KR102674605B1 - Method for mass production of maysin using E. coli - Google Patents

Abstract

The present invention includes (a) a plasmid into which the 6-C-glycosyltransferase gene (GtF6CGT1) of Gentiana triflora is inserted and a plasmid into which the glucose 2″-O-rhamnosyltransferase gene (UGT91L1) of Gentiana triflora is inserted. Culturing recombinant E. coli transformed with one or more plasmids selected from the group consisting of a medium; (b) adding luteolin to the culture medium in which the transformed recombinant E. coli is being cultured; and (c) cultivating the transformed recombinant E. coli in a culture medium to which the luteolin has been added. According to the present invention, by using E. coli, a small amount of maycin is contained in corn silk. There is an advantage in being able to produce large quantities of meishin.

Description

Method for mass production of maysin using E. coli}

The present invention relates to a method for mass production of maysin using E. coli. More specifically, since maysin, an important bioactive substance contained in corn silk, is contained in extremely small amounts, it is necessary to mass produce maysin. This is about a method of mass production of maycin in an E. coli cell factory using the biosynthesis gene.

Corn silk has long been widely used in various folk remedies such as nephritis treatment, urinary stone treatment, diuretic, and diabetes treatment in Korea, China, South America, and Europe.

Corn silk contains maysin, chlorogenic acid, neochlorogenic acid, 4-caffeoylquinic acid, and rhamnosyl isoorientin. It is known that

Maycin (rhamnosyl 6-C-(4-ketopucosyl)-5, 7, 3', 4' tetrahydroxyflavone) is a flavone C, luteolin, attached to a disaccharide residue at six carbon positions. -It is a glycoside. It was isolated as a natural product from Petrorhagia velutina and Zea mays and exhibits insecticidal and neuroprotective activities.

The specific maycin biosynthetic pathway in maize is unknown except that it occurs in the region where C-glucosyl flavones are produced and may be controlled by other general genetic mechanisms regulated by the pl locus.

Phlobaphene is a red pigment accumulated in certain floral organs of maize lineage plants, where phlobaphene biosynthesis is regulated by the maize p1 gene, and Myb- of the gene encoding a flavonoid biosynthetic enzyme that also regulates maysin synthesis in maize silk. Encodes a homologous transcriptional activator.

The description of the maycin pathway sequence (luteolin → isoorientin → rhamnosilyisoorientin → meisin) in maize was reported by McMullen et al. (2004) (J. Hered. 95, (2004) 225-233. ).

Conventional technologies related to the production of maysin include a method for producing a corn silk extract with a high maysin content (Korea Patent Publication No. 10-1201628), a high-yield extraction method of active substances containing maysin derived from corn silk (Korea Unveiled) Patent Publication No. 10-2015-0057084) is disclosed.

However, the above-described prior art has limitations in mass producing the maycin component contained in corn silk because it contains an extremely small amount. Therefore, the development of a method for mass producing the maycin is urgently needed. There is a situation.

Republic of Korea Patent Publication No. 10-1201628 Korean Patent Publication No. 10-2015-0057084

The purpose of the present inventors is to provide a method of mass producing maysin in an E. coli cell factory using maysin biosynthetic genes as an alternative to the fact that maysin, an important bioactive substance contained in corn silk, is contained in very small amounts. It is done.

Another object of the present invention is to provide recombinant E. coli capable of mass producing the above-mentioned maycin.

Another object of the present invention is to provide maisin mass-produced by the above method.

In order to achieve the above object, the present invention (a) a plasmid into which the 6-C-glycosyltransferase gene (GtF6CGT1) of Gentiana triflora is inserted and the glucose 2″-O-rhamnosyltransferase gene of Gentiana triflora ( Culturing recombinant E. coli transformed with one or more plasmids selected from the group consisting of plasmids into which UGT91L1) is inserted in a medium; (b) adding luteolin to the culture medium in which the transformed recombinant E. coli is being cultured; and (c) cultivating the transformed recombinant E. coli in a culture medium to which the luteolin has been added.

