초록
<P><B>Background</B></P><P>One of the main challenges for heterologous protein production by the methylotrophic yeast <I>Pichia pastoris</I> 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.</P><P><B>Results</B></P><P>Transient continuous cultures with a dilution rate of 0.023 h<SUP>-1</SUP> at 25°C were performed to quantitatively assess the benefits of a methanol/sorbitol co-feeding process with a Mut<SUP>+</SUP> strain in which the pAOX1-lacZ construct served as a reporter gene. Cell growth and metabolism, including O<SUB>2</SUB> consumption together with CO<SUB>2</SUB> 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 (qO<SUB>2</SUB>) 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 qO<SUB>2</SUB> 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.</P><P><B>Conclusion</B></P><P>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.</P>