Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures.
Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures.
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DOI：
10.1186/1475-2859-11-57
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发表时间：
2012-05-08
影响因子：
6.4
通讯作者：
Ferrer P
中科院分区：
文献类型：
--
作者：
Jordà J;Jouhten P;Cámara E;Maaheimo H;Albiol J;Ferrer P
巴斯德毕赤酵母是生产异源蛋白最有前景的酵母宿主之一。甲醇和多种碳源的混合饲料取代了甲醇作为唯一的碳源，已被证明可以提高产品生产率并减轻蛋白质生产产生的代谢负担。然而，对巴斯德毕赤酵母中心代谢和重组蛋白生产之间关系的系统定量研究仍然相当有限，特别是在利用混合碳源培养这种酵母时，从而阻碍了未来提高蛋白质生产的代谢网络工程策略。利用代谢通量分析(MFA)技术，利用13C-核磁共振的限制条件，分析了葡萄糖和甲醇混合饲料中巴斯德毕赤酵母中心代谢的代谢通量分布。为此，我们为生长在葡萄糖和甲醇混合物上的巴斯德毕赤酵母细胞的甲醇同化途径定义了新的通量比。利用这一实验方法，进一步分析了米根霉脂肪酶(ROL)在巴斯德毕赤酵母中的过度表达和分泌所造成的代谢负荷。这种蛋白以前已经被证明在巴斯德毕赤酵母中触发未折叠的蛋白反应。以葡萄糖：甲醇80：20(w/w)为碳源，在稀释率为0.09h−1的条件下，对两株产Rol菌和一株对照菌株进行了13C示踪实验。在本研究中进行的MFA显示，两个重组菌株与对照菌株相比，中心碳代谢中的碳通量发生了显著的重新分布，反映在糖酵解、TCA循环和NADH再生通量的增加，以及更高的甲醇异化速率。总体而言，进一步的基于13C的MFA开发已经实施，以表征在混合甲醇：多碳源上生长的甲醇营养酵母的中心代谢，从而为研究中心代谢和蛋白质生产之间的关系提供了新的工具。具体地说，这项研究指出，与重组蛋白分泌相关的构象压力对中枢代谢的影响有限但显著，即使在适度的生产水平上也会发生。
The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production. The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA) using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol) in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w) mix as carbon source. The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates. Overall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic yeasts when growing on mixed methanol:multicarbon sources has been implemented, thus providing a new tool for the investigation of the relationships between central metabolism and protein production. Specifically, the study points at a limited but significant impact of the conformational stress associated to secretion of recombinant proteins on the central metabolism, occurring even at modest production levels.
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影响因子：
3.8
作者：
Heyland, Jan;Fu, Jianan;Schmid, Andreas
通讯作者：
Schmid, Andreas
影响因子：
4.4
作者：
Dragosits, Martin;Stadlmann, Johannes;Mattanovich, Diethard
通讯作者：
Mattanovich, Diethard
影响因子：
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作者：
Jouhten, Paula;Rintala, Eija;Maaheimo, Hannu
通讯作者：
Maaheimo, Hannu
影响因子：
46.9
作者：
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通讯作者：
Callewaert, Nico
影响因子：
3.8
作者：
Lange, HC;Heijnen, JJ
通讯作者：
Heijnen, JJ