Elucidation of the biosynthesis pathway and heterologous construction of a sustainable route for producing umbelliferone

Coumarins play roles in many biological processes. Angelica decursiva is one of the major sources of coumarins in China. Due to increasing demand for coumarins in the marketplace, traditional extraction from plants is now considered economically insufficient and unsustainable. Microbial synthesis is a promising strategy for scalable production of coumarins. However, the biosynthetic pathway of coumarin remains poorly understood, and even more, the genes associated with this process have not been characterized in A. decursiva. RNA-seq was employed to elucidate the umbelliferone biosynthetic pathway. The results indicated that three enzymes, phenylalanine ammonia-lyase (PAL), 4-Coumarate: Coenzyme A Ligase (4CL), and p-coumaroyl CoA 2'-hydroxylase (C2’H) were involved in umbelliferone biosynthesis. Using the cloned genes, we generated a synthetic biology based microbial cell factory that produces coumarins from tyrosine utilizing Rhodotorula glutinis tyrosine ammonia lyase (RgTAL) to bypass cinnamic acid 4-hydroxylase (C4H). With metabolic engineering strategies, we deleted prephenate dehydratase (pheA), anthranilate synthase (trpE) and transcriptional regulatory protein (tyrR) and overexpressed six related genes involved in tyrosine biosynthesis, to drive the carbon flux from tyrosine. To overcome the limitation of 4CL, a virtual screening and site-specific mutagenesis-based protein engineering approach was applied. In addition, induction/culture conditions and different ions were employed to further improve the yield of umbelliferone. Finally, a yield of 356.59 mg/L umbelliferone was obtained. The current study elucidated the umbelliferone biosynthesis pathway in A. decursiva. The results also demonstrated the feasibility of integrating gene mining with synthetic biology techniques to produce natural compounds.