Deciphering molecular drivers of lactate metabolic shift in mammalian cell cultures

Lactate metabolism plays a critical role in mammalian cell bioprocessing, influencing cellular performance and productivity. The transition from lactate production to consumption, known as lactate metabolic shift, is highly beneficial and has been shown to extend culture lifespan and enhance productivity, yet its molecular drivers remain poorly understood. Here, we have explored the mechanisms that underpin this metabolic shift through two case studies, illustrating environmental- and genetic-driven factors. We characterised these study cases at process, metabolic and transcriptomic levels. Our findings indicate that glutamine depletion coincided with the timing of the lactate metabolic shift, significantly affecting cell growth, productivity and overall metabolism. Transcriptome analysis revealed dynamic regulation the ATF4 pathway, involved in the amino acid (starvation) response, where glutamine depletion activates ATF4 gene and its targets. Manipulating ATF4 expression through overexpression and knockdown experiments showed significant changes in metabolism of glutamine and lactate, impacting cellular performance. Overexpression of ATF4 increased cell growth and glutamine consumption, promoting a lactate metabolic shift. In contrast, ATF4 downregulation decreased cell proliferation and glutamine uptake, leading to production of lactate without any signs of lactate shift. These findings underscore a critical role for ATF4 in regulation of glutamine and lactate metabolism, related to phasic patterns of growth during CHO cell culture. This study offers unique insight into metabolic reprogramming during the lactate metabolic shift and the molecular drivers that determine cell status during culture.