UC Berkeley

The terminal alkyne functionality is widely used in organic synthesis, pharmaceutical science, and materials. It is also a useful moiety in the azide-alkyne [3+2] cycloaddition reaction (often known as “click chemistry”), which has recently emerged to be one of the most powerful tools in drug discovery and chemical biology. In particular, by tagging medicinally active natural products with alkynes and coupling them with subsequent click chemistry, researchers have enriched, visualized, and studied the mode of action of natural products. Despite the importance of the alkyne functionality, its applications in natural product research are limited by the underexplored alkyne biosynthetic tools and tagging strategies.

In this study, we first elucidated an unprecedented carrier protein–dependent terminal alkyne biosynthetic pathway in microbes that featured an acyl-acyl carrier protein (ACP) synthetase (JamA), a membrane-bound bifunctional desaturase/acetylenase (JamB), and an ACP (JamC). We further demonstrated that this enzymatic machinery can be exploited for the in situ generation and incorporation of terminal alkynes into two natural product scaffolds (a polyketide pyrone, and antimycin, respectively) in Escherichia coli. However, JamB showed stringent substrate specificities and weak catalytic activities, which restricted its broader applications in synthetic and chemical biology. We addressed these limitations in two ways. In silico analysis suggested that this carrier protein–dependent terminal alkyne biosynthetic mechanism could be widespread in bacteria. We screened additional gene cassettes that are homologous to jamABC and discovered a new terminal alkyne biosynthetic pathway comprised of TtuABC from Teredinibacter turnerae T7901. Different from JamABC, TtuABC displayed altered substrate specificities and improved catalytic activities. Using TtuABC, we further in situ generated and incorporated the alkyne functionality into another medicinally important natural product, a peptidyl epoxyketone. In a parallel effort, directed evolution was adopted to create a JamB variant with improved catalytic efficiency in E. coli. To quickly assess the protein engineering outcomes, we developed a new platform for quantifying extracellular alkyne-tagged metabolites through a fluorogenic click reaction. Overall, this study not only provides us with alkyne biosynthetic tools that are useful in synthetic and chemical biology, but it can also find applications in which enzymatic generation of terminal alkynes and in situ click chemistry is required or preferred.