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and sucrose, superseded enhanced thermostability in bacterial SuSys as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.