## 4 Conclusions  
The UGT-SuSy cascade system is expected to be successfully applied to the large-scale industrial production of SGs. Among _At_SUS1 and _At_SUS3 from _A. thaliana_, _St_SUS1 from _S. tuberosum_, and _Ac_SuSy from _A. caldus_, _At_SUS1 was the best candidate to set up the reaction coupling with _Stevia_ glycosyltransferase UGT76G1. Arrangement of the gene sequence of _At_SUS1 and UGT76G1 on the expression plasmid may affect the expression of enzymes, thus affecting the yield of RebA from stevioside. Fusions of the _C_-terminal of SuSy and _N_-terminal of UGT76G1 (At1-P3-76G1) with the rigid linker (EAAAK)3 seem to have a less negative effect on the active conformation of enzymes. However, the fusion containing _At_SUS1 and UGT76G1 was not superior to the reactions with their free enzymes, probably due to the low K\({}_{\text{m}}\) (UDP) value of plant SuSys. The space effect created by fusion worked in the case that prokaryotic _Ac_SuSym was applied, which maintained a high K\({}_{\text{m}}\) (UDP) value (above 0.1 mM). When 2 mM UDP was added to the reaction catalyzed by Acm-R3-76G1, the highest RebA yield of 78.2% (18.8 \(\pm\) 0.6 g/L) was obtained from 20 g/L stevioside after reaction for 12 h.  
The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app12083911/s1](https://www.mdpi.com/article/10.3390/app12083911/s1)]([https://www.mdpi.com/article/10.3390/app12083911/s1](https://www.mdpi.com/article/10.3390/app12083911/s1)), (Figures S1-S3 and S5) SDS-PAGE analysis of enzyme expression in the different recombinant strains; (Figure S4) HPLC analysis of the 12-h reaction mixtures catalyzed by Ac-76G1 and Acm-76G1 at 30 \({}^{\circ}\)C (PDF).  
Conceptualization, Y.L.; Methodology, Y.L. and H.J.; Investigation, Y.T., P.S. and R.C.; Data curation, Y.T. and P.S.; Writing--original draft preparation, Y.T., P.S. and Y.L.; Writing--review and editing, Y.T. and Y.L.; Supervision, Y.L. and H.J.; Project administration, Y.L.; Funding acquisition, Y.L. All authors have read and agreed to the published version of the manuscript.  
This research was funded by the National Key R&D Program of China (2021 YFC2101500), NSFC (21878155), the Jiangsu Synergetic Innovation Center for Advanced Bio-manufacture, and PAPD.  
Not applicable.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Protein** & **MW (kDa)** & **Size (aa)** & **Probability of Solubility or Insolubility** \\ \hline UGT76G1 & 52.0 & 458 & 57\% insoluble \\ _At_SUS1 & 93.0 & 808 & 60\% soluble \\ _Ac_SuSym & 91.3 & 794 & 63\% soluble \\ 76G1-P3-At1 & 147.4 & 1289 & 50\% soluble \\ At1-P3-76G1 & 145.3 & 1269 & 52\% soluble \\ R3 (At1-R3-76G1) & 146.4 & 1281 & 54\% soluble \\ Acm-R3-76G1 & 144.6 & 1266 & 56\% soluble \\ \hline \hline \end{tabular} Note: aa, amino acids, MW, molecular weight, Da, dalton.  
\end{table}
Table 5: Prediction of the solubility of proteins.  
Data Availability Statement: The data presented in this study are available in the submitted article. Conflicts of Interest: The authors declare no conflict of interest. Abbreviations ADP adenosine diphosphate; GT, glycosyltransferase; HPLC, high-performance liquid chromatography; NDP, nucleoside diphosphate; RebA, rebaudioside A; SG, steviol glycoside; St, steviolide; SutSy, sucrose synthase; UDP, uridine diphosphate; UDPG, UDP-glucose; UGT, uridine diphosphate glycosyltransferase.