title: Cloning and heterologous expression of UDP-glycosyltransferase genes from Bacillus subtilis and its application in the glycosylation of ginsenoside Rh1.
abstract: Bacillus subtilis CCTCC AB 2012913 can transform ginsenoside Rh1 to 3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol. Based on its genome sequence, strain B. subtilis 168 contains three UDP-glycosyltransferase genes. Here, we cloned the three UDP-glycosyltransferase genes (ydhE1, yojK1 and yjiC1) from B. subtilis CCTCC AB 2012913 and expressed in Escherichia coli BL21 (DE3) with His-tag. The crude enzyme extracts were assayed, respectively, for their activities to transform ginsenoside Rh1. Extracts containing enzymes YojK1 and YjiC1 could use ginsenoside Rh1 as a substrate to produce 3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol, which had an additional glucopyranosyl linked with C-3 over the substrate. Enzyme YjiC1 was purified by affinity chromatography on Ni-NTA His Binding resin. The molecular mass of purified YjiC1 was c. 47 kDa as determined by SDS-PAGE. This is the first report of an in vitro biotransformation of ginsenoside Rh1 to 3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol using the recombinant UDP-glycosyltransferase.
  
  
  CONCLUSIONS
  The Chinese traditional medicinal plant Panax is reported to have multiple health benefits. Its main active ingredient is saponin, and different saponins have different activity spectrum. In the study, three UDP-glycosyltransferase genes, ydhE1, yojK1 and yjiC1, were cloned from Bacillus subtilis CCTCC AB2012913 and the three genes were expressed in Escherichia coli BL21 (DE3). The enzyme YjiC1 was purified and converted ginsenoside Rh1 to 3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-20(S)-protopanaxatriol in vitro. The compound is the first saponin possessing β-glucopyranosyl at both C-3 and C-6 sites. We showed that the in vitro biotransformation was effective, and the reaction condition was easy to control. Our research suggests that a diversity of saponins could be generated through efficient and directed enzymatic biotransformation.
authors.affiliation: Laboratory for Conservation and Utilization of Bio-resource, and Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming, China.
authors.forename: S L
authors.initials: SL
authors.lastname: Luo
authors.forename: L Z
authors.initials: LZ
authors.lastname: Dang
authors.forename: K Q
authors.initials: KQ
authors.lastname: Zhang
authors.forename: L M
authors.initials: LM
authors.lastname: Liang
authors.forename: G H
authors.initials: GH
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001711:Biotransformation; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D036145:Ginsenosides / Q000378:metabolism*; D006031:Glycosylation; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D005894:Panax; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012502:Sapogenins / Q000378:metabolism; D012503:Saponins / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D036145:Ginsenosides; D011994:Recombinant Proteins; D012502:Sapogenins; D012503:Saponins; C081552:protopanaxatriol; C425564:ginsenoside Rh1; D016695:Glycosyltransferases
keywords: 3-O-β-d-glucopyranosyl-6-O-β-d-glucopyranosyl-20(S)-protopanaxatriol; Bacillus subtilis; UDP-glycosyltransferase; biotransformation; ginsenoside Rh1