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\begin{document}

\title{A sustainable strategy for biosynthesis of Rebaudioside D using a novel
glycosyltransferase of Solanum tuberosum}



\author[1]{Siyuan Ma}%
\author[1]{Yuanyuan Ma}%
\affil[1]{Tianjin University}%


\vspace{-1em}



  
  \date{May 21, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Bioconversion of Rebaudioside D faces high-cost obstacles. Herein, a
novel glycosyltransferase StUGT converting Rebaudioside A to
Rebaudioside D was screened and characterized, which exhibits stronger
affinity and substrate specificity for Rebaudioside A than previously
reported enzymes. A whole-cell catalytic system was thus developed using
the StUGT strain. The production of Rebaudioside D was enhanced
significantly by enhancing cell permeability, and the highest production
of 6.12 g/L (yield=98.08\%) by cell catalyst was obtained by
statistical-based optimization. A new cascade process utilizing this
recombinant strain and E. coli expressing sucrose synthase was further
established to reduce cost through replacing expensive UDPG with
sucrose. A StUGT-GsSUS1 system exhibited high catalytic capability, and
5.27 g/L Rebaudioside D was achieved finally without UDPG addition by
systematic optimization. This is the best performance reported in
cell-cascaded biosynthesis, which paves a new cost-effective strategy
for sustainable synthesis of scarce premium sweeteners from biomass.%
\end{abstract}%



\sloppy


\textbf{A sustainable strategy for biosynthesis of Rebaudioside D using
a novel glycosyltransferase of \emph{Solanum tuberosum}}

\textbf{Siyuan Ma\textsuperscript{a}} , \textbf{Yuanyuan
Ma\textsuperscript{a,b,c}} *

a Department of Biochemistry, School of Chemical Engineering and
Technology, Tianjin University, Tianjin 300072, China

b School of Marine Science and Technology, Tianjin University, Tianjin
300072, China

c Biomass Conversion Laboratory, Tianjin R\&D Center for Petrochemical
Technology, Tianjin University, Tianjin 300072, China

* Correspondence to: Y. Ma, School of Chemical Engineering and
Technology, School of Marine Science and Technology, Tianjin R\&D Center
for Petrochemical Technology, Tianjin University, Tianjin 300072, China.

E-mail addresses: myy@tju.edu.cn

\section*{Abstract}

{\label{abstract}}

Bioconversion of Rebaudioside D faces high-cost obstacles. Herein, a
novel glycosyltransferase StUGT converting Rebaudioside A to
Rebaudioside D was screened and characterized, which exhibits stronger
affinity and substrate specificity for Rebaudioside A than previously
reported enzymes. A whole-cell catalytic system was thus developed using
the StUGT strain. The production of Rebaudioside D was enhanced
significantly by enhancing cell permeability, and the highest production
of 6.12 g/L (yield=98.08\%) by cell catalyst was obtained by
statistical-based optimization. A new cascade process utilizing this
recombinant strain and \emph{E. coli} expressing sucrose synthase was
further established to reduce cost through replacing expensive UDPG with
sucrose. A StUGT-GsSUS1 system exhibited high catalytic capability, and
5.27 g/L Rebaudioside D was achieved finally without UDPG addition by
systematic optimization. This is the best performance reported in
cell-cascaded biosynthesis, which paves a new cost-effective strategy
for sustainable synthesis of scarce premium sweeteners from biomass.

\textbf{Key words:} UDP-glycosyltransferase, StUGT, Rebaudioside D,
whole-cell catalysis, cascade reaction\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Graphical Abstract and Lay Summary}

Bioconversion of Rebaudioside D faces high-cost obstacles. This study
screens and characterizes a novel high-efficient glycosyltransferase
StUGT converting Rebaudioside A to Rebaudioside D. And a new cascade
process utilizing this recombinant strain and \emph{E. coli} expressing
sucrose synthase is established to reduce cost through replacing
expensive UDPG with sucrose.

\section*{1. Introduction}

{\label{introduction}}

Steviol glycosides (SGs) isolated from the leaves of \emph{Stevia
rebaudiana} consist of a diterpenoid steviol backbone that are decorated
with various sugar moieties at the steviol C13-hydroxyl and/or
C19-carboxylic acid positions. They are popular, near-zero calory,
high-stability, and high-sweetness (250--450 times sweetness than
sucrose) sweeteners. As an ideal substitute for sucrose, SGs are widely
used as sweeteners in the food, pharmaceutical and cosmetic industry. In
addition, they have various health functions such as improving immunity,
lowering blood pressure, and inducing an antidiabetic
effect.\textsuperscript{{[}1{]}} They have been approved a Generally
Recognized as Safe (GRAS) status by the Food and Drug Administration
(FDA) of the United States and also allowed to be used in other
countries, including European Union, Japan, China, and
Australia.\textsuperscript{{[}2{]}} Stevioside (Stv, 5--10\% of leaf dry
weight) and Rebaudioside A (Reb A, 2--4\%) are the major SGs in stevia
leaves. Others such as Rebaudioside D (Reb D) and Rebaudioside M (Reb M)
have a lower content (0.4--0.5\%).\textsuperscript{{[}3{]}} Among SGs,
Reb D has a cleaner sweet taste, better sugar properties, and a higher
market value when compared with Reb A and Stv.
\textsuperscript{{[}4,5{]}}

The SGs on the market are typically extracted from Stevia leaves by
maceration and heat extraction.\textsuperscript{{[}6{]}}The isolated
single components of Reb A or Reb D are more popular as advanced food
additives, due to their improved sweetness characteristics and ease of
use. However, the extraction process of single target components
consumes large amounts of water, chemicals, and time. Large-scale
production of Reb D by traditional extraction is difficult due to its
low content in stevia leaves and poor water
solubility.\textsuperscript{{[}7{]}} Therefore, biocatalytic production
of Reb D has been paid attention to reduce its environmental impact and
increase production throughput.

In \emph{S. rebaudiana} , UDP-glycosyltransferases (UGTs) catalyze the
synthesis of SGs.\textsuperscript{{[}8{]}} The diterpene steviol
produced from the gibberellic acid (GA) biosynthetic pathway is
sequentially glucosylated by UGT74G1, UGT85C2 and UGT91D2 to form Stv.
Then, UGT76G1 and UGT91D2 sequentially convert Stv to Reb A followed by
a conversion to Reb D.\textsuperscript{{[}9{]}} The Reb A is relatively
cheap and easy to be obtained, and Reb D production from Reb A catalyzed
by UGTs is studied. The engineered \emph{Oryza sativa} EUGT11 was shown
to convert Reb A to Reb D.\textsuperscript{{[}10{]}} \emph{Solanum
lycopersicum} UGTSL2 was also identified to glycosylate Reb A and
produce Reb D, but with a byproduct of Reb M2.\textsuperscript{{[}7{]}}
Currently, only a few UGTs can synthesize Reb D from Reb A via
glycosylation at C19. The UGTs belong to the Glycosyltransferases 1
(GT1) family consisting of 26,825 members, and their actual functions
still remain obscure.\textsuperscript{{[}11{]}} Therefore, it is
essential and challenging to explore novel and highly efficient UGTs
from nature for improving the biosynthesis of Reb D.

