Pichia pastoris x 33

A. fumigatus Z5 (CGMCC Accession No. 3309) was stored in our lab and used for the GH10 xylanase study. Fungal liquid cultivation was performed with 250-mL flasks by inoculating 1 × 10⁷ fresh conidia or 0.1 g fresh mycelia into 50 mL of Mandels’ salt solution (0.25 g·L⁻¹ yeast extract, 1 g·L⁻¹ MnSO₄·H₂O, 0.5 g·L⁻¹ urea, 0.5 g·L⁻¹ (NH₄)₂SO₄, 0.5 g·L⁻¹ CaCl₂, 0.5 g·L⁻¹ MgSO₄·7H₂O, 7.5 mg·L⁻¹ FeSO₄·7H₂O, 2.5 mg·L⁻¹ MnSO₄·H₂O, 3.6 mg·L⁻¹ ZnSO₄·7H₂O, 3.7 mg·L⁻¹ CoCl₂·6H₂O) supplemented with 2% (w/v) glucose or oat spelt xylan (Sigma-Aldrich, St. Louis, USA). Fungal cultures were incubated at 37 °C and 150 rpm. Pichia pastoris X33 (Invitrogen, Waltham, USA) was used as the host for protein heterologous expression. The YPDS medium (2% peptone, 1% yeast extract, 2% glucose, and 1 M sorbitol, pH 6.0) with 100 µg·mL⁻¹ Zeocin (Sigma-Aldrich, St. Louis, USA) was used for transformant screening. BMGY/BMMY (2% peptone, 1% yeast extract, 1.34% YNB, 4 × 10⁻⁵% biotin, 1% glycerol, or 0.5% methanol, pH 6.0) was used as the growth/induction medium for enzyme production by P. pastoris X33. Plasmids were stored in Escherichia coli Top10 (Invitrogen, Waltham, USA) cultivated in the low-salt Luria–Bertani medium (1% peptone, 0.5% yeast extract, 0.5% NaCl, pH 7.0).

Human C1-INH cDNA was kindly provided by Dr. Erik C. Hack (Sanquin Research, Amsterdam, The Netherlands) and expressed in Pichia pastoris X-33 (Invitrogen Life Technologies) through the expression vector pPICZαA^{61 (link)}.
For intracellular C1-INH quantification, cell lysates were prepared by adding 2.5 ml of CelLytic-Y reagent to each gram of yeast cell pellet. 4 grams of glass beads per gram of wet cell pellet were then added, and the suspension was vortexed several times, with 1-minute incubation on ice after each vortex. To pellet the cellular debris, the lysed cells were centrifuged for 10 minutes at 12,000 × g.
Recombinant C1-INH was purified from P. pastoris supernatant by cation exchange liquid chromatography on a MonoS 5/50 GL Tricorn column (GE Healthcare Life Science). Immediately after purification, the peak fractions were dialyzed against PBS containing 0.1% Polyethylene Glycol (PBS-0.1% PEG), concentrated and stored at −80 °C.
The recombinant C1-INH from P. pastoris was used to perform all the functional and structural studies detailed below.

Aspergillus fumigatus Z5 (CGMCC Accession No. 3309, China General Microbiology Culture Collection Center, Genome GenBank accession AZZA01000000) is stored in our lab and used for the study of lignocellulose degradation. The cultivation was performed according to Miao et al. (2015a (link)). Briefly, 1 × 10⁷ fresh conidia was added into 200 ml of Mandels’ salt solution supplemented with 2% (w/v) oat spelts xylan (Sigma, USA), and then incubated for 20 h at 50 °C and 150 rpm. The mycelia were then harvested and washed thoroughly with sterile water to be stored at − 80 °C for RNA extraction.
Pichia pastoris X33 (Invitrogen, USA) was used as the gene expression host. Plasmid construction and storage was based on the Escherichia coli Top10 (stored in our lab) cultivated in LLB (Low-salt Luria–Bertani) medium (1% peptone, 0.5% yeast extract, 0.5% NaCl, pH7.0). YPDS medium (2% peptone, 1% yeast extract, 2% glucose, 1 M sorbitol, pH 6.0) was used for transformants screening. BMGY/BMMY (2% peptone, 1% yeast extract, 1.34% YNB, 4 × 10⁻⁵% biotin, 1% glycerol or 0.5% methanol, pH 6.0) was used as the growth/induction medium for enzyme production.

