Biosynthesis and engineering of lanthipeptide natural products for novel applications

Natural products have played prominent roles in science and medicine due to their diverse chemical scaffolds and biological activities. Ribosomally synthesized and post-translationally modified peptides (RiPPs) have recently been recognized as a major class of natural products as a result of the genome sequencing efforts. The post-translational modifications endow them with diverse and rigid structures such as polycylic or macrocyclic scaffolds, which are valuable resources for developing new molecules with potential pharmaceutical applications. The studies described herein focus on the biosynthesis and engineering of lanthipeptide natural products, a large group of polycyclic RiPPs classified by their intramolecular thioether crosslinks, for expanded functionalities. Protein-protein interactions (PPI) have been involved in many important biological and pathological processes, and are challenging drug targets for small molecules due to the relatively large and complex interaction interfaces. Cyclic RiPPs provide promising scaffolds for developing PPI inhibitors due to their 3D-like structure that mimics the native ligand, and the stability against cellular degradation. In Chapter 2, it is demonstrated that the substrate-tolerant lanthipeptide biosynthetic pathway could be utilized for combinatorial production of cyclic peptide libraries in Escherichia coli. One library with a ring scaffold derived from a native lanthipeptide was successfully coupled to a genetic selection system to select for inhibitors against the UEV-p6 interaction during HIV virus budding from the host cell. The hit compound generated from the selection exhibited in vitro activity against the target PPI, demonstrating for the first time that an active lanthipeptide natural product could be successfully evolved from an originally inactive scaffold under defined selection pressure. In an effort to search for new lanthipeptide biosynthetic clusters, a new class of synthetases was discovered that is involved in the D-amino acid formation in lanthipeptides. Chapter 3 describes the first in vitro reconstitution and characterization of a dehydrogenase that carries out the asymmetric reduction of dehydroalanine to D-alanine. The enzyme displays great substrate tolerance allowing incorporation of D-alanines into a range of non-native substrates. As ribosomally synthesized peptides are generally limited to L-amino acid building blocks, methods to incorporate D-stereocenters for structure stabilization are valuable. This chapter demonstrated using E. coli as biosynthetic host to produce D-alanine-containing ribosomal peptides. The observed substrate tolerance of lanthipeptide synthetases has lead to the hypothesis that not only the enzyme, but also the substrates in part determine the outcome of product formation. Regarding the cyclization process, one possible mechanism would be thermodynamic control of ring formation, which requires the cyclization reaction to be reversible in order to obtain the desired ring topology. Chapter 4 demonstrates for the class I lanthipeptide cyclase NisC and class II lanthipeptide synthetase HalM2 that, indeed, the conjugate addition reactions are reversible and that the enzymes can open up all thioether rings in their products. The studies described in Chapter 5 focus on the mode of action of haloduracin (Halα and Halβ), a two-component lanthipeptide bacteriocin (termed lantibiotic) that act in synergy to exhibit antimicrobial activity. It was previously proposed for the two-component lantibiotics that the α and β peptides recognize lipid II at the cell surface, and the lipid II-α-β complex induces pore formation on the membranes. In this chapter, lipid II analogues were synthesized that demonstrated the Halα-lipid II interaction. Interestingly, leakage assay on model membranes suggested that lipid II is not required for the membrane disruption activity, and the mode of action model was revised to include that the association of the α and β peptides with membrane phospholipids induces membrane disruption