Earticle

High-dose radiotherapy exposure is associated with normal tissue damage. Moreover, accumulating evidence from the Japanese atomic bomb survivors, Chernobyl liquidators, US astronauts, and some other exposed groups suggest that non-cancer effects also have been observed after moderate/low dose exposure in humans, in particular cardiovascular, respiratory, renal, and brain damage. The late onset of cardiovascular and kidney disease may be highly associated with ionizing radiation, but underappreciated. The exact pathogenesis and/or mediators involved in these chronic disease remain under investigation. Typically, as the senescent cell fraction following irradiation is known to increase, we examined whether ionizing radiation causes the renal injury by inducing cellular senescence. We assessed the effects of ionizing radiation on mouse inner medullary collecting duct- 3 (mIMCD-3) cells and Madin-Darby Canine Kidney (MDCK) cells, which predominantly used in vitro renal epithelial cells. We found that irradiation on mIMCD-3 and MDCK cells induced cellular senescence. Notably, the gene expression level of neuraminidase Klotho, highly expressed in renal cells/tissues with anti-senescence activity, was considerably decreased in irradiated mIMCD-3 cells and also kidney tissues of radiation-exposed mice. However, the protein level of klotho was persistent after radiation exposure. One of the negative regulators of klotho, Tumor necrosis factor-α (TNF-α) was significantly increased in both of irradiated mIMCD-3 cells and kidney tissues of mice. Critically, a Disintegrin and metalloproteinase domain-containing protein 9/10/17 (ADAM9/10/17), which functions as ectodomain shedding enzyme of klotho, was decreased in irradiated mouse renal cells/tissues. Collectively, our data suggest that TNF-α-mediated inhibition of klotho expression with blocking of soluble klotho formation following irradiation may contribute to the development of renal dysfunction through the acceleration of radiation-induced cellular senescence.

Antibody has been extensively investigated and developed as new drugs for many diseases, In-depth biological and structural analyses are needed for developing antibody drugs compared to small molecule drugs. Therefore, wide investigation of antibody with liquid chromatography (LC) and mass spectrometry (MS) have gained attention for the needs due to its sensitivity. Profiling of N-linked glycans and glycosylation site analysis are one of the major parts among the antibody physico-chemical characterization. In present works, glycan profiling is performed through deglycosylation, labeling and purification with Waters RapiFluor-MS N-glycan kit, then UPLC-FLR analysis gives more profiling results compared to conventional 2-AB method. A total of forty-one N-glycan was identified and a total of sixteen N-glycan were relatively quantified within 1% RSD for commercial antibody drugs. Glycosylation sites are easily identified with glycopeptides analysis and PNGase F treatment. Both LC-MS based analytical methods improved understanding of the antibody drugs and will be applicable to non-commercial antibodies or other biological drugs including biosimilars.

Due to the significant growth of glycan-related research field, especially in the biopharmaceutical industry, commercially available tools for glycan structural analysis and glycan interaction assay are highly required. In this presentation,"@three kinds of glycan research tool will be introduced: IgG glycan sample preparation kit EZGlyco® and versatile glycan purification kit BlotGlyco®as well as Glycan Microarray for high-throughput glycan interaction assay tool.

Glycosylation of biotherapeutics is significantly associated with drug’s quality, safety, and potency. Thus, comprehensive characterization of glycosylation is vital to assess the biotherapeutic quality and establish the equivalency of biosimilars. However, current glycan analysis mainly focuses on the N-glycans due to the absence of analytical tools to liberate O-glycans with high sensitivity. We developed selective and sensitive method to profile native O-glycans on therapeutic glycoproteins. O-glycosylation on biotherapeutics including O-acetylation on a sialic acid was comprehensively characterized. Details such as O-glycan structure and O-acetylmodification site were obtained from tandem MS. This method may be applied to QC and batch analysis of biotherapeutics bearing multiple O-glycosylations.

