Identification of two new flavone 4'--methyltransferases and their application in biosynthesis of ()-hesperetin in .

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