(437b) Discovery of a Pathway for Halogenated, Terminal Alkene, and Terminal Alkyne Amino Acid Biosynthesis

Living systems have been able to construct an enormous range of functions from a relatively limited set of functional groups, especially compared to the functional group and chemical reaction diversity that is available to synthetic chemists. As such, several bio-orthogonal reactions have been developed; taking advantage of the absence of certain functional groups inside the cell that can then be used as a specific chemical handle for a reacting partner when incorporated into a metabolite or macromolecule of interest. One of the most useful of these bio-orthogonal functional groups has been the terminal alkyne, which can be used within the complex cellular milieu to react selectively with another bio-orthogonal group, the azide, to ligate the two reacting partners in a biocompatible copper-catalyzed azide alkyne cycloaddition (CuAAC or “Cu-Click”) reaction. Since its discovery, the CuAAC has found broad utility in a wide range of applications such as attachment of fluorescent probes, pull down and discovery of small molecule and protein binding partners, ligation to bioactive payloads (e.g. antibody-drug conjugates), or modifying macromolecular solubility and stability (e.g. PEGylation or tethering to solid supports). More recently, “Click” reactions for selective ligation of terminal-alkenes with tetrazines and tetrazoles have also been developed. However, the application of these reaction to living systems has thus far been constrained by the requirement that both substrates must be prepared by chemical synthesis and supplied exogenously, and thus is limited in application.

Here we present the discovery and characterization of a natural biosynthetic pathway for terminal alkyne amino acid, β-ethynylserine (βes), from Streptomyces cattleya. The enzymes uncovered in this pathway perform surprising biochemistry that can be leveraged for the production of various halo, terminal-alkene, and terminal-alkyne L-amino acids. The ability to genetically encode the biosynthesis of halo, alkene, or terminal alkyne amino acids can enable new applications, such as targeting proteomic tags to tissues in a living organism, while offering advantages in spatiotemporal control, reduced toxicity, and reduced background when compared to traditional feeding/injection. Additionally, engineering hosts that can produce these amino acids from glucose can reduce the costs for industrial-scale production of proteins and enzymes that contain non-canonical amino acids as building blocks. Taken together, this work paves the way for metabolically expanding the amino acid repertoire of living systems to include new amino acids that have unique chemistries and function.