High-Throughput Profiling of Prenylelongases Enables the Assembly of Modified Prenoids.

Modified (non-natural) prenoids are useful as chemical biology tools, designer fragrances, and building blocks of bioactive compounds. However, their laborious synthesis generally limits their widespread application. Although prenylelongases offer potential biocatalytic access to modified long-chain prenoids, these enzymes have remained underexplored since (i) they are difficult to assay in high throughput and (ii) their pyrophosphate substrates are challenging and expensive to synthesize. To address these gaps, we herein report how prenylelongases can be interrogated with continuous high-throughput assays using readily accessible monophosphates of their native and modified substrates, bypassing the need for laborious pyrophosphate synthesis. We demonstrate the utility of this assay platform for the biochemical characterization of a panel of diverse prenylelongases, their profiling with non-native substrates, and two exploratory engineering campaigns. Lastly, we demonstrate that wild-type PEs and their variants can be used to assemble modified prenoids selectively at the (semi)preparative scale. The methods and biochemical data herein will simplify the engineering of PEs toward expanded substrate scopes and facilitate biocatalytic access to modified prenoids and derivatives thereof.