In one embodiment of the present invention, the plasmid into which the 6-C-glycosyltransferase gene (GtF6CGT1) is inserted may be pIBR181-GtF6CGT1 shown in Figure 2a.

In one embodiment of the present invention, the plasmid into which the glucose 2″-O-rhamnosyltransferase gene (UGT91L1) is inserted may be pET32a-UGT91L1, shown in Figure 2b.

In one embodiment of the present invention, the medium may be Luria-Bertani (LB) agar broth medium supplemented with antibiotics.

In one embodiment of the present invention, the antibiotic may be ampicillin, kanamycin, and chloramphenicol.

In one embodiment of the present invention, the antibiotics may be supplemented with 90 to 110 μg/mL of ampicillin, 40 to 60 μg/mL of kanamycin, and 90 to 110 μg/mL of chloramphenicol.

In one embodiment of the present invention, the luteolin may be added 15 to 25 hours after culturing the transformed recombinant E. coli.

In one embodiment of the present invention, the luteolin may be added together with one or more carbon sources selected from the group consisting of glucose and glycerin.

In one embodiment of the present invention, the glucose may be 4 to 6% (w/v), and the glycerin may be 4 to 6% (w/v).

In one embodiment of the present invention, the culture in step (c) may be performed at 35 to 40°C for 24 to 72 hours.

In one embodiment of the present invention, the recombinant E. coli may be recombinant E. coli C-41(DE3).

In addition, in order to achieve the above object, the present invention provides recombinant E. coli transformed with a plasmid into which the 6-C-glycosyltransferase gene (GtF6CGT1) of Gentiana triflora is inserted.

In addition, in order to achieve the above object, the present invention provides recombinant E. coli transformed with a plasmid into which the glucose 2″-O-rhamnosyltransferase gene (UGT91L1) of Gentiana triflora is inserted.

In addition, in order to achieve the above object, the present invention provides a plasmid into which the 6-C-glycosyltransferase gene (GtF6CGT1) of Gentiana triflora is inserted and the glucose 2″-O-rhamnosyltransferase gene (UGT91L1) of Gentiana triflora. ) provides recombinant E. coli transformed with a plasmid into which is inserted.

Additionally, in order to achieve the above object, the present invention provides meisin mass-produced by the above method.

According to the present invention, there is an advantage of being able to produce large quantities of maycin, which is contained in small amounts in corn silk, by using E. coli.

Figure 1a shows the mayin synthesis process, and Figure 1b shows the process of mayin production in E. coli .
Figure 2a Figure 2d shows the plasmid gene map into which each gene is inserted, Figure 2a is the pIBR181-GtF6CGT1 plasmid gene map, Figure 2b is the pET32a-UGT91L1 plasmid gene map, Figure 2c is the pACYC-UGT708A6 plasmid gene map, and Figure 2d is the pET32a-UGT91L1 plasmid gene map. The pET28a-RHS1 plasmid gene map is shown.
Figure 3 shows the enzymes expressed from each expression vector gene, 1. RHS1, 2. UGT91L1, 3. GtF6CGT1, and 4. UGT708A6.
Figure 4 shows the results of supplying luteolin to Daejing bacteria expressing piBR181-GtF6CGT1 and pACYC-UGT708A6, respectively.
Figure 5 shows the mass spectrometry results of the result of feeding luteolin to E. coli expressing piBR181-GtF6CGT1.
Figure 6 shows the results of supplying luteolin to E. coli expressing piBR181-GtF6CGT1 and pET32a-UGT91L1.
Figure 7 shows the mass spectrometry results for P1 and P2.
Figures 8a to 8c show the production results of rhamnosyl-isoorientin according to the addition of glucose and glycerin. Figure 8a is the result of supplying only 0.5mM of luteolin, Figure 8b is the result of supplying only 0.5mM. As a result of adding 5% glucose to luteolin, Figure 8c shows the result of adding 5% glycerin to 0.5 mM luteolin.
Figure 9 shows the mass spectrometry results of the RHS1 enzyme reaction product with rhamnosylisoorientin.

