Combined (13)C-assisted metabolomics and metabolic flux analysis reveals the impacts of glutamate on the central metabolism of high β-galactosidase-producing Pichia pastoris.
Combined (13)C-assisted metabolomics and metabolic flux analysis reveals the impacts of glutamate on the central metabolism of high β-galactosidase-producing Pichia pastoris.
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DOI：
10.1186/s40643-016-0124-6
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发表时间：
2016
影响因子：
4.6
通讯作者：
Zhang S
中科院分区：
文献类型：
--
作者：
Liu P;Huang M;Guo M;Qian J;Lin W;Chu J;Zhuang Y;Zhang S
毕赤酵母是一种流行的重组蛋白表达系统，因其可实现高效的基因操作和高蛋白产量。充足的前体、能量和氧化还原辅因子的供应对于重组蛋白的高产量至关重要。在我们目前的工作中，我们发现谷氨酸的添加提高了毕赤酵母 G1HL 的重组 β-半乳糖苷酶 (β-gal) 产量。为了详细阐明谷氨酸对中枢代谢的影响，基于 LC-MS/MS 和 GC-MS 数据进行了 13C 辅助代谢组学和 13C 代谢通量组合分析。葡萄糖/谷氨酸（Glc/Glu）的细胞内氨基酸库大小明显更高。 Glc/Glu 上的 EMP 进入反应和下游 TCA 循环中的通量分别比 Glc 上高 50% 和 67%。虽然上游 TCA 循环的通量几乎保持不变，但 PPP 氧化分支的通量下降。添加谷氨酸会导致高产β-半乳糖苷酶毕赤酵母G1HL的中枢代谢发生显着变化。为了满足 Glc/Glu 上更高的 β-半乳糖苷酶产量对氧化还原辅助因子和能量的需求增加，巴斯德毕赤酵母 G1HL 通过抑制和/或激活关键节点中的酶以及能量和氧化还原状态来重新分配中心代谢的流量。本文的在线版本 (doi:10.1186/s40643-016-0124-6) 包含补充材料，可供授权用户使用。
Pichia pastoris is a popular recombinant protein expression system for its accessibility of efficient gene manipulation and high protein production. Sufficient supply of precursors, energy, and redox cofactors is crucial for high recombinant protein production. In our present work, we found that the addition of glutamate improved the recombinant β-galactosidase (β-gal) production by P. pastoris G1HL. To elucidate the impacts of glutamate on the central metabolism in detail, a combined 13C-assisted metabolomics and 13C metabolic flux analysis was conducted based on LC–MS/MS and GC–MS data. The pool sizes of intracellular amino acids were obviously higher on glucose/glutamate (Glc/Glu). The fluxes in EMP entry reaction and in downstream TCA cycle were 50 and 67% higher on Glc/Glu than on Glc, respectively. While the fluxes in upstream TCA cycle kept almost unaltered, the fluxes in PPP oxidative branch decreased. The addition of glutamate leads to a remarkable change on the central metabolism of high β-galactosidase-producing P. pastoris G1HL. To meet the increased demands of redox cofactors and energy for higher β-galactosidase production on Glc/Glu, P. pastoris G1HL redistributes the fluxes in central metabolism through the inhibitions and/or activation of the enzymes in key nodes together with the energy and redox status. The online version of this article (doi:10.1186/s40643-016-0124-6) contains supplementary material, which is available to authorized users.