Metabolic engineering of CHO cells towards cysteine prototrophy and systems analysis of the ensuing phenotype

IF 6.8 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Laura Greenfield , Mariah Brantley , Pauline Geoffroy , Jeffrey Mitchell , Dylan DeWitt , Fang Zhang , Bhanu Chandra Mulukutla
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Abstract

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.

面向半胱氨酸原生质的 CHO 细胞代谢工程及后续表型的系统分析。
中国仓鼠卵巢(CHO)细胞的生长和生产需要半胱氨酸。在强化工艺中,由于半胱氨酸在溶液中的溶解度有限且不稳定,因此补充高浓度的半胱氨酸是一项挑战。蛋氨酸可转化为半胱氨酸(CYS),但 CHO 细胞中的关键酶--胱硫醚 beta 合成酶(Cbs)和胱硫醚 gamma-lyase 酶(Cth)--并不活跃,导致细胞培养环境中积累了中间产物--同型半胱氨酸(HCY)。在这项研究中,Cbs 和 Cth 在 CHO 细胞中过度表达,以赋予细胞半胱氨酸原营养能力,即在无半胱氨酸环境中生长的能力。这些细胞池(CbCt)需要同型半胱氨酸和β-巯基乙醇(βME)才能在不含 CYS 的培养基中生长。为了增加细胞内的同型半胱氨酸水平,在 CbCt 细胞池中过表达 Gnmt。由此产生的细胞池(GnCbCt)在不含 CYS 的培养基中适应后,残留 HCY 和 βME 水平下降,能够在不含 HCY、βME 和 CYS 的环境中增殖。有趣的是,CbCt 池也能适应在无 HCY 和无 CYS 的条件下生长,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。此外,从 GnCbCt 细胞池中提取的单细胞克隆的 Cbs、Cth 和 Gnmt 表达水平差异很大,在无 CYS 喂养批次条件下培养时,其表现与在 CYS 补充喂养批次培养条件下培养的野生型(WT)细胞系相似。细胞内代谢组分析表明,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,HCY 和谷胱甘肽(GSH)水平恢复到接近 WT 水平。转录组分析表明,GnCbCt 细胞上调了几个编码转运体以及蛋氨酸分解和转硫化途径酶的基因,这些基因支持这些细胞有效地生物合成半胱氨酸。此外,'omics'分析表明,CbCt池在无CYS条件下处于铁蛋白应激状态,当抑制铁蛋白应激时,可增强这些细胞在无CYS条件下的生长和活力。
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来源期刊
Metabolic engineering
Metabolic engineering 工程技术-生物工程与应用微生物
CiteScore
15.60
自引率
6.00%
发文量
140
审稿时长
44 days
期刊介绍: Metabolic Engineering (MBE) is a journal that focuses on publishing original research papers on the directed modulation of metabolic pathways for metabolite overproduction or the enhancement of cellular properties. It welcomes papers that describe the engineering of native pathways and the synthesis of heterologous pathways to convert microorganisms into microbial cell factories. The journal covers experimental, computational, and modeling approaches for understanding metabolic pathways and manipulating them through genetic, media, or environmental means. Effective exploration of metabolic pathways necessitates the use of molecular biology and biochemistry methods, as well as engineering techniques for modeling and data analysis. MBE serves as a platform for interdisciplinary research in fields such as biochemistry, molecular biology, applied microbiology, cellular physiology, cellular nutrition in health and disease, and biochemical engineering. The journal publishes various types of papers, including original research papers and review papers. It is indexed and abstracted in databases such as Scopus, Embase, EMBiology, Current Contents - Life Sciences and Clinical Medicine, Science Citation Index, PubMed/Medline, CAS and Biotechnology Citation Index.
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