Regiospecific modifications of naringenin for astragalin production in Escherichia coli

Abstract

We report the production of astragalin (AST) from regiospecific modifications of naringenin (NRN) in Escherichia coli BL21(DE3). The exogenously supplied NRN was converted into dihydrokaempferol (DHK) and then kaempferol (KMF) in the presence of flavanone-3-hydroxylase (f3h) and flavonone synthase (fls1) from Arabidopsis thaliana, respectively. KMF was further modified to produce AST by 3-O-glucosylation utilizing the endogeneous UDP-glucose in presence of UGT78K1 from Glycine max. To increase the intracellular UDP-glucose concentration by channeling the carbon flux toward UDP-glucose at the branch point of glucose-6-phosphate (G6P), the chromosomal glucose phosphate isomerase (pgi) and D-glucose-6-phosphate dehydrogenase (zwf) were knocked-out in E. coli BL21(DE3). The two enzymes directly involved in the synthesis of UDP-glucose from G6P, phosphoglucomutase (nfa44530) from Nocardia farcinia and glucose-1-phosphate uridylyltransferase (galU) from E. coli K12 were overexpressed, which successfully diverted the carbon flow from glycolysis to the synthesis of UDP-glucose. Furthermore, to prevent the dissociation of UDP-glucose into UDP and glucose, the UDP-glucose hydrolase (ushA) was deleted. The E. coli ΔpgiΔzwfΔushA mutant harboring the UDP-glucose biosynthetic pathway and the aforementioned genes for the regiospecific glucosylation produced 109.3 mg/L (244 µM) of AST representing 48.8% conversion from 500 µM of NRN in 60 h without any supplementation of extracellular UDP-glucose.

Assembly of a novel biosynthetic pathway for production of the plant flavonoid fisetin in Escherichia coli

“…Vectors pACYCDuet-1, pETDuet-1, pCDFDuet-1 and pRSFDuet-1 (Novagen) were used for cloning and subcloning. Plasmids pCDF4cl2pc (Leonard, et al, 2006), pETCHSph-CHIms (Leonard and Koffas, 2007) and pCDFF3HatFLSat (Malla et al, 2013) were previously reported. …”

“…The cell pellet was collected by centrifugation and resuspended in 100 ml of M9 minimal media supplemented with 1 mM of IPTG and 0.1 mM liquiritigenin and cultured at 30 1C and 300 rpm. The extraction was carried out as previously described by Malla et al (2013). Briefly, the culture was extracted with an equal volume of ethyl acetate and organic layer was collected followed by evaporation of excess solvent to dryness.…”

Enhanced Production of Nargenicin A1 and Generation of Novel Glycosylated Derivatives

“…Recombinant plasmids containing genes encoding for UMP kinase (UMK-pET15b), acetate kinase (ACK-pET24ma), UDP-α-D-glucose synthase (GalUpET24ma), phosphomannomutase (PMM-pET24ma), N-acetyl-D-glucosamine kinase (N-AGK-pET32a), and glycosyltransferase (YjiC-pET28a) were used [17,18]. For easy manipulation, N-AGK and PMM (NA-PM), and GalU and YjiC (G-Y) were co-expressed in a single transformant.…”

“…For example, doxorubicin, vancomycin, avermectin, and nystatin are biologically potent because of their sugar moieties [15]. In this context, the development of cheap and efficient glycosylation technologies, useful both in the laboratory and in industry, is highly desirable [16], so engineered microbial cell-based in vivo systems [17] or in vitro reactions [16] using various nucleotide diphosphate (NDP) sugars are employed to generate the glyco-conjugate of various natural products (NP). But, for NP exhibiting the antimicrobial activities or exhibiting detrimental effect on cell growth, the in vivo systems may not be practical.…”

Biosynthesis of amino deoxy-sugar-conjugated flavonol glycosides by engineered Escherichia coli

“…Flavonoids belong to a large group of plant-based phenolic bioactive natural products having anti-oxidant, anti-inflammatory, anti-cancer, anti-estrogenic, and anti-diabetic properties [12][13][14][15]. Selected flavonoid glycosides produced by reconstructing sugar pathways in E. coli include kaempferol 3-O-glucoside (astragalin) [16], naringenin 7-O-xyloside [17], quercetin 3-O-xyloside [18], quercetin 3-O-rhamoside, kaempferol 3-O-rhamnoside, quercetin 3-O-alloside [19], quercetin 3-O-6-deoxytaloside [20], quercetin 3-O-arabinoside [21], quercetin 3-O-N-acetylglucosamine [22] and quercetin 3-O-glucoside-7-O-rhamnoside [23]. The cytoplasmic pool of these sugars has been elevated either by editing the NDP-sugar pathway genes in the genome or by introducing NDP-sugars pathway genes along with a glycosyltransferase (GT).…”