Genetically engineered bacterium for producing cyanidin 3-O-galactoside and application of genetically engineered bacterium
Through genetic engineering technology, E. coli was transformed, relevant coding genes were introduced, and efficient biosynthesis of cornflower 3-O-galactoside was achieved, which solved the high cost and instability of plant extraction methods, and improved production efficiency and product quality.
Patent Information
- Application Number
- CN202411515436.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-28
- Publication Date
- 2025-03-04
AI Technical Summary
The existing cornflower 3-O-galactoside production method - plant extraction method faces problems such as high cost, unstable supply of raw materials, complex extraction processes and product quality susceptible to impact.
Escherichia coli was transformed through genetic engineering technology, and encoding genes such as anthocyanin synthase and UDP galactosyltransferase were introduced to construct genetically engineered bacteria to achieve the biosynthesis of cornflower 3-O-galactosinoside.
It has achieved efficient biosynthesis of cornflower 3-O-galactoside, shortened production cycles, improved production efficiency, reduced dependence on land resources, and can meet the increasing demand for this compound in the food and medicine fields.
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Figure CN119552789A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of bioengineering, and in particular to a genetically engineered bacterium for producing cyanidin 3-O-galactoside and an application thereof. Background Art
[0002] Cyanidin 3-O-galactoside (C3Ga), as a natural pigment, is not only favored by the food industry for its bright color, but also has become a hot spot in scientific research for its rich physiological activity. C3Ga has a unique chemical structure and its molecular formula is C 21 H 21 O 11 + , molecular weight is 449.38, and its specific chemical name is 2-(3,4-dihydroxyphenyl)-5,7-dihydroxychrome-3-yl β-D-galactoside. This structural feature gives it excellent water solubility and diverse biological activities.
[0003] The antioxidant properties of C3Ga are particularly outstanding. It can effectively remove free radicals in the body, increase the activity of antioxidant enzymes, inhibit lipid peroxidation, and thus protect cells from oxidative stress damage. This feature gives it broad application prospects in the fields of health products, cosmetics, and functional foods. In addition, C3Ga has also been found to have significant anti-inflammatory effects, which can reduce inflammatory responses and promote tissue repair; its anti-cancer potential should not be underestimated. Through mechanisms such as regulating the cell cycle, inhibiting tumor cell proliferation, and inducing apoptosis, it shows its potential as an anti-cancer adjuvant drug. For diabetic patients, C3Ga can provide new ideas for the treatment of diabetes by regulating blood sugar levels and improving insulin resistance. More importantly, C3Ga can also protect the nervous system and fight against neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, showing neuroprotective functions.
[0004] In view of the various health benefits of C3Ga, it has been approved as a food ingredient by food safety regulatory agencies in many countries and regions. C3Ga is widely used in the processing of dairy products, beverages, alcoholic beverages, candies, cakes and other foods, which not only adds to the color appeal of the products, but more importantly, enhances the nutritional value and health attributes of the products, meeting the growing demand of consumers for healthy food.
[0005] Although C3Ga has broad application prospects, its existing production method - plant extraction - faces many challenges. First, the cost of plant raw materials is high and is greatly affected by regional, seasonal and climate changes, resulting in unstable raw material supply and large price fluctuations. Secondly, the extraction process is complicated and requires multiple chemical reactions and separation and purification steps. It not only has high energy consumption, but also has low extraction efficiency. Product quality is also easily affected by operating conditions. In addition, the chemical stability of C3Ga is relatively poor, and it is easily degraded by factors such as light, heat, and pH value, which further increases the difficulty and cost of quality control in the production process.
