Christian Doppler Laboratory for Production of Next-Level Biopharmaceuticals in E. coli, Institute of Bioprocess Science and Engineering, Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
Institute of Biophysics, Department of Bionanosciences, University of Natural Resources and Life Sciences, Muthgasse 11, 1190 Vienna, Austria.
Int J Mol Sci. 2023 Jan 30;24(3):2641. doi: 10.3390/ijms24032641.
Stress-associated changes in the mechanical properties at the single-cell level of () cultures in bioreactors are still poorly investigated. In our study, we compared peptide-producing and non-producing BL21(DE3) cells in a fed-batch cultivation with tightly controlled process parameters. The cell growth, peptide content, and cell lysis were analysed, and changes in the mechanical properties were investigated using atomic force microscopy. Recombinant-tagged somatostatin-28 was expressed as soluble up to 197 ± 11 mg g. The length of both cultivated strains increased throughout the cultivation by up to 17.6%, with nearly constant diameters. The peptide-producing cells were significantly softer than the non-producers throughout the cultivation, and respective Young's moduli decreased by up to 57% over time. A minimum Young's modulus of 1.6 MPa was observed after 23 h of the fed-batch. Furthermore, an analysis of the viscoelastic properties revealed that peptide-producing BL21(DE3) appeared more fluid-like and softer than the non-producing reference. For the first time, we provide evidence that the physical properties (i.e., the mechanical properties) on the single-cell level are significantly influenced by the metabolic burden imposed by the recombinant peptide expression and C-limitation in bioreactors.
在生物反应器中，（）培养物的单细胞水平的机械性能的应激相关变化仍然研究不足。在我们的研究中，我们比较了在严格控制工艺参数的分批补料培养中产生肽和不产生肽的 BL21(DE3)细胞。分析了细胞生长、肽含量和细胞裂解情况，并使用原子力显微镜研究了机械性能的变化。重组标记的生长抑素-28以可溶性形式表达，最高可达 197±11mg g。两种培养菌株的长度在整个培养过程中增加了 17.6%，直径几乎保持不变。在整个培养过程中，产肽细胞明显比非产肽细胞柔软，相应的杨氏模量随时间降低了高达 57%。在补料批处理 23 小时后，观察到最小杨氏模量为 1.6 MPa。此外，粘弹性分析表明，产肽 BL21(DE3)比非产肽参考品表现出更类似流体的性质和更柔软的性质。这是首次提供证据表明，在生物反应器中，重组肽表达和 C 限制所带来的代谢负担会显著影响单细胞水平的物理性质（即机械性质）。
