Reconstitution and Optimization of the Marmesin Biosynthetic Pathway in Yeast

Marmesin is essential in plant defense systems and exhibits various biological activities. In this study, we reconstituted the marmesin biosynthetic pathway in the Saccharomyces cerevisiae BY4741 chassis. We engineered the aromatic amino acid (AAA) biosynthetic pathways by introducing Escherichia coli-derived ppsA to improve the availability of the AAA precursor phosphoenolpyruvate, overexpressing the feedback inhibition resistance genes ARO4K229L and ARO7G141S to direct the metabolic flux toward the tyrosine branch, and deleting ARO10, PDC5, and PDC6 to reduce the byproducts from the Ehrlich pathway. The umbelliferone 6-dimethylallyltransferase (U6DT) and marmesin synthase (MS) involved in marmesin synthesis were optimized to increase marmesin production. Marmesin production was improved by truncating the transmembrane domains of PcU6DT, FcMS, and AtCPR1 and increasing the copy numbers of the genes encoding the truncated enzymes. Finally, a marmesin titer of 27.7 mg/L was obtained in shake flasks using the engineered yeast strain MU5. The constructed marmesin-producing strain provides the foundation for the green and large-scale production of pharmaceutically important furanocoumarins.