细胞生长、增殖和代谢过程中,个体间通过静电作用力或蛋白质间分子作用维持相对悬浮状态,有利于营养物质、底物和产物的传递。细胞培养完成或发酵结束后沉降方式收集细胞可以大幅度节约离心收集或板框过滤收集等方式所需要的设备和能量消耗。细胞外膜作为维持个体细胞相对悬浮状态的主要细胞结构,其多糖组成和附着在多糖中的膜蛋白可能为细胞个体悬浮提供了相应的分子间作用力。考察细胞外膜中占比最高的脂多糖的减量合成及其可能对细胞自凝集过程的影响,将有助于实现菌体细胞的可控沉降。该研究以乳酸单体高产菌种大肠杆菌LG101为出发菌株,通过阻断脂多糖部分合成途径,构建脂多糖减量合成的新菌种LGL4A06。系统评价了新菌种脂多糖减量合成情况,以及对细胞结构、生长性能、代谢性能和自凝集性能等影响。结果显示,相比出发菌株,菌株LGL4A06脂多糖的含量降低了39.34%,单位OD600菌体干重减少32.46%;与出发菌株不同,菌株LGL4A06在显微镜下细胞出现聚集,摇瓶培养条件下,菌体细胞最终积累量相当但比生长速率降低;摇瓶培养条件下的LGL4A06菌体细胞培养液,静置12 h菌体自凝集率可达83.20%,是出发菌株的13.9倍;通过不同温度和钙、镁离子添加对菌体自凝集特性的影响分析显示,自凝集特性与环境温度的相关性不显著,添加钙离子(10 mmol/L)和镁离子(10 mmol/L)静置4 h后,LGL4A06自凝集率分别提高了95.00%和83.00%,进一步提高了菌株自凝集速率。该研究证实通过减量合成脂多糖可以提高细胞自凝集效率,有助于实现菌体细胞的可控沉降,促进生物质分离过程,并降低发酵产品制备过程下游工艺的生产成本。
During cell growth, proliferation, and metabolism, relative suspension state can be maintained through electrostatic forces between cells and intermolecular forces between proteins. It’s beneficial for cells to uptake of nutrients and substrates. The collection of cells by sedimentation at the end of cell culture or fermentation could significantly reduce the equipment and energy consumption required for centrifugal collection or plate and frame filter collection. Lipopolysaccharide (LPS) is a primary component of the cell membrane. Its structure and composition are directly related to cell auto-aggregation capacity, and consequently LPS plays a significant role in the cell sedimentation process for separating biomass and broth. In this study, Escherichia coli LGL4A06 with less LPS synthesis ability was constructed from E. coli LG101 through partially blocking the LPS synthesis pathway. Compared with the E. coli LG101, LPS content and the biomass dry weight (with the same cell density) of LGL4A06 decreased by 32.46% and 39.34%, respectively. The auto-aggregation rate of LGL4A06 reached 83.40% after 12 h of standing, which was 13.9 times higher than E. coli LG101. However, a low growth rate of LGL4A06 was observed. The effect of metal ions and temperatures on auto-aggregation was investigated at different ranges. In the presence of Ca2+ and Mg2+(10 mmol/L), the auto-aggregation rate of LGL4A06 was increased by 95.00% and 83.00%, respectively. No significant effect on cell auto-aggregation was observed with increasing temperature from 4 ℃ to 37 ℃. The present investigation focused on decreasing LPS content and enhancing the auto-aggregation efficiency of cells, which could facilitate the biomass separation process and reduce the time consumption of the downstream process during fermentation.
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