Department of Molecular Genetics, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
ACS Synth Biol. 2022 May 20;11(5):1949-1957. doi: 10.1021/acssynbio.2c00088. Epub 2022 May 3.
The class II lanthipeptide mersacidin, a ribosomally synthesized and post-translationally modified peptide (RiPP), displays unique intramolecular structures, including a very small lanthionine ring. When applied in the growing field of RiPP engineering, these can add unique features to new-to-nature compounds with novel properties. Recently, a heterologous expression system for mersacidin in was developed to add its modification enzymes to the RiPP engineering toolbox and further explore mersacidin biosynthesis and leader-processing. The dedicated mersacidin transporter and leader protease MrsT was shown to cleave the leader peptide only partially upon export, transporting GDMEAA-mersacidin out of the cell. The extracellular protease AprE was shown to release active mersacidin in a second leader-processing step after transport. The conserved LanT cleavage site in the mersacidin leader is present in many other class II lanthipeptides. In contrast to mersacidin, the leader of these peptides is fully processed in one step. This difference with mersacidin leader-processing raises fundamentally interesting questions about the specifics of mersacidin modification and processing, which is also crucial for its application in RiPP engineering. Here, mutational studies of the mersacidin leader-core interface were performed to answer these questions. Results showed the GDMEAA sequence is crucial for both mersacidin modification and leader processing, revealing a unique leader layout in which a LanM recognition site is positioned downstream of the conserved leader-protease LanT cleavage site. Moreover, by identifying residues and regions that are crucial for mersacidin-type modifications, the wider application of mersacidin modifications in RiPP engineering has been enabled.
类 II 兰尼肽 mersacidin 是一种核糖体合成和翻译后修饰的肽（RiPP），具有独特的分子内结构，包括一个非常小的硫醚键环。当应用于 RiPP 工程的不断发展的领域时，这些结构可以为具有新颖特性的新天然化合物添加独特的特征。最近，在 中开发了一种 mersacidin 的异源表达系统，将其修饰酶添加到 RiPP 工程工具包中，以进一步探索 mersacidin 的生物合成和前导肽加工。专用的 mersacidin 转运蛋白和前导肽酶 MrsT 在出口时仅部分切割前导肽，将 GDMEAA-mersacidin 运出细胞。细胞外 蛋白酶 AprE 在运输后通过第二个前导肽加工步骤释放活性 mersacidin。许多其他类 II 兰尼肽的前导肽中都存在保守的 LanT 切割位点。与 mersacidin 不同，这些肽的前导肽在一步中完全加工。这种 mersacidin 前导肽加工与其他肽的差异提出了关于 mersacidin 修饰和加工的具体问题，这对于其在 RiPP 工程中的应用也至关重要。在这里，通过突变研究 mersacidin 前导核心界面来回答这些问题。结果表明，GDMEAA 序列对于 mersacidin 的修饰和前导肽加工都是至关重要的，揭示了一种独特的前导肽布局，其中 LanM 识别位点位于保守的前导肽酶 LanT 切割位点的下游。此外，通过确定对 mersacidin 型修饰至关重要的残基和区域，使得 mersacidin 修饰在 RiPP 工程中的更广泛应用成为可能。