The present invention, as a first embodiment, includes (a) a plasmid into which the 6-C-glycosyltransferase gene (GtF6CGT1) of Gentiana triflora is inserted and the glucose 2″-O-rhamnosyltransferase gene (UGT91L1) of Gentiana triflora . Culturing recombinant E. coli transformed with one or more plasmids selected from the group consisting of inserted plasmids in a medium; (b) adding luteolin to the culture medium in which the transformed recombinant E. coli is being cultured; and (c) cultivating the transformed recombinant E. coli in a culture medium to which the luteolin has been added.

Zea maise L., which expresses the salmon silk (sm) locus, contains an sm1 region that encodes a glucose-transforming enzyme, whereas the sm2 region, located on the maize chromosome, regulates a rhamnosyltransferase on the maize chromosome.

All derivatives, including maysin, apimaysin and methoxymaysin, are synthesized in a common pathway reported by Lee et al. (2009) (Genome. 52, (2009) 39-48).

Maycin, the main flavone in corn germ extract, has been individually recognized by the Ministry of Food and Drug Safety for its bioactivity class 2 functionality related to skin protection. The compound has various biological activities including anti-obesity, diabetes, kidney-related diseases, and neurodegenerative properties. It is related to.

The biosynthesis of this compound was very limited in terms of yield. Identification of the biosynthetic pathway genes showed the potential to expand and synthesize maysin also from microbial sources. Microbial platforms have always shown remarkable examples of being exploited for natural product biosynthesis and desirable modifications to express foreign genetic material to some extent.

Microbial cells, such as Escherichia coli , can be metabolically manipulated through the application of systems/synthetic biology tools to increase the production of desired products, pharmaceuticals, cosmetic ingredients, etc. from detectable amounts to even gram scale. Natural product pharmacophore engineering through simple biotransformation has been an effective tool through single or multiple gene expression in E. coli .

This study highlights the biosynthesis of the plant secondary metabolite maysin in E. coli . Based on a previous report (Plant Cell 28, (2016) 1297-1309), three genes were identified in maize: bifunctional flavone C- and O-glucosyltransferase (UGT708A6), glucose 2″-O -Rhamnosyltransferase (UGT91L1), and bifunctional UDP-rhamnose synthase/UDP-4-keto-6-deoxy for codon optimization and nucleotide sequence synthesis so that it can be expressed in E. coli for mesin biosynthesis. Glucose synthase (RHS1) was selected.

Additionally, flavone 6-C-glycosyltransferase (GtF6CGT1) from Gentiana triflora was used. These enzymes have been functionally validated through in vitro reactions to synthesize maysin.

Additionally, luteolin and apigenin were exogenously supplied to the recombinant E. coli strain to decorate rhamnosyl of 2"-glucose and 2"-(4-ketofucose sugar moiety) at the 6-C position. Meanwhile, sugar O-methyltransferase was also co-expressed to produce methoxymycin in E. coli .

In the mass production method of maysin of the present invention, the plasmid into which the 6-C-glycosyltransferase gene (GtF6CGT1) is inserted may be pIBR181-GtF6CGT1 shown in Figure 2a, but is not limited thereto.

In the mass production method of maysin of the present invention, the plasmid into which the glucose 2″-O-rhamnosyltransferase gene (UGT91L1) is inserted may be pET32a-UGT91L1 as shown in Figure 2b, but is not limited thereto.

In the mass production method of macin of the present invention, the medium may be Luria-Bertani (LB) agar broth medium supplemented with antibiotics, but is not limited thereto.

In the mass production method of maycin of the present invention, the antibiotic may be ampicillin, kanamycin, and chloramphenicol, but is not limited thereto.