[0006] In order to overcome the limitations of plant extraction methods, researchers began to explore the possibility of producing C3Ga by biosynthesis. Biosynthesis is regarded as an important direction for the future production of natural products due to its advantages of high efficiency, environmental protection and sustainability. At present, there have been reports on the successful synthesis of cyanidin 3-O-glucoside (another anthocyanin) by microorganisms, and related patents have been applied for. However, for C3Ga, although its structure is similar to that of cyanidin 3-O-glucoside, due to the differences in glycosylation position and sugar chain type, its biosynthetic pathway and regulatory mechanism are more complicated. So far, there has been no successful case report of direct synthesis of C3Ga by microorganisms. Summary of the invention
[0007] The purpose of the present invention is to overcome the existing technical difficulties, realize the production of cyanidin 3-O-galactoside by microbial method, and provide a genetically engineered bacterium for producing cyanidin 3-O-galactoside and its application.
[0008] The present invention provides a genetically engineered bacterium for producing cyanidin 3-O-galactoside, wherein Escherichia coli is used as a starting strain, and the genetically engineered bacterium is obtained by introducing genes, wherein the introduced genes include any one of the following groups:
[0009] (1) Anthocyanin synthase encoding gene, UDP galactosyltransferase encoding gene, UDP-galactose-4-epimerase encoding gene, UTP glucose-1 phosphate uridyltransferase encoding gene, phosphoglucomutase encoding gene, cytidylate kinase encoding gene, nucleoside diphosphate kinase encoding gene and membrane transporter encoding gene yadH;
[0010] (2) Anthocyanin synthase encoding gene, UDP galactosyltransferase encoding gene, UDP-galactose-4-isomerase encoding gene.
[0011] The anthocyanin synthase is derived from one of petunia (Petunia hybrida (Hook.) E.Vilm.), pear (Pyrus communis), blueberry (Vaccinium corymbosum), apple (Malus domestica) and grape (Vitis vinifera), the UDP galactosyltransferase is derived from purple carrot, and the UDP-galactose-4-epimerase, UTP glucose-1 phosphate uridyltransferase, phosphoglucomutase, cytidine kinase, nucleoside diphosphate kinase, and membrane transporter yadH are derived from Escherichia coli DH5α.
[0012] Preferably, the anthocyanidin synthase encoding gene and the UDP galactosyltransferase encoding gene are optimized according to the codon preference of Escherichia coli. For the encoding genes of anthocyanidin synthase and UDP galactose glycosyltransferase, those skilled in the art can obtain them according to the amino acid sequences of anthocyanidin synthase and UDP galactose glycosyltransferase. Based on the codon rules, the nucleotide sequences of the genes encoding the above proteins are not unique, but all the nucleotide sequences encoding the above anthocyanidin synthase and UDP galactose glycosyltransferase are within the scope of protection of the present invention.
[0013] Preferably, the GenBank number of the anthocyanin synthase encoding gene is any one of X70786.1, NC_084814.1, MH321466.1, NC_041794.1 and NC_081806.1.
[0014] The GenBank number of the UDP galactosyltransferase encoding gene is AKI23632.1,
[0015] The GenBank number of the UDP-galactose-4-epimerase encoding gene is NP_415280.3,
[0016] The GenBank number of the UTP glucose-1 phosphate uridyl transferase encoding gene is NP_415752.1,
[0017] The GenBank number of the phosphoglucomutase encoding gene is NP_415214.1,
[0018] The GenBank number of the cytidylate kinase encoding gene is NP_415430.1,
[0019] The GenBank number of the nucleoside diphosphate kinase encoding gene is NP_417013.1,
[0020] The GenBank number of the membrane transporter encoding gene yadH is NP_414670.1.
[0021] Preferably, the starting strain is Escherichia coli BL21 (DE3), Escherichia coli OverExpress TM C41(DE3), E. coli OverExpress TM C43(DE3), E. coli Tuner(DE3), E. coli Arctic Express TM coli T7 Express.
