ELIMINATION OF THE 'ESSENTIAL' WARBURG EFFECT IN MAMMALIAN CELLS THROUGH A MULTIPLEX GENOME ENGINEERING STRATEGY

摘要

The Warburg effect has posed a constant challenge in mammalian bioprocessing since the field began. Indeed, the predisposition of mammalian cells to secrete large quantities of lactic acid through the Warburg effect leads to premature cell death, reduced product yields, and often lower quality products. Thus, over the past decades, numerous innovations in the mammalian cell culture field have focused on mitigating lactate secretion, including through media optimization, feeding control, chemical inhibition, etc. Despite extensive efforts from many researchers, complete elimination of lactic acid production has not yet been obtained. Specifically, several independent efforts to knock out lactate dehydrogenase (the enzyme responsible for producing lactic acid from pyruvate) have been unsuccessful, as it has seemed essential for immortalized cell growth. Here I present our work in which we discovered a panel of genes involved in a genetic feedback circuit that controls lactic acid secretion in mammalian cells. Knocking out individual genes in serial was unsuccessful since LdhA and other targets are essential for CHO cell growth. However, we knocked out these genes simultaneously and overcame the "essentiality" of these genes, leading to the successful elimination of lactic acid secretion in Chinese hamster ovary cells. Since many hypotheses have been proposed regarding the essentiality of lactic acid secretion for rapid cell proliferation in cancer, immune cell activation, and embryonic development, we were interested to study how the complete elimination of the Warburg effect impacts CHO cells. Surprisingly, the cells show improved metabolic and growth phenotypes, despite the elimination of this fundamental metabolic activity. To understand how immortalized mammalian cells can cope without this seemingly essential metabolic process, we conducted a comprehensive analysis of these cell lines using time-course RNA-Seq, metabolomics, and analysis with a genome-scale metabolic network model developed for Chinese hamster ovary cells1. We further characterized its impact on recombinant drug production yields and quality. Thus, through a multiplex metabolic engineering effort and comprehensive systems biology analysis, we have been able to engineer out a leading challenge in protein biotherapeutic development and begin to understand now a cell can survive without a seemingly essential process.