UC Berkeley

The alkyne functionality has attracted much interest due to its diverse chemical and biological applications. However, the underdeveloped alkyne biosynthetic toolbox severely limits the applications of this functionality in drug discovery and chemical biology. De novo biosynthesis is a recently developing and one of the most promising strategies for installing the terminal alkyne functionality into various molecular scaffolds, including diverse bioactive natural products, through the in situ generation and incorporation of the terminal alkyne functionality. We recently elucidated an acyl carrier protein (ACP)-dependent alkyne biosynthetic pathway comprised of JamABC from Moorea producens JHB that produces a terminal alkyne-tagged polyketide using hexanoic acid as a starting substrate for terminal alkyne generation. However, little is known about ACP interactions with the other alkyne biosynthetic enzymes in the JamABC pathway, including an acyl-ACP ligase (JamA) and a membrane-bound bi-functional desaturase/acetylenase (JamB). In this study, we demonstrated that JamB has a more stringent interaction with ACPs than JamA and subsequently pursued two different strategies to pinpoint key protein-protein interactions between JamB and several non-cognate ACPs. We first employed rational engineering of a non-cognate ACP (TtuC from Teredinibacter turnerae T7901) through site-directed mutagenesis, which significantly improved its compatibility with JamB and suggested a possible electrostatic interaction at the ACP-JamB interface. We also utilized an error-prone PCR and screening protocol towards a second non-cognate ACP (PeACP from Pseudomonas entomophila L48) to identify two hot spots on that ACP that are important for interacting with JamB and yielded mutants which were better recognized by JamB than the wild-type ACP. This study thus not only provides 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.