Reconstruction of reverse transsulfuration pathway enables cysteine biosynthesis and enhances resilience to oxidative stress in Chinese Hamster Ovary cells

Abstract

Cysteine is a critically important amino acid necessary for mammalian cell culture, playing key roles in nutrient supply, disulfide bond formation, and as a precursor to antioxidant molecules controlling cellular redox. Unfortunately, its low stability and solubility in solution make it especially problematic as an essential medium component that must be added to Chinese hamster ovary and other mammalian cell cultures. Therefore, CHO cells have been engineered to include the capacity of endogenously synthesizing cysteine by overexpressing multiple enzymes, including cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CTH) and glycine N-methyltransferase (GNMT) to reconstruct the reverse transsulfuration pathway and overcome a key metabolic bottleneck. Some limited cysteine biosynthesis was obtained by overexpressing CBS and CTH for converting homocysteine to cysteine but robust metabolic synthesis from methionine was only possibly after incorporating GNMT which likely represents a key bottleneck step in the cysteine biosynthesis pathway. CHO cells with the reconstructed pathway exhibit the strong capability to proliferate in cysteine-limited and cysteine-free batch and fed-batch cultures at levels comparable to wildtype cells with ample cysteine supplementation, providing a selectable marker for CHO cell engineering. GNMT overexpression led to the accumulation of sarcosine byproduct, but its accumulation did not affect cell growth. Furthermore, pathway reconstruction enhanced CHO cells’ reduced and glutathione levels in cysteine-limited conditions compared to unmodified cells, and greatly enhanced survivability and maintenance of redox homeostasis under oxidative stress induced by addition of menadione in cysteine-deficient conditions. Such engineered CHO cell lines can potentially reduce or even eliminate the need to include cysteine in culture medium, which not only reduces the cost of mammalian media but also promises to transform media design by solving the challenges posed by low stability and solubility of cysteine and cystine in future mammalian biomanufacturing processes.