[0022] The present invention also provides a method for constructing the genetically engineered bacteria for producing cyanidin 3-O-galactoside, wherein the introduced genes include anthocyanidin synthase encoding gene, UDP galactosyltransferase encoding gene, UDP-galactose-4-epimerase encoding gene, UTP glucose-1 phosphate uridyltransferase encoding gene, phosphoglucomutase encoding gene, cytidylate kinase encoding gene, nucleoside diphosphate kinase encoding gene and membrane transporter encoding gene yadH, and the construction method comprises the following steps:
[0023] (1) cloning the sequences of anthocyanin synthase encoding gene, UDP galactosyltransferase encoding gene and UDP-galactose-4-epimerase encoding gene into an expression vector to obtain a recombinant expression vector 1;
[0024] (2) cloning the sequences of the UTP glucose-1-phosphate uridyl transferase encoding gene, the phosphoglucomutase encoding gene, the cytidylate kinase encoding gene, the nucleoside diphosphate kinase encoding gene and the membrane transporter encoding gene yadH into an expression vector to obtain a recombinant expression vector 2;
[0025] (3) The recombinant expression vector 1 in step (1) and the recombinant expression vector 2 in step (2) are co-transformed into Escherichia coli to obtain the genetically engineered bacteria that produce cyanidin 3-O-galactoside.
[0026] Specifically, pCDFDuet-1 is used as the expression plasmid for anthocyanin synthase, UDP galactosyltransferase, and UDP-galactose-4-epimerase encoding genes, and pACYCDuet-1 is used as the expression plasmid for UTP glucose-1 phosphate uridyltransferase, phosphoglucomutase, cytidine kinase, nucleoside diphosphate kinase, and membrane transporter yadH encoding genes.
[0027] The invention also provides the use of the genetically engineered bacteria in producing cyanidin 3-O-galactoside.
[0028] The present invention also provides a method for producing cyanidin 3-O-galactoside, which uses (+)-catechin as a substrate, ferments and cultures the genetically engineered bacteria, and extracts and obtains cyanidin 3-O-galactoside.
[0029] During fermentation, isopropyl-β-D-thiogalactoside (IPTG) was used to induce expression, and the induction condition was OD 600 Induction was performed when the pH was 0.6-0.8.
[0030] After induction at 18-30°C for 3-24 hours, the bacteria were collected, resuspended in the catalytic reaction solution, and catalyzed at 20-30°C for 24 hours.
[0031] During the catalytic reaction, the catalytic reaction liquid used includes the following components:
[0032] Ammonium chloride 1g / L, ammonium sulfate 2.7g / L, disodium hydrogen phosphate 6.8g / L, potassium dihydrogen phosphate 3g / L, sodium chloride 0.6g / L, magnesium sulfate heptahydrate 0.2g / L, vitamin B1 10mg / L, zinc sulfate heptahydrate 5mg / L, manganese sulfate 1mg / L, cobalt chloride hexahydrate 0.5mg / L, copper sulfate pentahydrate 0.5mg / L, sodium molybdate 0.5mg / L, boric acid 0.2mg / L, L-sodium ascorbate 0.5g / L, ferrous sulfate heptahydrate 2.8mg / L, α-ketoglutaric acid 14.6mg / L, orotic acid 15.6g / L and glutamic acid 2mg / L.
[0033] Specifically, the method comprises: culturing the recombinant Escherichia coli described above, inducing the expression of anthocyanidin synthase, UDP galactosyltransferase, UDP-galactose-4-epimerase, UTP glucose-1 phosphate uridyltransferase, phosphoglucomutase, cytidylate kinase, nucleoside diphosphate kinase, and membrane transporter yadH with an inducer, collecting the bacterial cells and using the collected bacterial cells to produce cyanidin 3-O-galactoside by whole-cell catalysis using (+)-catechin as a substrate.
[0034] Preferably, the induction is to induce the expression of anthocyanin synthase, UDP galactosyltransferase, UDP-galactose-4-epimerase, UTP glucose-1 phosphate uridyltransferase, phosphoglucomutase, cytidine kinase, nucleoside diphosphate kinase, and membrane transporter yadH with IPTG when OD600 is 0.6-0.8.
[0035] Further preferably, the induction is carried out at 18-30°C; the inducer is 0.1-1 mM / L IPTG.
