Eliminating an Essential Gene Underlying the Warburg Effect through a Multiplex Engineering Strategy

Mammalian cells are the predominant hosts for biotherapeutic production, and produce 6 of the top 10 pharmaceuticals by sales. However, major challenges exist that must be controlled to improve the safety, efficacy, and affordability of these pharmaceuticals. One major challenge plaguing industry stems from a complex phenotype of rapidly proliferating cells. Specifically, the cells secrete large quantities of lactic acid through the Warburg effect. This leads to premature cell death, reduced product yields, and lower quality products. In an effort to control the Warburg effect, numerous efforts have tried, and failed to eliminate the key enzymes involved in lactic acid secretion, since the enzymes have proven essential for immortalized cell growth. Here we found that the "essential" gene circuit controlling the Warburg effect can be successfully knocked-out through a multiplex genome editing strategy, allowing us to eliminate lactic acid secretion. Hypotheses surrounding the role of the Warburg effect in cancer and mammalian biotherapetic production suggest that the Warburg effect is needed to produce biomass and ATP for rapid proliferation. However, surprisingly, our 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. Finally, since the Warburg effect has previously inhibited cell growth and drug production, we investigated how the elimination of this gene circuit impacts the yield and quality of monoclonal antibody drugs. Thus, through a multiplex genome editing effort and comprehensive systems biology analysis, we have been able to target a gene circuit to engineer out a leading challenge in protein biotherapeutic development and begin to understand now a cell can survive without a seemingly essential process.