In the mass production method of maycin of the present invention, the antibiotic is ampicillin 90 to 110 μg/mL, preferably 95 to 105 μg/mL, more preferably 100 μg/mL, and kanamycin 40 to 60 μg/mL. may be 45 to 55 μg/mL, more preferably 50 μg/mL, and chloramphenicol may be 90 to 110 μg/mL, preferably 95 to 105 μg/mL, and more preferably 100 μg/mL, but is limited thereto. It doesn't work.

In the mass production method of maycin of the present invention, the luteolin may be added after 15 to 25 hours, preferably 18 to 22 hours, and more preferably 20 hours after culturing the transformed recombinant E. coli, but is limited thereto. It doesn't work.

In the mass production method of maycin of the present invention, the luteolin may be added together with one or more carbon sources selected from the group consisting of glucose and glycerin, but is not limited thereto.

In the mass production method of maycin of the present invention, the glucose may be 4 to 6% (w/v), preferably 4.5 to 5.5% (w/v), and preferably 5% (w/v). The glycerin may be 4-6% (w/v), preferably 4.5-5.5% (w/v), and preferably 5% (w/v), but is not limited thereto.

In the mass production method of maisin of the present invention, the culture in step (c) is performed at 35 to 40 ° C., preferably 36 to 38 ° C., more preferably at 38 ° C. for 24 to 72 hours, preferably 36 to 60 ° C. It may be performed over a period of time, but is not limited to this.

In the mass production method of maycin of the present invention, the recombinant E. coli may be recombinant E. coli C-41 (DE3), but is not limited thereto.

As a second embodiment, the present invention provides recombinant E. coli transformed with a plasmid into which the 6-C-glycosyltransferase gene (GtF6CGT1) of Gentiana triflora is inserted.

As a third embodiment, the present invention provides recombinant Escherichia coli transformed with a plasmid into which the glucose 2″-O-rhamnosyltransferase gene (UGT91L1) of Gentiana triflora is inserted.

The present invention, as a fourth embodiment, is a plasmid into which the 6-C-glycosyltransferase gene (GtF6CGT1) of Gentiana triflora is inserted and the glucose 2″-O-rhamnosyltransferase gene (UGT91L1) of Gentiana triflora is inserted. Recombinant E. coli transformed with a plasmid is provided.

As a fifth embodiment, the present invention provides meisin mass-produced by the above method.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

<Example>

1. Materials and Methods

(One). Chemicals and Reagents

Luteolin, Apigenin, and Chrysin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Oligonucleotide primers and Isopropyl β-D-1-thiogalactopyranoside (IPTG) were purchased from GeneChem Inc. (Daejeon, Korea). High-performance liquid chromatography (HPLC) grade acetonitrile and water were purchased from Mallinckrodt Baker (Phillipsburg, USA).

All restriction enzymes, pGEM-T®-easy vector, Taq polymerase, and T4 DNA ligase were purchased from Takara Bio (Shiga, Japan) and Promega (Madison, WI, USA). The remaining chemicals and reagents were of the highest chemical grade purchased from reliable commercial sources.

(2). Bacterial strains, cloning and culture conditions

The identified and characterized genes of the maysin biosynthetic pathway (Plant Cell 28 (2016) 1297-1309) were codon optimized, and nucleotides were commercially synthesized for functional expression in E. coli (General Biosystem Inc. USA ).

Bi-functional enzymes: flavone 6-C/7-O-glucosyltransferase (UGT708A6: 1428 bp long, GeneBank: NP_001132650), glucose 2"-O-rhamnosyltransferase (Sm2 ie UGT91L1) : 1284 bp long, GeneBank: DAA40920.1) and bifunctional enzymes: UDP-rhamnose synthase and UDP-4-keto-6-deoxyglucose synthase (Sm1 i.e. RHS1: 2031 bp long, GeneBank: AFW77498.1 ) was synthesized and cloned into different expression vectors.

