Genetically engineered bacterium and application thereof in production of cyanidin 3-O-glucoside

Abstract

The invention discloses a genetically engineered bacterium and application of the genetically engineered bacterium in production of cyanidin 3-O-glucoside, and relates to the technical field of bioengineering. The invention provides an application of escherichia coli in production of cyanidin 3-O-glucoside. The modified recombinant escherichia coli can be used for efficient biosynthesis of cyanidin 3-O-glucoside through whole-cell catalysis. Compared with plant extraction, the method has the advantages that the production cycle can be shortened, the cost of raw materials and energy consumption can be remarkably reduced, glycosylation sites can be accurately regulated and controlled, the product yield can be increased, and the requirements of cyanidin-3-O-glucoside in the fields of food and cosmetics can be met.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a genetically engineered bacterium and its application in the production of cyanidin 3-O-glucoside. Background Technology

[0002] Cyanidin-3-O-β-D-glucoside (C3G), with the molecular formula C 21 H 21 O 11 The molecular weight is 449.38. C3G is mainly found in dark-colored plants such as blueberries, mulberries, black rice, and purple sweet potatoes. It has functions such as anti-oxidation and anti-inflammation, eye protection and neuroprotection, and can be added as a natural pigment in food and cosmetics.

[0003] C3G exhibits significant antioxidant capacity, reducing oxidative damage by scavenging free radicals and regulating reductase activity. It also possesses anti-inflammatory effects, repairing the intestinal mucosal barrier and alleviating inflammatory bowel disease by regulating levels of colitis-related indicators and signaling pathways. In combating obesity, C3G reduces lipid accumulation by regulating lipid metabolism, particularly by decreasing lipid synthesis and increasing fatty acid oxidation. Furthermore, C3G can improve insulin resistance, lower blood glucose levels, and possesses some anti-cancer potential. C3G can also penetrate the blood-brain barrier, reducing β-amyloid protein deposition and slowing the progression of Alzheimer's disease.

[0004] Currently, cyanidin-3-O-glucoside has been approved as a food additive by the European Union and the US FDA, and is widely used in functional beverages (such as juice and sports drinks), dairy products (such as yogurt and cheese), baked goods (such as purple sweet potato bread), and dietary supplements. In the cosmetics field, its antioxidant properties are used in anti-aging serums and sunscreens; in the pharmaceutical field, its potential as a hypoglycemic drug or chemotherapy adjuvant is being explored.

[0005] Although cyanidin-3-O-glucoside has a wide range of applications, its reliance on plant extraction leads to high costs, and the raw materials are limited by climate and origin, resulting in an extraction rate of less than 1%. The purification process requires multiple chromatographic steps, is energy-intensive, and is easily affected by pH and temperature, leading to poor product stability. A biosynthetic approach, employing metabolic engineering to modify microorganisms (such as *E. coli* and yeast) and introducing plant-derived glycosyltransferases and related genes for glycoside synthesis, achieves anthocyanin synthesis. This method offers mild reaction conditions, high product specificity, and allows for large-scale production through optimized fermentation processes. Currently, according to literature reports, the highest yield of cyanidin-3-O-glucoside through semi-biosynthesis in *E. coli* reached 439 mg / L.

[0006] In the prior art, patent application CN116656710A discloses the application of the multidrug resistance transporter MdtH in the synthesis of cyanidin-3-O-glucoside. A recombinant expression vector for MdtH was constructed using the gene coding sequence of the multidrug resistance transporter MdtH, and cyanidin-3-O-glucoside synthesized from feed (+)-catechins was transported extracellularly by overexpression of MdtH in host cells. This invention improves the efficiency of biosynthetic production of cyanidin-3-O-glucoside through genetically engineered strains, providing a new strategy and approach for increasing the yield of polyphenolic flavonoids through transport regulation.

[0007] However, more avenues need to be explored to enhance cyanidin-3-O-glucoside for large-scale production. Summary of the Invention

[0008] In view of the shortcomings of the prior art, the purpose of this invention is to provide a recombinant Escherichia coli for producing cyanidin 3-O-glucoside, and a method for producing cyanidin 3-O-glucoside.

