## 3 Results and Discussion
### Comparison of the Construct Strategy for Expressing AtSUS1 and UGT76G1  
In comparison to different construct strategies to generate the recombinant strain for enzyme preparation, AtI-76G1 was selected, since _At_SUS1 has the higher specific activity (47.6 MU/mg) than that of the other two eukaryotic SuSys. To construct AtI-76G1, genes encoding _At_SUS1 and UGT76G1 were inserted in _NoI_/_Eco_RI and _Ndel_/_Xli_o_I of pRSFDuet-1, respectively. Another strain, named 76G1-AtI, was obtained by transformed the plasmid pRSF-76G1-AtUS1 in which the genes encoding _At_SUS1 and UGT76G1 were inserted in _Ndel_/_Xli_o_I and _NoI_/_Eco_RI of pRSFDuet-1, respectively. That means that these two genes belonging to an individual transcription unit exchanged their cloning positions. Although the protein soluble expression patterns look similar (Figure S2), the specific activities of two enzymes obtained from AtI-76G1 and 76G1-AtI were not the same, especially for UGT76G1 (Figure 1a). The yield of RebA was 86.5% for 76G1-AtI, which was 1.3 times of AtI-76G1 (65.4%), but AtI-76G1 has higher UGT activity than 76G1-AtI (Figure 1). These results indicated that the arrangement of the gene sequence of _At_SUS1 and UGT76G1 in the expression plasmid may exert an influence on RebA production, probably because the activity ratio of UGT and SuSy is a critical factor under the condition that both enzyme activities were not low. Recent studies show gene fusion strategies promote the folding, stability, bioactivity, and soluble expression of the fused proteins [35; 36; 37]. In theory, the fusion of two enzymes will shorten the space distance of enzymes, which is more conducive to the conversion of substrates. It was reported that a linker encoding for amino acids Pro-Pro-Pro (PPP) had been successfully applied in a fusion construction of the flavonoid O-glucosyltransferasetransferase OsUGT3 and _At_SUS1 [38]. Therefore, we introduced the PPP linker between _At_SUS1 and UGT76G1 in AtI-76G1 and 76G1-AtI, which were named AtI-P3-76G1 and 76G1-P3-AtI, respectively. It was shown that the fusion order of these two enzymes had a great influence on the soluble expression of fusion proteins, and thus, on the activities of UGT and SuSy, and the synthesis of RebA catalyzed by the fusion enzymes (Figure S3a and Figure 1). When the _C_-terminal of _At_SUS1 was fused with the _N_-terminal of UGT76G1(AtI-P3-76G1), the yield of RebA was 76.2%, which was 1.2 times that of AtI-76G1 at 5 h under the same conditions. It was still not better than that of 76G1-AtI (86.5%). However, when the _N_-terminal of _At_SUS1 was fused with the _C_-terminal of UGT76G to form the fusion protein 76G1-P3-AtI, the RebA yield of 76G1-P3-AtI was only 6.9%. Merely exchanging the two encoding genes on the expression plasmids caused a similar RebA yield between AtI-76G1 and 76G1-AtI (Figure 1b). However, the fusion proteins with the PPP linker showed distinct differences compared to the free enzyme  
Figure 1: RebA production catalyzed by the free enzymes of UGT76G1 and _At_SUS1, and their fusion proteins. (**a**) The special activities of UGT and SuSy. (**b**) RebA yield of four reactions catalyzed by AtI-76G1, 76G1-AtI, AtI-P3-76G1, and 76G1-P3-AtI. The reaction mixture (20 mL), consisting of approximately 5 mg/mL of the crude extract, 20 g/L stevioside, and 60 g/L sucrose, was incubated at pH 7.2 and 30 \({}^{\circ}\)C for 5 h.  
systems. The specific activity of UGT76G1 significantly decreased for At1-P3-76G1 (less than half of At1-76G1 and 76G1-At1), but the production of RebA was comparable with At1-76G1 and 76G1-At1. The specific activities of both UGT and SuSy declined sharply for 76G1-P3-At1, leading to almost no accumulation of RebA in the glycosyltransferase-catalyzed reaction. Therefore, the fusion order of UGT and SuSy has an important effect on their enzyme activity and productivity.