Niranjan Koirala

Assessing acceptor substrate promiscuity of YjiC-mediated glycosylation toward flavonoids.

The acceptor substrate promiscuity of YjiC, a UDP-glycosyltransferase from Bacillus licheniformis, was explored with seven different classes (flavonols, flavanols, flavones, flavanones, chalcone, stilbene, and isoflavonoids) of 23 flavonoid acceptors. For most of the polyphenols used in the reactions, the enzymatic bioconversion was significantly higher with the production of multiple glucosylated derivatives. This study highlights the highly flexible non-regiospecific glycosylation ability of YjiC toward polyphenolic compounds. The catalytic potential of YjiC could be useful to generate a library of natural product glucosides.

Methylation and subsequent glycosylation of 7,8-dihydroxyflavone

An O-methyltransferase SpOMT2884, originating from Streptomyces peucetius ATCC 27952, was cloned, expressed, and applied for the production of target metabolite from Escherichia coli. Biochemical characterization of the 25kDa recombinant protein by in vitro and in vivo experiments showed that SpOMT2884 was an S-adenosyl-l-methionine-dependent O-methyltransferase. SpOMT2884 catalyzed O-methylation of different classes of flavonoids such as flavones (7,8-dihydroxyflavone (7,8-DHF), luteolin), flavonols (quercetin, rutin), flavanone (naringenin), and isoflavonoids (daidzein, formononetin). Biotransformation of 7,8-DHF, a preferred substrate of SpOMT2884, in a grown-induced culture of E. coli BL21 (DE3) harboring the recombinant pET-28a-SpOMT2884 stoichiometrically converted 7,8-DHF into 7-hydroxy-8-methoxyflavone, which was confirmed by liquid chromatography, mass spectrometry and various nuclear magnetic resonance (NMR) spectroscopy analyses. In order to improve the biotransformation substrate, time and media parameters were optimized and the production was scaled up using a 3-L fermentor. The maximum yield of 7-hydroxy-8-methoxyflavone was 192μM (52.57mg/L), representing almost 96% bioconversion within 12h, when 200μM of 7,8-DHF was supplemented in the culture. Further, the 7-hydroxy-8-methoxyflavone was purified in large scale and was used as a substrate separately for in vitro glycosylation to produce glucose, galactose and 2-deoxyglucose conjugated at 7th hydroxyl position of 7-hydroxy-8-methoxyflavone. Biological activity showed that 7-hydroxy-8-methoxyflavone had long term cytoprotective and antioxidant effects compared to 7,8-DHF suggesting that methylation enhances the stability of substrate and glycosylation has proved to increase the water solubility.

Glucosylation of Isoflavonoids in Engineered Escherichia coli

A glycosyltransferase, YjiC, from Bacillus licheniformis has been used for the modification of the commercially available isoflavonoids genistein, daidzein, biochanin A and formononetin. The in vitro glycosylation reaction, using UDP-α-D-glucose as a donor for the glucose moiety and aforementioned four acceptor molecules, showed the prominent glycosylation at 4′ and 7 hydroxyl groups, but not at the 5th hydroxyl group of the A-ring, resulting in the production of genistein 4′-O-β-D-glucoside, genistein 7-O-β-D-glucoside (genistin), genistein 4′,7-O-β-D-diglucoside, biochanin A-7-O-β-D-glucoside (sissotrin), daidzein 4′-O-β-D-glucoside, daidzein 7-O-β-D-glucoside (daidzin), daidzein 4′, 7-O-β-D-diglucoside, and formononetin 7-O-β-D-glucoside (ononin). The structures of all the products were elucidated using high performance liquid chromatography-photo diode array and high resolution quadrupole time-of-flight electrospray ionization mass spectrometry (HR QTOFESI/MS) analysis, and were compared with commercially available standard compounds. Significantly higher bioconversion rates of all four isoflavonoids was observed in both in vitro as well as in vivo bioconversion reactions. The in vivo fermentation of the isoflavonoids by applying engineered E. coli BL21(DE3)/ΔpgiΔzwfΔushA overexpressing phosphoglucomutase (pgm) and glucose 1-phosphate uridyltransferase (galU), along with YjiC, found more than 60% average conversion of 200 μM of supplemented isoflavonoids, without any additional UDP-α-D-glucose added in fermentation medium, which could be very beneficial to large scale industrial production of isoflavonoid glucosides.

