1. Department of Bioscience and Biotechnology Ahn Byoung chan
Formation of flavones di-O-glucosides using a glycosyltransferase from Bacillus cereus
Hi. I’m byoungchan ahn from molecularbiology lab. My advice professor is joong hoon ahn. My presentation is Formation of flavones di-O-glucosides using a glycosyltransferase from Bacillus cereus.
Department of Bioscience and Biotechnology
Ahn Byoung chan

2. Purpose
-modification of simple chemicals using microorganisms or proteins expressed in heterologous system such as Escherichia coli or yeasts.
-create regioselective compounds more efficiently than chemical synthesis
Di-O-glucosylation of flavonoids using recombinant
glucosyltransferase, BcGT3 from Bacillus cereus.
Genome projects from innumerable organisms have been completed or are still in progress Many researchers have been thinking how to use this vast information
One of good application areas would be modification of simple chemicals using microorganisms or proteins expressed in heterologous system such as Escherichia coli or yeasts
Also Using biological systems for modifying chemicals could create regioselective compounds more efficiently than chemical synthesis .
And Various forms of flavonoid O-glycosides have been found in nature. They contain different kinds and/or different number. Even though flavonoid O-mono glycosides are prevalent, di or tri glucosides are also present. It was curious to know if flavonoid di-O-glucosides are able to synthesize using enzymes from microorganisms. Thus, we report di-O-glucosylation of flavonoids using recombinant UGT, BsGT3 from Bacillus

3. Introduction *Flavonoid
-a large group of low molecular weight phenolic phytochemicals
-secondary metabolites found in plants
-flavones, flavanones, isoflavones, flavonols, flavanonols, flavan-3 – ols
-acts as antioxidants, anti-inflammitory, anti-cancer
-stored as glycosylated form
Flavonoids are a large group of low molecular weight polyphenolic phytochemicals
and secondary metabolites found in plants.
Many flavonoids share a common phenyl benzopyrane skeleton and differ from one another only by the numbers and positions for hydroxyl or methylated functional groups. Flavonoid classes include flavones, flavanones, isoflavones, flavonols, flavanonols, flavan-3 – ols
They play a variety of roles such as UV protection, pathogen defense and coloration flavonoids also have an effect on humans as antioxidants, anti-inflammitory, anti-cancer or phytoestrogen.
The flavonoid aglycones, which have a variety of glycosylation sites, are converted into glycon by glycosyltransferases(GTs) in the last step of flavonoid biosynthesis. Flvonoids are stored as glyconforms.

4. Introduction *Glycosylation *Glycosyltransferase
- Attachment of sugar unit to small compounds
- essential step for storing and locating small compounds
- detoxification of xenobiotics and alternation of biological activity
- increase the solubility
*Glycosyltransferase
-mediate glycosylation
-classified into 86 families
- Family 1 is UDP-dependent glycosyltransferase (UGT)
Attachment of sugar unit to small compounds, called glycosylation
is one of the common reactions found in plants.
Glycosylation is an essential step for storing and locating small compounds.
In addition, glycosylation has a role in detoxification of xenobiotics and alternation of biological activity
Glycosyltransferases (GTs) involved in synthesizing the carbohydrate moieties of several biological compounds
And the glycosyltransferases(GTs) have been classified into 86 families

5. Introduction Family1 *UGTs -Family 1 GTs are called UGTs
BC2799 (0.0699)
BcGT3 (0.0712)
Bcer98_1566 (0.3088)
BTZK2382 (0.3357)
BCE0562 (0.4249)
BCE3442 (0.4123)
BCE5387 (0.4537)
BCE1326 (0.4586)
BCE1664 (0.4228)
BCE4907 (0.4047)
BCE5390 (0.4269)
BCE5586 (0.4287)
BF2727 (0.0460)
BT1294 (0.0480)
BVU_3560 (0.0922)
CHU_1581 (0.3571)
Family1
Family2
Family4
Family3
*UGTs
-Family 1 GTs are called UGTs
-Use uridine dinucleotide as glucose donor
-involved in glycosylation of the secondary metabolites including flavonoids
We could draw phylogenetic tree of GTs from various sources. And we could know Bcgt3 is UGTs
What is UGTs?
UGTs are Family 1 GTs
They are dependant on uridine dinucleotide as glucose donor
And they are involved in storing of the secondary metabolites including flavonoids.
So we can choose flavonoids as candidate for substrate.

6. Consensus sequence (PSPG)
UDP-glucose binding residue
and We performed alignment of bcgt3 with another bcgt and already known ugt from rice.
As a result of alignment, pspg-box (plant secondary product glucosyltransferase box ) were mached with each other. UDP-glucose binding residue, glutamine is also mached with other one. In addition, it contained a conserved histidine residue (His14) that serves as a base for the catalysis.