SGs production using crude enzyme usually increase the time and cost due
to the complex preparation process of crude extracts. The whole-cell
catalysis is a simple, rapid, cost-effective, and environmentally
friendly process. \textsuperscript{{[}12{]}}However, the literature
hosts few reports with respect to highly efficient whole-cell catalysis
for Reb D. We engineered a \emph{P. pastoris}secretively expressing
EUGT11, which can catalyze Reb A to produce Reb D with a 95\% conversion
rate. The secreted extracellular enzyme is likely to contact the
substrate to increase the reaction rate. But, an intracellular enzyme
has better stability but relatively low efficiency due to the cellular
surface barrier.\textsuperscript{{[}7{]}}Therefore, a whole-cell
catalytic system with intracellular enzymes is also worthy being
investigated and developed, nevertheless, the relevant research is still
lack at present. Besides, in the reported whole-catalysis system, only 1
g/L substrate was conducted and the titer of the product is relatively
low,\textsuperscript{{[}10,13{]}} which would result in a relatively
high product recovery cost.\textsuperscript{{[}14{]}} Therefore, the
improvement of product concentration should be considered for
industrial-scale production.

Transglycosylation catalyzed by UGTs requires an expensive uridine
diphosphate glucose (UDPG) as a sugar donor. Sucrose synthase (SuSy)
converts sucrose and uridine diphosphate (UDP) to UDPG and fructose.
\textsuperscript{{[}15,16{]}} As such, the UGT and SuSy can be coupled
to generate UDPG providing sugar donors to reduce the production cost of
Reb D.\textsuperscript{{[}17{]}}However, the synthesis of Reb D using a
cell mixture of UGT and SuSy has not been used, to the best of our
knowledge. A mixed whole-cell strategy would decrease the costs of
feedstocks and enzyme preparation and reduce the production time.

In general, Reb D bioconversion faces serious bottlenecks. Only a few
UGTs may catalyze the reaction, which causes a necessity to explore
novel high-efficiency enzymes. The Reb D biosynthesis cost is relatively
high due to time-consuming and complicated enzyme preparation
procedures, low concentrations of the final product, and the high cost
of reagents in the reaction system. Therefore, exploration of new and
highly efficient UGTs and catalytic strategies is imperative for
large-scale production of Reb D.

To reach this goal, this study screened and characterized a novel
glycosyltransferase for glycosylation of Reb A to Reb D. A
high-efficiency whole-cell catalytic technology for Reb D was then
developed using a mixed recombinant \emph{E. coli} expressing StUGT and
GsSUS1. As a whole, this study provides a novel bioconversion technology
for Reb D synthesis. The technology may decrease wastewater discharge,
increase cost efficiency, and establish a foundation for green and
scalable industrial production of premium sweeteners.

\section*{2. Materials and Methods}

{\label{materials-and-methods}}

\subsection*{2.1 Chemicals and reagents}

{\label{chemicals-and-reagents}}

Reb A and Reb D were supplied by Sinochem Health Company Ltd. (Qingdao,
China). UDPG was obtained from Canspec Inc. (Beijing, China). HPLC grade
of acetonitrile was purchased from Fisher Scientific (Beijing, China).
All other reagents were analytical grade and commercially available.

\subsection*{2.2 Protein sequence and structure
analysis}

{\label{protein-sequence-and-structure-analysis}}

UGTSL2 (GenBank: XP\_004250485.1) and EUGT11 (GenBank: XP\_015629141.1)
was used as a query to search against the National Center for
Biotechnology Information (NCBI) database to retrieve the homology
sequences, respectively. Multiple sequence alignment was performed with
ClustalX software (EMBL, Heidelberg, Germany). The secondary structure
of the StUGT (XM\_006367619.1) was analyzed by the online software
ESPript3 (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi).
Functional domains were predicted using conserved domain database (CDD).
A three-dimensional structure of StUGT was simulated by the SWISS-MODEL
online server (https://swissmodel.expasy.sorg/) using the crystal
structure of the OsUGT91C1 (PDB: 7ES2) as a template. And the structure
of StUGT was analyzed and visualized using Pymol software (DeLano
Scientific LLC). Model refinement and docking of the Reb A to StUGT were
performed using AutoGrid4 and AutoDock4, respectively.

\subsection*{2.3 Plasmid and strains}

{\label{plasmid-and-strains}}

The StUGT, CaUGT and BdUGT genes were codon-optimized and synthesized by
GenScript (Nanjing, China), and the synthesized genes were then inserted
into \emph{Nco} I and\emph{Xho} I sites of expression vector backbone
pET28a (+) using Gibson Assembly according to the manufacturer's
procedure (NEB, America), respectively. The resulting recombinant
vectors were named pETStUGT, pETCaUGT and pETBdUGT, and then transformed
into competent \emph{E. coli} BL21 (DE3) cells (Biomed, Beijing). The
transformants were selected on Luria Bertani (LB) plates supplemented
with 50 \selectlanguage{greek}μ\selectlanguage{english}g/mL kanamycin. And the positive colonies were verified by PCR
using T7 primers (T7 forward: 5'-TAATACGACTCACTATA-3' and T7 reverse:
5'-TAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTT TTTTGA-3') and sequencing
(Sangon, Shanghai). The resulting recombinant strains were named BL21
(pETStUGT), BL21 (pETCaUGT) and BL21 (pETBdUGT), respectively. All
plasmids and strains used in this study are listed (Table S1).

\subsection*{2.4 Expression and preparation of the
glycosyltransferases}

{\label{expression-and-preparation-of-the-glycosyltransferases}}

The three engineered \emph{E. coli} strains were inoculated into 5 mL of
LB medium (10 g/L of tryptone, 5 g/L of yeast extract, and 5 g/L of
NaCl) containing 50 \selectlanguage{greek}µ\selectlanguage{english}g/mL of kanamycin, and cultured at 37 \selectlanguage{ngerman}°C with 220
rpm overnight, respectively. One mL of overnight cultures was
transferred into 100 mL of Terrific Broth (TB) for culture at 37 °C.
When the OD\textsubscript{600} of cultures reached 0.6--0.8,
isopropyl-\selectlanguage{greek}β-\selectlanguage{english}D-1-thiogalactopyranoside (IPTG) was added with a final
concentration of 0.5 mM for induction of expression of target proteins.

When they were cultured for 12 h, 100 mL culture broth was taken and
centrifuged at 12,000 rpm for 10 min at 4 \selectlanguage{ngerman}°C for collection of the
corresponding cells. And the cell pellets were washed once with 10 mM of
pH 7.2 phosphate buffer solution (PBS, 50 mM
NaH\textsubscript{2}PO\textsubscript{4}, 300 mM NaCl) and suspended in
pH 7.2 PBS with a final concentration of 30 OD\textsubscript{600}. The
suspensions were ultrasonicated by an Ultrasonic Cell Disruptor (Model
JY92-2D, Scientz Industry, Ningbo, China) for 1 h (5 s plus on and 5 s
off, 300 W) on ice. Then, the supernatants were collected and used for
enzyme assay.

\subsection*{2.5 Expression optimization and purification of
StUGT}

{\label{expression-optimization-and-purification-of-stugt}}

The strain BL21 (pETStUGT) was inoculated and cultured until the
OD\textsubscript{600} reached 0.6--0.8 as described aforementioned. The
IPTG was then added with a final concentration of 0.5 mM and the
cultures were incubated at 18°C, 25°C, and 30 °C for 5, 12 and 17 h to
optimize the expression level of StUGT. The 20 \selectlanguage{greek}μ\selectlanguage{english}L and 50 \selectlanguage{greek}μ\selectlanguage{english}L of crude
enzyme solution was subjected to SDS-PAGE analysis and detection of
protein concentration, respectively. The 150 \selectlanguage{greek}μ\selectlanguage{english}L of crude enzyme
preparation was used for enzyme assay.