[0009] The specific technical solution of this invention is as follows: This invention provides a genetically engineered bacterium for producing cornflower-3-O-glucoside. Using *Escherichia coli* as the starting strain, the genetically engineered bacterium is obtained through gene introduction. The introduced genes include any one of the following groups: (1) Anthocyanin synthase encoding gene, UDP glucose transferase encoding gene, UTP glucose-1 phosphate uridine transferase encoding gene, phosphate glucose mutase encoding gene, cytidine kinase encoding gene, nucleoside diphosphate kinase encoding gene and membrane transport protein encoding gene YadH; (2) Anthocyanin synthase encoding gene, UDP glucose transferase encoding gene, UTP glucose-1 phosphate uridine transferase encoding gene, phosphate glucose mutase encoding gene, cytidine kinase encoding gene and nucleoside diphosphate kinase encoding gene.

[0010] Specifically, the anthocyanin synthase is derived from grapes ( Vitis vinifera The UDP-glucosyltransferase is derived from blueberries ( Vaccinium corymbosum ), Dutch Iris ( Iris hollandica ) or Arabidopsis thaliana ( Arabidopsis thaliana The UTP glucose-1 phosphate uridine transferase, phosphate glucose mutase, cytidine kinase, nucleoside diphosphate kinase, membrane transport protein YadH, and membrane transport protein MdtH are all derived from *Escherichia coli*. Escherichia coli DH5α.

[0011] Furthermore, the anthocyanin synthase encoding gene and the UDP-glucosyltransferase encoding gene are optimized according to the codon bias of *E. coli*. Those skilled in the art can obtain the encoding genes for anthocyanin synthase and UDP-glucosyltransferase based on their amino acid sequences. Based on codon rules, the nucleotide sequences encoding the above proteins are not unique, but all nucleotide sequences encoding the above anthocyanin synthase and UDP-glucosyltransferase are within the scope of protection of this invention.

[0012] Preferably, the anthocyanin synthase has a GenBank number of NP_001268147.1, the UDP glucose syltransferase has a GenBank number of any one of AYC35394.1, BAD83701.1, and CAC01718.1, the UTP glucose-1 phosphate uridine transferase has a GenBank number of NP_415752.1, the phosphoglucose mutase has a GenBank number of NP_415214.1, the cytidine kinase has a GenBank number of NP_415430.1, the nucleoside diphosphate kinase has a GenBank number of NP_417013.1, and the membrane transport protein YadH has a GenBank number of NP_414670.1.

[0013] Preferably, the starting strain is *Escherichia coli* BL21(DE3) or *Escherichia coli* OverExpress. TM C41(DE3), Escherichia coli OverExpress TM C43(DE3), Escherichia coli Tuner(DE3), Escherichia coli Arctic Express TM (DE3) and any one of Escherichia coli T7 Express.

[0014] This invention also provides a method for constructing the genetically engineered bacterium that produces cyanidin 3-O-glucoside. The introduced genes include anthocyanin synthase encoding genes, UDP glucose transferase encoding genes, UTP glucose-1-phosphate uridine transferase encoding genes, phosphoglucose mutase encoding genes, cytidine kinase encoding genes, nucleoside diphosphate kinase encoding genes, and the membrane transport protein encoding gene YadH. The construction method includes the following steps: (1) The sequences of the anthocyanin synthase encoding gene and the UDP glucosyltransferase encoding gene were cloned into the expression vector to obtain recombinant expression vector 1; (2) The sequences of the UTP glucose-1 phosphate uridine transferase encoding gene, the phosphate glucose mutase encoding gene, the cytidine kinase encoding gene, the nucleoside diphosphate kinase encoding gene and the membrane transport protein encoding gene YadH were cloned into the expression vector to obtain recombinant expression vector 2. (3) The recombinant expression vector 1 in step (1) and the recombinant expression vector 2 in step (2) are transformed into Escherichia coli to obtain the genetically engineered bacteria that produce cyanidin 3-O-glucoside.

[0015] Specifically, pET-28a(+) was used as the expression plasmid for the genes encoding anthocyanin synthase and UDP glucose-syltransferase, and pACYCDuet-1 was used as the expression plasmid for the genes encoding UTP glucose-1 phosphate uridine transferase, phosphate glucose mutase, cytidine kinase, nucleoside diphosphate kinase, and membrane transport protein YadH.

[0016] The present invention also provides the application of the genetically engineered bacteria in the production of cyanidin 3-O-glucoside.

[0017] The present invention also provides a method for producing cyanidin 3-O-glucoside, using (+)-catechin and glucose as substrates, fermenting and culturing the genetically engineered bacteria, and extracting cyanidin 3-O-glucoside.