7. Expression of BcGT3 pGEX BcGT3
Ptac
Glutathione S-transferase
So, The open reading frame of BcGT-3 was subcloned into E. coli expression vector pGEX 5X-3 and expressed in E. coli as glutathione s-transferase fusion proteins. enzymes expressed successfully and purified using GST affinity column and this confirmed correct size by SDS- PAGE. like this.
SDS gelelectrophoresis analysis of recombinant protein
Lane M : Marker of standard protein
Lane 1 : E.coli.lysate before induction
Lane 2 : E.coli.lysate after induction
Lane 3 : Soluble protein after sonication.
Lane 4 : BcGT3 protein using GST affinity column

8. Analysis of reaction product using HPLC
Kaempferol-3-O-glucoside
Naringenin
In order to know whether BcGT-3 could utilize flavonoids as substrate, several flavonoids including isoflavone (genistein), flavanone (naringenin), flavone (luteolin), and flavonol (kaempferol and quercetin) were tested.
Glucosylation position of naringenin reaction product was determined by comparing HPLC retention time and UV-spectrum of the reaction product with those of authentic compounds. HPLC retention time and UV-spectra of naringenin reaction product with BcGT-3 were indistinguishable from those of the authentic naringenin 7-O-glucoside, indicating that the reaction product is a naringenin 7-O-glucoside. The genistein reaction product was determined to be genistein 7-O-glucoside.
Genistein and naringenin gave one reaction product whose molecular weight was increased by 162 Da, indicating that one glucose molecule was attached. On the other hand, flavone and flavonols gave more than one reaction products. Analysis of these compounds using mass spectrometer revealed that mono and di-O-glucosides were produced.
Genistein-7-O-glucoside

9. LC/MS analysis of reaction product
For example, This is analysis of kaempferol reaction product using HPLC showed two peaks. LC/MS analysis of these showed that the molecular weight of the first peak at 7.8 min increased 324-Da compared to that of kaempferol, indicating that two glucose residues were attached and the molecular weight of the second peak was increased by 162-D
Molecular weight of kaempferol is 286 D and mw of glucose is 162.

10. Confirmation of the reaction product5 10 15 20 25
Min 50
100
mAU A B H G F E D C P4 P3 P2 P1
A . Products of Bcgt3 with kaempferol
B. Control of kaempferol
C . Products of Bcgt3 with luteolin
D . Control of luteolin
E . Products of Bcgt3 with naringenin
F. Control of naringenin
G . Products of Bcgt3 with quercetin
H . Control of quercetin
P1 , Kaempferol-3,7-diglucoside
P2 . Luteolin-7-glucoside
P3 . Naringenin-7-glucoside
P4 . Quercetin-3-glucoside
We confirm the reaction product.
First, The mono-O-glucoside of kaempferol was determined to be kaempferol 3-O-glucoside based on HPLC retention time and UV spectrum. Glycosylation positions of kaempferol di-O-glucoside were determined using nuclear resonance spectrometer. In the HMBC spectrum, two separated long-ranged couplings between C-3 (δ 133.4) of kaempferol and an anomeric proton (δ 5.47) of glucose, and C-7 (δ 162.8) of kaempferol and another anomeric proton (δ 5.07). Therefore, it was proved that kaempferol is connected with two glucoses through C-3 and C-7.
And Luteolin mono O-glucoside was glucosylated at 3’-hydroxyl group by comparing the retention time with that of authentic luteolin 3’-O-glucosie. glucosylation position of luteolin di-O-glucosides is likely at 7 and 3’ hydroxyl group.
Reaction of quercetin with BcGT-3 also produced di-O-glucosides. Like reaction with kaempferol, BcGT-3 first converted quercetin into quercetin 3-O-glucoside, which is eventually converted into quercetin 3, 7-O-diglucosides
Substrate preference of BcGT-3 was examined. Flavonols (quercetin and kaempferol) and flavones (luteolin) were better substrates than flavanones (naringenin). It suggests that double bond between carbon 2 and 3 is important.

11. Realative conversion rate (%)
Relative conversion rate of BcGT-3
Flavonoid
Structure
Realative conversion rate (%)
Quercetin
100
Luteolin
99.7
Kaempferol 99
Naringenin
75.8
This slide is a relative flavonoid conversion rate of BcGT-3
It shows, also Flavonols (quercetin and kaempferol) and flavones (luteolin) were better substrates than flavanones (naringenin).

12. Conversion of kaempferol
And Conversion of kaempferol into 3,7-O-diglucoside was monitored periodically.
As a result, we know kaempferol was completely used up and 35 μM of kaempferol 3-O-glucoside and 65 μM of kaempferol 3, 7-O-diglucoside were formed. After longer incubation (100 min), final concentration of kaempferol 3-O-glucoside and kaempferol 3, 7-O-diglucoside was 20 and 80 μM, respectively.

13. Mechanism kaempferol BcGT3 2 UDP-glucose 2 UDP
Kaempferol-3,7-diglucoside
BcGT3
This slide is UGTs mechanism, UDP-sugar give a sugar to substrate, and enzyme catalyze glycosylation into substrate.

14. Summary
Microbial UDP-glycosyltransferases can convert many small lipophilic compounds into glycons using uridine-diphosphate-activated sugars.
The glycosylation of flavonoids affects solubility, stability and bioavailability.
BcGT3 tested for activity on several substrates.
Kaempferol was one of the most effective substrate tested. Based on HPLC, LC/MS, and NMR analysis of reaction products, BcGT-3 can be used for the synthesis of kaempferol 3, 7- O-diglucose
Microbial UDP-glycosyltransferases can convert many small lipophilic compounds into glycons using uridine-diphosphate-activated sugars.
and The glycosylation of flavonoids affects solubility, stability and bioavailability.
So we cloned the glycosyltransferase from bacillus cereus, and it tested for activity on several substrates.
As a result Kaempferol was one of the most effective substrate tested. Based on HPLC, LC/MS, and NMR analysis of reaction products, BcGT-3 can be used for the synthesis of kaempferol 3, 7- O-diglucose

15. Thank you
Thank you