Reconstruction of reverse transsulfuration pathway enables cysteine biosynthesis and enhances resilience to oxidative stress in Chinese Hamster Ovary cells
Reconstruction of reverse transsulfuration pathway enables cysteine biosynthesis and enhances resilience to oxidative stress in Chinese Hamster Ovary cells
复制标题
逆转硫途径的重建使中国仓鼠卵巢细胞能够进行半胱氨酸生物合成并增强对氧化应激的抵抗力
DOI：
10.1016/j.ymben.2023.02.010
复制
发表时间：
2023
影响因子：
8.4
通讯作者：
Betenbaugh, Michael J.
中科院分区：
文献类型：
--
作者：
Chen, Yiqun;Betenbaugh, Michael J.
半胱氨酸是哺乳动物细胞培养所必需的极其重要的氨基酸，在营养供应、二硫键形成以及作为控制细胞氧化还原的抗氧化剂分子的前体中发挥着关键作用。不幸的是，它在溶液中的稳定性和溶解度较低，使其作为必须添加到中国仓鼠卵巢和其他哺乳动物细胞培养物中的基本培养基成分尤其成问题。因此，CHO细胞经过改造，通过过表达多种酶（包括胱硫醚β-合酶（CBS）、胱硫醚γ-裂解酶（CTH）和甘氨酸N-甲基转移酶（GNMT））来具有内源合成半胱氨酸的能力，以重建反向转硫途径并克服关键的代谢瓶颈。通过过表达 CBS 和 CTH 将同型半胱氨酸转化为半胱氨酸获得了一些有限的半胱氨酸生物合成，但只有在并入 GNMT 后才可能实现来自蛋氨酸的稳健代谢合成，这可能代表半胱氨酸生物合成途径中的关键瓶颈步骤。具有重建途径的 CHO 细胞在半胱氨酸限制和无半胱氨酸分批和补料分批培养物中表现出强大的增殖能力，其水平与补充充足半胱氨酸的野生型细胞相当，为 CHO 细胞工程提供了选择标记。 GNMT过表达导致肌氨酸副产物的积累，但其积累并不影响细胞生长。此外，与未修饰的细胞相比，通路重建增强了 CHO 细胞在半胱氨酸限制条件下的还原谷胱甘肽水平，并大大增强了在半胱氨酸缺乏条件下添加甲萘醌诱导的氧化应激下的生存能力和氧化还原稳态的维持。这种工程化的CHO细胞系有可能减少甚至消除培养基中包含半胱氨酸的需要，这不仅降低了哺乳动物培养基的成本，而且有望通过解决未来哺乳动物生物制造过程中半胱氨酸和胱氨酸的低稳定性和溶解度带来的挑战来改变培养基设计。
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.