[0036] Preferably, the cells are collected 24 hours after induction, and the collected cells are resuspended in a catalytic reaction solution, and whole-cell catalysis is performed at 20-30°C for 24 hours.
[0037] The beneficial effects of the present invention are as follows: the present invention provides a new use of Escherichia coli in the production of cyanidin 3-O-galactoside. The modified recombinant Escherichia coli can be used for whole-cell catalysis and efficient biosynthesis of cyanidin 3-O-galactoside. Compared with plant extraction, the present invention can significantly shorten the production cycle (from one year to one week), improve production efficiency, reduce the use of land resources, and meet the increasing demand for cyanidin 3-O-galactoside in the fields of food and medicine. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Figure 1 It is a map of the recombinant expression vector pET28a-T7-PhANS-Dc3GT-EcgalE.
[0039] Figure 2 This is a map of the recombinant expression vector pET28a-T7-PcANS-Dc3GT-EcgalE.
[0040] Figure 3 This is a map of the recombinant expression vector pET28a-T7-MdANS-Dc3GT-EcgalE.
[0041] Figure 4 This is a map of the recombinant expression vector pET28a-T7-VcANS-Dc3GT-EcgalE.
[0042] Figure 5 This is a map of the recombinant expression vector pET28a-T7-VvANS-Dc3GT-EcgalE.
[0043] Figure 6 This is a map of the recombinant expression vector pCDFDuet-T7-VvANS-Dc3GT-EcgalE.
[0044] Figure 7 This is a map of the recombinant expression vector pACYCDuet-T7-EcgalU-Ecpgm-Eccmk-Ecndk-EcyadH.
[0045] Figure 8 Qualitative analysis of recombinant Escherichia coli fermentation products by liquid chromatography and liquid chromatography-mass spectrometry.
[0046] Fig. 9 Analysis of cyanidin 3-O-galactoside production by different recombinant Escherichia coli.
[0047] Fig.10 Comparison of cyanidin 3-O-galactoside production in recombinant Escherichia coli at different induction temperatures and induction times.
[0048] Fig.11 Comparison of cyanidin 3-O-galactoside production by recombinant Escherichia coli at different catalytic temperatures. DETAILED DESCRIPTION
[0049] The primers used in the examples of the present invention are shown in Table 1.
[0050]
[0051]
[0052] Example 1 Construction of recombinant Escherichia coli
[0053] 1. Obtain the target gene nucleic acid sequence from the NCBI database. The coding sequence of anthocyanin synthase is one of X70786.1, NC_084814.1, MH321466.1, NC_041794.1 and NC_081806.1, and the coding sequence of UDP galactosyltransferase is AKI23632.1. The GenBank number of the UDP-galactose-4-epimerase encoding gene is NP_415280.3, the GenBank number of the UTP glucose-1 phosphate uridyl transferase encoding gene is NP_415752.1, the GenBank number of the phosphoglucomutase encoding gene is NP_415214.1, the GenBank number of the cytidylate kinase encoding gene is NP_415430.1, the GenBank number of the nucleoside diphosphate kinase encoding gene is NP_417013.1, and the GenBank number of the membrane transporter encoding gene yadH is NP_414670.1. According to the codon preference of Escherichia coli, anthocyanidin synthase and UDP galactosyl transferase were codon optimized to synthesize related gene fragments. The remaining genes were directly amplified from the Escherichia coli genome.
