title: A quantitative study of methanol/sorbitol co-feeding process of a Pichia pastoris Mut⁺/pAOX1-lacZ strain.
abstract: BACKGROUND
  One of the main challenges for heterologous protein production by the methylotrophic yeast Pichia pastoris at large-scale is related to its high oxygen demand. A promising solution is a co-feeding strategy based on a methanol/sorbitol mixture during the induction phase. Nonetheless, a deep understanding of the cellular physiology and the regulation of the AOX1 promoter, used to govern heterologous protein production, during this co-feeding strategy is still scarce.
  
  
  RESULTS
  Transient continuous cultures with a dilution rate of 0.023 h(-1) at 25°C were performed to quantitatively assess the benefits of a methanol/sorbitol co-feeding process with a Mut+ strain in which the pAOX1-lacZ construct served as a reporter gene. Cell growth and metabolism, including O2 consumption together with CO2 and heat production were analyzed with regard to a linear change of methanol fraction in the mixed feeding media. In addition, the regulation of the promoter AOX1 was investigated by means of β-galactosidase measurements. Our results demonstrated that the cell-specific oxygen consumption (qO2) could be reduced by decreasing the methanol fraction in the feeding media. More interestingly, maximal β-galactosidase cell-specific activity (>7500 Miller unit) and thus, optimal pAOX1 induction, was achieved and maintained in the range of 0.45 ~ 0.75 C-mol/C-mol of methanol fraction. In addition, the qO2 was reduced by 30% at most in those conditions. Based on a simplified metabolic network, metabolic flux analysis (MFA) was performed to quantify intracellular metabolic flux distributions during the transient continuous cultures, which further shed light on the advantages of methanol/sorbitol co-feeding process. Finally, our observations were further validated in fed-batch cultures.
  
  
  CONCLUSIONS
  This study brings quantitative insight into the co-feeding process, which provides valuable data for the control of methanol/sorbitol co-feeding, aiming at enhancing biomass and heterologous protein productivities under given oxygen supply. According to our results, β-galactosidase productivity could be improved about 40% using the optimally mixed feed.
authors.forename: Hongxing
authors.initials: H
authors.lastname: Niu
authors.forename: Laurent
authors.initials: L
authors.lastname: Jost
authors.forename: Nathalie
authors.initials: N
authors.lastname: Pirlot
authors.forename: Hosni
authors.initials: H
authors.lastname: Sassi
authors.forename: Marc
authors.initials: M
authors.lastname: Daukandt
authors.forename: Christian
authors.initials: C
authors.lastname: Rodriguez
authors.forename: Patrick
authors.initials: P
authors.lastname: Fickers
mesh_terms: D061249:Batch Cell Culture Techniques; D018533:Biomass; D064688:Metabolic Flux Analysis; D000432:Methanol / Q000378:metabolism*; D010843:Pichia / Q000254:growth & development / Q000378:metabolism*; D013012:Sorbitol / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D013012:Sorbitol; D001616:beta-Galactosidase; D000432:Methanol
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