[0018] Preferably, during fermentation culture, isopropyl-β-D-thiogalactoside (IPTG) is used for induction expression, and the induction conditions are OD... 600 Induction was performed when the concentration was 0.6-0.8. Further, after induction at 20℃ for 6-24 hours, the bacterial cells were collected, resuspended in the catalytic reaction solution, and catalyzed at 20℃ for 12-48 hours. The catalytic reaction solution used in the catalytic reaction includes the following components: ammonium chloride 1 g / L, ammonium sulfate 2.7 g / L, disodium hydrogen phosphate 68 g / L, potassium dihydrogen phosphate 3 g / L, sodium chloride 0.6 g / L, magnesium sulfate heptahydrate 0.2 g / L, vitamin B1 10 mg / L, zinc sulfate heptahydrate 5 mg / L, manganese sulfate 1 mg / L, cobalt chloride hexahydrate 0.5 mg / L, copper sulfate pentahydrate 0.5 mg / L, sodium molybdate 0.5 mg / L, boric acid 0.2 mg / L, sodium L-ascorbate 0.5 g / L, ferrous sulfate heptahydrate 2.8 mg / L, α-ketoglutarate 14.6 mg / L, orotic acid 15.6 g / L, glutamic acid 2 mg / L, and glucose 5–80 g / L.

[0019] Specifically, the method includes: culturing the recombinant Escherichia coli described above, inducing the expression of anthocyanin synthase, UDP glucosyltransferase, UTP glucose-1 phosphate uridine transferase, phosphoglucose mutase, cytidine kinase, nucleoside diphosphate kinase, and membrane transport protein YadH with an inducer, collecting bacterial cells, and using the collected bacterial cells to produce cyanidin 3-O-glucoside via whole-cell catalysis using (+)-catechin as a substrate.

[0020] Preferably, the induction is at OD 600 When the concentration of β-hydroxypropyl phosphate (β-Pp) is 0.6–0.8, the expression of anthocyanin synthase, UDP-glucosyltransferase, UTP-glucose-1-phosphate uridine transferase, phosphoglucose mutase, cytidine kinase, nucleoside diphosphate kinase, and membrane transporter YadH is induced by IPTG. More preferably, the induction is performed at 20°C; the inducer is 1 mM IPTG. Preferably, bacterial cells are collected after 6–24 h of induction, and the collected cells are resuspended in the catalytic reaction solution and subjected to whole-cell catalysis at 20°C for 12–48 h. Most preferably, bacterial cells are collected after 12 h of induction, achieving the highest yield of 868.18 mg / L, an 8.5% increase compared to the pre-optimized induction time.

[0021] The collected bacterial cells were resuspended in the catalytic reaction solution and subjected to whole-cell catalysis at 20°C for 48 h. The highest yield of 926.47 mg / L was reached at 48 h, which was 7% higher than that before the catalytic time was optimized.

[0022] The beneficial effects of this invention are as follows: This invention provides the use of *Escherichia coli* in the production of cyanidin 3-O-glucoside. The modified recombinant *E. coli* can be used for the efficient whole-cell catalytic biosynthesis of cyanidin 3-O-glucoside. Compared with plant extraction, this invention can shorten the production cycle, significantly reduce raw material and energy costs, precisely control the glycosylation site, and improve product yield, meeting the demand for cyanidin-3-O-glucoside in the food and cosmetic fields. Attached Figure Description

[0023] Figure 1 The spectrum of the recombinant expression vector pET28a-T7-VvANS-Vc3GT.

[0024] Figure 2 The spectrum of the recombinant expression vector pET28a-T7-VvANS-Ih3GT.

[0025] Figure 3 The spectrum of the recombinant expression vector pET28a-T7-VvANS-At3GT.

[0026] Figure 4The map of the recombinant expression vector pACYCDuet-T7-EcgalU-Ecpgm-Eccmk-Ecndk.

[0027] Figure 5 The map of the recombinant expression vector pACYCDuet-T7-EcgalU-Ecpgm-Eccmk-Ecndk-EcYadH.

[0028] Figure 6 The spectrum of the recombinant expression vector pACYCDuet-T7-EcMdtH.

