Ppic9

Three colonies harboring each of the zeocin resistance plasmids were grown to an OD₆₀₀ (optical density measured at 600 nm) of 1 in 10 mL of YPD containing zeocin while LA3 was grown in 10 mL of YPD. Cells were collected by centrifugation at 2000 × g for 5 min. The cell pellet was resuspended with 1 mL 0.25% sodium dodecyl sulfate (SDS) and incubated at 98°C for 8 min according to previous work [26 (link)]. Finally, cellular debris was removed by centrifugation and the DNA was diluted 10-fold in nuclease-free water before the qPCR reactions.
qPCR reactions used primers qZEO-F and qZEO-R for plasmid quantification and qHIS-F and qHIS-R as an internal single-copy control. The assays used iTaq Universal SYBR Green Supermix (Bio-Rad) in a Rotor-Gene Q (Qiagen) thermal cycler in technical triplicates. The analysis used the absolute quantification method and standard curves that ranged from 1x10³ to 1x10⁷ copies of the gene of interest. pPIC9 (Invitrogen) and pPICH linearized plasmids were used for the construction of standard curves.

Talaromyces leycettanus JCM12802, the donor strain, was purchased from Japan Collection of Microorganisms RIKEN BioResource Center (Tsukuba, Japan). Escherichia coli Trans I-T1 (TransGen, Beijing, China) was used for routine gene cloning. T. reesei AST1116 and P. pastoris GS115 (Invitrogen, Carlsbad, CA, United States) were used as hosts for gene expression. pPIC9 (Invitrogen) and pTrEno plasmids of were used to drive Tlswo gene expression in P. pastoris and in T. reesei, respectively. The pTrEno plasmid was constructed described by Linger et al. (2015) (link).

Pichia pastoris strain GS115, Escherichia coli strain DH5α, and expression vector pPIC9 were purchased from Invitrogen (Life Technologies). MDBK and Daudi cells were obtained from Wuhan Boster. The plasmid pPIC9K-Kex-IFNα2b-Fcγ1 with the coding gene of fused human IFN-α2b and the wild-type human IgG1 Fc fragment was constructed and preserved in our laboratory [13 ]. WISH cells, the conventional IFN-α and the anti-IFN-α monoclonal antibody were obtained from Anke Biotechnology Group. HPR-conjugated anti-mouse antibodies were purchased from Cell Signaling. The 14-L fermenter (New Brunswick BioFlo115) used in pilot-scale fermentation was obtained from Eppendorf. A flex stand system with 0.45-μm hollow fiber membrane filtration cartridges and an AKTA Avant system with MabSelect and S-200 HR columns were obtained from GE Healthcare.

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.

T. leycettanus JCM12802 from the Japan Collection of Microorganisms (JCM, Japan) was used as the donor. It was cultivated at 40°C in potato-dextrose broth (PDB) or complex medium containing 5 g/L (NH₄)₂SO₄, 1 g/L KH₂PO₄, 0.5 g/L MgSO₄·7H₂O, 0.2 g/L CaCl₂, 10 mg/L FeSO₄·7H₂O, 30 g/L corncob, 30 g/L soybean meal and 30 g/L wheat bran. Escherichia coli Trans1-T1 used as the cloning host was cultivated in liquid Luria-Bertani (LB) medium with ampicillin at 37°C. The expression host P. pastoris GS115 (Invitrogen, Carlsbad, CA) was cultivated in the yeast peptone dextrose (YPD) medium at 30°C. Minimal dextrose (MD) medium or minimal methanol (MM) medium, buffered glycerol complex (BMGY) medium, and buffered methanol complex (BMMY) medium were prepared according to the manual of the Pichia Expression kit (Invitrogen) and used for transformant selection, cultivation and induction, respectively. The vectors pGEM-T Easy (Promega, Madison, WI) and pPIC9 (Invitrogen) were used for cloning and expression, respectively.

The GenBank accession numbers of Ochrobactrum sp. M231 MPH and OPHC2 are ACC63894 and CAE53631, respectively. Escherichia coli strain Top10 and the plasmid pGEM-T were purchased from Promega Corp. (Madison, WI, USA). E. coli strain BL21(DE3) and the expression plasmid pET-30a(+) were purchased from Novagen (Darmstadt, Germany). P. pastoris strain GS115 and the vector pPIC9 were purchased from Invitrogen (Carlsbad, CA).
E. coli cells were cultured aerobically at 37°C in Luria-Bertani medium. Minimal dextrose (MD) medium, buffered complex glycerol medium (BMGY), yeast extract peptone dextrose medium (YPD), and buffered complex menthol medium (BMMY) were prepared according to the manufacturer’s instructions (Invitrogen).

N. fischeri P1 (CGMCC 3.15369, the China General Microbiological Culture Collection, Beijing, China) was grown in cellulase inducing medium (w/v) containing 1% wheat bran, 0.5% (NH₄)₂SO₄, 0.1% KH₂PO₄, 0.05% MgSO₄ 7H₂O, 0.02% CaCl₂ and 0.001% FeSO₄ 7H₂O at 45°C for 3 days. Escherichia coli strain Trans1-T1 (TransGen, Beijing, China) grown at 37°C in Luria-Bertani medium supplemented with ampicillin (100 μg ml⁻¹) and pEASY-T3 (TransGen) were used for cloning experiment. P. pastoris strain GS115 and pPIC9 (Invitrogen, Carlsbad, CA, USA) were used for heterologous protein production. P. pastoris cells were propagated in YPD medium (yeast extract peptone dextrose medium: 1% yeast extract, 2% peptone, and 2% glucose) and subsequently grown in either MD (minimal dextrose medium: 1.34% YNB [yeast nitrogen base without amino acids], 4 × 10⁻⁵% biotin, 2% glucose, and 2% agarose) or BMGY (buffered glycerol-complex medium: 1% yeast extract, 2% peptone, 1.34% YNB, 4 × 10⁻⁵% biotin, and 1% glycerol). The induction medium was BMMY (buffered methanol-complex medium: 1% yeast extract, 2% peptone, 1.34% YNB, 4 × 10⁻⁵% biotin, and 0.5% methanol). Growth conditions were as described in the Pichia Expression kit (Invitrogen).