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

作者：Yiyun Wang, Ruiqiu Huang, Song Gao, Mingyu Yue, Xuan Zhang, Weizhu Zeng, Bin Tang, Jingwen Zhou, Dongliang Huang, Sha Xu · 发表于：Synthetic and Systems Biotechnology · 年份：2025 · DOI：10.1016/j.synbio.2025.03.003 · 被引用次数：13 · 研究领域：Microbial Metabolic Engineering and Bioproduction、Microbial Natural Products and Biosynthesis、Chromatography in Natural Products

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.