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- Title
Enhancing the thermostability and activity of glycosyltransferase UGT76G1 via computational design.
- Authors
Go, Seong-Ryeong; Lee, Su-Jin; Ahn, Woo-Chan; Park, Kwang-Hyun; Woo, Eui-Jeon
- Abstract
The diterpene glycosyltransferase UGT76G1, derived from Stevia rebaudiana, plays a pivotal role in the biosynthesis of rebaudioside A, a natural sugar substitute. Nevertheless, its potential for industrial application is limited by certain enzymatic characteristics, notably thermostability. To enhance the thermostability and enzymatic activity, we employed a computational design strategy, merging stabilizing mutation scanning with a Rosetta-based protein design protocol. Compared to UGT76G1, the designed variant 76_4 exhibited a 9 °C increase in apparent Tm, a 2.55-fold increase rebaudioside A production capacity, and a substantial 11% reduction in the undesirable byproduct rebaudioside I. Variant 76_7 also showed a 1.91-fold enhancement rebaudioside A production capacity, which was maintained up to 55 °C, while the wild-type lost most of its activity. These results underscore the efficacy of structure-based design in introducing multiple mutations simultaneously, which significantly improves the enzymatic properties of UGT76G1. This strategy provides a method for the development of efficient, thermostable enzymes for industrial applications. Diterpene glycosyltransferase UGT76G1 is a key enzyme in the biosynthesis of rebaudioside A, a naturally sweet-tasting compound, however, its poor thermostability hinders its industrial application. Here, the authors use a computational design strategy to generate a mutant of UGT76G1 that exhibits improved thermostability and enzymatic activity.
- Subjects
INDUSTRIAL enzymology; PROTEIN engineering; STEVIA rebaudiana; INDUSTRIAL capacity; DITERPENES
- Publication
Communications Chemistry, 2023, Vol 6, Issue 1, p1
- ISSN
2399-3669
- Publication type
Article
- DOI
10.1038/s42004-023-01070-6