Xie Li, Miao Junqing, Li Xiangchao, Yi Xiaoping, Chu Ju
State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East China University of Science and Technology 130 Mei Long Road, Xu Hui District Shanghai 200237 China
RSC Adv. 2019 Nov 7;9(61):35760-35770. doi: 10.1039/c9ra07418j. eCollection 2019 Oct 31.
HEK-293 cells are increasingly being used in the production of human adenovirus (HAdV) vaccines. However, the production of HAdV vaccine has not met the requirements of industrial production. Recently, we investigated the effects of various regulatory genes of the pyruvate metabolism node on the substance and energy metabolism and adenovirus reproduction in HEK-293 cells. Initially, single regulatory genes, including , , , , , and , were studied. We found that metabolic performance and adenovirus reproduction capacity in HEK-293 cells were improved, and maximum adenovirus titre was increased approximately 15-fold. Next, we co-overexpressed the key genes, including , and . The PYC2-A-P-L cells that had the appropriate co-overexpression levels of three genes had the most pronounced regulatory effect. The maximum cell density and maximum specific growth rate were increased by 21% compared with that in the control. The ΔLac/ΔGlc and ΔNH/ΔGln were decreased by 26% and 27%, respectively. The ATP production rate and the ATP/O ratio were increased by 110% and 20%, respectively. The level of reactive oxygen species (ROS) was reduced by 60%. The adenovirus reproductive ability of the PYC2-A-P-L cells was approximately 30-fold higher than that of the control. The results showed that proper overexpression of the , and genes can significantly improve the substance and energy metabolism efficiency in HEK-293 cells, maximize the metabolic balance of pyruvate, and ultimately improve HAdV reproduction. This study provides a method of regulation of pyruvate metabolism and polygenic metabolic engineering in mammalian cells cultured and suggests an effective method for efficient HAdV production.
人胚肾293（HEK - 293）细胞越来越多地用于生产人腺病毒（HAdV）疫苗。然而，HAdV疫苗的生产尚未达到工业生产的要求。最近，我们研究了丙酮酸代谢节点的各种调控基因对HEK - 293细胞中物质和能量代谢以及腺病毒繁殖的影响。最初，研究了单个调控基因，包括[此处原文缺失具体基因名称]、[此处原文缺失具体基因名称]、[此处原文缺失具体基因名称]、[此处原文缺失具体基因名称]、[此处原文缺失具体基因名称]、[此处原文缺失具体基因名称]和[此处原文缺失具体基因名称]。我们发现HEK - 293细胞的代谢性能和腺病毒繁殖能力得到了改善，腺病毒最高滴度提高了约15倍。接下来，我们共过表达了关键基因，包括[此处原文缺失具体基因名称]、[此处原文缺失具体基因名称]和[此处原文缺失具体基因名称]。三个基因具有适当共过表达水平的PYC2 - A - P - L细胞具有最显著的调控作用。与对照相比，最大细胞密度和最大比生长速率提高了21%。ΔLac/ΔGlc和ΔNH/ΔGln分别降低了26%和27%。ATP产生速率和ATP/O比值分别提高了110%和20%。活性氧（ROS）水平降低了60%。PYC2 - A - P - L细胞的腺病毒繁殖能力比对照高约30倍。结果表明，[此处原文缺失具体基因名称]、[此处原文缺失具体基因名称]和[此处原文缺失具体基因名称]基因的适当过表达可以显著提高HEK - 293细胞中的物质和能量代谢效率，使丙酮酸的代谢平衡最大化，并最终提高HAdV的繁殖。本研究提供了一种在培养的哺乳动物细胞中调控丙酮酸代谢和多基因代谢工程的方法，并提出了一种高效生产HAdV的有效方法。
