title: Use of a mixture of glucose and methanol as substrates for the production of recombinant trypsinogen in continuous cultures with Pichia pastoris Mut+.
abstract: Pure methanol, which is required as an inducer of the AOX1 promoter and a carbon/energy source in processes for recombinant protein production by Pichia pastoris, is impracticable and therefore generally undesirable. As an alternative, a procedure using double carbon substrate was examined (11.7g(carbon)l(-1), 60%/40% carbon from glucose/methanol). The effects on methanol metabolism, extracellular formation of porcine trypsinogen, biomass growth and cell viability were analyzed. In contrast to batch cultures, where the glucose and methanol were utilized sequentially, in carbon/energy-limited continuous cultures (operated between dilution rates 0.03 and 0.20h(-1)) the repressive effect of glucose on methanol utilization was eliminated up to 0.15h(-1) (ca. 130% of μ(max) with methanol). With the mixture, the yield of biomass (1.54±0.12) g(CDW)g(carbon)(-1) was found to be 1.4 times larger than the yield with methanol alone. Despite the current widespread view that glucose has a repressive effect on the AOX1 promoter, the product was synthesized over the entire range of dilution rates, with maximum productivities of (0.70±0.12)mgg(CDW)(-1) h(-1) at 0.07h(-1). Thus, glucose was shown to be a feasible partial substitute for methanol in recombinant protein production by P. pastoris Mut(+) strain while enhancing process productivity.
authors: Institute of Chemical Technology Prague, Department of Fermentation Chemistry and Bioengineering, Technická 5, 166 28 Prague 6, Czech Republic. leona.paulova@vscht.cz
authors: Leona
authors: L
authors: Paulová
authors: Petr
authors: P
authors: Hyka
authors: Barbora
authors: B
authors: Branská
authors: Karel
authors: K
authors: Melzoch
authors: Karin
authors: K
authors: Kovar
mesh_terms: D000818:Animals; D018533:Biomass; D019149:Bioreactors; D002470:Cell Survival; D003470:Culture Media; D005947:Glucose / Q000378:metabolism*; D007700:Kinetics; D000432:Methanol / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013552:Swine; D014362:Trypsinogen / Q000096:biosynthesis*
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D014362:Trypsinogen; D005947:Glucose; D000432:Methanol