Kim S I, Nam Y S, Lee S Y
Graduate School of Biotechnology, Korea University, Seoul, Korea.
Mol Cells. 1997 Dec 31;7(6):788-94.
To improve protein production, a heterologous secretion vector system was constructed with the aid of the amyR2 region. The operator sequence (amyO) of the amyR2 region on the secretion vector was changed through site-directed mutagenesis to eliminate carbon-source-mediated catabolite repression. Three substitutional (AG, G5, G10), one deletional (delta HH), and one insertional (AGHF) mutant promoters were obtained. The expression level and the degree of catabolite repression of amyR2 and the mutant promoters were examined with a single copy system using an integrational promoter probe vector, pDH32. Under glucose-free culture conditions, expression levels from all mutant promoters except HH were 1.4 to 1.5 fold higher than that from amyR2. While the expression of the amyR2 promoter was repressed by 90% in the presence of 2% glucose, expression levels of the mutant promoters were repressed by only 1% to 50%. To evaluate the advantage of the mutant promoters in production of foreign proteins by the heterologous secretion system, beta-lactamase and human pancreatic secretory trypsin inhibitor (hPSTI) were expressed by the mutant promoters. When B. subtilis LKS87 was used as a host strain, the production of the target proteins using the respective mutant promoter was increased by about 1.5 fold under glucose-free culture conditions. Under the high glucose culture conditions, secretion of target proteins produced from the mutant promoters increased 1.5 to 2 fold, whereas those by the amyR2 promoter were reduced to between 50% and 60%. The additive effect of degUh mutation on protein production was not observed under high glucose culture conditions. In addition, such culture conditions inhibited proteolytic degradation of secreted target proteins after the stationary growth phase even in B. subtilis LKS88 (degUh mutant). Thus, our results indicated that the mutant promoters, which are resistant to glucose-mediated catabolite repression, are very useful for over-production of foreign proteins under the high glucose culture conditions using the heterologous expression-secretion system in B. subtilis.
为提高蛋白质产量，借助amyR2区域构建了一种异源分泌载体系统。通过定点诱变改变分泌载体上amyR2区域的操纵序列（amyO），以消除碳源介导的分解代谢物阻遏。获得了三个替换型（AG、G5、G10）、一个缺失型（delta HH）和一个插入型（AGHF）突变启动子。使用整合型启动子探针载体pDH32的单拷贝系统检测了amyR2和突变启动子的表达水平及分解代谢物阻遏程度。在无葡萄糖培养条件下，除HH外的所有突变启动子的表达水平比amyR2高1.4至1.5倍。虽然在2%葡萄糖存在时amyR2启动子的表达被抑制了90%，但突变启动子的表达水平仅被抑制1%至50%。为评估突变启动子在异源分泌系统生产外源蛋白中的优势，用突变启动子表达了β-内酰胺酶和人胰腺分泌性胰蛋白酶抑制剂（hPSTI）。当枯草芽孢杆菌LKS87用作宿主菌株时，在无葡萄糖培养条件下，使用相应突变启动子生产目标蛋白的产量提高了约1.5倍。在高葡萄糖培养条件下，突变启动子产生的目标蛋白分泌增加了1.5至2倍，而amyR2启动子产生的目标蛋白分泌减少到50%至60%。在高葡萄糖培养条件下未观察到degUh突变对蛋白质生产的累加效应。此外，即使在枯草芽孢杆菌LKS88（degUh突变体）中，这种培养条件也抑制了稳定生长期后分泌的目标蛋白的蛋白水解降解。因此，我们的结果表明，对葡萄糖介导的分解代谢物阻遏具有抗性的突变启动子对于在高葡萄糖培养条件下使用枯草芽孢杆菌中的异源表达-分泌系统过量生产外源蛋白非常有用。
