The cloning of lpmo9A gene from P. anserina strain S mat+, encoding PaLPMO9A (protein ID CAP73254), was described by Bey et al. [14 (link)] Genes encoding PaLPMOC (protein ID CAP68173), PaLPMO9D (protein ID CAP66744), PaLPMO9E (protein ID CAP67740), PaLPMO9F (protein ID CAP71839), PaLPMO9G (protein ID CAP73072), and PaLPMO9H (protein ID CAP61476) were codon optimized for P. pastoris (GenScript, Piscataway, USA) and further inserted into the vector pPICZαA (Invitrogen, Cergy-Pontoise, France) using XhoI and XbaI restriction sites in frame with the (His)6 tag (located at the C terminus of recombinant proteins). P. pastoris strain X33 and the pPICZαA vector are components of the P. pastoris Easy Select Expression System (Invitrogen). All media and protocols are described in the Pichia expression manual (Invitrogen). Recombinant expression plasmids were sequenced to check the integrity of the corresponding sequences. Transformation of competent P. pastoris X33 was performed by electroporation with SacI-linearized pPICZαA recombinant plasmids as described in [45 (link)]. Zeocin-resistant P. pastoris transformants were then screened for protein production. The best-producing transformant was grown in a 1 l of BMGY containing 1 ml.l−1 of Pichia trace minerals 4 (PTM4) salts (2 g.l−1 CuSO4.5H2O, 3 g.l−1 MnSO4.H2O, 0.2 g.l−1 Na2MoO4.2H2O, 0.02 g.l−1 H3BO3, 0.5 g.l−1 CaSO4.2H2O, 0.5 g.l−1 CaCl2, 12.5 g.l−1 ZnSO4.7H2O, 22 g.l−1 FeSO4.7H2O, biotin 0.2 g.l−1, H2SO4 1 ml.l−1) in shaken flasks at 28 °C in an orbital shaker (200 rpm) for 16 h to an OD600 of 2–6. Expression was induced by transferring cells into 200 ml of BMMY containing 1 ml.l−1 of PTM4 salts at 20 °C in an orbital shaker (200 rpm) for another 3 days. Each day, the medium was supplemented with 3 % (v/v) methanol. Bioreactor production of the best-producing transformant was carried out in a 2-l bioreactor Tryton (Pierre Guerin, Mauze, France) according to the P. pastoris fermentation process guidelines (Invitrogen).
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