Lei Lin's research while affiliated with Nanjing Tech University and other places

Identification of sucrose synthase from Micractinium conductrix to favor biocatalytic glycosylation. Sucrose synthase (SuSy, EC 2.4.1.13) is a unique glycosyltransferase (GT) for developing cost-effective glycosylation processes. Up to now, some SuSys derived from plants and bacteria have been used to recycle uridine 5-diphosphate glucose in the reactions catalyzed by Leloir GTs. In this study, after sequence mining and experimental verification, a SuSy from Micractinium conductrix (McSuSy), a single-cell green alga, was overexpressed in Escherichia coli, and its enzymatic properties were characterized. In the direction of sucrose cleavage, the specific activity of the recombinant McSuSy is 9.39 U/mg at 37 • C and pH 7.0, and the optimum temperature and pH were 60 • C and pH 7.0, respectively. Its nucleotide preference for uridine 5-diphosphate (UDP) was similar to plant SuSys, and the enzyme activity remained relatively high when the DMSO concentration below 25%. The mutation of the predicted N-terminal phosphorylation site (S31D) significantly stimulated the activity of McSuSy. When the mutant S31D of McSuSy was applied by coupling the engineered Stevia glycosyltransferase UGT76G1 in a one-pot two-enzyme reaction at 10% DMSO, 50 g/L rebaudioside E was transformed into 51.06 g/L rebaudioside M in 57 h by means of batch feeding, with a yield of 76.48%. This work may reveal the lower eukaryotes as a promising resource for SuSys of industrial interest.

Sucrose synthase (SuSy, EC 2.4.1.13) is a unique glycosyltransferase (GT) for developing cost-effective glycosylation processes. Up to now, some SuSys derived from plants and bacteria have been used to recycle uridine 5’-diphosphate glucose in the reactions catalyzed by Leloir GTs. In this study, after sequence mining and experimental verification, a SuSy from Micractinium conductrix (McSuSy), a single-cell green alga, was identified. In the direction of sucrose cleavage, the optimum temperature and pH of the recombinant McSuSy were 60 °C and pH 7.0. The mutations of the predicted N-terminal phosphorylation site (S31D) and the QN motif (K684T and N685D) significantly stimulated the activity of McSuSy. When the mutant S31D/684T/685D of McSuSy, with the highest activity, was applied by coupling the engineered yeast glycosyltransferase UGT51 in a one-pot two-enzyme reaction, 8 mM protopanaxadiol was transformed into 6.02 mM (3.75 g/L) ginsenoside Rh2 within 3 h at 37 °C. The yield was comparable to the control reaction of AtSuSy1 from Arabidopsis thaliana. This work reveals the lower eukaryotes as a promising resource for SuSys of industrial interest.

Rebaudioside M (RebM) is a desired natural non-caloric and high potency sweetener with very low content in the leaves of Stevia rebaudiana. In this study, based on the crystal structure of UDP-dependent glycosyltransferase UGT76G1, a key enzyme responsible for RebM production in S. rebaudiana, a computational strategy combining protein structure, molecular docking, and single-point saturation mutation was adopted to engineer UGT76G1 to promote the transformation of rebaudioside E (RebE) into RebM via a two-step continuous glycosylation. An S195Q mutant was verified to have better performance, with a catalytic efficiency 1.2-fold higher towards RebE and 2-fold higher towards rebaudioside D (RebD) than wild-type UGT76G1. RebM (12.8 ± 0.6 g/L) and RebD (10.5 ± 0.1 g/L) were generated from RebE (20 g/L) catalyzed by the S195Q mutant coupling with a sucrose synthase from Micractinium conductrix at 40 °C for 32 h. This work would expand the application of structure-based enzyme design for glycosyltransferase engineering.