## 1 Introduction  
Glycosylation of small molecular compounds refers to the addition of glycosyl residues to the \(C\), _O_-, _N_-, and _S_-positions of some small molecules, such as antibiotics, vitamins, and spices, to improve their physical chemistry and biology characteristics [1, 2]. The formation of glycoside bonds during glycosylation is popularly obtained by chemical or enzymatic methods. Compared with complicated chemical manipulation, enzyme-mediated glycosylation is capable of recognizing certain chemical groups to synthesize specific products with simple steps and not introducing extra-toxic chemical reagents due to the splendid region- and stereoselectivity of enzymes, especially in the glycosylation of compounds with complex conformations. In nature, glycosylation of small molecular compounds is usually catalyzed by glycosyltransferases (GTs), among which uridine diphosphate glycosyltransferases (UGTs) are pivotal members [3, 4]. Uridine diphosphate (UDP)-activated sugar molecules are widely used as donors in glycosylation of natural products, such as stevisosides [5], triterpenoids [6], flavonoids [7], and ginsengides [8]. UGTs also contribute to the metabolic engineering applications for producing these natural products [9]. However, such glycosylation reactions are restricted by catalytic abilities of GTs and the availability of nucleoside diphosphate (NDP)-sugar donor [10, 11]. Sucrose synthase (SuSy, EC 2.4.1.13) belonging to the GT family, which reversibly catalyzes NDP and sucrose to produce NDP-glucose and fructose, is one of the most promising candidates addressing the supply of NDP-glucose [12]. Most eukaryotic SuSy-mainly participate in the physiological metabolism of plants, especially for the synthesis or decomposition of sucrose [13; 14]. A few SuSys have also been found in prokaryotes-like bacteria in recent years, which have higher thermal stability and prefer adenosine diphosphate (ADP), while plant-derived SuSys prefer UDP [13]. SuSys could work with UGTs in vitro to constitute the coupling system for regenerating UDP-glucose (UDPG) that is provided to the glycosylation reaction catalyzed by UGTs [14]. The one-pot two-enzyme reactions by coupling the activities of UGT and SuSy (UGT-SuSy) were applied to numerous chemical compounds such as \(C\)-glycosylation of phloretin [11], \(O\)-glycosylation of stoichiometric, and quercetin [15; 16]. Generally, the catalytic efficiency of free enzymes in the reaction that would be affected by several factors can be enhanced to some extent by organizing them into a multi-enzyme complex using co-immobilization technology [17], scaffolding technology [18], or fusion technology [19]. Among them, gene fusion technology, widely used in protein soluble expression, purification, imaging, and biopharmaceuticals, is also an effective and relatively simple strategy for setting up a multi-enzyme catalytic system in vitro [20]. UGT76G1, a UGT was initially discovered in _Stevia rebuudiana_ for the conversion of stevioside into rebaudioside A (RebA), a sweetener with low calories and intense sweetness [21]. As has been known so far in the biosynthesis of steviol glycosides (SGs), UGT76G1 uses UDPG as the glycosyl donor to form the \(\beta\)-1,3-glycosidic bond at the steviol C\({}_{13}\)- and C\({}_{19}\)-bound glucose involving eight different reactions [5]. It is the only enzyme directly responsible for the production of rebaudioside M, which is generally recognized as the best SG sweetener [22]. In our previous research, we have set up a UGT-SuSy reaction involving UGT76G1 and a SuSy from _Arabidopsis thaliana_ (_A_tSUS1) [23]. UDPG and UDP in crude extracts from the recombinant cells were used without additional supply [15; 24]. In the present study, we try to compare the performance of four different originated SuSys in cooperation with UGT76G1, as well as the coupling details, such as the gene construct strategy of two enzymes. As a result, a fusion of SuSy and UGT was investigated for increased effective production of RebA from stevioside.