甜味剂莱鲍迪苷D的高效生物催化合成

摘要

An engineered E.coli strain 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 strain E.coli BL21 (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 glycosyl-transferase 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 100mmol/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.%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1d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％.