FOOD SCIENCE ›› 2018, Vol. 39 ›› Issue (22): 116-122.doi: 10.7506/spkx1002-6630-201822018

• Bioengineering • Previous Articles     Next Articles

Construction of in situ UDPG Regeneration Strain for Biosynthesis of Rebaudioside D

FEI Liwen1, WANG Yong2,*   

  1. (1. Key Laboratory of Agricultural Genetics and Breeding of Shanghai, National R&D Center for Edible Fungi Processing, National Engineering Research Center of Edible Fungi, Key Laboratory of Edible Fungi Resources and Utilization (South), Ministry of Agriculture, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China; 2. Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China)
  • Online:2018-11-25 Published:2018-11-21

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

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