Prytz Ingela, Sandén Anna Maria, Nyström Thomas, Farewell Anne, Wahlström Asa, Förberg Cecilia, Pragai Zoltan, Barer Mike, Harwood Colin, Larsson Gen
The Swedish Centre for Bioprocess Technology, Stockholm Centre for Physics, Astronomy and Biotechnology, SE-106 91 Stockholm, Sweden.
Biotechnol Bioeng. 2003 Sep 5;83(5):595-603. doi: 10.1002/bit.10716.
A high-level production system using the universal stress promoters uspA and uspB in a fed-batch cultivation based on minimal medium was designed. In development it was shown that a standard industrial fed-batch protocol could not be used for this purpose since it failed to induce the levels of product as compared to the basal level. Instead, a batch protocol followed by a low constant feed of glucose was shown to give full induction. The levels of the product protein, beta-galactosidase, corresponded to approximately 25% of the total protein. Higher levels were found using the uspA than uspB vectors where uspA showed considerably higher basal level. The data indicate that the sigma(70) regulated promoter, uspA, although affected by the alarmone guanosine tetraphosphate, ppGpp, worked partly in a similar manner to constitutive promoters. An industrial high cell density fed-batch cultivation on the basis of the suggested fed-batch protocol and the uspA promoter gave a final beta-galatosidase concentration of 7 g/L and a final cell concentration of 65 g/L. The heterogeneity in production of the individual cell was measured by fluorescence microscopy. The data show that there is a process time independent heterogeneity in production, which is suggested to be caused by heterogeneity in the substrate uptake rate of the individual cell.
设计了一种基于基本培养基的补料分批培养中使用通用应激启动子uspA和uspB的高级生产系统。在开发过程中发现，标准的工业补料分批培养方案不能用于此目的，因为与基础水平相比，它未能诱导出产物水平。相反，先进行分批培养方案，然后以低恒定速率补加葡萄糖，结果显示能实现完全诱导。产物蛋白β-半乳糖苷酶的水平约占总蛋白的25%。使用uspA载体时的水平高于uspB载体，其中uspA显示出相当高的基础水平。数据表明，σ70调控的启动子uspA虽然受警报素四磷酸鸟苷（ppGpp）影响，但部分作用方式与组成型启动子相似。基于所建议的补料分批培养方案和uspA启动子进行的工业高细胞密度补料分批培养，最终β-半乳糖苷酶浓度为7 g/L，最终细胞浓度为65 g/L。通过荧光显微镜测量单个细胞生产过程中的异质性。数据表明，生产过程中存在与时间无关的异质性，这被认为是由单个细胞底物摄取速率的异质性引起的。
