A quantitative study of methanol/sorbitol co-feeding process of a Pichia pastoris Mut⁺/pAOX1-lacZ strain.
A quantitative study of methanol/sorbitol co-feeding process of a Pichia pastoris Mut⁺/pAOX1-lacZ strain.
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DOI：
10.1186/1475-2859-12-33
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发表时间：
2013-04-08
影响因子：
6.4
通讯作者：
Fickers P
中科院分区：
文献类型：
--
作者：
Niu H;Jost L;Pirlot N;Sassi H;Daukandt M;Rodriguez C;Fickers P
巴斯德毕赤酵母规模化生产异源蛋白的主要挑战之一与其高需氧量有关。一个有希望的解决方案是在诱导阶段基于甲醇/山梨醇混合物的共喂策略。然而，在这种共喂策略中，对用于控制异源蛋白质生产的AOX1启动子的细胞生理学和调控的深入了解仍然很少。在25℃，稀释率为0.023×h-1的瞬时连续培养条件下，以pAOX1-lacZ为报告基因的Mut+菌株与甲醇/山梨醇共喂过程的效益被定量评估。在混合培养基中甲醇组分呈线性变化的情况下，分析了细胞的生长和代谢，包括O2消耗、CO2和产热。此外，通过β-半乳糖苷酶测定研究了启动子AOX1的调控。我们的结果表明，降低进料介质中的甲醇含量可以降低细胞比耗氧量(QO2)。更有趣的是，最大的β-半乳糖苷酶细胞比活(>；7500Miller单位)和最优的pAOX1诱导，获得并保持在0.45mol.75Cmo1/Cmol甲醇组分的范围内。此外，在此条件下，qO2最多可降低30%。在简化代谢网络的基础上，利用代谢通量分析(MFA)对瞬时连续培养过程中细胞内代谢通量的分布进行了量化，进一步揭示了甲醇/山梨醇共喂工艺的优势。最后，我们的观察结果在补料批次培养中得到了进一步的验证。这项研究为控制甲醇/山梨醇共喂过程提供了有价值的数据，旨在提高给定供氧条件下的生物量和异源蛋白质的产量。根据我们的结果，使用优化的混合饲料，β-半乳糖苷酶的产量可以提高约40%。
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. 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. 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.
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影响因子：
6.4
作者：
Jordà J;Jouhten P;Cámara E;Maaheimo H;Albiol J;Ferrer P
通讯作者：
Ferrer P
影响因子：
6.4
作者：
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