title: Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter.
abstract: The codon sequence optimized bovine prochymosin B gene was cloned under the control of the alcohol oxidase 1 promoter (AOX1) in the vector pPIC9K and integrated into the genome of the methylotrophic yeast Pichia (Komagataella) pastoris (P. pastoris) strain GS115. A transformant clone that showed resistance to over 4 mg G418/ml and displayed the highest milk-clotting activity was selected. Cell growth and recombinant bovine chymosin production were optimized in flask cultures during methanol induction phase achieving the highest coagulant activity with low pH values, a temperature of 25°C and with the addition of sorbitol and ascorbic acid at the beginning of this period. The scaling up of the fermentation process to lab-scale stirred bioreactor using optimized conditions, allowed to reach 240 g DCW/L of biomass level and 96 IMCU/ml of milk-clotting activity. The enzyme activity corresponded to 53 mg/L of recombinant bovine chymosin production after 120 h of methanol induction. Western blot analysis of the culture supernatant showed that recombinant chymosin did not suffer degradation during the protein production phase. By a procedure that included high performance gel filtration chromatography and 3 kDa fast ultrafiltration, the recombinant bovine chymosin was purified and concentrated from fermentation cultures, generating a specific activity of 800 IMCU/Total Abs(280 nm) and a total activity recovery of 56%. This study indicated that P. pastoris is a suitable expression system for bioreactor based fed-batch fermentation process for the efficient production of recombinant bovine chymosin under methanol-inducible AOX1 promoter.
authors.affiliation: Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECH), Universidad Nacional de San Martín (UNSAM) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), San Martín, Buenos Aires, Argentina.
authors.forename: Diego Gabriel
authors.initials: DG
authors.lastname: Noseda
authors.forename: Matías Nicolás
authors.initials: MN
authors.lastname: Recúpero
authors.forename: Martín
authors.initials: M
authors.lastname: Blasco
authors.forename: Gastón Ezequiel
authors.initials: GE
authors.lastname: Ortiz
authors.forename: Miguel Angel
authors.initials: MA
authors.lastname: Galvagno
mesh_terms: D042931:Aldehyde Oxidase / Q000235:genetics*; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D019149:Bioreactors; D002417:Cattle; D012085:Chymosin / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013012:Sorbitol / Q000378:metabolism; D013696:Temperature
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D013012:Sorbitol; D042931:Aldehyde Oxidase; D012085:Chymosin; D001205:Ascorbic Acid
keywords: Bovine chymosin; Fed-batch fermentation process; Methanol-inducible AOX1 promoter; Optimization; Pichia (Komagataella) pastoris; Purification