Dynamic assembly and subcellular localization of class I lanthipeptide biosynthesis complexes

Lanthipeptides belong to a family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) containing (methyl)lanthionine residues. Commonly, class I lanthipeptides are synthesized by a putative membrane-associated lanthionine synthetase complex, which consists of the dehydratase LanB, the cyclase LanC, and the ABC transporter LanT. Despite intensive mechanistic studies on the individual proteins, the details on how these proteins interact are still not elucidated. In this thesis, we described the subcellular localization and assembly process of the nisin biosynthetic machinery NisBTC in the coccoid bacterium Lactococcus lactis by mutational analyses and fluorescence microscopy. Notably, the dynamic recruitment of NisT by NisBC from the cell membrane to the old cell pole was observed. Employing a platform we developed for nisin production in the rod-shaped bacterium Bacillus subtilis, the formation dynamics of the NisBTC complex were further investigated, which are likely associated with proteins residing in lipid rafts. From the above systems, the intact NisBTC complex was successfully isolated using pull-down assays. Furthermore, the cooperative actions of such a complex were systematically characterized. Besides, we evaluated the cross-functionality within LanBTC synthetase complexes from the nisin and subtilin systems. To solve the structure of the ABC transporter LanT, we developed an optimized two-step purification strategy and performed crystallization trials of two homologous transporters, i.e. NisT and SpaT. Overall, these comprehensive studies emphasize the crucial role of larger assemblies of multi-proteins during the lanthipeptide biosynthesis, and lay a foundation for further structural and functional characterization of the LanBTC complex