Heterologous Biosynthesis of Curcuminoids in Mammalian Cells

The plant molecule curcumin possesses useful medicinal properties. The abilities to scavenge free radicals, activate antioxidant response genes, and downregulate inflammatory pathways make curcumin a potent anti-inflammatory molecule. However, therapeutic use of curcumin is drastically limited by its poor bioavailability, low aqueous solubility, and rapid metabolism. To overcome these limitations, mammalian cells were engineered to express a set of plant derived enzymes catalyzing the biosynthesis of curcumin from tyrosine. Use of CRISPR/Cas9 gene integration can allow for stable curcumin production in-vitro to reduce cellular levels of reactive oxygen species. To facilitate CRISPR delivery, polymer-based vectors are preferable due to their high carrying capacity. Polyethylenimine (PEI) is considered the “gold standard” of polymer-based gene delivery. Despite this, PEI demonstrates low transfection efficiency in serum. Previous work in this lab determined that modifying PEI through succinylation improves gene delivery. In this study, the transfection efficiencies of various percent modified succinylated polyethylenimine (zPEI) types were evaluated using pGL3 firefly luciferase to determine optimal polyplex composition. From this analysis, zPEI-2% demonstrated the best transfection efficiency at the desired low polymer:DNA weight ratios. To evaluate CRISPR introduction HEK cells were transfected with optimized polyplexes containing plasmids for green fluorescent protein (GFP) and Cas9. Stability of CRISPR integration was analyzed by assessing GFP fluorescence over multiple passages. GFP assays and fluorescence imaging proved cells treated with the Cas9 enzyme obtained lasting gene expression. Additionally, cells treated with zPEI-2% polyplexes outperformed those treated with uPEI polyplexes. Finally, optimized polyplexes facilitated CRISPR introduction of the TAL/4CL1/CUS plasmid encoding curcumin producing enzymes. Fluorescence analysis of curcumin production produced inconsistent results, indicating the need to improve this method.