Cheng Fang, Takala Timo M, Saris Per E J
Department of Applied Chemistry and Microbiology, University of Helsinki, Helsinki, Finland.
J Mol Microbiol Biotechnol. 2007;13(4):248-54. doi: 10.1159/000104754.
The lantibiotic nisin is produced by Lactococcus lactis. In the biosynthesis of nisin, the enzyme NisB dehydrates nisin precursor, and the enzyme NisC is needed for lanthionine formation. In this study, the nisA gene encoding the nisin precursor, and the genes nisB and nisC of the lantibiotic modification machinery were expressed together in vitro by the Rapid Translation System (RTS). Analysis of the RTS mixture showed that fully modified nisin precursor was formed. By treating the mixture with trypsin, active nisin was obtained. However, no nisin could be detected in the mixture without zinc supplementation, explained by the fact that NisC requires zinc for its function. The results revealed that the modification of nisin precursor, which is supposed to occur at the inner side of the membrane by an enzyme complex consisting of NisB, NisC, and the transporter NisT, can take place without membrane association and without NisT. This in vitro production system for nisin opens up the possibility to produce nisin variants that cannot be producedin vivo. Moreover, the system is a promising tool for utilizing the NisB and NisC enzymes for incorporation of thioether rings into medical peptides and hormones for increased stability.
羊毛硫抗生素乳链菌肽由乳酸乳球菌产生。在乳链菌肽的生物合成过程中，NisB酶使乳链菌肽前体脱水，而羊毛硫氨酸的形成需要NisC酶。在本研究中，编码乳链菌肽前体的nisA基因以及羊毛硫抗生素修饰机制的nisB和nisC基因通过快速翻译系统（RTS）在体外共同表达。对RTS混合物的分析表明，形成了完全修饰的乳链菌肽前体。用胰蛋白酶处理该混合物后，获得了活性乳链菌肽。然而，在不添加锌的混合物中未检测到乳链菌肽，这是因为NisC发挥功能需要锌。结果表明，乳链菌肽前体的修饰本应通过由NisB、NisC和转运蛋白NisT组成的酶复合物在膜内侧发生，但该修饰可以在没有膜结合和NisT的情况下进行。这种乳链菌肽的体外生产系统为生产体内无法产生的乳链菌肽变体开辟了可能性。此外，该系统是一种很有前景的工具，可利用NisB和NisC酶将硫醚环掺入医用肽和激素中以提高稳定性。
