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Nagendra Prasad Kurumbang,Tae Jin Oh,Kwangkyoung Liou,Hei Chan Lee,Jae Kyung Sohng 한국당과학회 2008 한국당과학회 학술대회 Vol.2008 No.1
Metabolic engineering is a powerful tool for improvement and introduction of new cellular processes in a host strain which is mostly done by genetic engineering. We constructed E. coliΔpgi mutant by disrupting the pgi and inserting neomycin/kanamycin resistant marker. The pgi encoded enzyme glucose phosphate isomerase (Pgi) is responsible for the conversion of glucose-6-phosphate to fructose-6-phosphate and vice-versa. The mutant is a useful host for expression of a gene or gene cluster in which glucose-6-phosphate serves as a precursor. 2-deoxy-scyllo-inosose (DOI) is a first intermediate in biosynthesis of 2-deoxystreptamine (DOS)-containing aminoglycoside such as ribostamycin, neomycin and butirosin. Subsequent amination, dehydrogenation and again amination of DOI results DOS, another stable intermediate to be isolated. N-acetylglucosaminylation to DOS by a glycosyltransferase and deacetylation by a deacetylase gives paromamine, first and stable pseudosaccharide intermediate. In this study five genes required for the conversion of glucose-6-phosphate to paromamine were taken from butirosin gene cluster and cloned in expression vectors. The recombinants were transformed in E. coli BL21 (DE3) and its mutant E. coliΔpgi successively and together so as to get the desired product. After fermentation the products were extracted and analyzed by TLC and HPLC-MS.
Exploration of Glycosylated Flavonoids from Metabolically Engineered E. coli
Dinesh Simkhada,Nagendra Prasad Kurumbang,Hei Chan Lee,송재경 한국생물공학회 2010 Biotechnology and Bioprocess Engineering Vol.15 No.5
Flavonoids glycosylated with UDP-glucuronic acid and UDP-xylose are spatially distributed in nature. To produce these glycosides, E. coli was engineered to overexpress biosynthetic gene clusters of UDP-sugars (galU from E. coli K12, UDP-glucose dehydrogenase (calS8),and UDP-glucuronic acid decarboxylase (calS9) from Micromonospora echinospora spp. calichensis). Flavonoids were glycosylated by overexpression of the glycosyltransferase gene (atGt-5) from Arabidopsis thaliana. Finally, metabolically engineered host E. coli (US89Gt-5) was generated. Production of flavonoid glycosides was observed in a biotransformation system consisting of flavonoids (naringenin and quercetin) exogenously fed to host cells. The glycosylated derivatives 7-O-glucuronyl naringenin (m/z+449), 7-O-xylosyl naringenin (m/z+ 405), and 7-O-glucuronyl quercetin (m/z+ 479) were detected and confirmed by ESI-MS/MS, ESI-MS/MS and LC/MS-MS analysis, respectively.
Metabolic engineering of E. coli for the production of glycosylated flavonoids
Dinesh Simkhada,EuiMin Kim,Nagendra Prasad Kurumbang,Tae-Jin Oh,Hei Chan Lee,Jae Kyung Sohng 한국당과학회 2008 한국당과학회 학술대회 Vol.2008 No.1
Glycosylation of flavonoid play crucial roles in stabilization of antocyanins and cyanidins; storage of flavonoid and terpenoids; and regulation of hormones. In addition, glycosylation has been recognized as one of the important mechanisms for detoxification of exogenous compounds. Here in this research, we have metabolically engineered the E. coli BL21DE3 (Δ pgi mutant) host to generate four different engineered host to produce glycosylated flavonoid. E. coli BL21DE3 (Δ pgi mutant) was engineered by integration of GalU, expression of CalS8 (dehydrogenase) and CalS9 (decarboxylase) together by cloning in pDuet/ampr vector and expression of four different 3-O-glycosyltransferase and 7-O- glycosyltransferase gene from Arabidopsis thaliana. Engineered hosts are expected to produce glucosyl as well as xylosyl glycosylated flavonoids which are characterized by HPLC as well as LC-MS analysis.