Highly efficient production of rebaudioside D enabled by structure-guided engineering of bacterial glycosyltransferase YojK

Abstract

Owing to zero-calorie, high-intensity sweetness and good taste profile, the plant-derived sweetener rebaudioside D (Reb D) has attracted great interest to replace sugars. However, low content of Reb D in stevia rebaudiana Bertoni as well as low soluble expression and enzymatic activity of plant-derived glycosyltransferase in Reb D preparation restrict its commercial usage. To address these problems, a novel glycosyltransferase YojK from Bacillus subtilis 168 with the ability to glycosylate Reb A to produce Reb D was identified. Then, structure-guided engineering was performed after solving its crystal structure. A variant YojK-I241T/G327N with 7.35-fold increase of the catalytic activity was obtained, which allowed to produce Reb D on a scale preparation with a great yield of 91.29%. Moreover, based on the results from molecular docking and molecular dynamics simulations, the improvement of enzymatic activity of YojK-I241T/G327N was ascribed to the formation of new hydrogen bonds between the enzyme and substrate or uridine diphosphate glucose. Therefore, this study provides an engineered bacterial glycosyltransferase YojK-I241T/G327N with high solubility and catalytic efficiency for potential industrial scale-production of Reb D.

A sustainable strategy for biosynthesis of Rebaudioside D using a novel glycosyltransferase of Solanum tuberosum

“…We overcame this difficulty by expanding new candidate analogous enzymes and accurately designing cell combinations according to the characterization of catalysts. The present knowledge does not encompass the exploration of cell cascade catalysis of UGT and Susy, despite the utilization of their crude enzyme solutions for cascade synthesis of Reb D. [ 24 ] Process with mixed strains is convenient for screening of an appropriate catalyst combination, adjustment of ratio of cells and industrial‐scale optimization. But, a whole‐cell system is intricate and unstable, referring to the transmembrane transport of substances and cell viability state etc.…”

“…The K m of StUGT for Reb A was the lowest compared with those of wild-type glycosyltransferases Yojk, UGTSL2, and EUGT11 (Table S3). [24,27,28] As such, StUGT might have a higher affinity for Reb A, and the reaction by StUGT exhibits a relatively high catalytic efficiency without by-products. The high efficiency and lack of by-products suggest that StUGT has favorable product specificity and is a potential candidate for the process of producing Reb D for the food industry.…”

“…[7,10] The optimum temperature is 35 • C for the Yojk from Bacillus subtilis. [24] It may be due to that the enzymes derived from plant share similar characteristics.…”

Molecular simulation for food protein–ligand interactions: A comprehensive review on principles, current applications, and emerging trends

“…Structure-guided engineering can be used to improve the activity of enzymes. As an example, Guo et al (2022) used molecular docking to obtain the critical residues, which involved enzyme-substrates binding and the pocket shaping and would affect the migration of substrate. Based on the results, they performed alanine-scanning mutagenesis and finally found that variant YojK-I241T/G327N exhibited the best catalytic efficiency.…”

A phylogeny-based directed evolution approach to boost the synthetic applications of glycosyltransferases

“…56 It is known that the residues histidine-aspartate of GTs are the key sites to form the catalytic center: histidine acts as a catalytic base for the deprotonation of the acceptor to allow nucleophilic attack of the donor, while the acidic residue aspartate facilitates the proton transfer. 22 Through the multiple sequence alignment analysis of BarGT-3 with the typical crystal structures of enzymes in the GT1 family (YjiC PDB ID: 7BOV 12 and YojK PDB ID: 7VM0 57 ), H14 and D106 were identified as the two key catalytic residues of BarGT-3 (ESI Fig. S16 †).…”