RHS1 was cloned into the pET28a vector into the restriction sites of BamHI and

For expression and production of the enzyme glucose 6-C-glycosyltransferase (GtF6CGT1) in pIBR181, bifunctional UDP-rhamnose synthase/UDP-4-keto-6-deoxyglucose synthase (RHS1) and Escherichia coli C41 (DE3) was used in the glucose 2"-O-rhamnosyltransferase (UGT91L1) pET32 vector, and E. coli XL-1 Blue (MRF' Invitrogen, USA) was used for all DNA manipulations.

All recombinant strains were grown on Luria-Bertani (LB) agar plates and broth media supplemented with ampicillin (100 μg/mL), kanamycin (50 μg/mL), and chloramphenicol (100 μg/mL) wherever required.

Fresh recombinant E. coli (DE3) containing pIBR181-GtF6CGT1, pET28a-RHS1, pACYC-UGT708A6 and pET32a-UGT91L1, respectively, were prepared by heat shock transformation method. Another strain was constructed by biological transformation of piBR181-GtF6CGT1 and pET32a-UGT91L1 plasmids in C-41(DE3) host.

(3). Protein expression and analysis

Seed cultures of these four strains were prepared in LB broth medium supplemented with the corresponding antibiotics and cultured overnight at 37°C in a shaking incubator at 200 rpm. Then, 500 μL of the seed culture was transferred to fresh 100 mL LB medium containing each antibiotic in a 500 mL conical flask.

When the optical density at 600 nm (OD) reached 0.6, the culture was induced with 0.4 mM isopropyl β-D-1-Ithiogalactopyranoside (IPTG) and incubated at 20 °C in a shaking incubator at 200 rpm. was cultured for about 20 hours. The cell pellet was then harvested by centrifugation at 842xg (3,000 rpm) for 10 min, washed twice (vortexed and then centrifuged) with buffer (50mM Tris-HCl and 10% glycerol (pH 7.5)), and resuspended. did.

Cell pellets were lysed with 1 mL of the same buffer using a Fisher Scientific Sonic Dismembrator Model 500 (5-9 sec pulses on and off, 360 sec total, 20% amplitude) in an ice bath and lysed in a clear solution by high-speed centrifugation at 12,000 rpm. Seafood was collected. (13,475 xg) for 30 min at 4°C. Crude protein obtained was further analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Proteins were stored in buffer containing 50mM Tris-HCl, pH 7.5, and 10% glycerol at -20°C. Crude protein concentration was determined using the Bradford method using bovine serum albumin as a standard (Bradford et al. 1976).

(4). Biosynthesis of isoorientin by a cell factory system

First, the activities of UGT708A6 and GtF6CGT1 enzymes were confirmed by conducting in vivo reactions in separate 50mL flasks. Seed cultures of E. coli carrying UGT708A6 and GtF6CGT1 were prepared in LB broth supplied with each antibiotic and incubated overnight at 37°C in a shaking incubator at 200 rpm.

After inoculation of 500 μL of the seed, the culture was poured into two new 50 ml LB medium supplemented with each antibiotic in a 250 ml conical flask and incubated at 37°C in a shaking incubator at 200 rpm.

Soon the optical density at 600 nm (OD ) reached 0.6, and the culture was induced with 0.4 mM IPTG for approximately 20 hours at 20°C in a shaking incubator at 200 rpm. Then, luteolin (100 μM) was added to both induced cultures.

After 20 hours, 1 ml of the culture sample was taken and mixed with 1 ml methanol while vortexing for 10 minutes and centrifuged at 14,000 rpm to obtain a clear sample. Clear samples were analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC)-photodiode array (PDA).

(5). Biosynthesis of rhamnosylisoorientin by a cell factory system.

As a result of analyzing the activity results of GtF6CGT1 and UGT708A6 by HPLC-PDA, the function of GtF6CGT1 was found to be more effective. Therefore, recombinant strains carrying piBR181-GtF6CGT1 and pET32a-UGT91L1 plasmids in E. coli C-41(DE3) host were cultured in 50 mL LB broth.