[0029] Figure 7 Qualitative analysis of recombinant Escherichia coli fermentation products was performed using liquid chromatography and liquid chromatography-mass spectrometry; where A is cyanidin-3-O-glucoside standard; B is recombinant Escherichia coli fermentation broth; C is cyanidin-3-O-glucoside standard m / z 449; and D is recombinant Escherichia coli fermentation broth m / z 449.

[0030] Figure 8 Analysis of cyanidin 3-O-glucoside production in different recombinant Escherichia coli strains.

[0031] Figure 9 To analyze the yield of cyanidin-3-O-glucoside in recombinant Escherichia coli under the action of different membrane transport proteins.

[0032] Figure 10 To compare the yield of cyanidin 3-O-glucoside in recombinant Escherichia coli under different amounts of catechin and glucose.

[0033] Figure 11 Comparison of cyanidin-3-O-glucoside production in recombinant Escherichia coli at different induction times.

[0034] Figure 12 Comparison of cyanidin-3-O-glucoside yield in recombinant Escherichia coli at different fermentation times. Detailed Implementation

[0035] The primers used in the embodiments of this invention are shown in Table 1.

[0036] Table 1

[0037] Example 1: Construction of Recombinant Escherichia coli The target gene nucleic acid sequences were obtained from the NCBI database. The GenBank accession number for anthocyanin synthase (ANS) is NP_001268147.1; the GenBank accession number for UDP-glucosyltransferase (3GT) is one of AYC35394.1, BAD83701.1, or CAC01718.1; the GenBank accession number for UTP glucose-1-phosphouridine transferase is NP_415752.1; the GenBank accession number for phosphoglucose mutase is NP_415214.1; the GenBank accession number for cytidine kinase is NP_415430.1; the GenBank accession number for nucleoside diphosphate kinase is NP_417013.1; the GenBank accession number for membrane transporter YadH is NP_414670.1; and the GenBank accession number for membrane transporter MdtH is NP_415583.4. Codon optimization was performed on anthocyanin synthase and UDP-glucosyltransferase based on the codon preference of *E. coli*, and related gene fragments were synthesized. The optimized anthocyanin synthase gene sequence is shown in SEQ ID NO.1, and the optimized UDP-glucosyltransferase gene sequences are shown in SEQ ID NO.2-4, respectively. The remaining genes were directly amplified from the *E. coli* genome.

[0038] UDP-glucosyltransferase and anthocyanin synthase genes from different sources were amplified and inserted into the vector pET28a using the Gibson assembly method. The specific construction is as follows: VvANS was amplified using primers P3 / P4, and Vc3GT, Ih3GT, and At3GT were amplified using primers P5 / P6, P7 / P8, and P9 / P10, respectively. The pET28a backbone was amplified using primers P1 / P2 and recombined with VvANS, Vc3GT / Ih3GT / At3GT. The resulting vector was as follows. Figure 1-3 The recombinant expression vectors shown are pET28a-T7-VvANS-Vc3GT, pET28a-T7-VvANS-Ih3GT, and pET28a-T7-VvANS-At3GT.

[0039] The genes for UTP glucose-1 phosphouridine transferase (galU) (Gene ID: 945730), phosphoglucose mutase (pgm) (Gene ID: 945271), cytidine kinase (cmk) (Gene ID: 945535), and nucleoside diphosphate kinase (ndk) (Gene ID: 945611) were amplified and inserted into the vector pACYCDuet-1 using the Gbison assembly method. The specific construction is as follows: EcgalU was amplified using primers P13 / P14, Ecpgm using primers P15 / P16, Eccmk using primers P17 / P18, and Ecndk using primers P19 / P20. The EcgalU and Ecpgm fragments were then extended using primers P13 / P16 to obtain the EcgalU-Ecpgm fragment. Similarly, the Eccmk and Ecndk fragments were extended using primers P17 / P20 to obtain the Eccmk-Ecndk fragment. Finally, the pACYCDuet-1 backbone was amplified using primers P12 / 21, and the Eccmk-Ecndk fragment was recombinated to obtain the recombinant expression vector pACYCDuet-T7-Eccmk-Ecndk. Using primers P20 / 22, pACYCDuet-T7-Eccmk-Ecndk was amplified using a template to obtain the T7-Eccmk-Ecndk fragment. Then, using primers P11 / P12, the pACYCDuet-1 backbone was amplified to recombine with the fragments EcgalU-Ecpgm and T7-Eccmk-Ecndk, yielding the following result: Figure 4 The recombinant expression vector pACYCDuet-T7-EcgalU-Ecpgm-Eccmk-Ecndk was shown. This expression vector was then combined with the recombinant expression vectors pET28a-T7-VvANS-Vc3GT, pET28a-T7-VvANS-Ih3GT, and pET28a-T7-VvANS-At3GT, respectively, and transformed into *E. coli* BL21(DE3) using a CaCl2-treated *E. coli* chemical transformation method to obtain recombinant expression strains C3G-F1, C3G-F2, and C3G-F3.

