A Muti-Substrate Flavonol O-glucosyltransferases from Safflower.
A Muti-Substrate Flavonol O-glucosyltransferases from Safflower.
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DOI：
10.3390/molecules28227613
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发表时间：
2023-11-15
期刊：
影响因子：
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通讯作者：
Guo M
中科院分区：
文献类型：
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作者：
Qi S;He B;Wang H;Duan Y;Wang L;Gao Y;Guo M
为了探索红花中黄酮类苷的完整生物合成过程，明确可能参与的关键糖基转移酶，并开发合成黄酮类苷的高效生物催化剂，在红花中鉴定出具有黄酮- o -糖基转移酶活性的糖基转移酶CtUGT4。在大肠杆菌中异种表达了CtUGT4融合蛋白，并纯化了目的蛋白。重组蛋白在体外可催化槲皮素生成槲皮素-7- o -葡萄糖苷，山奈酚生成山奈酚-3- o，并以一系列黄酮、黄酮醇、二氢黄酮、查尔酮、查尔酮苷为底物生成新产物。在烟草瞬时表达体系中表达了CtUGT4，酶活结果表明，CtUGT4能催化山奈酚生成山奈酚-3- o -葡萄糖苷，并能催化槲皮素生成槲皮素-3- o -葡萄糖苷。在红花中过表达CtUGT4后，红花小花中槲皮素-3- o -芦丁苷含量显著增加，槲皮素-3- o -葡萄糖苷含量也有增加的趋势，初步证实了CtUGT4类黄酮- o -糖基转移酶的功能。本研究证实了红花CtUGT4的类黄酮- o -糖基转移酶功能，在体内和体外均显示了其对不同类黄酮底物的亲和力差异和催化位点的区域选择性，为进一步研究UGT基因的功能提供了线索，也为红花有效代谢产物定向改良的培养工程提供了新思路。
To explore the complete biosynthesis process of flavonoid glycosides in safflower, specifically the key glycosyltransferase that might be involved, as well as to develop an efficient biocatalyst to synthesize flavonoid glycosides, a glycosyltransferase CtUGT4, with flavonoid-O-glycosyltransferase activity, was identified in safflower. The fusion protein of CtUGT4 was heterologously expressed in Escherichia coli, and the target protein was purified. The recombinant protein can catalyze quercetin to form quercetin-7-O-glucoside, and kaempferol to form kaempferol-3-O in vitro, and a series of flavones, flavonols, dihydroflavones, chalcones, and chalcone glycosides were used as substrates to generate new products. CtUGT4 was expressed in the tobacco transient expression system, and the enzyme activity results showed that it could catalyze kaempferol to kaempferol-3-O-glucoside, and quercetin to quercetin-3-O-glucoside. After overexpressing CtUGT4 in safflower, the content of quercetin-3-O-rutinoside in the safflower florets increased significantly, and the content of quercetin-3-O-glucoside also tended to increase, which preliminarily confirmed the function of CtUGT4 flavonoid-O-glycosyltransferase. This work demonstrated the flavonoid-O-glycosyltransferase function of safflower CtUGT4 and showed differences in the affinity for different flavonoid substrates and the regioselectivity of catalytic sites in safflower, both in vivo and in vitro, providing clues for further research regarding the function of UGT genes, as well as new ideas for the cultivation engineering of the directional improvement of effective metabolites in safflower.
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影响因子：
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