Regulation of the pyruvate metabolism node by monogene and polygene engineering of HEK-293 cells

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 sub...

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Autores principales: Xie, Li, Miao, Junqing, Li, Xiangchao, Yi, Xiaoping, Chu, Ju
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Royal Society of Chemistry 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9074685/
https://www.ncbi.nlm.nih.gov/pubmed/35528064
http://dx.doi.org/10.1039/c9ra07418j
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author Xie, Li
Miao, Junqing
Li, Xiangchao
Yi, Xiaoping
Chu, Ju
author_facet Xie, Li
Miao, Junqing
Li, Xiangchao
Yi, Xiaoping
Chu, Ju
author_sort Xie, Li
collection PubMed
description 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 pkm2, pdhα, pyc2, mpc3, aralar1, ldha and pdk1, 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 pkm2, pyc2 and aralar1. 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(3)/ΔGln were decreased by 26% and 27%, respectively. The ATP production rate and the ATP/O(2) 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 aralar1, pkm2 and pyc2 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 in vitro and suggests an effective method for efficient HAdV production.
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spelling pubmed-90746852022-05-06 Regulation of the pyruvate metabolism node by monogene and polygene engineering of HEK-293 cells Xie, Li Miao, Junqing Li, Xiangchao Yi, Xiaoping Chu, Ju RSC Adv Chemistry 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 pkm2, pdhα, pyc2, mpc3, aralar1, ldha and pdk1, 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 pkm2, pyc2 and aralar1. 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(3)/ΔGln were decreased by 26% and 27%, respectively. The ATP production rate and the ATP/O(2) 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 aralar1, pkm2 and pyc2 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 in vitro and suggests an effective method for efficient HAdV production. The Royal Society of Chemistry 2019-11-07 /pmc/articles/PMC9074685/ /pubmed/35528064 http://dx.doi.org/10.1039/c9ra07418j Text en This journal is © The Royal Society of Chemistry https://creativecommons.org/licenses/by-nc/3.0/
spellingShingle Chemistry
Xie, Li
Miao, Junqing
Li, Xiangchao
Yi, Xiaoping
Chu, Ju
Regulation of the pyruvate metabolism node by monogene and polygene engineering of HEK-293 cells
title Regulation of the pyruvate metabolism node by monogene and polygene engineering of HEK-293 cells
title_full Regulation of the pyruvate metabolism node by monogene and polygene engineering of HEK-293 cells
title_fullStr Regulation of the pyruvate metabolism node by monogene and polygene engineering of HEK-293 cells
title_full_unstemmed Regulation of the pyruvate metabolism node by monogene and polygene engineering of HEK-293 cells
title_short Regulation of the pyruvate metabolism node by monogene and polygene engineering of HEK-293 cells
title_sort regulation of the pyruvate metabolism node by monogene and polygene engineering of hek-293 cells
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9074685/
https://www.ncbi.nlm.nih.gov/pubmed/35528064
http://dx.doi.org/10.1039/c9ra07418j
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