Metabolic Engineering of Yarrowia lipolytica for De Novo Biosynthesis of Umbelliferone

Umbelliferone, a key precursor for the biosynthesis of high-value furanocoumarin derivatives, is produced via the shikimate pathway. The oleaginous yeast Yarrowia lipolytica possesses an inherently robust shikimate pathway; however, de novo biosynthesis of umbelliferone in this microbial chassis has not been realized to date. Low intracellular pools of p-coumaric acid and suboptimal catalytic activity of downstream hydroxylation and cyclization enzymes represent the major bottlenecks limiting efficient umbelliferone production. In this study, we systematically screened five PAL/TAL variants, three 4CL isoforms, and two C2′H enzymes derived from diverse plant and microbial sources. The bifunctional enzyme RtPAL-TAL exhibited the highest productivity of p-coumaric acid, while the combination of At4CL3 and RgC2′H achieved the maximum umbelliferone accumulation. Expression of feedback-insensitive shikimate pathway mutant enzymes effectively relieved intrinsic pathway feedback inhibition, resulting in a 10.2-fold improvement in p-coumaric acid titer. All synthetic biosynthetic gene cassettes were stably integrated into the Y. lipolytica genome to generate the engineered strain YL-PU, which yielded 3.29 mg/L umbelliferone in shake-flask cultures. Further fed-batch fermentation in a 5 L bioreactor increased the umbelliferone titer to 125.52 mg/L at 84 h of cultivation. This work establishes the first de novo umbelliferone biosynthesis pathway in Y. lipolytica and identifies that C2′H represents a major candidate bottleneck under the tested configuration, in agreement with earlier findings. These findings provide a robust foundation for further metabolic engineering of the Y. lipolytica chassis to enable high-titer production of coumarins and other phenylpropanoid natural products.