Department of Chemical and Biological Engineering, Iowa State University, Ames, IA, 50011, USA.
J Ind Microbiol Biotechnol. 2019 Jun;46(6):843-853. doi: 10.1007/s10295-019-02158-6. Epub 2019 Feb 26.
The economic viability of bio-production processes is often limited by damage to the microbial cell membrane and thus there is a demand for strategies to increase the robustness of the cell membrane. Damage to the microbial membrane is also a common mode of action by antibiotics. Membrane-impermeable DNA-binding dyes are often used to assess membrane integrity in conjunction with flow cytometry. We demonstrate that in situ assessment of the membrane permeability of E. coli to SYTOX Green is consistent with flow cytometry, with the benefit of lower experimental intensity, lower cost, and no need for a priori selection of sampling times. This method is demonstrated by the characterization of four membrane engineering strategies (deletion of aas, deletion of cfa, increased expression of cfa, and deletion of bhsA) for their effect on octanoic acid tolerance, with the finding that deletion of bhsA increased tolerance and substantially decreased membrane leakage.
生物生产过程的经济可行性通常受到微生物细胞膜损伤的限制，因此需要寻找提高细胞膜稳定性的策略。抗生素对微生物细胞膜的破坏也是其常见的作用模式。膜不可渗透的 DNA 结合染料通常与流式细胞术一起用于评估细胞膜的完整性。我们证明，使用 SYTOX Green 原位评估大肠杆菌细胞膜的通透性与流式细胞术一致，其优势在于实验强度更低、成本更低，并且不需要预先选择采样时间。该方法通过对四种膜工程策略（aas 缺失、cfa 缺失、cfa 过表达和 bhsA 缺失）进行表征，以评估其对辛酸耐受性的影响，结果发现 bhsA 缺失可提高耐受性并显著降低膜泄漏。