The strain BL21 (pETStUGT) was cultured in LB medium containing 0.5 mM
IPTG at 18 \selectlanguage{ngerman}°C for 17 h. Cells were then collected by centrifugation at
12,000 rpm for 10 min and washed once with 10 mM of pH 7.2 PBS. To
optimize purification efficiency, pH 4.0, 6.0, 7.0 and 8.0 PBS was
prepared for affinity purification. The cell pellets were suspended in
pH 4.0--8.0 binding buffer (PBS with 2 mM imidazole) with a final
concentration of 30 OD\textsubscript{600}. The suspensions were
ultrasonicated by an Ultrasonic Cell Disruptor for 1 h (5 s plus on and
5 s off, 300 W) on ice. The supernatants were collected and then loaded
onto a Ni-NTA column (GE Healthcare, America). The target enzyme was
then washed twice using the pH 4.0--8.0 washing buffer (PBS containing
20 mM imidazole) and eluted with the same pH of the elution buffer (PBS
containing 150 mM imidazole) as described
previously.\textsuperscript{11} Subsequently, the elution fractions
containing the purified StUGT were desalted twice to remove the
imidazole and NaCl by using the PD-10 column (Merk, Germany). The
purified enzyme was then subjected to 10\% SDS-PAGE analysis and protein
concentration determination by Bradford
method.\textsuperscript{{[}18{]}}

\subsection*{2.6 Enzymatic assays}

{\label{enzymatic-assays}}

The glucosyltransferase assay was performed in 0.5 mL mixture containing
1 mM Reb A, 1 mM UDPG, 3 mM MgCl\textsubscript{2}, 50 mM pH 7.2 PBS and
2 nmol of purified protein (102 \selectlanguage{greek}μ\selectlanguage{english}g StUGT) or 150 uL crude enzyme. The
mixture was incubated at 30 \selectlanguage{ngerman}°C for 24 h, and 0.5 mL of 60\% (v/v)
acetonitrile was then added to quench the reaction. The Reb A and Reb D
contents were determined by High Performance Liquid Chromatography
(HPLC). The molar conversion rate of Reb A(C\textsubscript{RA}) and
molar yield of Reb D (Y\textsubscript{RD}) were then calculated as
follows:

C\textsubscript{RA} (\%) = (RA\textsubscript{t0}-RA\textsubscript{t}) /
RA\textsubscript{t0}

Y\textsubscript{RD} (\%) = RD\textsubscript{t} / RA\textsubscript{t0}

where RA\textsubscript{t0} represented the initial molar concentration
of Reb A, and the RA\textsubscript{t} and RD\textsubscript{t}represented
the final molar concentration of Reb A and Reb D after reaction,
respectively.

To determine the effect of reaction condition on catalytic efficiency,
the reaction parameters such as temperature, pH, metal ions and UDPG
were varied separately, while the others except the investigated
parameter were kept constant. And the relative activity was calculated
using the ratio of C\textsubscript{RA} each condition to the highest
that to compare the catalytic activities under different conditions. The
optimal temperature for StUGT was investigated at temperature ranging
from 25 °C to 50 °C. The optimal pH of StUGT was determined using 50 mM
citrate buffer (pH 5.0--6.0), potassium phosphate buffer (pH 6.0--8.0),
Tris-HCl buffer (pH 8.0--9.0) and glycine buffer (pH 9.0--10.5). The
concentrations of UDPG were adjusted to 1, 2, 3, 4 and 6 mM to test the
influence of the glycosyl donor concentration on glycosylation
efficiency. Divalent metal ions, including MgCl\textsubscript{2}, Ca
(NO\textsubscript{3})\textsubscript{2}, CuSO\textsubscript{4}, Pb
(CH\textsubscript{3}COO)\textsubscript{2}, FeSO\textsubscript{4},
BaCl\textsubscript{2}, ZnSO\textsubscript{4}, CoCl\textsubscript{2} and
MnCl\textsubscript{2} were added to the reaction mixtures at a final
concentration of 1 mM, 3 mM and 6 mM to optimize the yield of Reb D. One
unit (U) of glucosyltransferase activity was defined as amount of enzyme
catalyzing the bioconversion of 1mmol of Reb A per minute. The specific
activity was expressed as units per gram of protein.

\subsection*{2.7 HPLC and LC-MS analysis}

{\label{hplc-and-lc-ms-analysis}}

The 0.5 mL reaction system was mixed with 0.5 mL 60\% (v/v) acetonitrile
and centrifuged at 12,000 rpm for 10 min. The supernatants were taken up
and then filtered using a 0.22 \selectlanguage{greek}μ\selectlanguage{english}m nylon membrane filter for HPLC
analysis. HPLC was performed on an Agilent 1200 Series equipped with a
UV-vis detector (wavelength = 210 nm) and carried out at 40 \selectlanguage{ngerman}°C on a
column of LunaC18 column (5 \selectlanguage{greek}μ\selectlanguage{english}m, 100 \selectlanguage{ngerman}Å, Phenomenex). The mobile phase
consisting of acetonitrile and 10 mM sodium dihydrogen phosphate buffer
(pH 2.6) in the ratio of 32:68 (v/v) was used at a flow rate of 1
mL/min. The contents of Reb A and Reb D were detected based on an
external standard method. The HPLC fractions at the retention time of 4
min were collected and submitted to Liquid Chromatograph Mass
Spectrometer (LC-MS) analysis. Mass spectrometry for identification was
carried out on a LCMS-IT-TOF (Shimadzu, Japan) equipped with an Electron
Spray Ionization (ESI). ESI-MS was performed in negative ion mode under
the following operating parameters: a capillary voltage of 1.75 kV, an
atomizing gas flow of 10 L/min, a heating module temperature of 200 °C,
a CDL temperature of 200 °C and a drying gas pressure of 100 kPa.

\subsection*{2.8 Kinetic analysis of glucosyltransferase
StUGT}

{\label{kinetic-analysis-of-glucosyltransferase-stugt}}

The kinetic characteristics of purified StUGT were determined by varying
the Reb A concentration in the reaction mixture. The glycosylation
reaction was carried out in 0.5 mL mixture containing 3 mM UDPG, 3 mM
MgCl\textsubscript{2}, 2 nmol of purified protein, 50 mM pH 8.0
potassium phosphate buffer and different concentrations of Reb A
(0.1--1.0 mM) at 40 °C. After incubation for 1 hour, the reaction was
stopped and the supernatant was taken up for the component
determination. The reaction rate was calculated as micromoles of Reb A
converted per milligram of the purified StUGT per minute (\selectlanguage{greek}μ\selectlanguage{english}mol/mg/min).
The Reb D production rate versus substrate concentration was fitted to
Michaelis-Menten kinetics, and the K\textsubscript{m} and
K\textsubscript{cat} of StUGT for Reb A was calculated according to the
Lineweaver-Burk plot.\textsuperscript{{[}19{]}}

\subsection*{2.9 Whole-Cell catalysis
Assay}

{\label{whole-cell-catalysis-assay}}

The strain BL21 (pETStUGT) was cultured and collected as described
above. Then, the cells were washed once and resuspended in 10 mM
phosphate buffer (pH 7.2). The suspensions as whole-cell catalysts were
added to 0.5 mL of above reaction mixture with a final cell density of
60 OD\textsubscript{600} (22.8 g DCW/L). The reaction was conducted at
30 \selectlanguage{ngerman}°C for 24 h. In order to enhance the permeability of cell membrane,
the washed cells were suspended in 0.1--0.3 g/L surfactants
(Triton-X-100, CTAB and Tween-80) and incubated at 30 °C for 30 min. The
cells were then collected and washed once, and then resuspended to an
OD\textsubscript{600} of 100 (38 g DCW/L) in 10 mM PBS (pH 7.2). These
treated cells were added to aforementioned reaction mixture at a final
cell density of 60 OD\textsubscript{600} to investigate the catalytic
efficiency.

