Identification of two new flavone 4′-O-methyltransferases and their application in de novo biosynthesis of (2S)-hesperetin in Yarrowia lipolytica
Yiyun Wang, Ruiqiu Huang, Song Gao, Mingyu Yue, Xuan Zhang, Weizhu Zeng, Bin Tang, Jingwen Zhou, Dongliang Huang, Sha Xu
Abstract
Methyltransferases are pivotal enzymes in the biosynthesis of methylated flavonoids, including (2 S )-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, Crc OMT-2 and Cgt OMT-3, from Chinese citrus varieties Citrus reticulata ‘Chachiensis’ (CZG) and Citrus grandis Tomentosa (HZY). These enzymes displayed high substrate specificity for (2 S )-eriodictyol. A strain capable of de novo synthesis of (2 S )-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 (2 S )-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 S )-hesperetin during fed-batch fermentation with SAM supplementation, the highest yield reported to date. This study represents the first successful de novo biosynthesis of (2 S )-hesperetin in Y. lipolytica , providing valuable insights into the synthesis of other O-methylated flavonoids.