CHO cells are known to secrete growth inhibitory metabolites during growth and production phases, which hampers cellular performance and negatively impacts final productivity and product quality attributes. Previous studies have identified different metabolic by-products derived from CHO metabolism and demonstrated their negative impacts on growth and titer productivity. This work presents a control strategy that incorporated genetic engineering and inhibitory metabolites pathway analysis to regulate cellular metabolism. In this study, three different metabolic genes involving the metabolism of branched-chain amino acids (, and ) were cloned from CHO reversely synthesized cDNA to study for gene functionality towards regulating cellular metabolism. Identified metabolic genes were individually engineered into expression vector and transfected to cells at plasmid concentration of 40 µg/µL. For all engineered conditions, the peak VCD profile of cells on Day 4 realized a 18–20% increase with cumulative VCD profile realizing 16–19% increase in terms of total viable cells collected on harvest day. Increase in IgG1 titer production and also more complex glycosylation profiles formation were also observed from all engineered subclones. This study hereby successfully demonstrated rewiring cellular metabolism through up-regulation of key metabolic enzymes can effectively control the accumulation of process inhibitors which therefore allowed improvement desirable critical process attributes.

中文翻译：

---

Bcat1、Adh5 和 Hahdb 的代谢工程可控制代谢抑制剂并提高 CHO 细胞培养物的性能

众所周知，CHO 细胞在生长和生产阶段会分泌生长抑制代谢物，这会阻碍细胞性能并对最终生产力和产品质量属性产生负面影响。先前的研究已经确定了 CHO 代谢产生的不同代谢副产物，并证明了它们对生长和滴度生产率的负面影响。这项工作提出了一种结合基因工程和抑制代谢物途径分析来调节细胞代谢的控制策略。在这项研究中，从CHO反向合成的cDNA中克隆了涉及支链氨基酸代谢的三个不同的代谢基因（、和），以研究调节细胞代谢的基因功能。鉴定出的代谢基因被单独工程化到表达载体中，并以 40 µg/µL 的质粒浓度转染到细胞中。对于所有工程条件，第 4 天细胞的峰值 VCD 谱实现了 18-20% 的增加，累积 VCD 谱实现了收获日收集的活细胞总数的 16-19% 增加。从所有工程亚克隆中还观察到 IgG1 滴度产量的增加以及更复杂的糖基化谱的形成。这项研究成功证明，通过上调关键代谢酶来重新连接细胞代谢可以有效控制过程抑制剂的积累，从而改善所需的关键过程属性。