The cells treated with 0.2 g/L CTAB were added to the above reaction
system at a final cell density of 60 OD\textsubscript{600} to
investigate the influence of reaction parameters on catalytic
efficiency. The reaction parameters such as temperature, pH, and the
reaction time, StUGT biomass/RA ratio, shaking speed or RA/UDPG ratio
varied separately, while the others except the investigated parameter
were kept constant. The activity was measured at temperature ranging
from 25 °C to 50 °C. For determination of optimal reaction pH, the
reactions were performed in the four kinds of buffer with pH 5.5 to
10.5. The optimal reaction time was examined in the range of 2--48 h. To
optimize the ratio of StUGT biomass/RA, 24--96
OD\textsubscript{600}cells were used with 1, 2 and 3 g/L Reb A as
substrate, respectively. The shaking speed was set at 0, 50, 100, 150,
200 and 250 rpm, respectively. The ratio of RA/UDPG was set up ranging
from 2:1 to 1:4.

In order to determine the optimum combination of reaction conditions in
whole-cell bioconversion, four factors each at three levels were first
considered and an orthogonal layout of L9 (3\textsuperscript{4}) was
performed (Table S4). And then the catalytic parameters, including the
biomass, shaking speed, the ratio of RA/UDPG and reaction time of the
whole-cell catalysis were also optimized by the orthogonal test (Table
1). The influence of various parameters on Reb D yield was assessed by
the range analysis.

\subsection*{2.10 Fed-batch synthesis of Reb D by whole-cell
catalysis}

{\label{fed-batch-synthesis-of-reb-d-by-whole-cell-catalysis}}

The fed-batch reaction was carried out in optimal conditions as
described above. The initial reaction mixture (0.5 mL) containing 5 g/L
Reb A, 12.5 g/L UDPG, 50 mM PBS (pH 8.0), and 72
OD\textsubscript{600}(27.36 g DCW/L) StUGT cell catalysts was incubated
at 30°C for 60 h. In the fed-batch reaction, 5 g/L RA was added once at
16 h; in another reaction, 2.5 g/L Reb A was added twice at 16 and 30 h.
Samples were collected at different time intervals (16, 30, 36, 42, 48,
52, and 60 h) to measure Reb A and Reb D concentrations.

\subsection*{2.11 Whole-cell cascade
reaction}

{\label{whole-cell-cascade-reaction}}

The strain BL21 (pETStUGT) and BL21 (pETAtSUS1) expressing the sucrose
synthase AtSUS1 from \emph{Arabidopsis thaliana} as well as
BL21(pETGsSUS1) expressing GsSUS1 from \emph{Gossypium schwendimani}
were cultured and induced for expression as described above. The cells
were then suspended in 0.15 g/L CTAB and incubated at 30 °C for 30 min
for the improvement of cell permeability. The cells were collected and
resuspended to an OD\textsubscript{600} of 300 (114 g DCW/L) in 10 mM
phosphate buffer (pH 7.2) as whole-cell catalyst. The StUGT cell
catalysts were added to reaction mixture with a final cell density of 54
OD\textsubscript{600}(22.8 g DCW/L), and GsSUS1/AtSUS1 cell catalysts
(27--270 OD\textsubscript{600}) were added to the reaction mixture in
the ratios of 2:1, 1:1, 1:2, 1:3, 1:4, and 1:5, respectively. And the
0.5 mL of reaction mixture containing 2 g/L Reb A, 5 mM UDP, 50 mM PBS
(pH 8.0), 2.1 mM sucrose and the mixed cells was incubated at 30 °C for
24 h. Reactions were quenched by addition of 0.5 mL 60\% (v/v)
acetonitrile. The cascade efficiency was calculated as follows:

The cascade efficiency (\%) = Y\textsubscript{RDt} /
Y\textsubscript{RDt0}

where Y\textsubscript{RDt} indicated the Y\textsubscript{RD} in the
StUGT-SUS1 cascade system, and the Y\textsubscript{RDt0} indicated the
Y\textsubscript{RD} in the only StUGT system under the same reaction
conditions.

To further increase the Reb D yield by orthogonal test, four factors
each at three levels were first considered (Table S5). And then the
catalytic parameters, including the ratio of StUGT/GsSUS1, Reb
A/sucrose, and sucrose/UDP were also optimized by the orthogonal test
(Table S6).

\section*{3. Results and discussion}

{\label{results-and-discussion}}

\subsection*{3.1 Screening of novel UGT for Reb D synthesis by
bioinformatics
analysis}

{\label{screening-of-novel-ugt-for-reb-d-synthesis-by-bioinformatics-analysis}}

The glycosyltransferases UGTSL2 and EUGT11 catalyzing the bioconversion
of Reb A to Reb D were used as a query to search homologous
glycosyl-transferase. The UDP-glucuronosyltransferase from \emph{Solanum
tuberosum} (StUGT) and \emph{Capsicum annuum} (CaUGT) have 84.16\% and
74.66\% homology with UGTSL2, respectively. \emph{Brachypodium
distachyon} UDP-glucuronosyltransferase (BdUGT) shared the highest
homologous with the EUGT11 (Table S2). These three candidate enzymes
were expressed in \emph{E. coli} to characterize whether they could
convert Reb A to Reb D.

Their crude enzyme extracts were prepared for conversion of Reb A. No
any product was formed using BdUGT as a catalyst. Not only a 4-minute
product like Reb D but also other by-products were detected in the
CaUGT-catalyzed reaction mixture. Nevertheless, the peak of Reb A
catalyzed by the StUGT was disappeared completely and a new peak was
appeared at the retention time of 4 min (Figure S1). The 4-min fractions
were thus collected and analyzed by the LC-MS. The peak appearing at m/z
= 1151.4670 {[}M + H + Na{]}+ matched the Reb D peak (m/z = 1152.15 {[}M
+ H + Na{]}+). This indicates the product was Reb D with no evidence of
by-products forming. This is the first report on how glycosyltransferase
StUGT can specifically catalyze the bioconversion of Reb A to Reb D.
Therefore, the StUGT arouses our great interest.

