Brockmeier Ulf, Caspers Michael, Freudl Roland, Jockwer Alexander, Noll Thomas, Eggert Thorsten
Institut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf, im Forschungszentrum Jülich, D-52426 Jülich, Germany.
J Mol Biol. 2006 Sep 22;362(3):393-402. doi: 10.1016/j.jmb.2006.07.034. Epub 2006 Jul 26.
Efficient protein secretion is very important in biotechnology as it provides active and stable enzymes, which are an essential prerequisite for successful biocatalysis. Therefore, optimizing enzyme-producing bacterial strains is a major challenge in the field of biotechnology and protein production. In this study, the Gram-positive model bacterium Bacillus subtilis was optimized for heterologous protein secretion using a novel approach. Two lipolytic enzymes, cutinase from Fusarium solani pisi and a cytoplasmatic esterase of metagenomic origin, were chosen as reporters for heterologous protein secretion. In a systematic screening approach, all naturally occurring (non-lipoprotein) Sec-type signal peptides (SPs) from B. subtilis were characterized for their potential in heterologous protein secretion. Surprisingly, optimal SPs in cutinase secretion were inefficient in esterase secretion and vice versa, indicating the importance of an optimal fit between the SP and the respective mature part of the desired secretion target proteins. These results highlight the need for individually optimal signal peptides for every heterologous secretion target. Therefore, the SP library generated in this study represents a powerful tool for secretion optimization in Gram-positive expression hosts.
高效的蛋白质分泌在生物技术中非常重要，因为它能提供有活性且稳定的酶，而这是成功进行生物催化的必要前提。因此，优化产酶细菌菌株是生物技术和蛋白质生产领域的一项重大挑战。在本研究中，采用一种新方法对革兰氏阳性模式细菌枯草芽孢杆菌进行了异源蛋白质分泌的优化。选择了两种脂解酶，来自豌豆镰刀菌的角质酶和一种宏基因组来源的细胞质酯酶，作为异源蛋白质分泌的报告蛋白。在一种系统筛选方法中，对枯草芽孢杆菌所有天然存在的（非脂蛋白）Sec型信号肽（SPs）进行了表征，以确定它们在异源蛋白质分泌方面的潜力。令人惊讶的是，角质酶分泌中的最佳SPs在酯酶分泌中效率不高，反之亦然，这表明SP与所需分泌目标蛋白的各自成熟部分之间最佳匹配的重要性。这些结果凸显了针对每个异源分泌目标需要单独的最佳信号肽。因此，本研究中生成的SP文库是革兰氏阳性表达宿主中分泌优化的有力工具。
