(6f) Biosynthesis of Alkyne, Alkene, and Halogenated Amino Acids for Expanded Genetic Codes

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.

In this talk, I present the discovery and characterization of the natural, biosynthetic pathway for a terminal alkyne amino acid, β-ethynylserine (βes), from Streptomyces cattleya. Comparative genomics and comparative metabolomics were used to identify the genomic cluster and pathway intermediates, while analytical techniques were used to characterize the structure of individual chemical species. The enzymes uncovered in this pathway allow for the production of various halo, terminal alkene, and terminal alkyne L-amino acids from basic carbon sources, that can be genetically encoded and leveraged for novel applications. For example, we envision using elements of this pathway as proteomic tags that can be spatially and temporally targeted in living, complex hosts. Additionally, the βes pathway opens up the possibility of engineering industrial hosts that can seamlessly integrate halo, alkene, and alkyne amino acids into biomolecules, such as enzymes and natural product therapeutics, at scale. Taken together, this work presents an effort to bridge together advances in chemical biology (bioorthogonal reactions and genetic code expansion) with synthetic biology approaches of host engineering.

Marchand, J. A., Neugebauer, M. E., Ing, M. C., Lin, C.-I., Pelton, J. & Chang, M. C. Y. Discovery of a pathway for terminal-alkyne amino acid biosynthesis. Nature. (2019) doi:10.1038/s41586-019-1020-y.

Neugebauer, M. E., Sumida, K. H., Pelton, J. G., Mcmurry, J. L., Marchand, J. A. & Chang, M. C. Y. (2019). A family of radical halogenases for the engineering of amino-acid-based products Monica. Nat. Chem. Biol. (2019). doi: 0.1038/s41589-019-0355-x