For a better understanding of the StUGT function, bioinformatics
analysis of StUGT was conducted. Multiple sequence alignment and CDD
analyses revealed that StUGT belonged to the glycosyltransferase 1 (GT1)
family (Figure 1a). The model in Figure 1b indicates StUGT has
N-terminal and C-terminal domains. The C-terminal domain has a plant
secondary product glycosyltransferase (PSPG) motif composed of 44 amino
acid residues (residues 318--361), which involves the binding of
UDP-sugar donors (Figure 1a). \textsuperscript{{[}20{]}} The more
flexible N-terminal domain participates in the binding of various
acceptors of sugar residues. \textsuperscript{{[}21{]}} The amino acid
residues Glu67 and Glu70 at the StUGT N-terminus form bidentate hydrogen
bonds with the hydroxyl groups of the Reb A (Figure 1c). The analysis of
the secondary and spatial model also suggests StUGT has the potential to
transfer glucose from the glycosyl donor UDPG to Reb A to produce Reb D
(Figure 1d).

\subsection*{\texorpdfstring{3.2 Functional characterization of StUGT
in\emph{E.
coli}}{3.2 Functional characterization of StUGT inE. coli}}

{\label{functional-characterization-of-stugt-in-e.-coli}}

The expression of StUGT was induced under various conditions and the
crude enzyme was all prepared from the same numbers of cells for
SDS-PAGE analysis and protein concentration determination (Figure S2).
The most intense band of StUGT was observed in the sample taken from
cultures incubated at 18°C for 17 h. Under these culture conditions, the
total protein concentration and conversion rate of Reb A
(C\textsubscript{RA}) were the highest. All subsequent inductions were
carried out at 18°C for 17 h to maximize conversion.

We purified the StUGT by affinity chromatography (Figure 2a) using a pH
4.0--8.0 buffer to optimize the purification efficiency. Each purified
StUGT showed a single band on the SDS-PAGE gel with an approximate
molecular mass of 51 kDa, which was consistent with the predicted value.
C\textsubscript{RA} from the StUGT purified at pH levels of 4.0, 6.0,
7.0, and 8.0 were 85.40\%, 89.36\%, 70.48\%, and 64.39\%, respectively
(Figure S3). When the pH of the purification buffer was 4.0 or 6.0, the
catalytic activity was significantly higher than in other conditions.
This may be due to the distribution of total net charges on the StUGT at
those pH levels where the pH of the purification buffer is close to the
isoelectric point (pI = 5.18) of the target protein. As a result, the
distribution maintains the catalytic activity of active
sites.\textsuperscript{{[}22{]}} Due to the increased activity, the
StUGT was purified using a pH 6.0 buffer for subsequent experiments. The
effect of purification buffer pH on purification efficiency has rarely
been investigated; thus, our investigation of this effect in this study
may provide valuable information that can be used for the improvement of
purification efficiency.

\subsection*{3.3 Biochemical characteristics of the
StUGT}

{\label{biochemical-characteristics-of-the-stugt}}

The biochemical characteristics of enzymes are very crucial for their
industrial application. The activity of purified StUGT gradually
increased when the temperature was increased from 25°C to 40°C, but it
reached a peak at 40°C (Figure 3a). The catalytic activity at 45--50°C
decreased to 14.96--38.17\% of the highest catalytic activity. This
indicates a loss of activity at a higher temperature. The optimal
temperature of 40°C for StUGT converting Reb A is similar to both UGTSL2
and EUGT11.\textsuperscript{{[}7,10{]}}

The pH of the reaction impacts structural stability and catalytic
activity of the enzyme. The activity was increased with a pH ranging
from 5.0 to 8.0, with the maximum activity occurring at pH 8.0. The
activity then showed a downward trend from pH 8.0 to 10.5 (Figure 3b).
The dissociation state of the active groups at pH 8.0 may be optimum for
the combination of enzymes and substrates.

UDPG is a glycosyl donor in the glycosylation reaction for Reb D, so the
catalytic activity of StUGT was enhanced significantly when the
concentration of UDPG increased from 1 mM to 3 mM (Figure 3c). The 3 mM
UDPG concentration reached 98\% of the highest catalytic activity. The
maximum C\textsubscript{RA} of 100\% was observed at 4 mM UDPG, and the
maximum activity still remained at 4--6 mM UDPG. However, a 3 mM
concentration of UDPG is appropriate due to cost considerations.

Metal ions form complexes with enzymes and act as structural regulators
and usually play a key role in protein folding and
catalysis.\textsuperscript{{[}23{]}}Therefore, metal ions were added to
the reaction system to explore and expand the catalytic efficiency. Some
metal ions, Cu\textsuperscript{2+}, Zn\textsuperscript{2+}, and
Co\textsuperscript{2+}, severely inhibited the catalytic activity of the
StUGT (Figure 3d). However, the addition of Pb\textsuperscript{2+},
Mg\textsuperscript{2+}, Ca\textsuperscript{2+}, Ba\textsuperscript{2+},
and Mn\textsuperscript{2+} improved the Reb D production by 5.28\%,
10.01\%, 7.63\%, 9.86\%, and 9.86\%, respectively. This indicates these
ions exhibit a positive effect on the enzymatic activity. The influence
of concentrations of these ions on activity was thus also tested (Table
2). The Reb D yield (Y\textsubscript{RD}) reached a maximum of 86.61\%
in the presence of 6 mM MgCl\textsubscript{2}. Mg\textsuperscript{2+} as
a cofactor promotes the binding of pyrophosphate groups in the sugar
group donor and the active site of the enzyme to improve the catalytic
activity of StUGT.\textsuperscript{{[}24{]}}

Based on the optimal conditions discussed above, the specific activity
and kinetic parameters of purified StUGT toward Reb A glycosylation were
determined (Figure S4). The specific activity of the purified StUGT was
1024.53 U/g protein, and the maximum velocity (V\textsubscript{max}) and
the Michaelis constant (K\textsubscript{m}) value were 0.1174 \selectlanguage{greek}μ\selectlanguage{english}mol
min\textsuperscript{--1} mg\textsuperscript{--1} and 0.1073 mM,
respectively. Since the literature lacks reports discussing
glycosyltransferases catalyzing Reb A to Reb D, these parameters enable
more functional studies of UGTs. A low K\textsubscript{m} value
represents a high affinity for the enzyme with a substrate. The
K\textsubscript{m} of StUGT for Reb A was the lowest compared with those
of wild-type glycosyltransferases Yojk, UGTSL2, and EUGT11 (Table
S3).\textsuperscript{{[}25-27{]}} 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.

\subsection*{\texorpdfstring{3.4 Construction of the \emph{E.
coli}whole-cell biocatalyst
system}{3.4 Construction of the E. coliwhole-cell biocatalyst system}}

{\label{construction-of-the-e.-coli-whole-cell-biocatalyst-system}}

The strain expressing StUGT was directly used to construct a catalysis
system for Reb D synthesis (Figure 2b), where C\textsubscript{RA} of
13.45\% by the cells was lower than that by the purified StUGT. The poor
permeability of cell may limit enzyme--substrate contact to decrease
catalytic efficiency.\textsuperscript{{[}28{]}} As such, we treated
cells using surfactants (CTAB, SDS, Tween-80, and Triton-X-100) and
organic solvents (acetone, isopropyl alcohol, ethanol, and toluene) to
enhance permeability. Treatment with 0.1--0.3 g/L of surfactants, except
for SDS, significantly improved catalytic activity (Figure 4a), while
the treatment with the organic solvents and SDS reduced catalytic
activity (data not presented). The CTAB enhanced cell activity more than
Tween 80 and Triton-X-100 at the same concentrations (Figure 4a). The
cells treated with 0.2 g/L CTAB exhibited the highest catalytic
efficiency (62.83\% of C\textsubscript{RA}), which was 367\% higher than
that of the control.

