Expression of recombinant cytoplasmic yeast pyruvate carboxylase for the improvement of the production of human erythropoietin by recombinant BHK-21 cells
Expression of recombinant cytoplasmic yeast pyruvate carboxylase for the improvement of the production of human erythropoietin by recombinant BHK-21 cells
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DOI：
10.1016/s0168-1656(01)00409-6
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发表时间：
2002-02-28
影响因子：
4.1
通讯作者：
Wagner, R
中科院分区：
文献类型：
--
作者：
Irani, N;Beccaria, AJ;Wagner, R
最近，在 BHK-21 细胞的细胞质中表达的重组酵母丙酮酸羧化酶被证明可以重建糖酵解和 TCA 之间缺失的联系，从而增加葡萄糖进入 TCA 的流量，并导致细胞内 ATP 含量更高。现在，这些代谢工程细胞还被带有人类促红细胞生成素基因的质粒转染。将重组 BHK-21 细胞的 EPO 产量和底物比生产力与不含 PYC2 基因但用编码选择基因和 EPO 表达的质粒转染的对照细胞进行比较。在连续灌注的生物反应器中，表达 PYC2 的克隆显示出 2 倍高的葡萄糖比生产率和 2 倍高的产物浓度。此外，PYC2 表达使细胞对培养基中的低葡萄糖浓度更具抵抗力。它们可以在 0.05-1 g l(-1) 的葡萄糖限制条件下以几乎最高的生产率进行生产，从而保证分批补料生产系统中乳酸积累的减少。由于表达 PYC2 的细胞具有葡萄糖消耗减少的特点，因此可以维持生物反应器中延长的生产阶段。基于生产时细胞活力不低于 80% 的要求，由于葡萄糖的利用更加经济，并且通过谷氨酰胺限制驱动的分批培养延长了细胞的活力期，因此与对照相比，EPO 的生产时间可以延长 2 天 (30%)。 (C) 2002 Elsevier Science B.V. 保留所有权利。
Recently, a recombinant yeast pyruvate carboxylase expressed in the cytoplasm of BHK-21 cells was shown to reconstitute the missing link between glycolysis and TCA, thus increasing the flux of glucose into the TCA and resulting in a higher intracellular ATP content. Now, these metabolically engineered cells have been additionally transfected with a plasmid bearing the gene for human erythropoietin. EPO yield and substrate-specific productivity of the recombinant BHK-21 cells have been compared to control cells without the PYC2-gene but transfected with the plasmid coding for the expression of the selection genes and EPO. PYC2-expressing clones showed a 2-fold higher glucose-specific productivity and a 2-fold higher product concentration in a continuously perfused bioreactor. Moreover, the PYC2 expression enabled the cells to become more resistant to low glucose concentrations in the culture medium. They could produce at nearly maximum productivity under glucose-limiting conditions of 0.05-1 g l(-1) that guaranteed a reduced accumulation of lactate in fed-batch production systems. Due to the fact that PYC2-expressing cells are characterized by reduced glucose consumption, a prolonged production phase in bioreactors can be maintained. Based on the demand not to fall short of 80% cell viability for the production, EPO could be produced for 2 days (30%) longer compared to the control due to a more economic exploitation of glucose, and the prolonged viability period of the cells using a batch cultivation driven by glutamine limitation. (C) 2002 Elsevier Science B.V. All rights reserved.