Engineering Yarrowia lipolytica as a green yeast cell factory for de novo biosynthesis of daidzein and purein

Abstract

Isoflavones are natural polyphenolic secondary metabolites found in legumes and play crucial roles in human health. Puerarin (PIN) is a glycosylated derivative of the isoflavone daidzein (DEIN) 8-C, predominantly found in the traditional Chinese medicinal plant Pueraria lobata. However, PIN production relies primarily on plant extracts, which is often hindered by high cost and low yields. In the present study, the complete biosynthetic pathway for PIN was constructed in Yarrowia lipolytica. Initially, de novo DEIN was synthesized by the introduction of Ge2-HIS and GmHID into a liquiritigenin-producing Y. lipolytica strain, achieving a yield of 40 mg/L. Next, the expression of the P450 enzymes Ge2-HIS and crCPR was optimized by replacing various strong promoters, resulting in a DEIN titer of 86 mg/L. The optimal copy number ratio of Ge2-HIS to crCPR was determined by increasing the DEIN yield to 201 mg/L. To achieve the de novo synthesis of PIN, the glycosyltransferase PlUGT43 was integrated into a high-yield DEIN strain, resulting in a PIN concentration of 67 mg/L. Finally, through high-density fermentation in a 5-L bioreactor, the PIN concentration reached 482 mg/L. This is the first reported synthesis of both DEIN and PIN in Y. lipolytica and the highest titer of DEIN and PIN from microbial biosynthesis to date. This study provides valuable insights into the efficient microbial biosynthesis of isoflavones and their derivatives.