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Identification of novel glycoside hydrolases via whole genome sequencing of Niabella ginsenosidivorans for production of various minor ginsenosides.

ABSTRACT: In this study, many bacterial strains were screened for the production of minor ginsenosides, but based on conversion competence among the strains, the strain Niabella ginsenosidivorans BS26T has the good ginsenoside-transforming ability. Therefore, the strain BS26T was selected for complete genome sequence analysis to determine the target (glycoside hydrolase) functional genes. Whole genome analysis of strain BS26T showed 43 glycoside hydrolase genes in total. To determine the target functional gene, 12 sets of six different glycoside hydrolases (3 set of ?-glucosidase; 3 set of trehalase; 3 set of arabinofuranosidase; 2 set of xylosidase; and one set of each ?-galactosidase and ?-fucosidase, respectively) were selected and cloned in E. coli BL21 (DE3) using the pGEX4T-1 vector and were characterized. Among these 12 sets of clones, only one, ?-glucosidase (BglNg-767), showed ginsenoside conversion ability. The BglNg-767 comprised 767 amino acids and belonged to glycoside hydrolase family 3 (GH3). The recombinant GST-BglNg-767 was capable of altering the ginsenosides Rb1, Rd, and gypenoside XVII (Gyp-XVII) to F2; Rb2 to C-O; Rb3 to C-Mx1, and Rc to C-Mc1. Besides, complete genome sequence analysis of strain BS26T also indicates 30 endopeptidase genes, which may be responsible for self-hydrolysis of the proteins. Therefore, using SDS-PAGE analysis, we predict that the difference between the molecular weight of the expressed protein (around 90 kDa) and the predicted amino-acid sequence (102.7 kDa) is due to self-hydrolysis of the proteins.

SUBMITTER: Siddiqi MZ

PROVIDER: S-EPMC6556516 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Identification of novel glycoside hydrolases via whole genome sequencing of Niabella ginsenosidivorans for production of various minor ginsenosides.

Siddiqi Muhammad Zubair MZ Hashmi Majid S MS Oh Jung-Mi JM Chun Sungkun S Im Wan-Taek WT

3 Biotech 20190610 7

In this study, many bacterial strains were screened for the production of minor ginsenosides, but based on conversion competence among the strains, the strain Niabella ginsenosidivorans BS26T has the good ginsenoside-transforming ability. Therefore, the strain BS26T was selected for complete genome sequence analysis to determine the target (glycoside hydrolase) functional genes. Whole genome analysis of strain BS26T showed 43 glycoside hydrolase genes in t ...[more]

PMID: 31192083

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Project description:BACKGROUND:Several studies have reported that ginsenoside Rg3(S) is effective in treating metastatic diseases, obesity, and various cancers, however, its presence in white ginseng cannot be estimated, and only a limited amount is present in red ginseng. Therefore, the use of recombinant glycosidases from a Generally Recognized As Safe (GRAS) host strain is a promising approach to enhance production of Rg3(S), which may improve nutritional activity, human health, and quality of life. METHOD:Lactobacillus ginsenosidimutans EMML 3041T, which was isolated from Korean fermented pickle (kimchi), presents ginsenoside-converting abilities. The strain was used to enrich the production of Rg3(S) by fermenting protopanaxadiol (PPD)-mix-type major ginsenosides (Rb1, Rb2, Rc, and Rd) in four different types of food-grade media (1, MRS; 2, Basel Food-Grade medium; 3, Basel Food-Grade medium-I, and 4, Basel Food-Grade medium-II). Due to its tendency to produce Rg3(S), the presence of glycoside hydrolase in Lactobacillus ginsenosidimutans was proposed, the whole genome was sequenced, and the probable glycoside hydrolase gene for ginsenoside conversion was cloned. RESULTS:The L. ginsenosidimutans EMML 3041T strain was whole genome sequenced to identify the target genes. After genome sequencing, 12 sets of glycoside hydrolases were identified, of which seven sets (?,?-glucosidase and ?,?-galactosidase) were cloned in Escherichia coli BL21 (DE3) using the pGEX4T-1 vector system. Among the sets of clones, only one clone (BglL.gin-952) showed ginsenoside-transforming abilities. The recombinant BglL.gin-952 comprised 952 amino acid residues and belonged to glycoside hydrolase family 3. The enzyme exhibited optimal activity at 55 °C and a pH of 7.5 and showed a promising conversion ability of major ginsenoside Rb1?Rd?Rg3(S). The recombinant enzyme (GST-BglL.gin-952) was used to mass produce Rg3(S) from major ginsenoside Rb1. Scale-up of production using 50 g of Rb1 resulted in 30 g of Rg3(S) with 74.3% chromatography purity. CONCLUSION:Our preliminary data demonstrated that this enzyme would be beneficial in the preparation of pharmacologically active minor ginsenoside Rg3(S) in the functional food and pharmaceutical industries.

Dataset Information

Identification of novel glycoside hydrolases via whole genome sequencing of Niabella ginsenosidivorans for production of various minor ginsenosides.

Publications

Identification of novel glycoside hydrolases via whole genome sequencing of <i>Niabella ginsenosidivorans</i> for production of various minor ginsenosides.

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