The alkyne functionality has attracted much interest due to its diverse chemical and biological applications. We recently elucidated an acyl carrier protein (ACP)-dependent alkyne biosynthetic pathway, however, little is known about ACP interactions with the alkyne biosynthetic enzymes, an acyl-ACP ligase (JamA) and a membrane-bound bifunctional desaturase/ acetylenase (JamB). Here, we showed that JamB has a more stringent interaction with ACP than JamA. In addition, sitedirected mutagenesis of a non-cognate ACP significantly improved its compatibility with JamB, suggesting a possible electrostatic interaction at the ACP-JamB interface. Finally, error-prone PCR and screening of a second non-cognate ACP identified hot spots on the ACP that are important for interacting with JamB and yielded mutants which were better recognized by JamB. Our data thus not only provide insights into the ACP interactions in alkyne biosynthesis, but it also potentially aids in future combinatorial biosynthesis of alkyne-tagged metabolites for chemical and biological applications. Figure 4. Relative JamB in vivo activities toward ACPs. The compounds 1 and 2 were produced in an approximately 1:5 ratio by the strain XZ1, and this efficiency was set to be a relative JamB activity of 100%. Error bars represent standard deviations from at least three experiments. 4258 Figure 8. Modeled structures of PeACP and JamB. Left: A modeled ribbon diagram of PeACP. The five PeACP residues as hot spots for providing JamB compatibility upon mutation are shown as sticks. Right: Modeled surface structure of the JamB-PeACP complex. The two hot spots of the JamB-PeACP binding interface that were identified in the error-prone PCR and screening are marked. [Color figure can be viewed at wileyonlinelibrary.com] AIChE Journal