Nagao Jun-ichi, Aso Yuji, Shioya Kouki, Nakayama Jiro, Sonomoto Kenji
Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan.
J Mol Microbiol Biotechnol. 2007;13(4):235-42. doi: 10.1159/000104749.
Lanthionine-containing peptide antibiotics called lantibiotics are produced by a large number of Gram-positive bacteria. Nukacin ISK-1 produced by Staphylococcus warneri ISK-1 is type-A(II) lantibiotic. Ribosomally synthesized nukacin ISK-1 prepeptide (NukA) consists of an N-terminal leader peptide followed by a C-terminal propeptide moiety that undergoes several post-translational modification events including unusual amino acid formation by the modification enzyme NukM, cleavage of leader peptide and export by the dual functional ABC transporter NukT, finally yielding a biologically active peptide. Unusual amino acids in lantibiotics contribute to biological activity and also structural stability against proteases. Thus, lantibiotic-synthesizing enzymes have a high potentiality for peptide engineering by introduction of unusual amino acids into desired peptides with altering biological and physicochemical properties, e.g., activity and stability, termed lantibiotic engineering. We report the establishment of a heterologous expression of nukacin ISK-1 biosynthetic gene cluster by the nisin-controlled expression system and discuss our recent progress in understanding of the biosynthetic enzymes for nukacin ISK-1 such as localization, molecular interaction in biophysical and biochemical aspects. Substrate specificity of the lantibiotic-synthesizing enzymes was evaluated by complementation of the biosynthetic enzymes (LctM and LctT) of closely related lantibiotic lacticin 481 for nukacin ISK-1 biosynthesis. We further explored a rapid and powerful tool for introduction of unusual amino acids by co-expression of hexa-histidine-tagged NukA and NukM in Escherichia coli.
一类名为羊毛硫抗生素的含羊毛硫氨酸的肽抗生素由大量革兰氏阳性细菌产生。沃氏葡萄球菌ISK-1产生的Nukacin ISK-1是A型（II）类羊毛硫抗生素。核糖体合成的Nukacin ISK-1前肽（NukA）由一个N端前导肽和一个C端前肽部分组成，该前肽部分会经历多个翻译后修饰事件，包括修饰酶NukM形成异常氨基酸、前导肽的切割以及双功能ABC转运蛋白NukT的输出，最终产生具有生物活性的肽。羊毛硫抗生素中的异常氨基酸有助于生物活性以及对蛋白酶的结构稳定性。因此，通过将异常氨基酸引入所需肽中以改变生物和物理化学性质（例如活性和稳定性），羊毛硫抗生素合成酶在肽工程方面具有很高的潜力，这被称为羊毛硫抗生素工程。我们报道了通过乳链菌肽控制表达系统建立Nukacin ISK-1生物合成基因簇的异源表达，并讨论了我们最近在理解Nukacin ISK-1生物合成酶方面的进展，例如其定位、生物物理和生化方面的分子相互作用。通过互补密切相关的羊毛硫抗生素乳酸乳球菌素481的生物合成酶（LctM和LctT）以进行Nukacin ISK-1生物合成，评估了羊毛硫抗生素合成酶的底物特异性。我们还通过在大肠杆菌中共表达六组氨酸标签的NukA和NukM，进一步探索了一种引入异常氨基酸的快速且强大的工具。
