Microbial Cell Factories | |
A quantitative study of methanol/sorbitol co-feeding process of a Pichia pastoris Mut+/pAOX1-lacZ strain | |
Research | |
Marc Daukandt1  Christian Rodriguez1  Nathalie Pirlot1  Laurent Jost1  Hosni Sassi2  Patrick Fickers2  Hongxing Niu2  | |
[1] Eurogentec S.A., Rue du Bois Saint Jean, n°14, 4102, Seraing, Belgium;Unité de Biotechnologies et Bioprocédés, Université libre de Bruxelles, 50 CP 165/61, Av. F.-D. Roosevelt, 1050, Brussels, Belgium; | |
关键词: Pichia pastoris; pAOX1; β-galactosidase; Methanol/sorbitol co-feeding; Metabolic flux analysis; Transient continuous culture; Fed batch culture; | |
DOI : 10.1186/1475-2859-12-33 | |
received in 2012-12-21, accepted in 2013-03-22, 发布年份 2013 | |
来源: Springer | |
【 摘 要 】
BackgroundOne 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.ResultsTransient 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.ConclusionThis 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.
【 授权许可】
CC BY
© Niu et al.; licensee BioMed Central Ltd. 2013
【 预 览 】
Files | Size | Format | View |
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RO202311100825525ZK.pdf | 672KB | download |
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