Glycan-associated recognition events in cells and organisms are not only critical for diverse physiological processes, but they are also involved in various pathological processes. Therefore, elucidation of glycan-mediated biomolecular interactions and their consequences is of great importance in basic biological research and biomedical applications. In 2002, we and others were the first to employ high-density glycan microarrays in efforts aimed at the high-throughput assessments of glycan-associated recognition events. As they include many glycans attached in a dense and orderly manner to a solid surface, glycan microarrays allow for multiple parallel analyses of glycan-protein binding events by utilizing only small amounts of glycan samples. Therefore, this microarray technology has become a leading edge tool in studies aimed at elucidating roles played by glycans and glycan binding proteins in biological systems. In this presentation, I will discuss our efforts on the construction of glycan microarrays and their applications in studies of glycan-associated interactions. Immobilization strategies of functionalized and unmodified glycans on derivatized glass surfaces will be discussed. Although others have developed immobilization techniques, our efforts have focused on improving the efficiencies and operational simplicity of microarray construction. The microarray-based technology has been most extensively used for rapid analysis of the glycan binding properties of proteins. In addition, glycan microarrays have been employed to determine glycan–protein interactions quantitatively, detect pathogens and rapidly assess substrate specificities of carbohydrate-processing enzymes. More recently, the microarrays have been employed to identify functional glycans that elicit cell surface lectin-mediated cellular responses.

Polypeptide N-acetyl-galactosaminyltransferases (GALNTs) have been shown to play diverse roles in several biological processes, but their function in organ-tropic metastasis remains unknown. Here, we find that GALNT14expression is specifically associated with pulmonary metastasis in breast cancer patients. Furthermore, we demonstrate that GALNT14 promotes lung metastasis by facilitating two important steps during the metastatic process, i.e., initiation of metastatic colonies and their subsequent growth into overt metastases. Mechanistic studies suggest that GALNT14 not only augments self-renewal of breast cancer cells within the lung microenvironment but also allows breast cancer cells to exploit macrophage-derived growth factors for their continuous growth. The mediators of GALNT14 involved in these processes will also be discussed.

Bechet’s disease (BD) is an immune disease which showed a chronic and relapsing systemic vasculitis of unknown etiology. It is characterized by recurrent ulcers and inflammations on oral, genital, uvea area and further vascular, central nervous system (CNS), and gastrointestinal tracks. Bechet’s disease has a wide spectrum of symptoms ranging from benign episodes to more serious complications such as blindness. Although many diagnostic criteria have been developed and revised by experts in the field, diagnosing BD is still complicated and challenging. To discover the diagnostic biomarkers, serum N-glycans from Bechet patient (n=100) and healthy control (n=100) have been compared. Instead of conventional global profiling of serum N-glycome, quantitative ratio between structural isomers were analyzed by UPLC-triple quadruple mass spectrometry. Three acidic glycans with the composition of 5:4:0:1, 5:4:0:2, and 5:4:1:1 (Hex:HexNAc:Fuc:NeuAc) were monitored in MRM mode, then six, two and three isomers were found, respectively. Among eleven bi-antennary glycan isomers, four of them showed high diagnostic efficacy having AUC of ROC curve over 0.98. When the sensitivity was fixed to 90%, corresponding specificities were over 92% to 96%. Another four isomers showed the AUC from 0.84 to 0.94 showing 64%~84% specificity with the sensitivity of 90%. Marker glycans exhibits unique correlations with biological characteristics of cohorts. Two isomers of 5:4:0:1 and one of 5:4:0:2 isomer were able to completely distinguish male and female groups in healthy control cohort. Glycan expression level in female groups were 6 to 10 times higher than those observed in male groups resulting 1.0 AUC in ROC curve. Expression level in patient cohort were 5 to 10-folds higher than those in control group showing complete segregation. Correlation between the glycans were also clear. Groups of isomers with each composition exhibits Pearson’s product over ±0.90 within or between the groups. This study present the novel approach for the disease marker discovery that tracing the isomeric structure of specific N-glycan. As results, the markers with undoubtable quantitative deviation and the correlations reflecting their biological characteristics have been found.

