采用来自水稻的UDP-糖基转移酶EUGT11并结合染色体改造构建了大肠杆菌工程菌;用于全细胞催化莱鲍迪苷A(Rebaudioside A;RA)生产莱鲍迪苷D(Rebaudioside D;RD)。为了增加宿主细胞内源性糖配体葡萄糖尿苷二磷酸(uridine diphosphate glucose;UDPG)的供给以满足糖基化反应需要;以大肠杆菌BL21(DE3)为出发菌对UDPG 代谢通径进行改造;获得了一系列改造菌株SG1、SG2、SG3和SG4。其中以SG4为宿主过表达可高效催化RA合成RD的糖基转移酶EUGT11进行全细胞催化;RD产量高。随后以此工程菌为对象;对可能影响RD催化合成的条件进行了研究;结果表明50 mL培养菌液离心所收集新鲜菌体可在100 mmol/L pH 8.0磷酸钠缓冲液、80 mmol/L 柠檬酸钠、体积分数0.1% Triton X100、500 g/L蔗糖、5 mmol/L ZnCl2、42 ℃转化1 d的条件下催化1 mmol/L RA底物生成1112.21 mg/L RD;转化率可达98.5%。

An engineeredE.colistrain was developed to produce the noncaloric sweetener rebaudioside D (RD) from rebaudioside A (RA) through whole cell biocatalysis. In order to increase endogenous production of the essential active sugar donor of the catalytic reaction; uridine diphosphate glucose (UDPG); host strainE.coliBL21(DE3) was subjected to chromosomal modifications and the resultant genetically modified host strains were named as SG1; SG2; SG3 and SG4; respectively. These modified hosts were transformed with plasmid pYF09 harboring the glycosyltransferase EUGT11 that catalyze the biosynthesis of RD from RA; and the resultant engineered cells were then used to catalyze the biosynthesis of RD. The strain pYF09-SG4 was selected for further studies on whole cell catalysis conditions. The results showed that using fresh cells collected from 50 mL liquid culture and under conditions 100 mmol/L pH 8.0 sodium phosphate buffer 5 mmol/L ZnCl2; 80 mmol/L sodium citrate; 0.1% volumetric percent of Triton X100; 500 g/L sucrose; temperature 42 ℃; and 1 d incubation time; 98.5% of 1 mmol/L RA was catalyzed to RD with a yield of 1 112.21 mg/L.