中国仓鼠卵巢(CHO)细胞在补料分批培养中需要半胱氨酸才能生长和生产力。在强化过程中,由于其有限的溶解度和在溶液中的不稳定性,以高浓度补充半胱氨酸是一个挑战。蛋氨酸可以转化为半胱氨酸(CYS),但关键酶,胱硫醚β-合酶(Cbs)和胱硫醚γ-裂解酶(Cth),在CHO细胞中不活跃,导致中间体的积累,同型半胱氨酸(HCY),在细胞培养环境中。在这项研究中,Cbs和Cth在CHO细胞中过表达以赋予半胱氨酸原养型,即,在无半胱氨酸环境中生长的能力。这些池(CbCt)需要高半胱氨酸和β-巯基乙醇(βME)才能在无CYS的培养基中生长。为了增加细胞内同型半胱氨酸水平,Gnmt在CbCt池中过表达。得到的细胞池(GnCbCt),在无CYS培养基中适应后,残留HCY和βME水平降低,能够在无HCY的环境中扩散,无βME和无CYS环境。有趣的是,CbCt池也能够适应在无HCY和无CYS条件下生长,尽管倍增时间明显高于GnCbCt细胞,但不能完全适应无βME的条件。Further,来自GnCbCt细胞池的单细胞克隆具有广泛的Cbs表达水平,Cth和Gnmt,当在无CYS的分批补料条件下培养时,类似于在补充CYS的补料分批培养中培养的野生型(WT)细胞系进行。细胞内代谢组学分析显示,在无CYS条件下,CbCt池中的HCY和谷胱甘肽(GSH)水平较低,但在无CYS条件下培养的GnCbCt细胞中恢复到更接近WT水平。转录组分析表明,GnCbCt细胞上调了几个编码转运蛋白的基因以及蛋氨酸分解代谢和转硫途径酶,这些酶支持这些细胞有效地生物合成半胱氨酸。Further,组学分析表明,CbCt池在无CYS条件下处于铁胁迫下,which,当被抑制时,增强了这些细胞在无CYS条件下的生长和活力。
Chinese hamster ovary (CHO) cells require
cysteine for growth and productivity in fed-batch cultures. In intensified processes, supplementation of
cysteine at high concentrations is a challenge due to its limited solubility and instability in solution. Methionine can be converted to
cysteine (CYS) but key enzymes, cystathionine beta-synthase (Cbs) and cystathionine gamma-lyase (Cth), are not active in CHO cells resulting in accumulation of an intermediate, homocysteine (HCY), in cell culture milieu. In this study, Cbs and Cth were overexpressed in CHO cells to confer
cysteine prototrophy, i.e., the ability to grow in a
cysteine free environment. These pools (CbCt) needed homocysteine and beta-mercaptoethanol (βME) to grow in CYS-free medium. To increase intracellular homocysteine levels, Gnmt was overexpressed in CbCt pools. The resultant cell pools (GnCbCt), post adaptation in CYS-free medium with decreasing residual HCY and βME levels, were able to proliferate in the HCY-free, βME-free and CYS-free environment. Interestingly, CbCt pools were also able to be adapted to grow in HCY-free and CYS-free conditions, albeit at significantly higher doubling times than GnCbCt cells, but couldn\'t completely adapt to βME-free conditions. Further, single cell clones derived from the GnCbCt cell pool had a wide range in expression levels of Cbs, Cth and Gnmt and, when cultivated in CYS-free fed-batch conditions, performed similarly to the wild type (WT) cell line cultivated in CYS supplemented fed-batch culture. Intracellular metabolomic analysis showed that HCY and glutathione (GSH) levels were lower in the CbCt pool in CYS-free conditions but were restored closer to WT levels in the GnCbCt cells cultivated in CYS-free conditions. Transcriptomic analysis showed that GnCbCt cells upregulated several genes encoding transporters as well as methionine catabolism and transsulfuration pathway enzymes that support these cells to biosynthesize cysteine effectively. Further, \'omics analysis suggested CbCt pool was under ferroptotic stress in CYS-free conditions, which, when inhibited, enhanced the growth and viability of these cells in CYS-free conditions.