Identification of two new flavone 4′-O-methyltransferases and their application in de novo biosynthesis of (2S)-hesperetin in Yarrowia lipolytica

Methyltransferases are pivotal enzymes in the biosynthesis of methylated flavonoids, including (2<i>S</i>)-hesperetin. However, existing flavonoid 4'-<i>O</i>-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, <i>Crc</i>OMT-2 and <i>Cgt</i>OMT-3, from Chinese citrus varieties <i>Citrus reticulata</i> 'Chachiensis' (CZG) and <i>Citrus grandis</i> Tomentosa (HZY). These enzymes displayed high substrate specificity for (2<i>S</i>)-eriodictyol. A strain capable of <i>de novo</i> synthesis of (2<i>S</i>)-hesperetin was developed by integrating the novel F4'OMTs and other biosynthetic pathway genes at high copy numbers into <i>Yarrowia lipolytica</i>. The engineered strain achieved a remarkable production titre of (2<i>S</i>)-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 (2<i>S</i>)-hesperetin during fed-batch fermentation with SAM supplementation, the highest yield reported to date. This study represents the first successful <i>de novo</i> biosynthesis of (2<i>S</i>)-hesperetin in <i>Y. lipolytica</i>, providing valuable insights into the synthesis of other O-methylated flavonoids.