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Production of rebaudioside D from stevioside using a UGTSL2 Asn358Phe mutant in a multi-enzyme system.
Microbial Biotechnology ( IF 4.8 ) Pub Date : 2020-02-03 , DOI: 10.1111/1751-7915.13539
Liangliang Chen 1 , Ruxin Cai 1 , Jingyuan Weng 1 , Yan Li 1 , Honghua Jia 1 , Kequan Chen 1 , Ming Yan 1 , Pingkai Ouyang 1
Affiliation  

Rebaudioside D is a sweetener from Stevia rebaudiana with superior sweetness and organoleptic properties, but its production is limited by its minute abundance in S. rebaudiana leaves. In this study, we established a multi‐enzyme reaction system with S. rebaudiana UDP‐glycosyltransferases UGT76G1, Solanum lycopersicum UGTSL2 and Solanum tuberosum sucrose synthase StSUS1, achieving a two‐step glycosylation of stevioside to produce rebaudioside D. However, an increase in the accumulation of rebaudioside D required the optimization of UGTSL2 catalytic activity towards glucosylation of rebaudioside A and reducing the formation of the side‐product rebaudioside M2. On the basis of homology modelling and structural analysis, Asn358 in UGTSL2 was subjected to saturating mutagenesis, and the Asn358Phe mutant was used instead of wild‐type UGTSL2 for bioconversion. The established multi‐enzyme reaction system employing the Asn358Phe mutant produced 14.4 g l−1 (1.6 times of wild‐type UGTSL2) rebaudioside D from 20 g l−1 stevioside after reaction for 24 h. This system is useful for large‐scale rebaudioside D production and expands our understanding of the pathways involved in its synthesis.

中文翻译:


在多酶系统中使用 UGTSL2 Asn358Phe 突变体从甜菊苷生产莱鲍迪苷 D。



莱鲍迪甙 D 是一种来自甜叶菊的甜味剂,具有卓越的甜味和感官特性,但其产量因其在甜叶菊叶中含量极少而受到限制。在本研究中,我们利用莱鲍迪安娜UDP-糖基转移酶UGT76G1、番茄UGTSL2和马铃薯蔗糖合酶StSUS1建立了多酶反应体系,实现了甜菊苷的两步糖基化生产莱鲍迪苷D。莱鲍迪苷 D 的积累需要优化 UGTSL2 对莱鲍迪苷 A 糖基化的催化活性并减少副产物莱鲍迪苷 M2 的形成。在同源建模和结构分析的基础上,对UGTSL2中的Asn358进行饱和诱变,用Asn358Phe突变体代替野生型UGTSL2进行生物转化。所建立的采用Asn358Phe突变体的多酶反应系统在反应24小时后从20gl -1甜菊糖中产生14.4gl -1 (野生型UGTSL2的1.6倍)莱鲍迪苷D。该系统对于大规模莱鲍迪甙 D 生产非常有用,并扩展了我们对其合成所涉及途径的理解。
更新日期:2020-02-03
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