Modification of nucleocytoplasmic proteins with O-GlcNAc regulates a wide variety of cellular processes and has been linked to human diseases. The enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) add and remove O-GlcNAc, but the mechanisms regulating their expression remain unclear. Here, we demonstrate that retention of the fourth intron of OGT is regulated in response to O-GlcNAc levels. We further define a conserved intronic splicing silencer (ISS) that is necessary for OGT intron retention. Deletion of the ISS in colon cancer cells leads to increases in OGT, but O-GlcNAc homeostasis is maintained by concomitant increases in OGA protein. However, the ISS-deleted cells are hypersensitive to OGA inhibition in culture and in soft agar. Moreover, growth of xenograft tumors from ISS-deleted cells is compromised in mice treated with an OGA inhibitor. Thus, ISS-mediated regulation of OGT intron retention is a key component in OGT expression and maintaining O-GlcNAc homeostasis.

Anoikis is a form of anchorage-dependent apoptosis, and cancer cells adopt anokis-resistance molecular machinery to conduct metastasis. Here, we report that N-acetylglucosaminyltransferase V gene expression confers anoikis resistance during cancer progression. Overexpression of MGAT5 (N-acetylglucosaminyltransferase V) protected detached cancer cells from apoptotic death, and suppression or knockout of the gene sensitized cancer cells to the apoptotic death. The gene expression also stimulated anchorage-dependent as well as anchorage-independent colony formation of cancer cells following anoikis stress treatments. Importantly, treatment with the lectin from Sambucus sieboldiana significantly sensitized anoikis-induced cancer cell deaths in vitro as well as in vivo. We propose that the lectin alone or an engineered form could offer a new therapeutic treatment option for cancer patients with advanced tumors.

Glycosylation, which is susceptible to the biochemical environments, is one of the most important post translational modifications in eukaryotic proteins. Moreover, glycans are involved in many important biochemical events. Especially, sialic acids which are expressed as outer terminal units on all vetebrate are play fundamental roles in cell-to-cell and cell-to-microenvionment interactions. N-acetylneuraminic acid (NeuAc) and N-acetylglycolylneuraminic acid (NeuGc) are predominant sialic acids on most mammalian cells. Unlike other mammalian except chicken, human cannot biosynthesize the NeuGc due to irreversible mutation on gene CMAH encoding CMP-N-acetylneuraminic acid hydroxylase that convert CMP-NeuAc to CMP-NeuGc. When the NeuGc is metabolically incorporated into human tissue from dietary sources (especially red meat), the anti-NeuGc antibodies which leads immune response will be produced and circulated through blood. The evidence showed that the such anti-NeuGc antibodies may cause systemic inflammation and finally can promote tumor progression. Therefore, quantitation of the non-human sialic acid NeuGc in food and therapeutic glycoproteins is of high importance. Here, we introduce analytical methods for rapid and highly sensitive identification and quantitation of NeuGc in food contents containing non-human glycan epitope using LC-MS/MS.

Functional roles of starch besides a carbohydrate energy source have been recently focused on the regulation of gut microbiota which can derive to various prebiotic benefits such as anti-obesity and hypoglycemic effects as a diabetes-preventive strategy. Our attempts were to identify prebiotic starch sources and investigate the prebiotic functions. Firstly, the effects of various starches on the gut microbiota were investigated using normal chow diets formulated based on AIN93G but replaced corn starch to potato, rice, and wheat starch. Potato starch-based diet prevented the development of obesity and insulin resistance through the slowest digestion property of potato starch by mammalian mucosal α-glucosidase and also it caused obviously changed microbial community. For instance, the ratio of Bacteroidetes to Firmicutes was significantly increased by potato starch-based diet compared to diets based on other starches and predictably an increase in the abundance of Akkermansia which has known to be beneficial for diabetes treatment was found. These indicate that digestion property of starches may be a key to regulate the gut microbial community. Secondly, we investigated the prebiotic effects of resistant starch whose proportion was increased by structural modification of chestnut starch by amylosucrase from Deinococcus geothermalis (DGAS) in diet-induced obesity animal model. Chest starch modified by DGAS (DMCS) showed significant anti-obesity effects related with short chain fatty acids (SCFA) receptor GPR43-mediated suppression of insulin signaling pathway in white adipose tissue. Concurrently, the mechanisms of energy expenditure were activated by DMCS, mediated by the significant changes in microbial community such as Ruminococcus, Ruminococcaceae, Prevotella etc and the metabolites such as SCFA particularly acetic acid which were significantly correlated with the expression of genes involved in GPCR signaling, fatty acid oxidation, and the ATP-formation pathway.

