Scheme 1
It is known that SuSy has a broad substrate spectrum for different NDP “acceptors”.[8] In the past five decades, more attention has been focused on plant SuSys with uridine 5’-diphosphate (UDP) preference, which is conducive to the production of UDPG.[4, 9] Prokaryotic SuSys are diversified in nucleotide substrate preference, such as some recently characterized SuSys from Thermosynechococcus elongatus (Te SuSy),Nitrosomonas Europaea (Ne SuSy), Acidithiobacillus caldus (Ac SuSy), and Denitrovibrio acetiphilus(Da SuSy), which are more inclined to use adenosine 5’-diphosphate (ADP) as nucleotide.[10, 11] However, bacterial SuSys showed better thermostability than plant SuSys, which could be more suitable for application in large-scale industrial production by increasing reaction temperature to avoid microbial contamination.[12, 13] The optimum temperature of plant SuSys is between 40 and 55 °C, but the enzyme stability decreased significantly above 30 °C,[3, 14, 15] while that of bacterial SuSys is between 60 and 80 °C.[10, 11] SuSy from moderately thermophilic Acidithiobacillus caldushas the best thermostability reported so far with the optimum temperature at 60 °C and maintains 96% activity after incubating at this temperature for 15 minutes.[11] In 2016, Gutmann et al. used Ac SuSy (A . caldus) to overcome the limitation of pH and thermodynamics, and 144 g/L UDPG was synthesized with the highest yield of 86%.[3] In this case, biocatalyst production, excessive sucrose, and a pH of 5.0 are crucial for high yield.[16]
The conversion efficiency of the glycosylation reaction is largely due to the removal of UDP, a product inhibitor of Leloir GT, where SuSy plays an indispensable role in the depletion of UDP in the SuSy-GT cascade.[17] To obtain a bacterial SuSy variant suitable for UDPG regeneration during glycosylation reactions, the affinity of Ac SuSy for UDP has been significantly improved by introducing plant residues at positions of a putative nucleotide binding motif (QN motif).[13] The comparison was made between the L637M-T640V double mutant of Ac SuSy that has a 60-fold decreased Michaelis-Menten constant (K m) for UDP, and the SuSy from Glycine max (Gm SuSy) by coupling them respectively with the glycosyltransferase Os CGT in a one-pot reaction for the synthesis of C -glucoside nothofagin.[5] Fitness in terms of kinetics, expressed by the relatively low K m values for UDP and sucrose, superseded enhanced thermostability in bacterial SuSys as the selection criterion, which made plant SuSys the strongly preferred choice.[5]
Thanks to the ever-increasing numbers of sequences deposited in databases and the rapid development of data mining algorithms,[18-20] more SuSys would be uncovered as competitive substitutes to support the development of efficient SuSy-GT cascades. In the present study, by sequence mining, we focused on SuSys from lower eukaryotes like green algae, and their characters are still poorly understood. A candidate SuSy-encoding sequence derived from Micractinium conductrix (Mc SuSy) was code-optimized synthesized and heterologous overexpressed in Escherichia coliBL21(DE3). The recombinant SuSy was characterized, and the site-directed mutagenesis was conducted at the predicted N -terminal phosphorylation site (S31) and the QN motif of Mc SuSy. Then, the selected mutant S31D/684T/685D with enhanced activity and the engineered glycosyltransferase UGT51 (UGT51m) from Saccharomyces cerevisiaewere co-expressed in E. coli . A SuSy-GT coupled system was constructed by the recombinant enzymes, to transform protopanaxadiol (PPD) into ginsenoside Rh2, a trace ginseng saponin with diverse pharmacological effects.[21] A control experiment was performed under the same conditions using UGT51m coupling with SuSy from Arabidopsis thaliana(At SuSy1).[22] This work may provide a biocatalyst with potential advantages for the establishment of cost-effective SuSy-GT cascade biotransformation in biocatalytic glycosylation.