As mentioned above, 50 mL cultures were prepared, induced with 0.4 mM IPTG at 20°C in a shaking incubator at 200 rpm, and then 100 μM luteolin was supplied 20 hours later.

Similarly, after 20 h, 1 ml culture sample was taken and mixed with 1 ml methanol while vortexing for 10 min and centrifuged at 14,000 rpm for 30 min to obtain a clear sample. Clear samples were analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC)-photodiode array (PDA).

(6). Large-scale production of rhamnosylisoorientin in microbial cells.

Large-scale production of rhamnosyl-isoorientin was performed in a fermentor using E. coli C-41(DE3) strains containing piBR181-GtF6CGT1 and pET32a-UGT91L1 plasmids.

Seed cultures were prepared in 15ml test tubes with the necessary antibiotics added, and cultured in a shaking incubator at 37°C for about 5 hours. Then, 500 μL of seed was transferred to three 500 mL individual flasks supplied with the required amount of antibiotics in 100 mL LB broth medium, and cultured overnight at 37°C in a shaking incubator at 200 rpm.

Afterwards, 300 mL of the seed was transferred to an autoclave fermenter with 3L-LB broth medium containing the required amount of antibiotics at 37°C while maintaining air and pH 7.5. When the optical density (OD) reached 0.6, 0.4mM IPTG was added and the culture was incubated at 20°C for approximately 20 hours.

Afterwards, 0.5mM luteolin was supplied to the culture while 5% glucose was supplied. Additionally, the fermentor reaction was performed under the same conditions by supplying 5% glycerin instead of glucose. Afterwards, samples were collected at 24, 48, and 72 hours, and the products were analyzed using reversed-phase high-performance liquid chromatography (RP-HPLC)-photodiode array (PDA).

Then, the culture was harvested with twice the volume of ethyl acetate, and the extracted crude product was dried using a rotary evaporator.

(7). Biosynthesis of maysin by in vitro reaction

A bifunctional UDP-rhamnose synthase/UDP-4-keto-6-deoxyglucose synthase (RHS1) was expressed in Escherichia coli C-41(DE3). This freshly prepared strain was cultured in 50 ml LB broth for protein expression using the method described above.

Coenzyme RHS1 was used in the reaction. The reaction was performed in a total volume of 15 μL using 100 mM Tris-HCl (pH 7.5) containing 10 mM MgCl2·6H ₂ O, 3 mM NAD ⁺ , 3 mM NADH, 49 μL distilled water, and 5 mM rhamnosylisoorientin.

Similarly, another set of reactions was performed under different conditions in the same set, changing the phosphate buffer instead of Tris-HCl and the cofactors NADP ⁺ and NADPH instead of NAD ⁺ and NADH. The reaction mixture was incubated at 37°C for 20 hours.

(8). Analytical procedure od Meishin

Reverse-phase HPLC-PDA analysis was performed at 290 nm UV absorbance using a C18 column packed with YMC-PACK ODS-AQ. The 4.6 mm internal diameter, 150 mm length and 5 μm particle size was coupled to a photodiode array (PDA) in binary conditions, maintained in solvent A (0.05% trifluoroacetic acid buffer-TFA) at a flow rate of 1 mL/min for a 25 min program. ) It consists of water and 100% acetonitrile (CAN).

Acetonitrile was set at 0-20% (0-8 min), 40-75% (8-15 min), 75-100% (15-20 min) and 100-20% (25 min). Purification of the compounds was performed by preparative HPLC with a C ₁₈ column (YMC-PACK ODS-AQ; 150 mm length, 20 mm internal diameter, 10 μm particle size) coupled to a UV detector using a 36 min binary program. Flow was 10 mL/min throughout the program with ACN flow for 0-20 min (40%), 20-30 min (75%), and 30-36 min (0%).