α-Amylase first hydrolyzes starch structures to linear maltooligosaccharides and branched α -limit dextrins, then complete hydrolysis to glucose takes place through the mucosal α -glucosidases. In this study, we hydrolyzed waxy corn starch (WCS) by human pancreatic α -amylase to determine the digestion and structural properties of different size fractions of the branched α-limit dextrins. The α-amylolyzed WCS was separated by size exclusion chromatography (SEC), and the analyzed chromatograms showed four main hydrolyzate fractions. The first three eluted peaks (regions I-III) corresponded to branched α-limit dextrins, while region IV was the linear maltooligosaccharides. Region I, II, and III had multiple (>2), two, and one α-1,6 linkages, respectively, and region I was the most slowly hydrolyzed to glucose by mucosal α-glucosidases (hydrolysis rate: Region I< II < III < IV). This study shows the possibility of producing slowly digestible oligosaccharides that may decrease postprandial glycemic response and control glucose delivery to the body, to address metabolic syndrome-associated diseases.

Macrophages, the cells of the innate immune system, play a key role in all three types of xenograft rejection. However, the mechanism underlying their direct effects is incompletely understood. To study the direct effects of macrophages, established human model system combined the U937 monocyte cell line and mini-pig adipose derived mesenchymal stem cells (mp AD-MSCs). Treatment of mp AD-MSCs with macrophage secretion medium (Macro SM) markedly reduced the cell viability, increased lactate dehydrogenase release, and induced apoptotic cell death. The proteomic analysis identified 17 proteins in Macro SM. Based on the gene ontology analysis tool, matrix metalloproteinase (MMP)-9 was selected as the protein affecting MSC viability. To confirm the effect of MMP-9, cells were treated with recombinant human MMP-9 that resulted in decreased cell viability and increased lactate dehydrogenase release. These results suggest that MMP-9 may decrease the cell viability by damaging the cell membrane in the co-culture model.

Fucosylation is one of the most common glycosylation and has been associated with the development of hepatocellular carcinoma (HCC). Alpha-fetoprotein (AFP) is clinically used as serological marker to diagnose HCC. Although the level of AFP increased in HCC patients, its sensitivity for diagnosis of HCC is poor because AFP levels are also increased in liver diseases, such as liver cirrhosis. Here, we introduced detection of AFP glycopeptides by parallel reaction monitoring (PRM) mass spectrometry (MS) combined with immunoprecipitation. Also, desialylation of AFP glycopeptides was used to improve MS detection limit (LOD < 2 ng/mL) and to obtain reliable signal (CV < 20%) from 0.1 μL serum. Finally, we compared serum samples from HCC with liver cirrhosis using relative percentage of fucosylated glycopeptide in AFP (AFP-fuc%). AFP-fuc% showed an area under the ROC curve (AUC = 0.949, p value < 0.0001) to discriminate between HCC and liver cirrhosis patients. These results suggest the potential of PRM-based AFP-fuc% in early diagnosis of HCC using serum samples.