[0054] 2. Anthocyanin synthases from different sources were amplified with UDP galactosyltransferase and UDP-galactose-4-epimerase genes, and inserted into the vector pET28a using the Gibson assembly method. The specific construction is as follows: PhANS, PcANS, MdANS, VcANS, and VvANS were amplified using primers P3 / P4, P10 / P11, P13 / P14, P16 / P17, and P19 / P20, respectively. Dc3GT was amplified using primers P5 / P6, and EcgalE was amplified using primers P7 / P8. The pET28a backbone was amplified using primers P1 / P2 and recombined with PhANS, Dc3GT, and EcgalE. The pET28a backbone was amplified using primers P9 / P2 and recombined with PcANS, Dc3GT, and EcgalE. Primers P12 / P2 were used to amplify the pET28a backbone and recombined with MdANS, Dc3GT, and EcgalE. Primers P15 / P2 were used to amplify the pET28a backbone and recombined with VcANS, Dc3GT, and EcgalE. Primers P18 / P2 were used to amplify the pET28a backbone and recombined with VvANS, Dc3GT, and EcgalE. Figures 1 to 5 The recombinant expression vectors pET28a-T7-PhANS-Dc3GT-EcgalE, pET28a-T7-PcANS-Dc3GT-EcgalE, pET28a-T7-MdANS-Dc3GT-EcgalE, pET28a-T7-VcANS-Dc3GT-EcgalE, and pET28a-T7-VvANS-Dc3GT-EcgalE shown were respectively transformed into Escherichia coli BL21 (DE3) by chemical transformation of Escherichia coli treated with CaCl2 to obtain recombinant strains BL21 (DE3)-C3Ga-Ph, BL21 (DE3)-C3Ga-Pc, BL21 (DE3)-C3Ga-Md, BL21 (DE3)-C3Ga-Vc, and BL21 (DE3)-C3Ga-Vv.
[0055] 3. The codon-optimized sequence of anthocyanin synthase (NC_081806.1) from grape (Vitis vinifera) and the UDP galactosyltransferase and UDP-galactose-4-epimerase genes were amplified and inserted into the vector pCDFDuet-1 using the Gibson assembly method. The specific construction is as follows: The pCDFDuet backbone was amplified using primers P 18 / P2 and recombined with VvANS, Dc3GT, and EcgalE. Figure 6 The recombinant expression vector shown is pCDFDuet-T7-VvANS-Dc3GT-EcgalE.
[0056] 4. Amplify the genes of UTP glucose-1 phosphate uridyltransferase, phosphoglucomutase, cytidylate kinase, nucleoside diphosphate kinase and membrane transporter yadH, and insert them into the vector pACYCDuet-1 using the Gibson assembly method. The specific construction is as follows: EcgalU and Ecpgm were amplified using primers P22 / P23 and P24 / P25, respectively, and the pACYCDuet backbone was amplified using primers P1 / P21 and recombined with EcgalU and Ecpgm to obtain pACYCDuet-T7-ECgalU-Ecpgm. Eccmk, Ecndk, and EcyadH were amplified using primers P28 / P29, P30 / P31, and P32 / P33, respectively. Using primers P26 / P27, pACYCDuet-T7-ECgalU-Ecpgm was used as a template for amplification to obtain the linearized vector pACYCDuet-T7-ECgalU-Ecpgm backbone, which was then recombined with Eccmk, Ecndk, and EcyadH. Figure 7 The recombinant expression vector shown is pACYCDuet-T7-EcgalU-Ecpgm-Eccmk-Ecndk-EcyadH.
[0057] 5. The recombinant expression vector pACYCDuet-T7-EcgalU-Ecpgm-Eccmk-Ecndk-EcyadH and the recombinant expression vector pCDFDuet-T7-VvANS-Dc3GT-EcgalE were co-transformed into Escherichia coli BL21 (DE3) by the chemical transformation method of Escherichia coli treated with CaCl2 to obtain recombinant Escherichia coli BL21 (DE3)-C3Ga-Vv-2.
