Regulation of alcohol oxidase of a recombinant Pichia pastoris Mut+ strain in transient continuous cultures
Regulation of alcohol oxidase of a recombinant Pichia pastoris Mut+ strain in transient continuous cultures
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DOI：
10.1016/j.jbiotec.2007.04.004
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发表时间：
2007-06-30
影响因子：
4.1
通讯作者：
von Stockar, Urs
中科院分区：
文献类型：
--
作者：
Jungo, Carmen;Marison, Ian;von Stockar, Urs
在甲醇营养型毕赤酵母中，醇氧化酶（AOX）是参与甲醇异化的关键酶。杂合蛋白通常在AOX1启动子的控制下表达，AOX1启动子驱动醇氧化酶1在野生型菌株中的表达。本研究探讨了重组巴斯德毕赤酵母Mut（+）菌株在以甘油和甲醇为唯一碳源的培养物中以及在以两种底物为混合底物的培养物中醇氧化酶的调节。目的是更好地了解从甘油生长到甲醇生长的转变，这是标准高细胞密度巴斯德毕赤酵母培养物生产外源蛋白的关键步骤。恒化培养物中的营养转移表明，在甘油上生长后，使用甘油和甲醇的混合进料比使用含有甲醇作为唯一碳源的进料允许更快地诱导醇氧化酶和更快地适应细胞代谢。本研究的结果还表明，在低残留甲醇浓度下操作的巴斯德毕赤酵母培养物期间避免瞬时甲醇积累是多么关键。实际上，恒化器培养期间的脉冲实验表明，在甲醇限制或双重甲醇和甘油限制的生长条件下进行的培养物中甲醇浓度的突然增加导致培养物的洗脱，因为甲醇的消耗速率太高，这导致有毒中间体的排泄。高速率的甲醇消耗是由于在低残留甲醇浓度下观察到的高比AOX活性。(c)2007 Elsevier B.V.保留所有权利。
In the methylotrophic yeast Pichia pastoris, alcohol oxidase (AOX) is a key enzyme involved in the dissimilation of methanol. Heterologous proteins are usually expressed under the control of the AOX1 promoter, which drives the expression of alcohol oxidase 1 in the wild-type strain. This study investigates the regulation of the alcohol oxidase enzyme of a recombinant R pastoris Mut(+) strain in cultures on glycerol and methanol as sole carbon sources and in mixed substrate cultures on both substrates. The aim was to have a better insight in the transition from growth on glycerol to growth on methanol, which is a key step in standard high cell density P. pastoris cultures for the production of foreign proteins. Nutrient shifts in chemostat cultures showed that after growth on glycerol use of mixed feeds of glycerol and methanol allowed faster induction of alcohol oxidase and faster adaptation of cellular metabolism than with a feed containing methanol as sole carbon source.The results of this study showed also how critical it is to avoid transient methanol accumulation during P. pastoris cultures operated at low residual methanol concentrations. Indeed, pulse experiments during chemostat cultures showed that sudden increase in methanol concentrations in cultures performed under methanol-limited or dual methanol and glycerol-limited growth conditions leads to wash-out of the culture because of too high consumption rate of methanol, which leads to excretion of toxic intermediates. High rate of methanol consumption was due to high specific AOX activities observed at low residual methanol concentrations. (c) 2007 Elsevier B.V. All rights reserved.