Single vessel multi-enzyme UDP-α-D-glucose recycling system was coupled with a forward glucosylation reaction to produce glucose moiety conjugated novel derivatives of different tetracycline antibiotic analogues. Among five tetracycline analogues used for the reaction, four molecules (chlorotetracycline, doxytetracycline, meclotetracycline, and minotetracycline) were accepted by a glycosyltransferase enzyme, YjiC, from Bacillus licheniformis to produce glucoside derivatives. However, the enzyme was unable to conjugate sugar unit to rolitetracycline. All glucosides of tetracycline derivatives were characterized by ultraviolet absorbance maxima, ultra-pressure liquid chromatography coupled with photo-diode-array, and high-resolution quadruple time-of-flight electrospray mass spectrometry analyses. These synthesized glucosides are novel tetracycline derivatives. The conversion of tetracycline analogues to respective glucosides has been found to be very low yet. This gives the clue that the enzyme has low preferences toward tetracycline molecules. Attachment of bulky group in rolitetracycline might have prevented the molecule to reach the catalytic cleft of GT enzyme. Results of this research could become a basis to explore the possibility of synthesizing glucosylated derivatives of different tetracyclines using GT enzyme.

Resveratrol glycosides have been produced using the flexible glycosyltransferase gene YjiC from Bacillus licheniformis DSM-13. The reaction mixture containing purified YjiC, nucleotide diphosphate (NDP) D- and L-sugars as sugar donor and Resveratrol as acceptor substrate was incubated at 37 ℃for 5 hours.The spectrometric analysis HPLC-PDA, mass and nuclearmagnetic resonance analysis of the reaction mixture showed the production of glucose, galactose, 2-deoxyglucose, viosamine, rhamnose, and fucosesugar-conjugated resveratrol glycosides. α-D-glucose as the sugar donor gave the higher(90%) bioconversion of resveratrol producing four different glucosides of resveratrol, resveratrol 3-O-β -D-glucoside, resveratrol 4′-O-β-D-glucoside, resveratrol 3,5-O-β-D-diglucoside and resveratrol 3,5,4′-O-β-D-triglucoside. In the case of other NDP-sugar donors, the conversion rates and the number of products formed were varied. While using TDP-2-deoxyglucose as sugar donor, Resveratrol 3-O-β-D-2-deoxyglucoside, resveratrol 3,5-O-β-D-di-2-deoxyglucoside were found to be produced, however ,monoglycosides resveratrol 4′O-β-D-galactoside, resveratrol 4′O- β-D-viosaminoside, resveratrol 3′O-β -L-rhamnoside, resveratrol 3′O- β-L-fucoside were produced from respective sugar donors. On the whole, ten diverse glycoside derivatives of medicinally important resveratrol were generated. This demonstrated the capacity of YjiC to produce structurally diverse resveratrol glycosides.

Phenolic compounds are used as preservatives for cosmetics and antioxidants as well as for various biological activities such as anti-inflammation, anti-cancer, antioxidant and immunomodulation, and have many physiologically active functions. Hydroquinone inhibits skin melanin formation and brightens dark skin. Resorcinol is used to treat rough skin or cured skin, to disinfect the skin to prevent infection, to treat pain and itching caused by minor wounds and other skin irritations, and to treat other skin conditions. Catechol is reported to be an inhibitor of the pro-inflammatory pathway and can be considered a candidate for treating neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and other inflammatory diseases. Despite the importance of these phenolic compounds, the use of these compounds is not well soluble in water, they are easily oxidized by light in aqueous solutions, and are generally limited due to toxicity and side effects. Amylosucrase (AS) from Deinococcus geothermalis (DGAS) was selected in the GH-13 line to address this problem. The advantage of the amylosucrase enzyme is that it has excellent activity, such as ADP- or UDP-glucose, but is industrially useful because it uses relatively inexpensive glycosyl donors such as sucrose as compared to expensive glycosyl donors. DGAS produced dihydroxybenzene glucoside, which was confirmed by high performance liquid chromatography (HPLC). Each reaction efficiency is hydroquinone glucoside (90%), resorcinol glucoside (70%), and catechol glucoside (90%). The major glycoside products are identified based on mass spectrometry.

