Abstract
Methyltransferases are pivotal enzymes in the biosynthesis of methylated flavonoids, including (2S)-hesperetin. However, existing flavonoid 4'-O-methyltransferase (F4'OMT) enzymes typically exhibit low substrate specificity and catalytic efficiency, which hinders microbial synthesis. To overcome this limitation, this study screened and identified two novel F4'OMTs, CrcOMT-2 and CgtOMT-3, from Chinese citrus varieties Citrus reticulata 'Chachiensis' (CZG) and Citrus grandis Tomentosa (HZY). These enzymes displayed high substrate specificity for (2S)-eriodictyol. A strain capable of de novo synthesis of (2S)-hesperetin was developed by integrating the novel F4'OMTs and other biosynthetic pathway genes at high copy numbers into Yarrowia lipolytica. The engineered strain achieved a remarkable production titre of (2S)-hesperetin (130.2 mg/L), surpassing the yields of previously reported F4'OMTs. Furthermore, availability of the cofactor S-adenosylmethionine (SAM) was optimised to enhance methyltransferase catalytic efficiency, enabling the engineered strain to produce 178.2 mg/L of (2S)-hesperetin during fed-batch fermentation with SAM supplementation, the highest yield reported to date. This study represents the first successful de novo biosynthesis of (2S)-hesperetin in Y. lipolytica, providing valuable insights into the synthesis of other O-methylated flavonoids.