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首页> 外文期刊>Biotechnology Progress >Improving glucose and gluamiue metabolism of human HEK 293 and Trchoplusia ni insect cells engineered to express a cytosolic pyruvate carboxylase enzyme
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Improving glucose and gluamiue metabolism of human HEK 293 and Trchoplusia ni insect cells engineered to express a cytosolic pyruvate carboxylase enzyme

机译：改善工程化表达胞质丙酮酸羧化羧化酶的人HEK 293和Trchoplusia ni昆虫细胞的葡萄糖和谷氨酰胺代谢

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Metabolic engineering has been defined as a directed improvement of product formation or cellular properties by modification of specific biochemical pathways or introduction of new enzymatic reactions by recombinant DNA technology. The use of metabolic flux analysis (MFA) has helped in the understanding of the key limitation in the metabolic pathways of cultured animal cells. The MFA of the major nutrients glucose and glutamine showed that the flux of glucose to the TCA cycle and its subsequent utilization is limited. as a result of the lack of certain key enzymes in the pathway. One of the key enzymes controlling this flux is pyruvate carboxylase. Introduction of this enzyme into mammalian cells has been shown to improve the utilization of glucose and limit the production of lactate and ammonia, which are deleterious to cell growth. In the present work a yeast pyruvate carboxylase gene has been introduced into mammalian (HEK 293) and insect (Trichoplusia ni High-Five) cells, resulting in the cytosolic expression of the enzyme. In both cases the resulting transfected cells were able to utilize glucose and glutamine more efficiently and produce lower amounts of lactate and ammonia. Differences in the amino acid utilization pattern were also observed, indicating changes in the basic metabolism of the cells. The performance of the transfected cells as expression systems for adenovirus and baculovirus vectors, respectively, has also been examined. The results obtained and their impact on the process development for protein and viral vector production are discussed.
机译：代谢工程已经定义为通过修饰特定的生化途径或通过重组DNA技术引入新的酶促反应来直接改善产品形成或细胞特性。代谢通量分析（MFA）的使用有助于理解培养动物细胞代谢途径中的关键限制。主要营养元素葡萄糖和谷氨酰胺的MFA表明，葡萄糖向TCA循环的通量及其后续利用受到限制。由于该途径缺乏某些关键酶。控制该通量的关键酶之一是丙酮酸羧化酶。已证明将该酶引入哺乳动物细胞可提高葡萄糖的利用率，并限制对细胞生长有害的乳酸和氨的产生。在本工作中，酵母丙酮酸羧化酶基因已被引入哺乳动物（HEK 293）和昆虫（Trichoplusia ni High-Five）细胞中，从而导致该酶的胞质表达。在这两种情况下，所得的转染细胞都能够更有效地利用葡萄糖和谷氨酰胺，并产生较少量的乳酸和氨。还观察到氨基酸利用模式的差异，表明细胞基本代谢的变化。还检查了转染的细胞分别作为腺病毒和杆状病毒载体表达系统的性能。讨论了获得的结果及其对蛋白质和病毒载体生产工艺开发的影响。

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