The cell permeability is crucial for catalytic function. An appropriate
permeability improvement is difficult to obtain due to the divergent
composition of the cell wall/membrane of different cell
catalysts.\textsuperscript{{[}29{]}} Therefore, proper cell treatment
should be considered to establish an efficient whole-cell catalytic
system.

\subsection*{3.5 Optimization of whole-cell catalysis
efficiency}

{\label{optimization-of-whole-cell-catalysis-efficiency}}

The biological reaction conditions have~considerable influence on the
biocatalytic activity.\textsuperscript{{[}30{]}} Tracking the effect of
reaction temperature, pH, and reaction time on catalytic efficiency
enables optimal performance. The highest catalytic activity was obtained
at 30\selectlanguage{ngerman}°C (Figure 3a). The catalytic activity decreased when the
temperature increased from 30°C to 50°C. The higher temperatures may
contribute to cell degradation and
inactivation.\textsuperscript{{[}31{]}} The optimal temperature of 30°C
can easily be maintained during industrial processes.

The peak activity by the whole-cell catalyst was observed at a pH of
8.0, which coincides with the optimal pH used with purified StUGT
(Figure 3b). In the pH range of 6.5--9.5, the whole-cell biocatalysts
could retain more than 70\% of its optimal activity, while the purified
enzyme only retained about 30\% of its optimal activity. As such, the
whole-cell catalysis has a wider pH tolerance than the purified enzyme
due to how the outer barrier of cell wall and membrane protects enzymes
from adverse experimental conditions.\textsuperscript{{[}32{]}}

With regard to reaction time, the catalytic activity increased as the
time increased from 0 to 14 h, with the highest
C\textsubscript{RA}obtained at 14 h (Figure 4b). The activity at 20 h
retained 98.8\% of the maximum, which was deemed insignificant (P
\textgreater{} 0.05). This result demonstrates the catalysis reaction is
rapid and stable.

The interactive effects of the abovementioned reaction condition
parameters were further investigated by the L9 (3\textsuperscript{4})
orthogonal test. The range value R indicated the order of significance
for each factor upon Y\textsubscript{RD} was as follows: pH
\textgreater{} CTAB concentration \textgreater{} temperature
\textgreater{} reaction time (Table S4). Accordingly, the optimum
reaction condition was pH 8.0, 30°C, 0.15 g/L CTAB, and 16 h reaction
time. Under the optimal conditions, C\textsubscript{RA} and
Y\textsubscript{RD} could reach 90.07\% and 68.53\%, respectively,
suggesting adequate Reb D production. Cell catalysis is more easily
influenced by factors such as cell density and stirring speed than a
purified enzyme is, but the production is rapidly optimized using
statistical methods. In short, C\textsubscript{RA} improved by 34.32\%
more than the maximum value of the single-factor experiments. These
results highlight the importance and impact of a systematic and
efficient statistical strategy to optimize complex catalytic systems.

\subsection*{3.6 Whole-cell catalytic synthesis of high-titer Reb
D}

{\label{whole-cell-catalytic-synthesis-of-high-titer-reb-d}}

To obtain high-titer products, the substrate concentration was
correspondingly increased from 1g/L to 3 g/L, and the effect of the
ratio of catalyst and glycosyl donor to substrate on catalytic
efficiency was investigated. When the substrate concentration was
constant, C\textsubscript{RA} increased gradually with increasing
biomass of StUGT where it reached a maximum of 96 OD\textsubscript{600}.
An increase in C\textsubscript{RA} had no significant impact in the
range of 72--96 OD\textsubscript{600} (Figure 4c), possibly due to that
the excessive accumulation of cells in the reaction system obstructs the
movement of substances into and out of the
cells.\textsuperscript{{[}33{]}}

With substrate concentrations of 1, 2, and 3 g/L, the catalytic
efficiency reached the maximums at the 72:1, 48:1, and 36:1 ratios of
StUGT biomass/Reb A (Figure S5a), respectively. This demonstrates the
ratio of catalyst to substrate for obtaining a maximum
C\textsubscript{RA} decreased as the substrate concentration increased.
This relationship highlights how the StUGT cells exhibit good catalytic
performance for high-concentration substrates. The ratio of UDPG to
substrate is important for glycosylation. C\textsubscript{RA} reached a
maximum with a RA/UDPG mass ratio of 1:2 (Figure S5b). The low content
of UDPG possibly leads to insufficient glycosyl donors, while excessive
high-concentration UDPG inhibits the activity of
UGTs.\textsuperscript{{[}34{]}} The catalytic efficiency at 0--250 rpm
was also investigated. C\textsubscript{RA} reached a maximum at 200 rpm
and then gradually decreased with increasing speeds (Figure S5c). This
may be due to how the increased shaking speed disrupts the structure of
the enzyme.\textsuperscript{{[}31{]}} When the system contains
high-titer substrates, the cell catalysts and glycosyl donors increase
proportionally with respect to the substrate concentration making the
interaction space between substrate and enzyme closer. Accordingly, the
shaking speed and reaction time need to be adjusted to obtain highly
efficient conversion.

To investigate the reciprocal effect of high-titer substrate, cell
catalyst concentration (biomass), and shaking speed, an L9
(3\textsuperscript{4}) orthogonal design was performed using 3 g/L Reb A
as the substrate (Table 1). Based on the range analysis, the factor that
exhibited the greatest influence on the Reb D production was RA/UDPG
ratio, followed by the StUGT biomass, shaking speed, and reaction time.
The optimal combination for the maximum production was a RA/UDPG ratio
of 1:2, 72 OD\textsubscript{600} cells, a shake speed of 0 rpm, and a 20
h reaction time (Table 1). The confirmatory tests under these optimal
conditions highlight the highest Y\textsubscript{RD} could reach was
94.53\%, which is significantly higher than that of the first orthogonal
test (68.53\% of Y\textsubscript{RD}).

A total of 2--10 g/L Reb A was then added in the optimal system for high
titer production of Reb D. Y\textsubscript{RD} increased gradually with
Reb A amounts increasing from 1 to 6 g/L (Figure 4d) to reach a maximum
yield of 6.12 g/L (Y\textsubscript{RD} of 94.41\%). The yield then
exhibited a downward trend with increasing Reb A concentrations (Figure
4d). When the formed Reb D exceeded 4 g/L, it would precipitate due to
the low solubility. Reb D found in solution and precipitate form were
both calculated as part of the yield.

The Reb D production using over 8 g/L Reb A was significantly lower than
when 6 g/L of Reb A was utilized as the substrate. This phenomenon stems
from substrate inhibition. Two fed-batch reactions were initiated to
avoid substrate inhibition. A 5 g/L Reb A sample was used as the initial
substrate, and 5 g/L Reb A was added subsequently a single time or two
doses of 2.5 g/L Reb A were added twice to achieve a final concentration
of 10 g/L (Figure 5). Y\textsubscript{RD} in the two fed reactions was
increased to 5.88 g/L, which was 68\% higher than the yield in the batch
reaction using 10 g/L Reb A as a substrate (3.50 g/L). As such, the
inhibitory effects might be circumvented by a fed-batch reaction.

