ENGINEERED METABOLIC PATHWAYS
FOR THE SYNTHESIS OF PLANT NATURAL PRODUCTS
The structural and chemical diversity of plant natural products (PNPs) offers an enormous chemical space from which molecules with beneficial characteristics can be discovered and produced. To date, thousands of PNPs have been exploited for human benefit with applications ranging from drugs and nutraceuticals to industrial chemicals. However, agricultural and geographic variations and extraction of the desired compound(s) poses a significant challenge for cost-effective products due to the high cost associated with downstream processing and purification. Furthermore, native metabolic pathways for product synthesis often limit the attainable product titers, rates, and yields due to intrinsic inefficiencies and negative cross-talk between product-forming and growth-sustaining reactions. To address these limitations, we are working on engineering microbial-based platforms for the efficient synthesis of PNPs. This includes a synthetic pathway for the production of isoprenoids (PNAS June 25, 2019 116 (26) 12810-12815) and a PKS-independent platform for the synthesis of polyketide backbones (Manuscript in Review).(PNAS June 25, 2019 116 (26) 12810-12815).
Our current efforts focus on developing microbial platforms for the synthesis of isoprenoid and polyketide products as well as their derivatives, including compounds like prenylated aromatics (PAs) that are currently sourced from plants. We have used plant enzymes for product synthesis in Escherichia coli, such as the functional expression of a polyketide synthase and olivetolic acid cyclase from Cannabis sativa, which when combined with engineering pathways for precursor generation enabled olivetolic acid production (ACS Synthetic Biology 7: 1886, 2018). However, in many cases, such as the integral membrane aromatic prenyltransferases (aPTases) involved in the production of PAs, functional heterologous expression of the plant enzymes in microbial systems can prove a major challenge. We have demonstrated how the use of soluble, bacterial-derived enzymes catalyzing the condensation reaction between a prenyl chain and an aromatic ring can be used in place of plant aPTases to enable the synthesis of PAs in bacteria. Key to our approach was the engineering of soluble aPTases through protein modeling and rational design, resulting in significant improvement to their catalytic efficiency. Expression of engineered aPTases coupled with exogenous addition of aromatic substrates and pyrophosphate supply through an engineered mevalonate pathway enabled the synthesis of an array of PA compounds, including medicinally important cannabigerovarinic, cannabigerolic, and grifolic acids (Biotechnol. Bioeng. doi:10.1002/bit.26932, 2019). Finally, we have also engineered non-natural routes for the generation of the prenyl and polyketide moieties used in the synthesis of PAs. This includes a synthetic pathway for the production of isoprenoids and a PKS-independent platform for the synthesis of polyketide backbones (PNAS June 25, 2019 116 (26) 12810-12815). These new systems enable the efficient synthesis of isoprenoids, polyketides, and their derivatives, including PAs, in microorganisms.