High-Level <i>De Novo</i> Production of (2<i>S</i>)-Naringenin in <i>Yarrowia lipolytica</i> Using Metabolic and Enzyme Engineering

(2S)-Naringenin, a type of natural flavonoid found in rutaceae plants, has antibacterial, anti-inflammatory, antioxidant, and lipid-lowering effects. However, biosynthesizing (2S)-naringenin results in an inadequate precursor supply and low catalytic efficiency of chalcone synthase (CHS). Here, Yarrowia lipolytica was developed by enzyme and metabolic engineering for high-level (2S)-naringenin production. In enzyme engineering, the catalytic pockets were identified by molecular docking, alanine scanning and iterative mutation were performed according to the conformation obtained, and the mutation results were simulated by molecular dynamics. The results showed that the mutation caused the enzyme and substrate to bind more closely, thus increasing the titer of (2S)-naringenin. In metabolic engineering, the number of copies of CHS and CHI (encoding chalcone isomerase), the key genes in the metabolic pathway of (2S)-naringenin, was increased to four to promote the synthesis of (2S)-naringenin, which resulted in the production of (2S)-naringenin reaching 246.4 mg/L. In addition, by introducing key genes of the shikimate pathway and highly active mutants to remove feedback inhibition and by introducing unnatural ways to enhance malonyl-CoA supply, these strategies resulted in a titer of 615.0 mg/L for (2S)-naringenin. However, p-coumaric acid still accumulated, and a multicopy integration tool was further used to integrate the synthesis genes of the downstream metabolic pathway to improve the conversion of the precursor to (2S)-naringenin. Through the combination of enzyme engineering and metabolic engineering, the titer of (2S)-naringenin increased from the initial 24.1 to 776.3 mg/L. Finally, using fed-batch fermentation, a remarkable amount of (2S)-naringenin (8.65 g/L) was obtained. This study reports the highest quantity of (2S)-naringenin synthesized in Y. lipolytica while facilitating green and sustainable methodologies for industrial production.