Probing 3-Hydroxyflavone for In Vitro Glycorandomization of Flavonols by YjiC

ABSTRACT The glycosylation of five different flavonols, fisetin, quercetin, myricetin, kaempferol, and 3-hydroxyflavone, was achieved by applying YjiC. 3-Hydroxyflavone was selected as a probe for in vitro glycorandomization of all flavonols using diverse nucleotide diphosphate-d/l-sugars. This study unlocked the possibilities of the glycodiversification of flavonols and the generation of novel compounds as future therapeutics.

Metabolic engineering of E. coli for the production of isoflavonoid‐7‐O‐methoxides and their biological activities

Isoflavonoid representatives such as genistein and daidzein are highly potent anticancer, antibacterial, and antioxidant agents. It have been demonstrated that methylation of flavonoids enhanced the transporting ability, which lead to facilitated absorption and greatly increased bioavailability. In this paper, genetically engineered Escherichia coli was reconstructed by harboring E. coli K12‐derived metK encoding S‐adenosine‐l‐methionine (SAM) synthase (accession number: K02129) for enhancement of SAM as a precursor and Streptomyces avermitilis originated SaOMT2 (O‐methyltransferase, accession number: NP_823558) for methylation of daidzein and genistein as preferred substrates. The formation of desired products via biotransformation including 4′‐O‐methyl‐genistein and 4′‐O‐methyl‐daidzein was confirmed individually by using chromatographical methods such as high‐performance liquid chromatography, liquid chromatography/time‐of‐flight/mass spectrometry (LC‐TOF‐MS), and nuclear magnetic resonance (NMR), and NMR (1H and 13C). Furthermore, substrates concentration, incubation time, and media parameters were optimized using flask culture. Finally, the most fit conditions were applied for fed‐batch fermentation with scale‐up to 3 L (working volume) to obtain the maximum yield of the products including 164.25 µM (46.81 mg/L) and 382.50 µM (102.88 mg/L) for 4′‐O‐methyl genistein and 4′‐O‐methyl daidzein, respectively. In particular, potent inhibitory activities of those isoflavonoid methoxides against the growth of cancer line (B16F10, AGS, and HepG2) and human umbilical vein endothelial cells were investigated and demonstrated. Taken together, this research work described the production of isoflavonoid‐4′‐O‐methoxides by E. coli engineering, improvement of production, characterization of produced compounds, and preliminary in vitro biological activities of the flavonoids being manufactured.

Functional Analysis of the GlcP Promoter in Streptomyces peucetius var. caesius

In Streptomyces, carbon utilization is of significant importance for the expression of genes involved in morphological differentiation and antibiotic production. Glucose is mainly transported by GlcP, a membrane protein encoded by glcp. In Streptomyces coelicolor, this protein is encoded by sco5578. However, there is little information about the physiology of the GlcP promoter in Streptomyces. The aim of the present work was to clone and perform a functional analysis of the sp7066 promoter (ortholog of sco5578) from Streptomyces peucetius var. caesius. Hydrophobicity and cellular location analysis of the putative amino acid sequence of the cloned gene predicted SP7066 would be a membrane protein with a topology of six plus six transmembrane segments interrupted by a large cytoplasmic loop. In silico analysis of the upstream region of the sp7066 transcription initiation site predicted the sequences 5′-AGGAATAGT-3′ and 5′-TTGACT-3′ for regions -10 and -35 of sp7066 promoter. To reflect sp7066 expression, the promoter sequence was amplified, subcloned, and fused to the egfp reporter gene. Immunoblot analysis revealed that D-glucose and its analog 2-deoxyglucose were able to induce sp7066 expression. This effect was not modified by the presence of equimolar concentrations of D-galactose or N-acetylglucosamine. No expression of egfp was detected with the use of other carbon sources such as L-arabinose, D-fructose, and glycerol. Based on these analyses, we conclude that D-glucose is a preferred carbon source in S. peucetius var. caesius and that the sp7066 expression product, a putative non-PTS glucose permease, likely is a H+/symporter, localized to the membrane, and shows a strong specificity for D-glucose for inducing expression.