植物糖基转移酶的功能表征及类黄酮糖苷的微生物生产

Functional characterization of plant glycosyltransferases and microbial production of flavonoid glycosides

摘要:

目的 研究茶树(Camellia sinensis)、野菊花(Chrysanthemum indicum)以及拟南芥(Arabidopsis thaliana)来源的3个UDP-葡萄糖基转移酶CsUGT75L12、CiUGT11和UGT73B1的底物特异性并比较它们的催化效率。方法 利用大肠杆菌(Escherichia coli) BL21(DE3)表达植物糖基转移酶重组蛋白，将体外纯化得到的重组蛋白分别与黄酮、黄烷酮等6个黄酮类化合物进行体外酶促反应，通过液相质谱联用(LC-MS)及标准品比对确定糖苷产物，借助高效液相色谱(HPLC)峰面积计算底物转化率。结果 体外酶促反应结果显示，3个糖基转移酶CsUGT75L12、CiUGT11和UGT73B1具有宽泛的底物特异性，对柚皮素、圣草酚、异樱花素、橙皮素、芹菜素和金合欢素6个黄酮类化合物具有催化活性，生成的主要产物是类黄酮-7-O-葡萄糖苷。其中，CiUGT11和UGT73B1对橙皮素和柚皮素的转化率分别达到了96%。利用高效的糖基转移酶基因CiUGT11和UGT73B1在大肠杆菌中成功异源合成橙皮素-7-O-葡萄糖苷和柚皮素-7-O-葡萄糖苷。结论 植物糖基转移酶CsUGT75L12、CiUGT11和UGT73B1具有宽泛的底物识别能力，主要作用于黄酮类化合物的C7-OH位置，其中CiUGT11和UGT73B1对6个黄酮类化合物具有较高的催化活性，本研究为微生物高效生产类黄酮糖苷化合物提供了候选基因元件。

Abstract:

Objective To systematically investigate the substrate promiscuity and catalytic performance of three UDP-glycosyltransferases: CsUGT75L12 (Camellia sinensis), CiUGT11 (Chrysanthemum indicum), and UGT73B1 (Arabidopsis thaliana).Methods The recombinant proteins of plant glycosyltransferases were heterologously expressed in Escherichia coli BL21(DE3) and purified for in vitro enzymatic assays. In vitro enzymatic reactions of the purified recombinant proteins were performed with six flavonoids including flavones (apigenin and acacetin) and flavanones (naringenin, eriodictyol, isosakuranetin, and hesperetin). The enzymatic products were characterized by HPLC and LC-MS and the conversion rates were calculated through comparative HPLC peak area analysis.Results CsUGT75L12, CiUGT11, and UGT73B1 exhibited broad substrate promiscuity towards the six tested flavonoids. The primary products were identified as flavonoid-7-O-glucosides. Notably, CiUGT11 and UGT73B1 demonstrated exceptional catalytic efficiency, achieving >96% conversion rates for hesperetin and naringenin. Leveraging this activity, we engineered CiUGT11 and UGT73B1 with high efficiency to produce hesperetin-7-O-glucoside and naringenin-7-O-glucoside through precursor feeding in E. coli.Conclusion The three glycosyltransferases display remarkable versatility in flavonoid recognition, with conserved preference for the C7-OH position. CiUGT11 and UGT73B1 show high catalytic efficiency for six flavonoids. These findings provide candidate gene elements for the efficient microbial production of flavonoid glycosides.