Engineering regioselectivity of glycosyltransferase for efficient polydatin synthesis
Fucheng Zhu1, Jingli Dai2, Zixu Yan2
1College of Biological and Pharmaceutical Engineering, Anhui Engineering Laboratory for Conservation and Sustainable Utilization of Traditional Chinese Medicine Resources, Anhui Province Key Laboratory for Quality Evaluation and Improvement of Traditional Chinese Medicine, West Anhui University, Lu'an city 237012, China.
Food Chemistry
|August 4, 2024
Summary
Molecular evolution enhanced a Bacillus subtilis uridine diphosphate-dependent glycosyltransferase (UGT) for improved resveratrol glycosylation. A triple mutant efficiently produced polydatin, a functional food ingredient, via enhanced enzyme-substrate interactions.
Area of Science:
- Biotechnology
- Enzyme Engineering
- Food Science
Background:
- Resveratrol is a valuable functional ingredient with limited use due to poor bioavailability and solubility.
- Glycosylation can enhance resveratrol's properties, but regioselective synthesis of specific glycosides like polydatin is challenging.
- Uridine diphosphate-dependent glycosyltransferase (UGT) from Bacillus subtilis 168 (UGTBS) shows potential for resveratrol glycosylation but lacks precise regioselectivity.
Purpose of the Study:
- To engineer UGTBS for improved regioselective glycosylation of resveratrol, specifically targeting the 3-OH group for polydatin synthesis.
- To elucidate the molecular mechanisms behind the enhanced activity and regioselectivity of the engineered enzyme.
- To establish an efficient production method for polydatin using the engineered UGTBS.
Main Methods:
- Molecular evolution techniques were employed to introduce mutations into UGTBS.
- A triple mutant (Y14I/I62G/M315W) was generated and characterized.
- Enzyme kinetics, molecular docking, and fed-batch cascade reactions were utilized to assess enzyme performance and optimize polydatin production.
Main Results:
- The triple mutant Y14I/I62G/M315W demonstrated significantly improved regioselectivity for resveratrol 3-OH glycosylation.
- Polydatin constituted 91% of the total product synthesized by the mutant enzyme.
- Molecular docking and kinetic studies revealed enhanced hydrogen bonding and altered binding pocket conformation contributing to increased affinity and stability.
- A fed-batch cascade reaction achieved a high yield of nearly 20 mM polydatin.
Conclusions:
- The engineered UGTBS mutant Y14I/I62G/M315W is highly effective for the regioselective synthesis of polydatin.
- The improved enzyme offers a viable strategy for the industrial manufacture of polydatin.
- This study highlights the potential of protein engineering to overcome limitations in functional ingredient production.