The purpose of this experiment is to investigate the glycosylation using enzymes. E. coli BL21 (DE3) was used as a host for protein overexpression. Phloroglucinol (PG) is used as an antiseptic and the treatment of gallstones, cramps and gastrointestinal disorders. According to HPLC results, the glycosylation of PG quickly generated in a few hours. Pyrogallol is a medicinal product that is used in psoriasis and antimicrobial agents. We progressed this reaction with ascorbic acid which is antioxidant for reaction stability of phloroglucinol and pyrogallol. 2,4-dihydroxybenzoic acid are belong to flavor modulators. The pharmacological action of 2,5-dihydroxybenzoic acid has a wide range of biological activities such as anti-inflammatory, antirheumatic and antioxidant properties. When salicylic acid is glycosylated, we anticipate both antivirus and antipyretic effects against influenza. In the case of p-aminosalicylic acid, when glycosylated, we anticipate alleviation of various side effects mentioned above as a result of enhanced water solubility of drug. The glycosylation of salicylic acid by amylosucrase was not detected, but we observed through HPLC that the level of glycosylation of p-aminosalicylic acid, 2,4-dihydroxybenzoic acid, and 2,5-dihydroxybenzoic acid, although slow in reaction rate, increased over a time period.

Kaempferol is a natural flavonol with numerous bioactivities. It reduces the risk of various cancers and possess skin-lightening effect. Due to these efficacies, its possible application in food, medicine and cosmetics industry are increasing. However, like most flavonoids it has limited application due to water solubility and stability. Glycosylation has been an important approach to diversify and extend novel biological functions in natural products and secondary metabolites. Hence, in this study, considering these demerits, we synthesized astragalin (kaempferol glucoside) from kaempferol using uridine 5′-diphosphate (UDP)-3-O-glycosyltransferase. To enhance the pool of UDP-glucose necessary for UGT78K1, UDP-glucose biosynthetic genes along with glucose facilitator diffusion protein (glf) and glucokinase (glk) were expressed in a multi-monocistronic vector pIBR to produce astragalin in significant titer.

Generally glycosylated natural compounds have the specific contribution to the pharmacological properties. Particularly, C-glycosylflavones are involved in UV protection, defense against pathogens and inhibition of caterpillar growth. C-glycosylflavones are biosynthesized from flavone via C-glycosylation of 2-hydroxyflavone or flavone. Two plant C-glucosyltransferases (CGTs) UGT708D1 from Glycine max and GtUF6CGT1 from Gentian triflora were used for glucosylation of chrysin. UDP-glucose pool was enhanced by introducing heterologous UDP-glucose biosynthestic genes i.e., glucokinase (glk), phosphoglucomutase (pgm2), and glucose 1-phosphate uridylyltransferase (galU), along with glucose facilitator diffusion protein (glf), in a multi-monocistronic vector pIBR. Moreover, the bioengineered E. coli strains enhanced the production by approximately 1.4–fold, thus producing 10 mg/L and 14 mg/L by UGT708D1 and GtUF6CGT1, respectively, without supplementation of additional UDP-glucose in the medium. HPLC analysis of fermentation broth extract showed 50 % (42 mg/L) conversion. Thus in this study, we successfully biosynthesized C-glycosylflavone in vivo and the product was confirmed by LC-MS and NMR.

