莱鲍迪苷D(Rebaudioside D,Reb D)是高甜度、低热量的天然甜味剂,兼具商业与健康价值,为合成莱鲍迪苷M(Rebaudioside M,Reb M)的前体,其研究可推动高品质甜味剂开发。该研究通过构建糖基转移酶EUGT11与蔗糖合成酶AtSUS3的重组大肠杆菌菌株,进一步建立双酶偶联体系,实现尿苷二磷酸葡萄糖(uridine diphosphate-glucose,UDPG)的循环利用,驱动莱鲍迪苷A(Rebaudioside A,Reb A)高效生物合成Reb D。优化双酶催化体系反应条件结果表明,在3 mL含100 mmol/L NaH2PO4-Na2HPO4缓冲液(pH 7.0)的糖基化偶联反应体系中,含粗酶提取物2.4 mg/mL,Reb A 1.2 g/L和蔗糖60 g/L,30 ℃下反应20 h后,Reb D的产量达到最大值1 205.81 mg/L,产率提高至86.06%。该研究有效提升了Reb D的合成效率,也为低成本规模化生产优质甜菊糖苷奠定了技术基础。
Rebaudioside D (Reb D), a natural high-intensity, low-calorie sweetener, serves as a crucial precursor for synthesizing Rebaudioside M (Reb M), offering significant commercial and health benefits.Research on Reb D is therefore crucial for advancing the development of high-quality sweetener.In this study, a dual-enzyme coupled system was established by constructing a recombinant Escherichia coli strain co-expressing glucosyltransferase EUGT11 and sucrose synthase AtSUS3.This system facilitated the recycling of uridine diphosphate glucose (UDPG), enabling the efficient bioconversion of Reb A to Reb D.Under optimized conditions in a 3 mL glycosylation reaction system containing 100 mmol/L NaH2PO4-Na2HPO4 buffer (pH 7.0), supplementation with 2.4 mg/mL crude enzyme extract, 1.2 g/L Reb A, and 60 g/L sucrose, followed by incubation at 30 ℃ for 20 h, resulted in a maximum Reb D yield of 1 205.81 mg/L, corresponding to an 86.06% conversion rate.This study significantly enhances the synthesis efficiency of Reb D and establishes a robust technical foundation for the cost-effective, large-scale production of high-purity steviol glycosides.
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