[0058] Example 2 Analysis of the ability of recombinant Escherichia coli to synthesize cyanidin 3-O-galactoside
[0059] 1. This example uses the recombinant E. coli BL21(DE3)-C3Ga-Ph, BL21(DE3)-C3Ga-Pc, BL21(DE3)-C3Ga-Md, BL21(DE3)-C3Ga-Vc, and BL21(DE3)-C3Ga-Vv prepared in Example 1 for analysis. The recombinant E. coli is streaked on LB solid medium and cultured overnight at 37°C. A single clone is picked and inoculated into 2 ml LB liquid medium and cultured overnight at 37°C and 220 rpm. The initial OD 600 Inoculate 20 ml LB medium at 0.1 and culture at 37°C and 220 rpm until OD 600When the pH value is 0.6-0.8, add IPTG at a final concentration of 1 mM for induction, and culture at 30°C 200 rpm for 3 hours. Then collect the bacterial solution, centrifuge at 4000 rpm for 5 minutes, discard the supernatant, add 4 ml of catalytic reaction solution to resuspend the bacteria, take 320 μl and place it in a 14 ml shaking tube, add (+)-catechin at a final concentration of 0.5 mM as a substrate for catalysis, and incubate at 30°C 200 rpm for 24 hours.
[0060] The catalytic reaction liquid has the following components: ammonium chloride 1g / L, ammonium sulfate 2.7g / L, disodium hydrogen phosphate 6.8g / L, potassium dihydrogen phosphate 3g / L, sodium chloride 0.6g / L, magnesium sulfate heptahydrate 0.2g / L, vitamin B1 10mg / L, zinc sulfate heptahydrate 5mg / L, manganese sulfate 1mg / L, cobalt chloride hexahydrate 0.5mg / L, copper sulfate pentahydrate 0.5mg / L, sodium molybdate 0.5mg / L, boric acid 0.2mg / L, L-sodium ascorbate 0.5g / L, ferrous sulfate heptahydrate 2.8mg / L, α-ketoglutaric acid 14.6mg / L, orotic acid 15.6g / L and glutamic acid 2mg / L.
[0061] 2. After incubation, the reaction solution was centrifuged at 12000rpm for 5 minutes, and the supernatant was used for extracellular yield analysis. The precipitate was added with 1% hydrochloric acid solution for ultrasonic extraction and centrifuged again. The supernatant was used for intracellular yield analysis. The sum of extracellular and intracellular yields was the total yield. The qualitative results of liquid chromatography and liquid chromatography-mass spectrometry were as follows Figure 8 As shown, recombinant Escherichia coli can synthesize cyanidin 3-O-galactoside using (+)-catechin as substrate. Fig. 9 The results showed that anthocyanin synthases from different sources had different catalytic activities towards the substrate catechins. The C3Ga yield of the strain BL21(DE3)-C3Ga-Md corresponding to the anthocyanin synthase from apple was the lowest, only 2.64 mg / L, while the C3Ga yield of the strain BL21(DE3)-C3Ga-Vv corresponding to the anthocyanin synthase from grape was the highest, reaching 19.92 mg / L. The yields of the two differed by 6.5 times.
[0062] Example 3 Analysis of cyanidin 3-O-galactoside production by recombinant Escherichia coli at different induction temperatures and induction times
[0063] 1. This example uses the recombinant E. coli BL21(DE3)-C3Ga-Vv-2 prepared in Example 1 for analysis. The recombinant E. coli is streaked on LB solid medium and cultured overnight at 37°C. A single clone is picked and inoculated into 2 ml LB liquid medium and cultured overnight at 37°C and 220 rpm. The initial OD 600 Inoculate 20 ml LB medium at 0.1 and culture at 37°C and 220 rpm until OD 600When the pH value is 0.6-0.8, add IPTG at a final concentration of 1mM for induction. Cultivate at 18℃ and 30℃ at 200rpm for 24h. Then collect the bacterial liquid at 3h, 9h and 24h, centrifuge at 4000rpm for 5min, discard the supernatant, add 4ml of catalytic reaction liquid to resuspend the bacteria, take 320μl and place it in a 14ml shaking bacterial tube, add (+)-catechin at a final concentration of 2mM as a substrate for catalysis, and incubate at 30℃ at 200rpm for 24h.
[0064] 2. After incubation, the reaction solution was centrifuged at 12000rpm for 5 minutes, and the supernatant was used for extracellular yield analysis. The precipitate was added with 1% hydrochloric acid solution and ultrasonically extracted, and then centrifuged again, and the supernatant was used for intracellular yield analysis. The sum of extracellular and intracellular yields was the total yield.
