关键词: 甜菊糖苷, 莱鲍迪苷D, 尿苷二磷酸葡萄糖, 蔗糖合酶, UDP-糖基转移酶, 生物催化

Abstract: Sucrose synthase 3 from Arabidopsis thaliana (AtSUS3) was used to regenerate uridine diphosphate glucose, an activated glucose donor required in biotransformation of rebaudioside D (RD) from rebaudioside A (RA). The AtSUS3 gene was cloned from the cDNA of A. thaliana and assembled into plasmid pET28a to construct a expression plasmid pLW105. Co-transformation of pLW105 and plasmid pYF09 harboring UDP-glucosyltransferase EUGT11 gene into Escherichia coli (E. coli) BL21(DE3) generated the engineered strain pLW105+pYF09-BL21(DE3) co-expressing both enzymes. Using the whole cells of this strain as a biocatalyst, more than 80% of RA was conversed to RD without exogenous addition of UDPG, yielding about 930 mg/L RD. Then a plasmid harboring the genes encoding both enzymes was constructed and the resulting engineered strain E. coli BL21(DE3) showed the same biotransformation efficiency as the strains containing two plasmids. In order to decrease the amount of sucrose used in the bioconversion reaction, the cell lysate instead of the whole cells was used to catalyze the biotransformation of RD. Using the cell lysate of the engineered strain pLW108-BL21(DE3), about 93% RA was conversed to RD with a yield of about 1 051 mg/L at 5 g/100 mL sucrose concentration, which used only 1/8 of the amount of sucrose used in whole cell catalysis with a comparable RD yield.

Key words: steviol glycosides, rebaudioside D, uridine diphosphate glucose (UDPG), sucrose synthase, uridine diphosphate glycosyltransferase (UDP-glycosyltransferase), biocatalysis