P pastoris gs115

Strain A. fumigatus Af293 was obtained from the Fungal Genetics Stock Center, which genome has been published in 2005. Escherichia coli Trans1-T1 (TransGen Biotech, Beijing, China) was cultivated in Luria-Bertani (LB) medium at 37°C for gene cloning and sequencing. P. pastoris GS115 (Invitrogen, Carlsbad, CA) cultivated in yeast peptone dextrose (YPD) medium at 30°C was used for gene expression. The plasmids pGEM-T Easy (Promega, Madison, WI) and pPIC9 (Invitrogen) were used as cloning and expression vectors, respectively. Beechwood xylan, barley β-glucan, carboxymethyl cellulose sodium (CMC-Na) and locust bean gum were purchased from Sigma-Aldrich (St. Louis, MO). Soluble wheat arabinoxylan was obtained from Megazyme (Wicklow, Ireland). The DNA purification kit, LA Taq DNA polymerase and restriction endonucleases were purchased from TaKaRa (Otsu, Japan). SV Total RNA Isolation System was purchased from Promega.T4 DNA ligase was from New England Biolabs (Hitchin, UK). All chemicals were of analytical grade and commercially available.

The pPICZαC_GA carrying the insert was linearized by SacI and transformed into P. pastoris GS115 (Invitrogen) competent cells. The competent cells were prepared by using the Pichia EasyComp Transformation kit according to the instruction guidelines (Invitrogen). Linearized vector pPICZαC, without insert, was also transformed into P. pastoris GS115 and used as a control. After transformation, Pichia cells were plated onto yeast extract peptone dextrose sorbitol (YPDS) agar plate containing 150 μg mL⁻¹ zeocin and incubated for 3 to 8 days at 30°C. Transformed colonies were confirmed by colony PCR using 5′AOX1 and 3′AOX1 primers and glucoamylase gene specific primers. Putative Mut⁺ colony was selected based on PCR using AXO1 primers. Several Mut⁺ colonies from YPDS agar plate were selected and independently blotted onto Buffered Methanol-complex Medium (BMMY) (containing 1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) YNB, 4 × 10⁻⁵% (w/v) biotin, 0.5% (v/v) methanol, and 100 mM potassium phosphate buffer pH 6.0) agar plate containing 1% (w/v) soluble starch. Glucoamylase producing transformants were identified by the formation of decolorization zone or clear halo around the Pichia colonies after the addition of iodine solution.

N. fischeri P1 CGMCC 3.15369 [15] (link) was grown in a β-glucosidase inducing medium containing 10 g/l wheat bran, 10 g/l CMC-Na, 5 g/l (NH₄)₂SO₄, 1 g/l KH₂PO₄, 0.5 g/l MgSO₄·7H₂O, 0.2 g/l CaCl₂, and 10 mg/l FeSO₄·7H₂O at 45°C and pH 5.0 for 4 days. The total β-glucosidase activity in N. fischeri culture supernatants was 11.9±0.2 U/ml.
Escherichia coli Trans1-T1 (TransGen, Beijing, China) was cultivated in Luria-Bertani (LB) medium with 100 µg/ml ampicillin at 37°C for gene cloning and sequencing. P. pastoris GS115 (Invitrogen, Carlsbad, CA) cultivated in yeast peptone dextrose (YPD) medium at 30°C was used for heterologous protein expression. The plasmids pEASY-T3 (TransGen) and pPIC9 (Invitrogen) were used as cloning and expression vectors, respectively.

For PCR experiments, standard protocols were applied with a PCR amplification kit (TaKaRa, Cat. # R011). Fungal RNA was extracted by means of the RNAsimple Total RNA Kit (TIANGEN Cat. # DP419). Plasmid DNA was isolated from E. coli using the TIANprep Rapid Mini Plasmid Kit (TIANGEN Cat. # DP105–03). DNA fragments separated in an agarose gel were extracted with the Universal DNA Purification Kit (TIANGEN Cat. # DP214–03). Multiple fragments were assembled via the ClonExpress^TM II One Step Cloning Kit (Vazyme Biotech Co., Ltd., China). Strains P. pastoris GS115 and E. coli TOP10 and yeast vectors pPICZ B and pPIC3.5 K were purchased from Invitrogen. Transformation of yeast cells and screening of transformants were executed according to Pichia protocols³⁹ . Yeast two-hybrid (Y2H) assay were described in detail in supplementary data file (Supplementary Fig. S7).

E. coli Trans1-T1 (TransGen, Beijing, China) and P. pastoris GS115 (Invitrogen, Carlsbad, CA, USA) were used as the hosts for gene cloning and expression, respectively. The pEasy-Blunt Simple Cloning Vector was purchased from TransGen.
MD medium contained 1.34% yeast nitrogen base (YNB), 4 × 10⁻⁵% biotin, 2% dextrose, and 2% agar; BMDY, BMRY, and BMDRY media (pH 6.0) contained 300 mM potassium phosphate, 1.34% YNB, 4 × 10⁻⁵% biotin, and 4 × 10⁻³% histidine, plus either 2% dextrose (BMDY), 2% rhamnose (BMRY), or 1% dextrose and 1% rhamnose (BMDRY).
The primers used for PCR are listed in Supplementary Table S1.

The shuttle plasmid pPIC9K (Invitrogen) was used for gene cloning and expression. E. coli JM109 (stored in our laboratory) was used for plasmid amplification and bacterial transformation; P. pastoris GS115 (Invitrogen) was the host bacteria for gene expression. A. niger CGMCC 3.7193 was stored in the China General Microbial Culture Collection Management Center (CGMCC) and used to clone the HypZn gene.

The recombinant plasmid used in this work was pPIC9-pg63, which contained the gene pg63 (HQ446162) from the strain Penicillium sp. CGMCC 166923 . Site-directed mutagenesis was carried out using the specific primers (Additional file 6) and two-step polymerase chain reactions (PCRs). All mutants were verified by double-stranded plasmid sequencing. Escherichia coli Trans I-T1 (TransGen, Beijing, China) and P. pastoris GS115 (Invitrogen, Carlsbad, CA) were used for plasmid amplification and heterologous expression, respectively. The pEASY-T3 vector (TransGen) and pPIC9 (Invitrogen) were used for plasmid construction, respectively. Culture media for His⁺ transformants selection and P. pastoris growth and induction were prepared according to the manual of the Pichia Expression kit (Invitrogen). Mono-, di-, and trigalacturonic acid (GalpA, GalpA2 and GalpA3) and the substrate PGA were purchased from Sigma-Aldrich (St. Louis, MO). All other standard chemicals were of analytical grade.