[0065] like Fig.10 As shown in the figure, the yield of cyanidin 3-O-galactoside was significantly different under different induction temperatures and times. Under 30°C, as the induction time increased, the C3Ga yield gradually decreased, and the highest was 42.02 mg / L at 3 hours of induction. Under 18°C, as the induction time increased, the C3Ga yield gradually increased, and the highest was 134.46 mg / L at 24 hours of induction. By optimizing the induction temperature and time, the yield of cyanidin 3-O-galactoside was increased to 3.2 times before optimization.
[0066] Example 4 Analysis of cyanidin 3-O-galactoside production by recombinant Escherichia coli at different catalytic temperatures
[0067] 1. This example uses the recombinant E. coli BL21(DE3)-C3Ga-Vv-2 prepared in Example 1 for analysis. The recombinant E. coli is streaked on LB solid medium and cultured overnight at 37°C. A single clone is picked and inoculated into 2 ml LB liquid medium and cultured overnight at 37°C and 220 rpm. The initial OD 600 Inoculate 20 ml LB medium at 0.1 and culture at 37°C and 220 rpm until OD 600 When the pH value is 0.6-0.8, add IPTG at a final concentration of 1mM for induction. Cultivate at 18℃ 200rpm for 24h. Then collect the bacterial solution, centrifuge at 4000rpm for 5min, discard the supernatant, add 4ml of catalytic reaction solution to resuspend the bacteria, take 320μl and place it in a 14ml shaking tube, add (+)-catechin at a final concentration of 2mM as a substrate for catalysis, and incubate at 20℃, 25℃ and 30℃ at 200rpm for 24h.
[0068] 2. After incubation, the reaction solution was centrifuged at 12000rpm for 5 minutes, and the supernatant was used for extracellular yield analysis. The precipitate was added with 1% hydrochloric acid solution and ultrasonically extracted, and then centrifuged again, and the supernatant was used for intracellular yield analysis. The sum of extracellular and intracellular yields was the total yield.
[0069] like Fig.11 As shown in the figure, the catalytic temperature has a significant effect on the yield of cyanidin 3-O-galactoside of recombinant Escherichia coli. The C3Ga yield shows a trend of increasing first and then decreasing as the catalytic temperature decreases. The yield is the highest at a catalytic temperature of 25°C, reaching 173.43 mg / L, which is 28.9% higher than the yield before temperature optimization. The above results show that the C3Ga yield can be further increased by optimizing the catalytic temperature.
Claims
1. A genetically engineered bacterium for producing cyanidin 3-O-galactoside, characterized in that: The genetically engineered bacteria are obtained by introducing genes into Escherichia coli as a starting strain, and the introduced genes include any one of the following groups: (1) Anthocyanin synthase encoding gene, UDP galactosyltransferase encoding gene, UDP-galactose-4-epimerase encoding gene, UTP glucose-1 phosphate uridyltransferase encoding gene, phosphoglucomutase encoding gene, cytidylate kinase encoding gene, nucleoside diphosphate kinase encoding gene and membrane transporter encoding gene yadH; (2) Anthocyanin synthase encoding gene, UDP galactosyltransferase encoding gene, UDP-galactose-4-isomerase encoding gene.
2. The genetically engineered bacteria for producing cyanidin 3-O-galactoside according to claim 1, characterized in that: The anthocyanin synthase encoding gene and the UDP galactosyltransferase encoding gene are optimized according to the codon preference of Escherichia coli.
