Purification and optimization of sequential biosynthetic enzymes for co-localization onto a nanocarrier

Flavonoids are polyphenolic plant secondary metabolites with many purposes, including providing pigmentation in plants and anti-oxidant and anti-cancerous activities in humans. There are over 10,000 different flavonoids known, making them one of the largest groups of natural products. One major sub-class of flavonoids is the flavan-3-ols, which are known for their health benefits and the ability to form proanthocyanidins, or condensed tannins. Many of the intermediates in this pathway are unstable and have not been isolated in planta. Another natural product of interest is sclareol, a diterpene alcohol, which is important in the fragrance and flavor industry as a precursor to Ambrox, and ambergris analog that can be used as a fixative in high end perfumes. In this study we are aiming to create a synthetic complex using a polymer nanocarrier to co-localize two sequential enzymes in a pathway. The first system tried included the final two enzymes in flavan-3-ol biosynthesis, anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR). Each of these enzymes, along with the preceding enzyme, dihydroflavonol 4-reductase (DFR) has been recombinantly expressed in E. coli with a specific tag for binding to the nanocarrier. However, due to difficulty achieving ANS activity a backup system involved in sclareol synthesis was developed. Two enzymes, NgCPS and sSsSS, were used to convert geranylgeranyl-pyrophosphate (GGPP) to sclareol in a coupled assay. Each enzyme was purified with either a 6xHis tag or monomeric DM3 streptavidin tag for attachment to the nanocarrier. Assays to measure each enzymes activity have been designed, and our goal now is to test each enzyme’s activity before and after co-localization onto the nanocarrier.