In the two fed-batch reactions, Reb D titer, before feeding, reached
80\% of the maximum, and the production rate (0.28 g/L/h) before feeding
was significantly higher than that after feeding (Figure 5). Basically,
the production process of Reb D became sluggish after feeding. Whether
in a batch or feed-batch reaction, the product was no longer formed when
Reb D reached a concentration close to 5.8 g/L, indicating that it is a
threshold for dynamic equilibrium and the feeding does not significantly
improve the yield. This stems from how excessively high concentrations
of Reb D inhibit the reaction process, but the product inhibition could
be alleviated by simultaneously removing the fermentation
products.\textsuperscript{{[}35{]}}

The maximum Reb D yield of 6.12 g/L was obtained from 6 g/L Reb A in the
batch reaction. Presently, whole cell systems can convert 0.12--1.11 g/L
Reb D from Reb A and with a Reb D yield of
10\%--98.27\%.\textsuperscript{{[}13,36{]}} The higher Reb D production
in this system (6.12 g/L) supports the use of this new system as an
optimized and efficient catalytic system. High-titer products are
difficult to gain successfully because the system usually contains
plentiful substances such as substrates, catalysts, and ions that
harmfully contribute to volume exclusion and Donnan equilibrium
effects.\textsuperscript{{[}37{]}} To overcome these challenges, we
developed a cell catalytic platform to produce high-titer Reb D, which
would promote high-titer production using cell catalysts.

\subsection*{3.7 Reb D production by StUGT and sucrose
synthase}

{\label{reb-d-production-by-stugt-and-sucrose-synthase}}

To reduce production costs, a system using whole-cell catalysts of StUGT
and sucrose synthase was constructed to regenerate UDPG as donors for
glycosylation (Figure 6a). The SuSy has a relatively broad substrate
spectrum, and the candidate SuSy can convert sucrose and UDP to
eliminate the need to synthesize costly
UDPG.\textsuperscript{{[}17{]}}Among the 48,300 different types of SuSy,
only a few, such as AtSUS1, exhibit the proper
function.\textsuperscript{{[}38{]}} Therefore, the AtSUS1 sequence was
used as a query to obtain a homologous SuSy from\emph{Gossypium
schwendimani} (GsSUS1). This shares the highest amino acid sequence
identity (85\%) with AtSUS1 and is a novel putative unknown function
SuSy.

The authors previously constructed recombinant \emph{E. coli} expressing
AtSUS1 and GsSUS1 (unpublished) to develop the coupled system. The StUGT
cells were combinatorically used with the various amounts of biomass of
AtSUS1 and GsSUS1 cells. The two cascade-reaction system converted Reb A
to Reb D, and Y\textsubscript{RD} in the StUGT-GsSUS1 was significantly
higher than that in the StUGT-AtSUS1 system (Figure 6b). When the ratio
of StUGT/SUS1 was changed from 2:1 to 1:3, the production showed a
noticeable upward trend where it reached a maximum at the ratio of 1:3.
This is 61.67\% of the maximum Y\textsubscript{RD} in the StUGT reaction
(Figure 6c). The Reb D decreased slightly as the ratio decreased from
1:3 to 1:5. This trend may stem from excessive whole-cell catalyst. As a
whole, a cascade system using sucrose as the glucosyl donor was
developed, and the function of GsSUS1 was also revealed for the first
time. However, the StUGT-GsSUS1 system could benefit from further
optimizing the catalytic conditions.

The orthogonal design was performed using 3 g/L Reb A as a substrate to
optimize the production. The optimal condition for Reb D production was
a StUGT/GsSUS1 cell ratio of 1:2, a pH of 8.0, a 200 rpm shake speed,
and a 20 h reaction time (Table S5). The reaction time had the largest
effect on Y\textsubscript{RD} as shown by its extreme value. The optimal
reaction time of 20 h was the longest among the investigated reaction
times, which may be because the cascade reaction requires multiple
catalytic steps. A relatively long reaction time is conducive to high
conversion. Therefore, extending the reaction time was considered in the
following experiment.

The Reb A concentration was increased to 6 g/L to improve the economy of
industrial production, and an orthogonal test was performed to
investigate the effect of the ratio between the reaction components on
the cascade reaction. The optimal combination for maximum production was
a StUGT/GsSUS1 cell ratio of 1:2.5, a RA/sucrose ratio of 1:5, a
sucrose/UDP ratio of 1:1, and a 28 h reaction time (Table S6). The
maximum Y\textsubscript{RD} was 3.57 g/L, and the cascade efficiency
reached 84.98\%, which was 37.79\% higher than the single-factor
experiments. The sucrose/UDP ratio affected the results more than any
other factor. Y\textsubscript{RD} was highest when using a 1:1 ratio of
sucrose/UDP, but it decreased significantly when using a 1:2 ratio. This
may be because the high concentrations of UDP inhibit catalytic
activity.

As a final effort to maximize efficiency, the biomass of StUGT/GsSUS1 in
the system was increased from 54/135 to 72/180
OD\textsubscript{600}under the above optimal reaction conditions.
Y\textsubscript{RD}improved to 5.27 g/L and could reach 88.10\% of the
pure StUGT reaction system (Figure 6d). The high-performance conversion
results from the high-activity catalyst and the other optimized
parameters.

Developing an efficient cascade catalytic system consisting of the
poorly studied enzymes is difficult. We overcame this difficulty by
expanding new candidate analogous enzymes and accurately designing cell
combinations according to the characterization of catalysts. This work
provides the first example of efficient production of high-titer Reb D
by a whole-cell cascade model using novel SuSy and UGT. This system may
serve as a guideline to build other cascade glycosylation systems to
establish green and scalable industrial production of Reb D.

\section*{4. Conclusions}

{\label{conclusions}}

A novel glycosyltransferase StUGT with transglycosylation function of
Reb A to Reb D was identified. The StUGT exhibited high substrate
specificity and strong Reb A affinity. A whole-cell catalytic platform
for high-titer production was developed, and a final 6.12 g/L Reb D was
achieved from a 6 g/L Reb A feedstock, which is the highest production
by whole-cell catalysis to date. We also developed a StUGT-GsSUS1
whole-cell-based cascade catalysis using cheap sucrose as a glycosyl
donor, and 5.27g/L Reb D was obtained by systematic optimization. This
report revealed a novel glycosyltransferase and established a
high-efficiency low-cost biotransformation technology.

\textbf{AUTHOR CONTRIBUTIONS}

\textbf{Si-yuan Ma:} Methodology, Formal analysis, Investigation,
Writing -- original draft. \textbf{Yuan-yuan Ma:} Conceptualization,
Formal analysis, Funding acquisition, Project administration,
Validation, Resources, Supervision, Writing -- review and editing.

\textbf{CONFLICT OF INTEREST STATEMENT}

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.

\textbf{DATA AVAILABILITY STATEMENT}

No data was used for the research described in the article.

\textbf{ACKNOWLEDGMENTS}

We would like thank Prof. Hao Song for his guidance, and thank Dr.
Jiefang Hong for his help in protein expression and HPLC analysis. We
thank Yatong Li for her help in the LC-MS catalysis experiments. We also
appreciate Lifang Yu and Zhenyang Wang for their help in polishing this
manuscript. We thank LetPub (www.letpub.com) for its linguistic
assistance during the preparation of this manuscript. This work was
supported by the National Natural Science Foundation of China
(30900033), Sinochem Health Company Ltd. (2020GFW-0163), and the
Independent Innovation Foundation of Tianjin University (2020XZC-0034;
2021XZC-0002).

