Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Gothenburg, Sweden.
Appl Environ Microbiol. 2021 Jul 13;87(15):e0030121. doi: 10.1128/AEM.00301-21.
Recombinant protein production is a known source of oxidative stress. However, knowledge of which reactive oxygen species are involved or the specific growth phase in which stress occurs remains lacking. Using modern, hypersensitive genetic HO-specific probes, microcultivation, and continuous measurements in batch culture, we observed HO accumulation during and following the diauxic shift in engineered Saccharomyces cerevisiae, correlating with peak α-amylase production. In agreement with previous studies supporting a role of the translation initiation factor kinase Gcn2 in the response to HO, we find that Gcn2-dependent phosphorylation of eIF2α increases alongside translational attenuation in strains engineered to produce large amounts of α-amylase. Gcn2 removal significantly improved α-amylase production in two previously optimized high-producing strains but not in the wild type. Gcn2 deficiency furthermore reduced intracellular HO levels and the Hac1 splicing ratio, while expression of antioxidants and the endoplasmic reticulum (ER) disulfide isomerase increased. These results suggest protein synthesis and ER oxidative folding are coupled and subject to feedback inhibition by HO. Recombinant protein production is a multibillion dollar industry. Optimizing the productivity of host cells is, therefore, of great interest. In several hosts, oxidants are produced as an unwanted side product of recombinant protein production. The buildup of oxidants can result in intracellular stress responses that could compromise the productivity of the host cell. Here, we document a novel protein synthesis inhibitory mechanism that is activated by the buildup of a specific oxidant (HO) in the cytosol of yeast cells upon the production of recombinant proteins. At the center of this inhibitory mechanism lies the protein kinase Gcn2. By removing Gcn2, we observed a doubling of recombinant protein productivity in addition to reduced HO levels in the cytosol. In this study, we want to raise awareness of this inhibitory mechanism in eukaryotic cells to further improve protein production and contribute to the development of novel protein-based therapeutic strategies.
重组蛋白的生产是氧化应激的已知来源。然而，对于涉及哪些活性氧物种或应激发生的特定生长阶段，人们仍然知之甚少。使用现代、高敏感的 HO 特异性遗传探针、微量培养和分批培养中的连续测量，我们观察到工程化酿酒酵母在双相型转变过程中以及之后 HO 的积累，这与峰值α-淀粉酶的产生相关。与支持翻译起始因子激酶 Gcn2 在对 HO 反应中起作用的先前研究一致，我们发现，在工程化生产大量α-淀粉酶的菌株中，与翻译衰减同时增加的是 Gcn2 依赖性的 eIF2α 磷酸化。在两种先前优化的高产菌株中，Gcn2 的缺失显著提高了α-淀粉酶的产量，但在野生型中则没有。Gcn2 缺陷进一步降低了细胞内 HO 水平和 Hac1 剪接比，同时增加了抗氧化剂和内质网（ER）二硫键异构酶的表达。这些结果表明，蛋白质合成和 ER 氧化折叠是耦合的，并受到 HO 的反馈抑制。
重组蛋白的生产是一个价值数十亿美元的产业。因此，优化宿主细胞的生产力具有重要意义。在几种宿主中，氧化剂是作为重组蛋白生产的不必要副产物产生的。氧化剂的积累会导致细胞内应激反应，从而影响宿主细胞的生产力。在这里，我们记录了一种新的蛋白质合成抑制机制，该机制是在酵母细胞的细胞质中产生特定氧化剂（HO）时被激活的，当生产重组蛋白时。这种抑制机制的核心是蛋白激酶 Gcn2。通过去除 Gcn2，我们观察到重组蛋白生产力提高了一倍，同时细胞质中的 HO 水平也降低了。在这项研究中，我们希望提高人们对真核细胞中这种抑制机制的认识，以进一步提高蛋白质生产，并为新型蛋白质治疗策略的发展做出贡献。
