Microbial production of next-generation stevia sweeteners.
Microbial production of next-generation stevia sweeteners.
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DOI：
10.1186/s12934-016-0609-1
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发表时间：
2016-12-07
影响因子：
6.4
通讯作者：
Møller BL
中科院分区：
文献类型：
--
作者：
Olsson K;Carlsen S;Semmler A;Simón E;Mikkelsen MD;Møller BL
甜菊糖苷转移酶UGT76G1是一种变色龙酶，用于下一代优质甜菊糖苷D （Reb D）和Reb M （Reb M）的靶向生物合成。这些甜菊糖苷分别含有5个和6个葡萄糖单位，与甜菊糖苷和莱鲍迪糖苷a（甜菊叶中含量最多的甜菊糖苷）相比，甜菊糖苷具有较低的甜味阈值，较高的最大甜味强度，并大大减少了苦味。在导致Reb D和Reb M产生的代谢糖基化网格中，UGT76G1被发现催化8种不同的反应，所有反应都涉及甜菊醇c13 -和c19结合葡萄糖的1,3-糖基化。其中4个反应生成Reb D和Reb M，另外4个反应生成不需要的副产物。在这项工作中，通过靶向优化UGT76G1对已经1,2-二糖基化的甜菊醇糖苷进行1,3糖基化，减少了副产物的形成。UGT76G1的优化基于同源性建模，能够识别底物结合口袋中存在的关键目标氨基酸。然后对这些残基进行位点饱和诱变，并对包含1748个UGT76G1变体的突变文库进行筛选，以增加Reb D或M的积累，以及减少副产物的积累。该筛选是在一个酿酒酵母菌株中进行的，该菌株表达了雷鲍迪苷生物合成途径中除UGT76G1外的所有酶。突变文库筛选发现对Reb D和Reb m积累有积极影响的突变，并对引入突变对代谢网格中其他反应的影响进行了表征。这种筛选使鉴定变异成为可能，如UGT76G1Thr146Gly和UGT76G1His155Leu，它们减少了不需要的副产物的积累，并增加了所需的Reb D或Reb M甜味剂的特定积累。甜叶菊甜味剂生物合成途径的关键酶的改进代表了下一代甜叶菊甜味剂商业化生产的重要一步。本文的在线版本（doi:10.1186/s12934-016-0609-1）包含补充材料，可供授权用户使用。
The glucosyltransferase UGT76G1 from Stevia rebaudiana is a chameleon enzyme in the targeted biosynthesis of the next-generation premium stevia sweeteners, rebaudioside D (Reb D) and rebaudioside M (Reb M). These steviol glucosides carry five and six glucose units, respectively, and have low sweetness thresholds, high maximum sweet intensities and exhibit a greatly reduced lingering bitter taste compared to stevioside and rebaudioside A, the most abundant steviol glucosides in the leaves of Stevia rebaudiana. In the metabolic glycosylation grid leading to production of Reb D and Reb M, UGT76G1 was found to catalyze eight different reactions all involving 1,3-glucosylation of steviol C 13- and C 19-bound glucoses. Four of these reactions lead to Reb D and Reb M while the other four result in formation of side-products unwanted for production. In this work, side-product formation was reduced by targeted optimization of UGT76G1 towards 1,3 glucosylation of steviol glucosides that are already 1,2-diglucosylated. The optimization of UGT76G1 was based on homology modelling, which enabled identification of key target amino acids present in the substrate-binding pocket. These residues were then subjected to site-saturation mutagenesis and a mutant library containing a total of 1748 UGT76G1 variants was screened for increased accumulation of Reb D or M, as well as for decreased accumulation of side-products. This screen was performed in a Saccharomyces cerevisiae strain expressing all enzymes in the rebaudioside biosynthesis pathway except for UGT76G1. Screening of the mutant library identified mutations with positive impact on the accumulation of Reb D and Reb M. The effect of the introduced mutations on other reactions in the metabolic grid was characterized. This screen made it possible to identify variants, such as UGT76G1Thr146Gly and UGT76G1His155Leu, which diminished accumulation of unwanted side-products and gave increased specific accumulation of the desired Reb D or Reb M sweeteners. This improvement in a key enzyme of the Stevia sweetener biosynthesis pathway represents a significant step towards the commercial production of next-generation stevia sweeteners. The online version of this article (doi:10.1186/s12934-016-0609-1) contains supplementary material, which is available to authorized users.
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影响因子：
14.8
作者：
Gietz, R. Daniel;Schiestl, Robert H.
通讯作者：
Schiestl, Robert H.
DOI：
10.1001/jama.2014.732
发表时间：
2014-02-26
期刊：
JAMA
影响因子：
--
作者：
Ogden CL;Carroll MD;Kit BK;Flegal KM
通讯作者：
Flegal KM
影响因子：
0.8
作者：
Misra, Himanshu;Soni, Manish;Jain, D. C.
通讯作者：
Jain, D. C.
影响因子：
14.9
作者：
Davis IW;Leaver-Fay A;Chen VB;Block JN;Kapral GJ;Wang X;Murray LW;Arendall WB 3rd;Snoeyink J;Richardson JS;Richardson DC
通讯作者：
Richardson DC
DOI：
10.1107/s0907444909042073
发表时间：
2010-01
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Chen VB;Arendall WB 3rd;Headd JJ;Keedy DA;Immormino RM;Kapral GJ;Murray LW;Richardson JS;Richardson DC
通讯作者：
Richardson DC