[0040] The genes for UTP glucose-1 phosphourydyltransferase, phosphoglucose mutase, cytidine kinase, nucleoside diphosphate kinase, and the membrane transport protein YadH (Gene ID: 944836) or MdtH (Gene ID: 946920) were amplified and inserted into the vector pACYCDuet-1 using the Gbison assembly method. The specific construction is as follows: Using primers P25 / P29, pACYCDuet-T7-EcgalU-Ecpgm-Eccmk-Ecndk was amplified using this template to obtain the fragment T7-EcgalU-Ecpgm-Eccmk-Ecndk(YadH). YadH was amplified using primers P30 / P31. The pACYCDuet backbone was amplified with the fragment T7-EcgalU-Ecpgm-Eccmk-Ecndk(YadH) and YadH recombination using primers P12 / P23 to obtain the following... Figure 5 The recombinant vector pACYCDuet-T7-EcgalU-Ecpgm-Eccmk-Ecndk-EcYadH is shown. MdtH is amplified using primers P28 / P27. The pACYCDuet backbone is then amplified with MdtH using primers P24 / P26, yielding the desired result. Figure 6 The recombinant vector pACYCDuet-T7-EcMdtH is shown. The recombinant expression vectors pACYCDuet-T7-EcMdtH and pACYC-T7-ECgalU-Ecpgm-T7-Eccmk-T7-Ecndk-EcYadH were co-transformed into E. coli BL21(DE3) with the recombinant expression vector pET28a-T7-VvANS-Ih3GT via CaCl2-treated E. coli chemical transformation, yielding recombinant E. coli C3G-F4 and C3G-F5.

[0041] Example 2: Analysis of the synthetic capacity of recombinant Escherichia coli cyanidin-3-O-glucoside 1. In this embodiment, recombinant Escherichia coli C3G-F1, C3G-F2, and C3G-F3 prepared in Example 1 were used for analysis. The recombinant Escherichia coli were streaked on LB solid medium and cultured overnight at 37°C. Single colonies were picked and inoculated into 2 ml of LB liquid medium and cultured overnight at 37°C and 220 rpm. The initial OD was determined... 600 0.1 g was inoculated into 20 ml of LB medium and cultured at 37°C and 220 rpm until OD. 600When the bacterial concentration was 0.6-0.8, 1 mM IPTG was added for induction, and the culture was carried out at 20℃ and 200 rpm for 18 h. Subsequently, the bacterial culture was collected, centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and 4 ml of catalytic reaction solution was added to resuspend the bacterial cells. 320 μl of the resuspended cells were placed in a 14 ml shake tube, and 2 mM (+)-catechin was added as a substrate for catalysis. The cells were incubated at 20℃ and 200 rpm for 24 h.

[0042] The catalytic reaction solution used in the catalytic reaction includes the following components: ammonium chloride 1 g / L, ammonium sulfate 2.7 g / L, disodium hydrogen phosphate 68 g / L, potassium dihydrogen phosphate 3 g / L, sodium chloride 0.6 g / L, magnesium sulfate heptahydrate 0.2 g / L, vitamin B1 10 mg / L, zinc sulfate heptahydrate 5 mg / L, manganese sulfate 1 mg / L, cobalt chloride hexahydrate 0.5 mg / L, copper sulfate pentahydrate 0.5 mg / L, sodium molybdate 0.5 mg / L, boric acid 0.2 mg / L, sodium L-ascorbate 0.5 g / L, ferrous sulfate heptahydrate 2.8 mg / L, α-ketoglutarate 14.6 mg / L, orotic acid 15.6 g / L, glutamic acid 2 mg / L, and glucose 10 g / L.

[0043] 2. After incubation, the reaction solution is centrifuged at 12000 rpm for 5 min. The supernatant is used for extracellular yield analysis. The precipitate is extracted with 1% hydrochloric acid solution by sonication and centrifugation again. The supernatant is used for intracellular yield analysis. The sum of extracellular and intracellular yields is the total yield.

