Promiscuity of an Alcohol-Dependent Hemiterpene Pathway for the In Vivo Production of a Non-Natural Alkylated...

The prenyl motif determines the biological activity of many natural products. Yet, structural diversification of the prenyl site has been restricted due to the limitations of native biosynthetic pathways to hemiterpenes, the universal isoprenoid building blocks. Previously, we developed the artificial alcohol dependent hemiterpene (ADH) pathway comprising the acid phosphatase PhoN and the isopentenyl kinase IPK to provide natural isoprenoids assembled from hemiterpenes in vivo. Here, we revealed the broad specificity of the first enzyme of the ADH module, PhoN, and a downstream aromatic prenyltransferase. We then showed that the combined promiscuity of the ADH module and prenyltransferase were sufficient to produce a non-natural-alkylated tryptophan derivative in vivo when coupled with the previously described promiscuity of IPK. The short and modular ADH pathway provides a convenient and scalable approach to alkyl-pyrophosphates and facilitates probing the promiscuity of other downstream enzymes involved in isoprenoid biosynthesis without the tedious in vitro preparation of alkyl-pyrophosphates. This sets the stage to leverage the ADH pathway to forward engineer isoprenoid biosynthesis and expand its chemical space accessible to synthetic biology.