A cheap UDP-D-glucose producing system was developed by using sucrose as primary source. The system was coupled with the UDP generating glycosylation reaction of UDP-D-glucose dependent glycosyltransferase (GT) enzymes mediated reactions. As a result, the UDP produced as by-product of the GT-mediated reaction was recycled. YjiC, a UGT from Bacillus licheniformis DSM 13 was used as GT for transferring glucose from UDP-glucose to resveratrol while AtSUS1 from Arabidopsis thaliana was used to hydrolyze sucrose to UDP-glucose and fructose. The established UDP-recycling system is highly feasible for large scale production of different derivatives of resveratrol and other natural products glucosides using inexpensive starting materials. The effect of glucosylation of resveratrol on macrophage cell viability and immunomodulation properties have been studied in murine macrophage RAW 264.7 cells. During the study, nitric oxide (NO) and interleukin 6 (IL-6) expression and production after treatment with (E)-resveratrol, (E)-resveratrol 3-O-β-D-glucoside (R-3-G) and (E)-resveratrol 4ʹ -O-β-D-glucoside (R-4ʹ-G) in vitro at different concentrations. In cell viability assays of macrophages, two different resveratrol monoglucosides, R-3-G and R-4ʹ-G, exhibited significantly reduced cytotoxicity in comparison to (E)-resveratrol standard. The conjugation of glucose moiety on resveratrol was found to enhance the immunomodulating activity and viability of RAW 264.7 cells. Glycoside derivatives of resveratrol have shown many potentiality of medicinal and biological activities.

Glycosyltransferase from Bacillus licheniformis DSM13 (YjiC) was expressed in Escherichia coli BL21 (DE3) for enzymatic modification of emodin, aloe-emodin, alizarin, antraflavic acid and 2-amino-3-hydroxyanthraquinone in vivo. The recombinant strain successfully modified the above mentioned substrates to their glycosylated products by using their indigenous UDP-glucose for glycoconjugation. Additionally, their major glycosylated products exhibited the highest stability at pH 8.0 and temperature up to 70 °C and 60 °C respectively. Furthermore, the biological activities of emodin and its major glucoside (P1) were compared and their anti-cancer activities were assayed in several cancer cell lines. The results demonstrate that YjiC has the capacity to catalyze the glycosylation of these aromatic compounds and that glycosylation of anthraquinones enhances their aqueous solubility while retaining their biological activities.

Rhamnose is naturally occurring deoxy-sugar commonly bound to other sugars in nature. It is a common glycogenic component of glycosides in many plants and microbes. Indigenously available TDP-L-rhamnose from E. coli can be conjugated to natural products using specific glycosyltransferases. Current research focus on the enhancement in production of such potential anthraquinone deoxy-sugar conjugated products through microbial engineering and biotransformation approach. A recombinant E. coli strain was designed by expressing rhamnosyltransferase and anthraquinones were fed exogenously to produce several anthraquionone rhamnose-conjugated derivatives. Among all anthraquinones; Quinizarin, 1,4-dihydroxy-9,10-anthraquinone was efficiently bio-transformed yielding high production. These anthraquinones have important applications in the field of medicine, such as inhibitory effects against human intestinal bacteria. In addition, quinizarin exhibits inhibitory effects in the aflatoxin B1 biotransformation to the corresponding 8, 9-epoxides, which is responsible for the toxic and carcinogenic effects of aflatoxins.

Violacein is a purple pigment made by enzymatic oxidation and coupling of two molecules of L-tryptophan. It has attracted interest as antileukemic agent and also known for inhibiting the growth of NCI-H460 non-small-cell lung cancer and KM12 colon-cancer cell lines. For most potent antibiotics like avermectin, erythromycin, vancomycin, and doxorubicin, sugars are often essential for the pharmacological properties, especially for water solubility and/or the biological activity of the compounds. Previously increasing solubility of violacein by inclusion complex with β-cyclodextrin and biotransformation by oxidative enzymes to increase the solubility and biological activity of violacein has been reported. The naturally occurring glucosylated violacein analogs have not yet been reported, and no sugar biosynthetic genes have been found in the violacein biosynthetic gene cluster. Violacein glucosides with enhanced solubility and bioactivity is expected, which may be able to fulfill the demand of soluble violacein for in vivo assays. In this study, in order to increase the solubility and enhance the biological activity of violacein we applied two plasmids systems, using the violacein synthesis enzymes (vioABCDE-pETM6), and glycosylation enzyme (Yjic-pET28a). In addition, we tried using the E.coliΔpgiΔzwfΔushA mutant, that has high efficiency in production of UDP-glucose. These studies may be the strategic approach for the production of a glycosylated violacein in reasonable titer.