[0044] Qualitative analysis of the synthesized product was performed using liquid chromatography and liquid chromatography-mass spectrometry, and the results are as follows: Figure 7 As shown, the recombinant strain can synthesize cyanidin-3-O-glucoside using (+)-catechin. (As shown...) Figure 8 As shown, the catalytic activities of UDP-glucosyltransferases from different sources are different. Among them, the strain C3G-F3 corresponding to the UDP-glucosyltransferase from Arabidopsis thaliana has the lowest yield, only 161.62 mg / L, while the strain C3G-F2 corresponding to the UDP-glucosyltransferase from Iris tectorum has the highest yield, at 488.58 mg / L, which is 3 times that of the former.

[0045] Example 3: Analysis of cyanidin-3-O-glucoside production in recombinant Escherichia coli under the action of different membrane transport proteins This embodiment uses Escherichia coli C3G-F2, C3G-F4, and C3G-F5 prepared in Example 1 for analysis. The specific experimental methods are the same as in Example 2, and the results are as follows. Figure 9As shown, overexpression of YadH and MdtH both increased the yield of cyanidin-3-O-glucoside to varying degrees. Meanwhile, strains overexpressing UTP glucose-1-phosphourydyltransferase, phosphoglucosidase, cytidine kinase, nucleoside diphosphate kinase, and the membrane transport protein YadH exhibited the highest cyanidin-3-O-glucoside yield, at 644.15 mg / L, representing a 30% increase compared to the unmodified strain.

[0046] Example 4: Analysis of cyanidin 3-O-glucoside yield in recombinant Escherichia coli under different amounts of catechin and glucose addition. 1. In this embodiment, recombinant Escherichia coli C3G-F5 prepared in Example 1 was used for analysis. The recombinant E. coli was streaked on LB solid medium and cultured overnight at 37°C. Single colonies were picked and inoculated into 2 ml of LB liquid medium and cultured overnight at 37°C and 220 rpm. The initial OD was determined... 600 0.1 g was inoculated into 20 ml of LB medium and cultured at 37°C and 220 rpm until OD. 600 Induction was initiated by adding 1 mM IPTG at a final concentration when the bacterial concentration was between 0.6 and 0.8. The culture was then incubated at 20°C and 200 rpm for 18 h. Afterward, the 18-h bacterial culture was collected, centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and 4 ml of catalytic reaction solution was added to resuspend the cells. 320 μl of this solution was placed in a 14 ml shaker tube, and final concentrations of 1, 2, 3, 4, and 5 mM (+)-catechin and final concentrations of 0.5%, 1%, 3%, 5%, and 8% glucose were added as substrates for catalysis. The tubes were incubated at 20°C and 200 rpm for 24 h.

[0047] 2. After incubation, the reaction solution is centrifuged at 12000 rpm for 5 min. The supernatant is used for extracellular yield analysis. The precipitate is extracted with 1% hydrochloric acid solution by sonication and centrifugation again. The supernatant is used for intracellular yield analysis. The sum of extracellular and intracellular yields is the total yield.

[0048] like Figure 10 As shown, the yield of cyanidin-3-O-glucoside varied significantly with different amounts of catechin and glucose added. With increasing catechin addition, the yield of cyanidin-3-O-glucoside initially increased and then decreased, reaching a maximum of 699.48 mg / L at a concentration of 3 mM. With increasing glucose addition, the yield of cyanidin-3-O-glucoside initially increased and then gradually stabilized, reaching a maximum of 799.54 mg / L at a glucose addition of 3%. By optimizing the amounts of catechin and glucose added, the yield of cyanidin-3-O-glucoside was 1.1 times that before optimization.

[0049] Example 5: Analysis of cyanidin-3-O-glucoside yield in recombinant Escherichia coli at different induction times 1. In this embodiment, recombinant Escherichia coli C3G-F5 prepared in Example 1 was used for analysis. The recombinant E. coli was streaked on LB solid medium and cultured overnight at 37°C. Single colonies were picked and inoculated into 2 ml of LB liquid medium and cultured overnight at 37°C and 220 rpm. The initial OD was determined... 600 0.1 g was inoculated into 20 ml of LB medium and cultured at 37°C and 220 rpm until OD. 600 Induction was initiated by adding 1 mM IPTG at a final concentration when the bacterial concentration was between 0.6 and 0.8. The culture was then incubated at 20°C and 200 rpm. Subsequently, bacterial cultures were collected at 6 h, 12 h, 18 h, and 24 h, centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and 4 ml of catalytic reaction solution was added to resuspend the bacterial cells. 320 μl of this solution was placed in a 14 ml shaker tube, and 3 mM (+)-catechin and 3% glucose were added as substrates for catalysis. The tube was incubated at 20°C and 200 rpm for 24 h.