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\end{enumerate}

\textbf{Table 1} Orthogonal results for Reb D production by StUGT cells\selectlanguage{english}
\begin{longtable}[]{@{}lllllllllllllll@{}}
\toprule
\begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
Number\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
Number\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
A (biomass)\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
A (biomass)\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
B (shaker speed)\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
B (shaker speed)\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
B (shaker speed)\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
C (RA/UDPG)\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
C (RA/UDPG)\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
D (reaction time)\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
D (reaction time)\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
D (reaction time)\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
Results\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
Results\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
Results\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
RA conversion\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
RA conversion\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
RD yield\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 72\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 72\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 72\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 72\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0 rpm\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1:1.5\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1:1.5\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
12 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
12 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
12 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
84.24\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
84.24\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
73.42\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 72\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 72\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 72\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 72\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
100 rpm\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1:2\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1:2\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
16 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
16 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
16 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
93.01\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
93.01\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
84.87\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 72\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 72\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 72\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 72\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
200 rpm\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1:2.5\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1:2.5\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
20 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
20 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
20 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
97.42\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
97.42\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
91.88\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600}=96\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600}=96\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600}=96\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600}=96\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0 rpm\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1:2\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1:2\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
20 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
20 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
20 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
92.67\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
92.67\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
83.87\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600}=96\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600}=96\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600}=96\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600}=96\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
100 rpm\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1:2.5\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1:2.5\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
12 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
12 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
12 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
96.77\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
96.77\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
89.10\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600}=96\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600}=96\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600}=96\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600}=96\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
200 rpm\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1:1.5\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1:1.5\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
16 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
16 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
16 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
65.11\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
65.11\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
57.59\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
7\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 120\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 120\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 120\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 120\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0 rpm\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1:2.5\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1:2.5\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
16 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
16 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
16 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
96.54\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
96.54\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
87.47\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
8\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 120\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 120\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 120\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 120\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
100 rpm\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1:1.5\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1:1.5\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
20 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
20 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
20 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
62.49\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
62.49\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
52.77\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
9\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 120\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 120\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 120\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
OD\textsubscript{600} = 120\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
200 rpm\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1:2\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1:2\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
12 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
12 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
12 h\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
76.56\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
76.56\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
63.55\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
K\textsubscript{I}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
K\textsubscript{I}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
92.92\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
92.92\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
88.98\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
88.98\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
88.98\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
68.26\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
68.26\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
81.71\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
K\textsubscript{II}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
K\textsubscript{II}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
83.65\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
83.65\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
81.96\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
81.96\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
81.96\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
86.55\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
86.55\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
83.20\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
K\textsubscript{III}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
K\textsubscript{III}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
73.49\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
73.49\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
79.12\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
79.12\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
79.12\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
95.24\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
95.24\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
85.15\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
R\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
R\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
19.43\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
19.43\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
9.86\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
9.86\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
9.86\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
26.98\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
26.98\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
3.45\%\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

K\textsubscript{I}, K\textsubscript{II} and K\textsubscript{III} were
the average values of the results at each level in the orthogonal
experiment. The range R is the absolute value of the difference between
the maximum value and the minimum value of K\textsubscript{ij}.

\textbf{Table 2} Relative catalytic activity of StUGT at different
concentrations of metal ions\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\textbf{Concentration} \textbf{Metal Ion}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
1mM\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
3mM\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
6mM\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Ca\textsuperscript{2+}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
103.2\selectlanguage{ngerman}±1.5\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
103.4\selectlanguage{ngerman}±0.1\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
66.5\selectlanguage{ngerman}±2.6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Pb\textsuperscript{2+}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
109.7\selectlanguage{ngerman}±0.4\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
101.2\selectlanguage{ngerman}±3.4\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
19.4\selectlanguage{ngerman}±1.3\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Ba\textsuperscript{2+}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
112.9\selectlanguage{ngerman}±3.9\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
105.5\selectlanguage{ngerman}±0.1\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
109.9\selectlanguage{ngerman}±0.3\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Mn\textsuperscript{2+}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
114.6\selectlanguage{ngerman}±0.8\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
105.6\selectlanguage{ngerman}±0.1\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
103.3\selectlanguage{ngerman}±0.3\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Mg\textsuperscript{2+}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
92.1\selectlanguage{ngerman}±0.2\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
115.1\selectlanguage{ngerman}±1.4\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
115.6\selectlanguage{ngerman}±1.0\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
CK\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
100\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Figure legends}

\textbf{Figure 1} : Bioinformatics analysis of StUGT. (a) Multiple
sequence alignment of StUGT and its homologues. The sequences of UGTSL2
(XP\_004250485.1), EUGT11 (XP\_015629141.1), and UGT91D2 (KAG6582627.1)
were from NCBI. The predicted secondary structure of StUGT is shown
above each line of the alignment. \selectlanguage{greek}α, β, \selectlanguage{english}and \selectlanguage{greek}η \selectlanguage{english}indicate alpha-helix,
beta-sheet, and 310-helix, respectively. Conserved residues are
highlighted, the identical residues are colored in red, while similar
residues are in the blue frame. The PSPG motif (residues 318--361) is
labeled by a single blue bracket. (b) Structure modeling of StUGT. (c)
Molecular docking of StUGT with Reb A. Green and red sticks indicate Reb
A. Yellow dashed lines show the hydrogen bonds between Reb A and amino
acid residues Glu67 and Glu70 (grey). (d) Synthetic pathway of Reb D
from Reb A with StUGT.

\textbf{Figure 2} : Schematic illustration of expression, purification,
and catalysis of StUGT. (a) The expression and purification of StUGT.
(b) Strategy for whole-cell production of Reb D from Reb A.

\textbf{Figure 3} : Biochemical properties of the StUGT. Relative
activity of StUGT at different temperatures (a) and pH (b) by two forms
of catalytic reaction. (c) Y\textsubscript{RD} under various metal ions.
Blank group refers to the control without the addition of any metal
ions. (d) C\textsubscript{RA} of reaction at different UDPG
concentrations.

\textbf{Figure 4} : Optimization of Reb D synthesis using whole-cell
catalysis. (a) Relative activity of StUGT cells treated by CTAB,
Tween-80, and Triton-X-100. Relative activity at various reaction times
(b) and biomass (c). C\textsubscript{RA} and Y\textsubscript{RD} of
StUGT at various Reb A concentrations (d).

\textbf{Figure 5} : Fed-batch strategies of whole-cell bioconversion for
the production of Reb D from Reb A. The 5 g/L Reb A was added once at 16
h (a), and 2.5 g/L Reb A was added twice at 16 h and 30 h (b),
respectively.

\textbf{Figure 6:} Cascade reaction for the Reb D synthesis using
sucrose as the glycosyl donor. (a) A scheme of whole-cell cascade
reaction using StUGT and GsSUS1. (b) The Reb D yield under various cell
density ratios of StUGT/SUS1. (c) CRA and YRD of the StUGT-GsSUS1
reaction at high substrate concentration (6g/L). Control group refers to
the reaction system without the addition of GsSUS1 cells.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/figure1Ya-Suo-/figure1Ya-Suo-}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/figure2-tifYa-Suo-/figure2-tifYa-Suo-}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/figure3/figure3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/figure4/figure4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/figure5/figure5}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/figure6/figure6}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

