Identification of the genes involved in 1-deoxynojirimycin synthesis in Bacillus subtilis MORI 3K-85

Abstract

1-Deoxynojirimycin (DNJ), a D-glucose analogue with a nitrogen atom substituting for the ring oxygen, is a strong inhibitor of intestinal α-glucosidase. DNJ has several promising biological activities, including its antidiabetic, antitumor, and antiviral activities. Nevertheless, only limited amounts of DNJ are available because it can only be extracted from some higher plants, including the mulberry tree, or purified from the culture broth of several types of soil bacteria, such as Streptomyces sp. and Bacillus sp. In our previous study, a DNJ-producing bacterium, Bacillus subtilis MORI, was isolated from the traditional Korean fermented food Chungkookjang. In the present study, we report the identification of the DNJ biosynthetic genes in B. subtilis MORI 3K-85 strain, a DNJ-overproducing derivate of the B. subtilis MORI strain generated by γ-irradiation, xhe genomic DNA library of B. subtilis MORI 3K-85 was constructed in Escherichia coli, and clones showing α-glucosidase inhibition activity were selected. After DNA sequencing and a series of subcloning, we were able to identify a putative Operon which consists of gabT1, yktc1, and gutB1 genes predicted to encode putative transaminase, phosphatase, and oxidoreductase, respectively. When a recombinant plasmid containing this Operon sequence was transformed into an E. coli strain, the resulting transformant was able to produce DNJ into the culture medium. Our results indicate that the gabT1, yktc1, and gutB1 genes are involved in the DNJ biosynthetic pathway in B. subtilis MORI, suggesting the possibility of employing these genes to establish a large-scale microbial DNJ overproduction system through genetic engineering and process optimization.

Nectrisine Biosynthesis Genes in Thelonectria discophora SANK 18292: Identification and Functional Analysis

“…Also, route 2 is similar to the proposed biosynthetic pathway for the iminosugar deoxynojirimycin, with a 6-membered ring, in Bacillus amyloliquefaciens, in which amina- tion, dephosphorylation, and oxidation occur in the same order as in route 2 (29). Clustered genes for iminosugar biosynthesis are not yet known except for those in deoxynojirimycin biosynthesis, for which the gene cluster could contain gabT1, yktC1, and gutB1, coding for a putative aminotransferase, phosphatase, and dehydrogenase, respectively (28,29), analogous to necA, necB, and necC for nectrisine biosynthesis. However, necA, necB, and necC showed relatively low levels of identity (25%, 14%, and 17%, respectively) and similarity (48%, 31%, and 33%, respectively) with respect to gabT1, yktC1, and gutB1 from Bacillus amyloliquefaciens.…”

“…We hypothesized that necC is involved in nectrisine biosynthesis, since it catalyzes the conversion of 4-amino-4-deoxyarabinitol, which can be produced by T. discophora, to nectrisine. We also assumed that nectrisine biosynthesis genes might be clustered, since many of the biosynthesis genes for fungal secondary metabolites, including the iminosugar deoxynojirimycin (28,29), are known to be clustered (30)(31)(32)(33). Thus, in order to find nectrisine biosynthesis genes, cosmid clones of T. discophora genomic DNA containing necC were retrieved and sequenced (Fig.…”

Quantitative Analysis of 1-deoxynojirimycin in Mulberry Leaves

“…)된 이래 기능 (Kang et al, 2011). 또한 α-glucosidase 의 강력한 효소저해제인 1-deoxynojirimycin(DNJ)을 고효 율로 생산하는 균주의 선발 및 동정, 배양최적화 및 균 …”

Isolation of the Putative Biosynthetic Gene Cluster of 1-Deoxynojirimycin by <i>Bacillus amyloliquefaciens</i> 140N, Its Production and Application to the Fermentation of Soybean Paste

“…4,5) Several studies have therefore investigated the production of DNJ by using Bacillus spp. 6,7) Numerous studies have attempted to isolate and identify DNJ and its derivatives from microorganisms, whereas few studies have investigated the biosynthesis of DNJ. 8) These studies on DNJ biosynthesis have only been based on isotope-labeling experiments.…”

“…A promising operon has recently been isolated containing the putative transaminase (GabT1), phosphatase (Yktc1), and oxidoreductase (GutB1) which may be responsible for the proposed essential biosynthetic gene cluster of DNJ. 6,7) Given the biosynthesis of DNJ by Bacillus spp., this may be useful for directly adding DNJ to food products or for developing DNJ-containing functional foods. However, there have been few studies of the application of DNJ-producing Bacillus to functional foods.…”