High-resolution quadruple-of-flight electrospray ionization mass spectrometry (HRQTOF-ESI/MS) was performed in positive ion mode using an Acquity mass spectrometer (with UPLC; Waters, Milford, MA USA) coupled to a Synapt G2-S system (Waters). .

Purified compounds were structurally determined by an Avance II 900 Bruker (Germany) BioSpin NMR spectrometer equipped with a TCI CryoProbe (5 mm). All samples were exchanged with DO and dissolved in pure DO for 1H NMR (proton), 13C NMR (carbon), heteronuclear multiple bond correlation (HMBC), and heteronuclear signal-proton correlation (HSQC).

2. Test results

(One). Protein expression and SDS-PAGE analysis

To produce soluble recombinant proteins for in vitro reactions, four plasmids pET28a-RHS1, pET32a-UGT91L1, piBR181-GtF6CGT1, and pACYC-UGT708A6 (Figure 2) were transformed into E. coli C-41(DE3).

SDS-PAGE analysis of the soluble fractions of each protein RHS1, UGT91L1, GtF6CGT1, and UGT708A6 showed bands at approximately (75, 47, 55, and 55) kDa, respectively (Figure 3). Band size corresponds to the calculated molecular weight of each protein with hexahistidine-tagged fusion protein.

Soluble lysates of these proteins were applied to the Ni ⁺⁺ -NTA bead purification system. The purified protein was concentrated, quantified, and used for in vitro reactions.

(2). Microbial production of isoorientin and rhamnosylisoorientin

As a result of analyzing the RP-HPLC-PDA of a sample prepared by supplying luteolin to the culture medium containing UGT708A6 and GtF6CGT1 strains after 20 hours, the expected isoorientin peak did not appear in UGT708A6, but a distinct isoorientin peak was observed in GtF6CGT1. appeared (Figure 4).

Additionally, the same sample of GtF6CGT1 was subjected to high-resolution QTOF-ESI/MS mass in positive mode, and [M+H] ⁺ m/z ⁺ was found to be 449.1089 Da, which is similar to the mass of one glucose bound to luteolin. do. The chemical formula C ₂₁ H ₂₁ O ₁₁ ⁺ with the correct mass was 449.1078 (Figure 5).

Next, another sample was analyzed by RP-HPLC-PDA obtained by supplying luteolin to E. coli C-41(DE3) strain carrying two plasmids piBR181-GtF6CGT1 and pET32a-UGT91L1.

The HPLC-PDA chromatogram showed two peaks at retention times of 8.0 and 8.5 min at a wavelength close to that of luteolin. This may provide clues to the possible products of rhamnosylisoorientin and isoorientin, which were further identified by QTOF-ESI/MS in positive mode. According to QTOF-ESI/MS analysis, peak P1 with a retention time of 8.0 min has a [M+H] of 595.1655 Da, similar to one glucose and one rhamnose bound to luteolin with the chemical formula C ₂₇ H ₃₁ O ₁₅ ^{+ +} m/z ⁺ was shown.

The exact mass of 595.1657 and the peak at 8.5 minutes showed [M+H] ⁺ m/z ⁺ of 449.1089 Da, which is glucose attached to luteolin with the chemical formula C ₂₁ H ₂₁ O ₁₁ ⁺ and the exact mass of 449.1078. Similar to (Figures 6 and 7).

(3). Mass production of rhamnosylisoorientin

As mentioned in Methods and Materials above, a strain carrying two plasmids piBR181-GtF6CGT1 and pET32a-UGT91L1 in E. coli C-41(DE3) was cultured and induced with IPTG in a fermentor. Samples for analysis were taken from the fermenter at 24, 48 and 72 hours.

Additional HPLC-PDA and high-resolution QTOF-ESI/MS analyzes were performed. Based on analysis of the HPLC-PDA peak areas of the products, at 24 hours approximately 45% of the substrate was converted to products P1 and P2 at retention times of 8 and 8.5 min, respectively.