3. The genetically engineered bacteria for producing cyanidin 3-O-galactoside according to claim 1, characterized in that: The GenBank number of the anthocyanin synthase encoding gene is any one of X70786.1, NC_084814.1, MH321466.1, NC_041794.1 and NC_081806.1, The GenBank number of the UDP galactosyltransferase encoding gene is AKI23632.1, The GenBank number of the UDP-galactose-4-epimerase encoding gene is NP_415280.3, The GenBank number of the UTP glucose-1 phosphate uridyl transferase encoding gene is NP_415752.1, The GenBank number of the phosphoglucomutase encoding gene is NP_415214.1, The GenBank number of the cytidylate kinase encoding gene is NP_415430.1, The GenBank number of the nucleoside diphosphate kinase encoding gene is NP_417013.1, The GenBank number of the membrane transporter encoding gene yadH is NP_414670.
1.
4. The genetically engineered bacteria for producing cyanidin 3-O-galactoside according to claim 1, characterized in that: The starting strains are Escherichia coli BL21 (DE3), Escherichia coli OverExpress TM C41(DE3), E. coli OverExpress TM C43(DE3), E. coli Tuner(DE3), E. coli Arctic Express TM coli T7 Express.
5. The method for constructing a genetically engineered bacterium for producing cyanidin 3-O-galactoside according to any one of claims 1 to 4, characterized in that: The introduced genes include anthocyanidin synthase encoding gene, UDP galactosyltransferase encoding gene, UDP-galactose-4-epimerase encoding gene, UTP glucose-1 phosphate uridyl transferase encoding gene, phosphoglucomutase encoding gene, cytidylate kinase encoding gene, nucleoside diphosphate kinase encoding gene and membrane transporter encoding gene yadH, and the construction method includes the following steps: (1) cloning the sequences of anthocyanin synthase encoding gene, UDP galactosyltransferase encoding gene and UDP-galactose-4-epimerase encoding gene into an expression vector to obtain a recombinant expression vector 1; (2) cloning the sequences of the UTP glucose-1-phosphate uridyl transferase encoding gene, the phosphoglucomutase encoding gene, the cytidylate kinase encoding gene, the nucleoside diphosphate kinase encoding gene and the membrane transporter encoding gene yadH into an expression vector to obtain a recombinant expression vector 2; (3) The recombinant expression vector 1 in step (1) and the recombinant expression vector 2 in step (2) are co-transformed into Escherichia coli to obtain the genetically engineered bacteria that produce cyanidin 3-O-galactoside.
6. Use of the genetically engineered bacteria according to any one of claims 1 to 4 in the production of cyanidin 3-O-galactoside.
7. A method for producing cyanidin 3-O-galactoside, characterized in that: The genetically engineered bacteria according to any one of claims 1 to 4 are fermented and cultured with (+)-catechin as a substrate, and cyanidin 3-O-galactoside is extracted and obtained.
8. The method for producing cyanidin 3-O-galactoside according to claim 7, characterized in that: During fermentation, IPTG was used to induce expression, and the induction condition was OD 600 Induction was performed when the pH was 0.6-0.
8.
9. The method for producing cyanidin 3-O-galactoside according to claim 8, characterized in that: After induction at 18-30°C for 3-24 hours, the bacteria were collected, resuspended in the catalytic reaction solution, and catalyzed at 20-30°C for 24 hours.
10. The method for producing cyanidin 3-O-galactoside according to claim 9, characterized in that: The catalytic reaction liquid used includes the following components: Ammonium chloride 1g / L, ammonium sulfate 2.7g / L, disodium hydrogen phosphate 6.8g / L, potassium dihydrogen phosphate 3g / L, sodium chloride 0.6g / L, magnesium sulfate heptahydrate 0.2g / L, vitamin B1 10mg / L, zinc sulfate heptahydrate 5mg / L, manganese sulfate 1mg / L, cobalt chloride hexahydrate 0.5mg / L, copper sulfate pentahydrate 0.5mg / L, Sodium molybdate 0.5 mg / L, boric acid 0.2 mg / L, L-sodium ascorbate 0.5 g / L, ferrous sulfate heptahydrate 2.8 mg / L, α-ketoglutaric acid 14.6 mg / L, orotic acid 15.6 g / L and glutamic acid 2 mg / L.