Effective synthesis of Rebaudioside A by whole-cell biocatalyst Pichia pastoris

Rebaudioside A is an attractive non-nutritive natural sweetener. UDP-glycosyltransferase UGT76G1 from Stevia rebaudiana Bertoni has been found to effectively convert stevioside to rebaudioside A. In this study, sucrose synthase mbSUS from Vigna radiata was first reported to co-operate with glycosyltransferase. Compared with other sucrose synthases, mbSUS was selected for co-expression with UGT76G1 in Pichia pastoris GS115 because of its high efficiency in improving the synthesis of rebaudioside A. After optimizing the gene dose ratio of mbSUS and UGT76G1, which is an effective whole-cell biocatalyst GS115 (U76/mb3:1) was constructed. For the biosynthesis of rebaudioside A with whole-cell biocatalyst GS115 (U76/mb3:1), the reaction parameters were optimized and the limiting conditions were identified. Under optimal conditions, the yield of rebaudioside A reached 261.2 mM in a 1 L batch within 26 h. Based on its the high reaction efficiency, the whole-cell catalyst GS115 (U76/mb3:1) is a powerful biocatalyst with the potential for industrial-scale synthesis.