Protein Expression, Novo Nordisk A/S, Novo Nordisk Park 1, 2760-Måløv, Denmark.
Microb Cell Fact. 2011 Dec 7;10:104. doi: 10.1186/1475-2859-10-104.
The adaptation of unicellular organisms like Saccharomyces cerevisiae to alternating nutrient availability is of great fundamental and applied interest, as understanding how eukaryotic cells respond to variations in their nutrient supply has implications spanning from physiological insights to biotechnological applications.
The impact of a step-wise restricted supply of phosphate on the physiological state of S. cerevisiae cells producing human Insulin was studied. The focus was to determine the changes within the global gene expression of cells being cultured to an industrially relevant high cell density of 33 g/l cell dry weight and under six distinct phosphate concentrations, ranging from 33 mM (unlimited) to 2.6 mM (limited). An increased flux through the secretory pathway, being induced by the PHO circuit during low P(i) supplementation, proved to enhance the secretory production of the heterologous protein. The re-distribution of the carbon flux from biomass formation towards increased glycerol production under low phosphate led to increased transcript levels of the insulin gene, which was under the regulation of the TPI1 promoter.
Our study underlines the dynamic character of adaptive responses of cells towards a change in their nutrient access. The gradual decrease of the phosphate supply resulted in a step-wise modulated phenotypic response, thereby alternating the specific productivity and the secretory flux. Our work emphasizes the importance of reduced phosphate supply for improved secretory production of heterologous proteins.
单细胞生物（如酿酒酵母）对交替养分供应的适应具有重要的基础和应用意义，因为了解真核细胞如何响应其养分供应的变化，不仅有助于深入了解生理学机制，还能为生物技术应用提供启示。
本研究考察了逐步限制磷酸盐供应对生产人胰岛素的酿酒酵母细胞生理状态的影响。重点是确定在六种不同磷酸盐浓度（从 33 mM（无限制）到 2.6 mM（限制））下，培养到工业相关的高细胞密度（33 g/l 细胞干重）的细胞中，其整体基因表达的变化。在低磷补充时，PHO 回路诱导的分泌途径通量增加，被证明可以提高异源蛋白的分泌产量。在低磷酸盐条件下，碳通量从生物量形成重新分配到甘油产量增加，导致胰岛素基因的转录水平增加，该基因受 TPI1 启动子的调控。
我们的研究强调了细胞对营养物质获取变化的适应性反应的动态特征。磷酸盐供应的逐渐减少导致逐步调节的表型反应，从而交替调节特定生产力和分泌通量。我们的工作强调了减少磷酸盐供应对提高异源蛋白分泌生产的重要性。
