Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore.
J Biotechnol. 2010 May 17;147(2):116-21. doi: 10.1016/j.jbiotec.2010.03.018. Epub 2010 Apr 2.
We have established a liquid chromatography-mass spectrometry based metabolomics platform to identify extracellular metabolites in the medium of recombinant Chinese hamster ovary (CHO) fed-batch reactor cultures. Amongst the extracellular metabolites identified, malate accumulation was the most significant. The contributing factors to malate efflux were found to be the supply of aspartate from the medium, and an enzymatic bottleneck at malate dehydrogenase II (MDH II) in the tricarboxylic acid cycle. Subsequent metabolic engineering to overexpress MDH II in CHO resulted in increases in intracellular ATP and NADH, and up to 1.9-fold improvement in integral viable cell number.
我们建立了一个基于液相色谱-质谱的代谢组学平台，以鉴定重组中国仓鼠卵巢（CHO）补料分批培养物中培养基中的细胞外代谢物。在鉴定出的细胞外代谢物中，苹果酸的积累最为显著。导致苹果酸外排的因素被发现是来自培养基的天冬氨酸供应，以及三羧酸循环中天冬氨酸苹果酸酶 II（MDH II）的酶学瓶颈。随后在 CHO 中过表达 MDH II 的代谢工程导致细胞内 ATP 和 NADH 增加，整体活细胞数提高了 1.9 倍。
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