Plants possess myriads of secondary metabolites with a broad spectrum of health-promoting benefits. To date, plant extraction is still the primary route to produce high-value natural products which inherently suffers from economics and scalability issues. Heterologous expression of plant biosynthetic gene clusters in microbial host is considered as a feasible approach to overcoming these limitations. Oleaginous yeast produces a large amount of lipid bodies, the abundant membrane structure and the lipophilic environment provide the ideal environment for the regioselectivity and stereoselectivity of many plant-derived P450 enzymes. In this work, we used modular method to construct, characterize, and optimize the flavonoid pathways in Yarrowia lipolytica. We also evaluated various precursor biosynthetic routes and unleashed the metabolic potential of Y. lipolytica to produce flavonoids and hydroxylated flavonoids. Specifically, we have identified that chalcone synthase (CHS) and cytochrome P450 reductases (CPR) were the bottlenecks of hydroxylated flavonoid production. We determined the optimal gene copy number of CHS and CPR to be 5 and 2, respectively. We further removed precursor pathway limitations by expressing genes associated with chorismate and malonyl-CoA supply. With pH and carbon−nitrogen ratio (C/N) optimization, our engineered strain produced 252.4 mg/L naringenin, 134.2 mg/L eriodictyol, and 110.5 mg/L taxifolin from glucose in shake flasks. Flavonoid and its hydroxylated derivatives are most prominently known as antioxidant and antiaging agents. These findings demonstrate our ability to harness the oleaginous yeast as the microbial workhorse to expand nature's biosynthetic potential, enabling us to bridge the gap between drug discovery and natural product manufacturing.

Conventional plasmid-based gene expression tends to introduce genetic instability and gene copy number variations that lead to degenerated production. The limited number of auxotrophic markers in Yarrowia lipolytica also restricts our ability to perform iterative genetic modifications and manipulate long gene clusters. To overcome these limitations, we combined the high recombination efficiency of the Cre-loxP system and the high integration rate of 26s rDNA, and developed a versatile framework to iteratively integrate multicopy metabolic pathways in Y. lipolytica. We demonstrated the efficient genome integration of a plant-derived flavonoid pathway at random sites with multiple copies. Transient expression of Cre recombinase enabled efficient marker removal and allowed for the next round of genome integration. Investigating the recombination events demonstrated that the iterative integration is happening at sufficiently high rates (more than 80%) without disrupting the previous integration. Both the flavonoid precursor pathway and the plant-derived cytochrome c P450 enzymes were functionally integrated to improve flavonoid and hydroxylated flavonoid production. The engineered strains produced 71.2 mg/L naringenin, 54.2 mg/L eriodyctiol, and 48.1 mg/L taxifolin. The reported work provides a versatile platform to iteratively integrate functional gene clusters at high copy numbers. This work may streamline and expand our capability to build efficient microbial cell factories for high-value natural products and commodity chemical production in Y. lipolytica.

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