protein sequences of these 26 fungi were compared by BLASTP with e-value < 1e-5 and hit number < 500. Then, the BLASTP result was analyzed by OrthoMCL[73 (link)] with default parameters to get the orthologous genes, and 756 single-copy orthologous genes were determined. Multiple sequence alignments of these 756 genes were calculated by MAFFT v7.158b [74 (link)] software, and were combined into a long sequence for each species. Then, the conserved block regions of the alignment were picked out by Gblocks 0.91b with default parameters [75 (link)] of the software, and the final alignment length was 193323 aa. With the input of this alignment, phylogenetic tree was constructed by RAxML-8.0.26 [76 (link)] software with bootstrap 1000. Three fossil calibration points [77 (link)] were fixed in the molecule clock analysis: the most recent common ancestor (MRCA) of Coprinopsis cinerea, Laccaria bicolor and Schizophyllum commune were diverged at 122.74 MYA; the MRCA of Serpula lacrymans and Coniophora puteana were diverged at 104.23 MYA; the MRCA of Pichia stipitis, Aspergillus niger, Cryphonectria parasitica, Stagonospora nodorum and Trichoderma reesei were diverged at 517.55 MYA. Then, the divergence time of other nodes was calculated by r8s v1.80 [78 (link)] software with TN algorithm, PL method and the smoothing parameter value set to 1.8 through cross-validation. Based on the ultrametric tree, the orthologous gene family expansion was calculated by CAFE version 3 [25 (link)] software.

The A. niger strains used were N402 [57] (link) and AB4.1 pyrG[7] (link) or as specified otherwise. Strains were maintained on potato dextrose agar (Oxoid). All AB4.1 cultures were supplemented by 10 mM uridine (Sigma). Cultures were incubated at 28°C until they had conidiated. Spores were harvested into 0.1% (v/v) Tween 20 (Sigma). ΔxlnR and ΔcreA strains are A. niger AB4.1 pyrG containing a deletion of the respective open reading frame. Strains were constructed using the method developed by Scherer and Davis. [58] (link) based on recombination between a plasmid containing the flanking region of the gene of interest and the chromosome. As a selection/counter-selection marker we used the gene coding for the orotidine-5-phosphate decarboxylase [59] (link) (pyrG, from Aspergillus oryzae). After transformation of A. niger, cells were selected for uridine prototrophy, confirming integration of the plasmid into the chromosome. After purification of the transformants, release of the selective pressure for the integrated plasmid was achieved by propagating the clones twice on potato dextrose agar containing 10 mM uridine. Selection for cells that had excised the plasmid from the chromosome was done by plating them on media containing 4 mM of 5-fluoro-orotic acid (Melford) and 1.6 mM uridine. Deletion of creA or xlnR was confirmed by PCR using internal and external oligonucleotide primers and by sequencing around the respective loci.
Liquid batch cultures were inoculated with spores to a final concentration of 10⁶ spores/ml. A. niger was grown in 100 ml of minimal media [all l⁻¹: NaNO₃, 6 g; KCl, 0.52 g; MgSO₄.7H₂O, 0.52 g; KH₂PO₄, 1.52 g; Na₂B₄O₇.10H₂O, 0.008 mg; CuSO₄.5H₂O, 0.16 mg; FePO₄.H₂O, 0.16 mg; MnSO₄.4H₂O, 0.16 mg; NaMoO₄.2H₂O, 0.16 mg; ZnSO₄, 1.6 mg] with the appropriate carbon source added to a final concentration of 1% (w/v) in 250 ml Erlenmeyer flasks at 28°C, shaken at 150 rpm. The standard time-course consisted of growth for 48 h in 1% (w/v) glucose media, after which mycelia were removed by filtration through Miracloth (Merck), washed thoroughly with media devoid of carbon source, and transferred to fresh media containing 1% (w/v) ball-milled wheat straw as sole carbon source. Incubation was continued for 24 h. Glucose was then added exogenously to a final concentration of 1% (w/v) and incubation continued for 5 hours. Figure 1 shows an image of a mycelial clump that was magnified using a Nikon SMZ1000 stereomicroscope and the picture taken using a Nikon 4500 camera.

The polysaccharides were extracted as the method from Peng et al. with minor modification [15 (link)]. The crude cell wall material was suspended in 0.5% (w/v) ammonium oxalate and heated for 1 h in a boiling water bath, and the supernatants were combined as total pectin. The remaining pellet was suspended in 4 M KOH containing 1.0 mg mL^-1 sodium borohydride for 1 h at 25°C, and the combined supernatant was neutralized, dialyzed and lyophilized as hemicelluloses. The KOH non-extractable residue was further extracted with acetic-nitric acids for 1 h at 100°C and the remaining pellet was defined as crystalline cellulose. All samples were carried out in triplicate for wall fractionations.

The straw lignocellulose-degrading bacteria were isolated from soil samples collected at various sampling dates using the serial dilution plate method [25 (link)]. To initiate the isolation process, 1 g of soil obtained from the straw surface was thoroughly mixed and added to 9 mL of sterile water in a shaker set at 30 °C and 120 rpm for 20 min. Subsequently, the soil suspension was serially diluted and inoculated onto a straw agar medium consisting of 20 g/L of commercial straw powder (<1 mm) and 1 g/L of urea and adjusted to a pH of 7.0. Following a 7-day incubation period at 30 °C, single colonies were selected and cultured on the same medium to achieve purification. Genomic DNA extraction was performed employing a bacterial genomic DNA extraction kit (Tiangen Biochemical Technology Co. Ltd., Beijing, China). Bacteria and fungi were identified using the primers 27F/1492R and ITS1/ITS4, respectively. The obtained 16S rRNA and ITS gene sequences were compared using the Ezbiocloud and NCBI databases. After the removal of potential clonal duplicates, a total of 59 bacterial and 14 fungal strains were successfully isolated, and their glycerol stocks were prepared and stored at −80 °C.

R package SQMtools [64 (link)] was used to identify the taxonomic origin of genes related to lignocellulose degradation and nitrogen metabolism. Taxonomic information was extracted from the annotated dataset of each species using the subsetFun and subsetTax functions of SQMtools. Counts transformed into relative abundances were allocated to the corresponding substrates and bacterial phyla. Adjacency matrices were then generated and visualized using Circos webtool [65 (link)].