Li Yan, Li Yangyang, Wang Yu, Chen Liangliang, Yan Ming, Chen Kequan, Xu Lin, Ouyang Pingkai
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, Jiangsu, 211816, China.
Appl Biochem Biotechnol. 2016 Apr;178(8):1586-98. doi: 10.1007/s12010-015-1969-4. Epub 2016 Jan 6.
Rebaudioside A has superior taste quality among the steviol glycosides extracted from Stevia rebaudiana leaves. Given its high purity as a general-purpose sweetener, rebaudioside A has received significant attention and has been widely applied in food and beverages in recent decades. Stevioside is one of the major steviol glycosides and can be converted to rebaudioside A by the uridine-diphosphate dependent glucosyltransferase UGT76G1 in S. rebaudiana. To explore the applicability of and limits in producing rebaudioside A from stevioside through whole-cell biocatalysis, the engineered Saccharomyces cerevisiae expressing UGT76G1, using a newly constructed constitutive expression vector, was used as the whole-cell biocatalyst. Citrate was added to the reaction mixture to allow metabolic regulation when glucose was fed to provide the activated sugar donor UDP-glucose for glycosylation of stevioside in vivo. In an evaluation of the whole-cell reaction parameters involving cell permeability, temperature, pH, citrate and Mg(2+) concentrations, and glucose feeding, production of 1160.5 mg/L rebaudioside A from 2 g/L stevioside was achieved after 48 h without supplementation of extracellular UDP-glucose.
莱鲍迪苷A在从甜叶菊叶片中提取的甜菊糖苷中具有卓越的口感品质。鉴于其作为通用甜味剂的高纯度，莱鲍迪苷A在近几十年来受到了广泛关注，并已广泛应用于食品和饮料中。甜菊糖苷是主要的甜菊糖苷之一，在甜叶菊中可通过尿苷二磷酸依赖性葡萄糖基转移酶UGT76G1转化为莱鲍迪苷A。为了探索通过全细胞生物催化从甜菊糖苷生产莱鲍迪苷A的适用性和局限性，使用新构建的组成型表达载体表达UGT76G1的工程酿酒酵母作为全细胞生物催化剂。当加入葡萄糖以提供用于体内甜菊糖苷糖基化的活化糖供体尿苷二磷酸葡萄糖时，向反应混合物中添加柠檬酸盐以进行代谢调节。在对涉及细胞通透性、温度、pH、柠檬酸盐和Mg(2+)浓度以及葡萄糖添加的全细胞反应参数进行评估时，在不补充细胞外尿苷二磷酸葡萄糖的情况下，48小时后从2g/L甜菊糖苷中生产出了1160.5mg/L的莱鲍迪苷A。
