Ppiczαa expression vector

The xylanase gene from T. maritima and its ancestral sequences, TmxN3, TmxN2, and TmxN1, were artificially synthesized(Genscript, Nanjing, China) and cloned into the pPICZαA expression vector from Invitrogen. This allowed for fusion expression with the alpha-mate signal peptide. The resulting plasmids—pPIC-TmxN3, pPIC-TmxN2, pPIC-TmxN1, and pPIC-TmxB—were obtained and transformed into yeast Pichia pastoris via electroporation. The transformants were cultured overnight in liquid YPD medium containing 20 g/L peptone, 10 g/L yeast extract, and 20 g/L glucose, and then inoculated with 1.5 mL in 25 mL of BMGY medium, consisting of 1% yeast extract, 2% peptone, and 100 mM potassium phosphate (pH 6.0). Inductive expression was carried out for 72 h, and the resulting secreted expression enzymes were purified by Ni-agarose column.

Pseudomonas putida ATCC47054 containing the TreS gene was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). P. pastoris GS115 (Invitrogen, Carlsbad, CA, USA) was used as the host for cell-surface display. Saccharomy cescerevisiae CICC 32919, containing the Pir1p gene, was preserved in our laboratory. The pPICZαA expression vector, containing zeocin resistance, was purchased from Invitrogen (Carlsbad, CA, USA) and zeocin was obtained from Cayla(Toulouse, France). Restriction enzymes, T4 DNA ligase, and an agarose gel DNA purification kit were all purchased from TaKaRa (Dalian, China). Primers shown in Table S1 were used to amplify the target genes.

Cellulase was produced from Trichoderma reesei in Czapek dox media (0.2% KH₂PO₄, 0.42% (NH₄)₂SO₄, 0.03% MgSO_4, 0.03% CaCl₂, 0.1% peptone, 1% glucose, 0.2% trace element solution (0.1% MnSO₄, 0.2% CoCl₂, 0.14% ZnSO₄, 0.5% FeSO₄), 1% glucose, 0.02% urea, and 2% Tween 80). A 100 L bioreactor (KobioTech, South Korea) was operated at 400×g and 28 ºC for 8 days. Cellulase expression was induced by the addition of 1% (w/v) Avicel. The airflow rate and pH adjustment were auto-regulated to 1 vvm and pH 4.8, respectively.
GtAA9- auxiliary activity family 9 (AA9) copper-dependent lytic polysaccharide monooxygenases (LPMOs)- from Gloeophyllum trabeum were cloned and expressed in Pichia pastoris with the pPICZαA expression vector (Invitrogen, Carlsbad, CA, USA) [44 (link)]. The recombinant enzyme was purified with Ni–NTA column (Qiagen Hilden, Germany), and quantified by a Bradford assay.
Xylanase from T. longibrachiatum (Cat. X2629) and beta-glucosidase (AnBgls) from Aspergillus niger were purchased from Sigma-Aldrich and Megazyme Inc. (Lot 141001, Bray, Ireland), respectively.