Pichia pastoris X-33 (Invitrogen, Waltham, MA, USA, Cat. No. K1740-01) was used as the expression host. The amino acid sequences of AnTx, Ts6, and Vm24 were retrieved from the Uniprot database https://www.uniprot.org/ (accessed on 10 March 2021) (AnTx: P0C166, Ts6: P59936, and Vm24: P0DJ31). The sequences were reverse translated, and the gene cassettes were designed by adding the coding sequence of the 6x-HisTag at the N-terminus to facilitate the protein purification by Ni-NTA affinity chromatography. In addition, a factor Xa protease site (IEGR) was added in the case of Vm24, and an enterokinase protease site (DDDDK) was added in the case of AnTx and Ts6 to remove the His-tag of the expressed toxin if needed. The gene cassettes were codon-optimized for P. pastoris according to the codon usage database available at www.kazusa.or.jp/codon (accessed on 10 March 2021) and synthesized by Integrated DNA Technologies, Belgium. The designed gene sequences were cloned into the yeast expression vector pPICZαA (Invitrogen, Waltham, MA, United States) using PstI and SalI for AnTx and Vm24, and KpnI and EcoRI for Ts6. The in-frame ligation and nucleotide sequence of AnTx, Ts6, and Vm24 were confirmed by DNA sequencing.

Escherichia coli TOP10F' strain was used for the amplification of the expression construct pPICZαC/FoFaecMut (Eurofins Genomics, Luxembourg) and the transformants were selected on Low Salt Luria-Bertani medium (1% tryptone; 0.5% yeast extract; 0.5% NaCl pH 7.5) by Zeocin™ resistance (25 μg mL⁻¹). The resistant transformants were grown overnight at 37°C under shaking and plasmid DNA was isolated by the Plasmid DNA Extraction Mini Prep Kit (Fisher Molecular Biology, Rome, Italy). The recombinant plasmid pPICZαC/FoFaecMut was linearized with SacI restriction enzyme (NEB, Ipswich, MA, USA) to transform Pichia pastoris X-33 (Invitrogen, Carlsbad, CA, USA). The transformation of yeast was performed with 5 μg pure recombinant vector by Electroporation protocol according to the EasySelect™ Pichia Expression Kit (Invitrogen, Carlsbad, CA, USA). P. pastoris transformants were selected on YPDS agar (1% w/v yeast extract; 2% w/v peptone; 2% w/v dextrose; 1 M sorbitol; 2% w/v agar) containing Zeocin™ at final concentration of 100 μg mL⁻¹at 28°C. Thirty selected transformants were grown in BMGY and BMMY (1% w/v yeast extract; 2% w/v peptone; 100 mM potassium phosphate, pH 6.0; 1.34% w/v YNB; 4 × 10⁻⁵% w/v biotin; 1% v/v glycerol or 0.5% v/v methanol) at 28°C.

All chemical reagents in this study were obtained from commercial supplier Sinopharm Chemical Reagent Co. Ltd., Shanghai, China. Cellulosic substrates and commercial cellulase were purchased from Sigma-Aldrich, Mo, USA. Phosphoric acid-swelling cellulose (PASC)) was prepared from Avicel PH -101 (Kuo and Lee 2009 (link)). Fungal strain of Arthrobotrys sp. CX1 was isolated from the campus of Dalian Polytechnic University, China, that was cultured as previously described (Lan et al. 2016 (link)). Pichia pastoris X-33 and vector pPICαA were purchased from Invitrogen.

The strain used for gene cloning was Escherichia coli Trans1-T1 (TransGen, Beijing, China). Moniliophthora roreri is a manganese peroxidase-producing filamentous fungus. One of the MnPs (i.e., MrMnP used in this study) can be successfully recombinantly expressed in Pichia pastoris [21 (link),22 (link)]. The plasmid used to construct the expression vector was pPICZα(A) (Invitrogen, Carlsbad, CA, USA). Pichia pastoris X-33 (Invitrogen, Carlsbad, CA, USA) was used for recombinant expression of the MrMnP, and it was routinely cultured and maintained on YPD-agar plates (where YPD represents yeast potato dextrose, containing 1% yeast extract, 2% peptone, 2% glucose). A single colony of P. pastoris strains was inoculated in the YPD liquid medium in a shaker flask and cultured at 30 °C.