[0050] 2. After incubation, the reaction solution is centrifuged at 12000 rpm for 5 min. The supernatant is used for extracellular yield analysis. The precipitate is extracted with 1% hydrochloric acid solution by sonication and centrifugation again. The supernatant is used for intracellular yield analysis. The sum of extracellular and intracellular yields is the total yield.

[0051] like Figure 11 As shown, the yield of cyanidin-3-O-glucoside varied significantly under different induction times. With increasing induction time, the yield of cyanidin-3-O-glucoside first increased and then decreased, reaching a maximum of 868.18 mg / L at 12 h of induction. Optimizing the induction time significantly improved the yield of cyanidin-3-O-glucoside, increasing it by 8.5% compared to before optimization.

[0052] Example 6: Analysis of cyanidin-3-O-glucoside yield in recombinant Escherichia coli at different catalytic times 1. In this embodiment, recombinant Escherichia coli C3G-F5 prepared in Example 1 was used for analysis. The recombinant E. coli was streaked on LB solid medium and cultured overnight at 37°C. Single colonies were picked and inoculated into 2 ml of LB liquid medium and cultured overnight at 37°C and 220 rpm. Initial OD600 was 0.1, and the culture was inoculated into 20 ml of LB medium and cultured at 37°C and 220 rpm until OD600 reached 0.6-0.8. A final concentration of 1 mM IPTG was added for induction. The culture was then incubated at 20°C and 200 rpm for 12 h. The bacterial culture was then collected, centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and 4 ml of catalytic reaction solution was added to resuspend the bacteria. 320 μl of this solution was placed in a 14 ml shaker tube, and a final concentration of 3 mM (+)-catechin and 3% glucose were added as substrates for catalysis. The culture was incubated at 20°C and 200 rpm for 12-48 h.

[0053] 2. After incubation, the reaction solution is centrifuged at 12000 rpm for 5 min. The supernatant is used for extracellular yield analysis. The precipitate is extracted with 1% hydrochloric acid solution by sonication and centrifugation again. The supernatant is used for intracellular yield analysis. The sum of extracellular and intracellular yields is the total yield.

[0054] like Figure 12 As shown, the yield of cyanidin 3-O-glucoside gradually increased with the extension of catalytic time, with most of the catalysis completed at 24 h. After 24 h, the yield increased slowly, reaching a maximum yield of 926.47 mg / L at 48 h, which is a 7% increase compared to the yield before catalytic time optimization. Further yield improvement can be achieved by optimizing the catalytic time.

Claims

1. A genetically engineered bacterium that produces cornflower-3-O-glucoside, characterized in that, Using *Escherichia coli* as the starting strain, the genetically engineered bacteria were constructed by introducing genes, including any one of the following groups: (1) Anthocyanin synthase encoding gene, UDP glucose transferase encoding gene, UTP glucose-1 phosphate uridine transferase encoding gene, phosphate glucose mutase encoding gene, cytidine kinase encoding gene, nucleoside diphosphate kinase encoding gene and membrane transport protein encoding gene YadH; (2) Anthocyanin synthase encoding gene, UDP glucose transferase encoding gene, UTP glucose-1 phosphate uridine transferase encoding gene, phosphate glucose mutase encoding gene, cytidine kinase encoding gene and nucleoside diphosphate kinase encoding gene.

2. The genetically engineered bacterium for producing cyanidin-3-O-glucoside according to claim 1, characterized in that, The anthocyanin synthase is derived from grapes ( Vitis vinifera ), The UDP-glucosyltransferase is derived from blueberries ( Vaccinium corymbosum ), Dutch Iris ( Iris hollandica ) or Arabidopsis thaliana ( Arabidopsis thaliana ), The UTP glucose-1 phosphate uridine transferase, phosphate glucose mutase, cytidine kinase, nucleoside diphosphate kinase, and membrane transport protein YadH were all derived from *Escherichia coli*. Escherichia coli ).

