Abstract
Steviol glycosides (SGs) with zero calories and high-intensity sweetness are the best substitutes of sugar for the human diet. Uridine diphosphate dependent glycosyltransferase (UGT) UGT76G1, as a key enzyme for the biosynthesis of SGs with a low heterologous expression level, hinders its application. In this study, a suitable fusion partner, Smt3, was found to enhance the soluble expression of UGT76G1 by 60%. Additionally, a novel strategy to improve the expression of Smt3-UGT76G1 was performed, which co-expressed endogenous genes prpD and malK in Escherichia coli. Notably, this is the first report of constructing an efficient E. coli expression system by regulating prpD and malK expression, which remarkably improved the expression of Smt3-UGT76G1 by 200% as a consequence. Using the high-expression strain E. coli BL21 (DE3) M/P-3-S32U produced 1.97 g/L of Smt3-UGT76G1 with a yield rate of 61.6 mg/L/h by fed-batch fermentation in a 10 L fermenter. The final yield of rebadioside A (Reb A) and rebadioside M (Reb M) reached 4.8 g/L and 1.8 g/L, respectively, when catalyzed by Smt3-UGT76G1 in the practical UDP-glucose regeneration transformation system in vitro. This study not only carried out low-cost biotransformation of SGs but also provided a novel strategy for improving expression of heterologous proteins in E. coli.
Highlights
Nowadays, more and more countries are facing a serious public health burden from the consumption of high-calorie sugars such as glucose, fructose, and sucrose [1,2,3]
Through SDS-PAGE analysis results, we found that the soluble expression of UGT76G1 was relatively higher with pET32a-UGT76G1 or pET22b-UGT76G1 than that with plasmid pET26b-UGT76G1 (Figure S1A)
The soluble recombinant enzyme expressed by plasmid pET32a-UGT76G1 showed the highest activity which could completely convert St to rebadioside A (Reb A) in 12 h (Figure S1B)
Summary
More and more countries are facing a serious public health burden from the consumption of high-calorie sugars such as glucose, fructose, and sucrose [1,2,3]. Reb D (penta-glycoside) and Reb M (hexa-glycoside) which are trace components in stevia plants, but their sweetness potency is up to 350 times greater than that of sucrose and notably have no bitter aftertaste, are recognized to be high-quality sweeteners and the best substitute for sugar [10]. UGT91D2 and UGT76G1 directly catalyze the formation of 1, 2-β-d- and 1, 3-β-d-glucoside bonds at the C13- and C19- positions, respectively [18] With these four enzymes, the biosynthesis of almost all SGs including St, Reb A, Reb D and Reb M, could be identified [19]. Recombinant glycosyltransferase produced by the engineered strain constructed in this work has proven to be efficient in high yield of Reb A and Reb M in vitro enzymatic biotransformation
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