When the incubation time was further extended to 48 hours, the substrate gradually decreased, while the products P1 and P2 increased. In the same way, when the incubation period was further extended to 72 hours, 80% of the substrate was converted to products P1 and P2 (Figure 8).

Similarly, large-scale production was performed using the same strain in a fermenter using glucose and glycerin as carbon sources. As a result of analyzing the results of samples collected at 24 hours, 48 hours, and 72 hours, in the case of glycerin, the formation patterns of products P1 and P2 were almost identical, while in the case of glucose, P1 was more abundant than P2. However, the rate of conversion of substrate to product gradually decreases over time (Figures 8A to 8C).

(4). In vivo mesin synthesis and analysis

The crude enzyme of RHS1 was used in the reaction. The reaction was performed in a total volume of 15 μL using 100 mM Tris-HCl (pH 7.5) containing 10 mM MgCl ₂ ·6H ₂ O, 3 mM NAD ⁺ , 3 mM NADH, 49 μL distilled water, and 5 mM rhamnosylisoorientin. Similarly, another set of reactions was performed under different conditions in the same set, changing the phosphate buffer instead of Tris-HCl and the cofactors NADP ⁺ and NADPH instead of NAD ⁺ and NADH.

The reaction mixture was incubated at 37°C for 20 hours. In analysis by HPLC-PDA and QTOF-ESI/MS, trace amounts of maycin were detected as a result of performing the ramanosylisoorientin reaction as mentioned in Methods and Materials, with a mass of [M+H] of 577.1577 Da. ] ⁺ m/z ⁺ , which is similar to the mass of mesine 577.1552 Da and the chemical formula C ₂₇ H ₂₉ O ₁₄ ⁺ .

Mass spectrometry results showed that although Tris-HCl and phosphate buffer were used, maysin was formed in small amounts (FIG. 9).

As the specific parts of the present invention have been described in detail above, those skilled in the art will understand that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. It will be obvious.

Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents. Simple modifications or changes of the present invention can be easily used by those skilled in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.

Claims (15)

(a) Transformed with a plasmid into which the 6-C-glycosyltransferase gene (GtF6CGT1) of Gentiana triflora is inserted and a plasmid into which the glucose 2″-O-rhamnosyltransferase gene (UGT91L1) of Gentiana triflora is inserted. Culturing recombinant E. coli in a medium;
(b) adding 0.5 mM of luteolin to the culture medium in which the transformed recombinant E. coli is being cultured, along with at least one carbon source selected from the group consisting of glucose and glycerin, after 15 to 25 hours of cultivation; and
(c) A method for mass production of mesin using E. coli, comprising culturing the transformed recombinant E. coli in a culture medium to which the luteolin and carbon source have been added.
The method for mass production of mesin using E. coli according to claim 1, wherein the plasmid into which the 6-C-glycosyltransferase gene (GtF6CGT1) is inserted is pIBR181-GtF6CGT1 as shown in Figure 2a.
The method for mass production of mesin using E. coli according to claim 1, wherein the plasmid into which the glucose 2″-O-rhamnosyltransferase gene (UGT91L1) is inserted is pET32a-UGT91L1 as shown in Figure 2b.
The method of claim 1, wherein the medium is Luria-Bertani (LB) agar broth medium supplemented with antibiotics.
The method of claim 4, wherein the antibiotics are ampicillin, kanamycin, and chloramphenicol.
A method for mass production of maycin using E. coli, characterized in that:
The method of claim 4, wherein the antibiotics are supplemented with 90-110 μg/mL of ampicillin, 40-60 μg/mL of kanamycin, and 90-110 μg/mL of chloramphenicol.
delete delete The method of claim 1, wherein the glucose is 4 to 6% (w/v) and the glycerin is 4 to 6% (w/v).
The method for mass production of mesin using E. coli according to claim 1, wherein the culturing in step (c) is performed at 35 to 40°C for 24 to 72 hours.
The method for mass production of maycin using E. coli according to claim 1, wherein the recombinant E. coli is recombinant E. coli C-41(DE3).
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