© 2015 Nature America, Inc. The terminal alkyne is a functionality widely used in organic synthesis, pharmaceutical science, material science and bioorthogonal chemistry. This functionality is also found in acetylenic natural products, but the underlying biosynthetic pathways for its formation are not well understood. Here we report the characterization of what is to our knowledge the first carrier protein-dependent terminal alkyne biosynthetic machinery in microbes. We further demonstrate that this enzymatic machinery can be exploited for the in situ generation and incorporation of terminal alkynes into two natural product scaffolds in Escherichia coli. These results highlight the prospect for tagging major classes of natural products, including polyketides and polyketide/nonribosomal peptide hybrids, using biosynthetic pathway engineering.