3. The genetically engineered bacterium for producing cornflower-3-O-glucoside according to claim 2, characterized in that, The anthocyanin synthase encoding gene and the UDP glucosyltransferase encoding gene were optimized based on E. coli codon bias.

4. The genetically engineered bacterium for producing cyanidin-3-O-glucoside according to claim 2, characterized in that, The anthocyanin synthase has the GenBank accession number NP_001268147.

1. The UDP-glucosyltransferase has a GenBank number of any one of AYC35394.1, BAD83701.1, or CAC01718.

1. The GenBank accession number for the UTP glucose-1 phosphate uridine transferase is NP_415752.

1. The GenBank accession number for the phosphoglucosuric enzyme is NP_415214.

1. The GenBank accession number for the cytidine kinase is NP_415430.

1. The GenBank accession number for the nucleoside diphosphate kinase is NP_417013.

1. The GenBank accession number for the membrane transporter YadH is NP_414670.

1.

5. The genetically engineered bacterium for producing cyanidin-3-O-glucoside according to claim 1, characterized in that, The starting strains were *Escherichia coli* BL21(DE3) and *Escherichia coli OverExpress*. TM C41(DE3), Escherichia coli OverExpress TM C43(DE3), Escherichia coli Tuner(DE3), Escherichia coli Arctic Express TM (DE3) and any one of Escherichia coli T7 Express.

6. The method for constructing the genetically engineered bacteria for producing cyanidin 3-O-glucoside according to any one of claims 1 to 5, characterized in that, The imported genes include the anthocyanin synthase encoding gene, the UDP glucose transferase encoding gene, the UTP glucose-1 phosphate uridine transferase encoding gene, the phosphoglucose mutase encoding gene, the cytidine kinase encoding gene, the nucleoside diphosphate kinase encoding gene, and the membrane transport protein encoding gene YadH. The construction method includes the following steps: (1) The sequences of the anthocyanin synthase encoding gene and the UDP glucosyltransferase encoding gene were cloned into the expression vector to obtain recombinant expression vector 1; (2) The sequences of the UTP glucose-1 phosphate uridine transferase encoding gene, the phosphate glucose mutase encoding gene, the cytidine kinase encoding gene, the nucleoside diphosphate kinase encoding gene and the membrane transport protein encoding gene YadH were cloned into the expression vector to obtain recombinant expression vector 2. (3) The recombinant expression vector 1 in step (1) and the recombinant expression vector 2 in step (2) are transformed into Escherichia coli to obtain the genetically engineered bacteria that produce cyanidin 3-O-glucoside.

7. The use of the genetically engineered bacteria according to any one of claims 1 to 5 in the production of cyanidin 3-O-glucoside.

8. A method for producing cornflower 3-O-glucoside, characterized in that, Using (+)-catechin and glucose as substrates, the genetically engineered bacteria according to any one of claims 1 to 5 are fermented and cultured, and cyanidin 3-O-glucoside is extracted.

9. The method for producing cornflower 3-O-glucoside according to claim 8, characterized in that, During fermentation culture, IPTG was used to induce expression, and the induction conditions were OD... 600 Induction was performed when the concentration was between 0.6 and 0.

8.

10. The method for producing cornflower 3-O-glucoside according to claim 9, characterized in that, The bacterial cells were collected after induction at 20℃ for 6–24 h, and the collected bacterial cells were resuspended in the catalytic reaction solution and catalyzed at 20℃ for 12–48 h. The catalytic reaction solution used in the catalytic reaction includes the following components: Ammonium chloride 1 g / L, ammonium sulfate 2.7 g / L, disodium hydrogen phosphate 68 g / L, potassium dihydrogen phosphate 3 g / L, sodium chloride 0.6 g / L, magnesium sulfate heptahydrate 0.2 g / L, vitamin B1 10 mg / L, zinc sulfate heptahydrate 5 mg / L, manganese sulfate 1 mg / L, cobalt chloride hexahydrate 0.5 mg / L, copper sulfate pentahydrate 0.5 mg / L, sodium molybdate 0.5 mg / L, boric acid 0.2 mg / L, sodium L-ascorbate 0.5 g / L, ferrous sulfate heptahydrate 2.8 mg / L, α-ketoglutarate 14.6 mg / L, orotic acid 15.6 g / L, glutamic acid 2 mg / L, glucose 5–80 g / L.

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