Division of Systems and Synthetic Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, SE-412 96, Gothenburg, Sweden; Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, SE-412 96, Gothenburg, Sweden.
Division of Systems and Synthetic Biology, Department of Biology and Biological Engineering, Chalmers University of Technology, SE-412 96, Gothenburg, Sweden.
Metab Eng. 2022 Jul;72:311-324. doi: 10.1016/j.ymben.2022.04.005. Epub 2022 May 1.
High-level production of recombinant proteins in industrial microorganisms is often limited by the formation of misfolded proteins or protein aggregates, which consequently induce cellular stress responses. We hypothesized that in a yeast Alzheimer's disease (AD) model overexpression of amyloid-β peptides (Aβ42), one of the main peptides relevant for AD pathologies, induces similar phenotypes of cellular stress. Using this humanized AD model, we previously identified suppressors of Aβ42 cytotoxicity. Here we hypothesize that these suppressors could be used as metabolic engineering targets to alleviate cellular stress and improve recombinant protein production in the yeast Saccharomyces cerevisiae. Forty-six candidate genes were individually deleted and twenty were individually overexpressed. The positive targets that increased recombinant α-amylase production were further combined leading to an 18.7-fold increased recombinant protein production. These target genes are involved in multiple cellular networks including RNA processing, transcription, ER-mitochondrial complex, and protein unfolding. By using transcriptomics and proteomics analyses, combined with reverse metabolic engineering, we showed that reduced oxidative stress, increased membrane lipid biosynthesis and repressed arginine and sulfur amino acid biosynthesis are significant pathways for increased recombinant protein production. Our findings provide new insights towards developing synthetic yeast cell factories for biosynthesis of valuable proteins.
在工业微生物中高水平生产重组蛋白常常受到错误折叠的蛋白质或蛋白质聚集体的形成的限制，这会导致细胞应激反应。我们假设在酵母阿尔茨海默病（AD）模型中，淀粉样β肽（Aβ42）的过度表达，是与 AD 病理相关的主要肽之一，会诱导类似的细胞应激表型。我们使用这种人类 AD 模型，先前鉴定了 Aβ42 细胞毒性的抑制剂。在这里，我们假设这些抑制剂可用作代谢工程的靶点，以减轻细胞应激并提高酵母酿酒酵母中重组蛋白的生产。单独删除了 46 个候选基因，并单独过表达了 20 个基因。增加重组α-淀粉酶生产的阳性靶基因进一步组合，导致重组蛋白产量增加了 18.7 倍。这些靶基因参与多个细胞网络，包括 RNA 加工、转录、内质网-线粒体复合物和蛋白质展开。通过使用转录组学和蛋白质组学分析，并结合反向代谢工程，我们表明降低氧化应激、增加膜脂生物合成以及抑制精氨酸和硫氨基酸生物合成是增加重组蛋白生产的重要途径。我们的研究结果为开发用于生物合成有价值蛋白质的合成酵母细胞工厂提供了新的见解。
