BACKGROUND In recent years, the industrial workhorse Corynebacterium glutamicum has gained increasing interest as a host organism for the secretory production of heterologous proteins. Generally, the yield of a target protein in the culture supernatant depends on a multitude of interdependent biological and bioprocess parameters which have to be optimized. So far, the monitoring of such optimization processes depends on the availability of a direct assay for the respective target protein that can be handled also in high throughput approaches. Since simple assays, such as standard enzymatic activity assays, are not always at hand, the availability of a general protein secretion biosensor is highly desirable. RESULTS High level secretion of proteins via the Sec protein export pathway leads to secretion stress, a phenomenon that is thought to be caused by the accumulation of incompletely or misfolded proteins at the membrane-cell envelope interface. We have analyzed the transcriptional responses of C. glutamicum to the secretory production of two different heterologous proteins and found that, in both cases, the expression of the gene encoding a homologue of the extracytosolic HtrA protease was highly upregulated. Based on this finding, a C. glutamicum Sec secretion biosensor strain was constructed in which the htrA gene on the chromosome was replaced by the eyfp gene. The fluorescence of the resulting reporter strain responded to the secretion of different heterologous proteins (cutinase from Fusarium solani pisi and alkaline phosphatase PhoA from Escherichia coli) in a dose-dependent manner. In addition, three differently efficient signal peptides for the secretory production of the cutinase could be differentiated by the biosensor signal. Furthermore, we have shown that an efficient signal peptide can be separated from a poor signal peptide by using the biosensor signal of the respective cells in fluorescence activated cell sorting experiments. CONCLUSIONS We have succeeded in the construction of a C. glutamicum biosensor strain that allows for the monitoring of Sec-dependent secretion of heterologous proteins in a dose-dependent manner, independent of a direct assay for the desired target protein.

中文翻译：

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用于监测谷氨酸棒杆菌中 Sec 依赖性蛋白输出的分泌生物传感器。


背景技术近年来，工业主力谷氨酸棒杆菌作为分泌性生产异源蛋白质的宿主生物体越来越受到人们的关注。一般来说，培养物上清液中靶蛋白的产量取决于大量必须优化的相互依赖的生物学和生物过程参数。到目前为止，对此类优化过程的监测取决于是否可以对相应的靶蛋白进行直接测定，而这种直接测定也可以在高通量方法中进行处理。由于简单的测定（例如标准酶活性测定）并不总是可用，因此非常需要通用蛋白质分泌生物传感器的可用性。结果 通过 Sec 蛋白输出途径的高水平蛋白分泌会导致分泌应激，这种现象被认为是由膜-细胞包膜界面处不完全或错误折叠的蛋白积累引起的。我们分析了谷氨酸棒杆菌对两种不同异源蛋白分泌产生的转录反应，发现在这两种情况下，编码胞质外HtrA蛋白酶同源物的基因的表达高度上调。基于这一发现，构建了谷氨酸棒杆菌Sec分泌生物传感器菌株，其中染色体上的htrA基因被eyfp基因取代。所得报告菌株的荧光以剂量依赖性方式响应不同异源蛋白（茄病镰刀菌的角质酶和大肠杆菌的碱性磷酸酶 PhoA）的分泌。此外，可以通过生物传感器信号区分用于角质酶分泌产生的三种不同效率的信号肽。 此外，我们已经表明，通过在荧光激活细胞分选实验中使用各个细胞的生物传感器信号，可以将有效的信号肽与不良信号肽分开。结论我们已经成功构建了谷氨酸棒杆菌生物传感器菌株，该菌株允许以剂量依赖性方式监测异源蛋白的Sec依赖性分泌，而不依赖于对所需靶蛋白的直接测定。