A Chemoenzymatic Approach to Accessing Novel Isoprenoid Scaffolds

Project Summary
Isoprenoids represent a diverse class of compounds with a broad range of applications in medicine and industry.
Their extraction from natural sources is both challenging and potentially harmful to the environment, while the
enormous structural complexity of many isoprenoids makes traditional chemical synthesis nontrivial. Modern
metabolic engineering and synthetic biology approaches have overcome some of these difficulties, but issues
related to metabolic flux and the limited availability of the universal isoprenoid precursors complicate their
widespread implementation. The artificial pathways developed thus far have been solely focused on synthesizing
dimethylallyl and isopentenyl diphosphates and require additional enzymes for the generation of polyprenyl-
diphosphates (polyprenyl-PPs). Thus, the primary objective of this proposal is to develop an efficient strategy for
the synthesis of both natural and unnatural (poly)prenyl-PPs for downstream applications. This will be achieved
using two complementary methods: i) employing undecaprenol kinases and isopentenyl phosphate kinases; and
ii) employing hydroxyethylthiazole kinase, isopentenyl phosphate kinases, and farnesyl diphosphate synthase.
Additionally, the two methods will work in conjunction with isoprenoid methylatransferases to incorporate
additional diversity into the polyprenyl-PPs. The proposed studies include: i) structural and functional assessment
of selected enzymes, ii) catalyst engineering, and iii) optimization of coupled in vitro and in vivo platforms for
the generation of diversified libraries of select natural products. We expect these studies to generate: i) rules and
concepts to advance knowledge on structure-activity relationships in selected classes of enzymes; ii) an optimized,
enzyme-coupled platform to generate diversified substrates and isoprenoids; and iii) novel isoprenoid analogs
with potential therapeutic applications. Thus, the proposed work will offer unprecedented access to uniquely
bioactive isoprenoid libraries not readily accessible via traditional methods, and it stands to deepen our
fundamental understanding of four enzyme classes while also developing them into useful biocatalysts.