Genetically engineered bacterium for producing cyanidin-3-O-rutinoside as well as construction method and application of genetically engineered bacterium

Abstract

The invention discloses a genetically engineered bacterium for producing cyanidin-3-O-rutinoside as well as a construction method and application of the genetically engineered bacterium, and relates to the technical field of biological engineering. The invention provides an application of escherichia coli in production of cyanidin 3-O rutinoside. The modified recombinant escherichia coli can be used for efficient biosynthesis of cyanidin 3-O-rutinoside through whole-cell catalysis. Compared with plant extraction, the method has the advantages that the production conditions are mild, the product yield is increased, the specific synthesis of the product is realized, and an efficient and environment-friendly method with a wide development prospect is provided for the production and application of the cyanidin-3-O-rutinoside.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a genetically engineered bacterium that produces cyanidin-3-O-rutin, its construction method, and its application. Background Technology

[0002] Cyanidin-3-O-rutinoside (C3R) is a natural pigment widely found in plants. Its chemical name is Cyanidin-3-O-(α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside), and its molecular formula is C3R. 27 H 31 O 15 Its molecular weight is 595.52.

[0003] Cyanide 3-O-rutinoside is commonly found in anthocyanin-rich plant tissues, such as blueberries, blackberries, purple sweet potatoes, black rice, red grapes, and purple cabbage. It is also found in higher concentrations in berries (such as blackcurrants and elderberries) and some flowers (such as cornflowers and roses). It possesses antioxidant and anti-inflammatory, neuroprotective, and anti-aging properties. Cyanide 3-O-rutinoside can effectively scavenge free radicals and inhibit mitochondrial oxidative stress, reducing systemic inflammation and lowering the risk of breast and liver cancer metastasis. Furthermore, cyanide 3-O-rutinoside has certain effects in regulating gut microbiota and improving metabolic syndrome.

[0004] Cyanide 3-O-rutin has garnered significant attention in the high-end functional food and pharmaceutical sectors. In cosmetics, its stability and permeability make it a core ingredient in anti-wrinkle masks and repair serums. In agriculture, cyanide 3-O-rutin has been developed as a natural preservative to extend the shelf life of berries.

[0005] Currently, high-purity cyanidin-3-O-rutinoside is mainly obtained through plant extraction. For example, the method for separating and purifying cyanidin-3-O-rutinoside disclosed in patent application CN108659068A can isolate and prepare high-purity cyanidin-3-O-rutinoside monomers from mulberries. However, due to the low content in plants, the supply of raw materials is limited by the plant growth cycle and geographical distribution, resulting in seasonal fluctuations, high extraction costs, and limitations imposed by season and production location, making large-scale production difficult. A biosynthetic method, by modifying E. coli or yeast strains and introducing plant-derived glycosyltransferases and related genes for glycoside synthesis, can achieve anthocyanin synthesis. This method overcomes seasonal limitations, offers mild reaction conditions, has high product specificity, and allows for large-scale production through optimized fermentation processes.

[0006] In the prior art, patent application CN118813644A discloses overexpression SoMYB1 The content of anthocyanin metabolites, especially cyanidin-3-O-rutin, was significantly increased in genetically modified tobacco.

[0007] Currently, there are no reports on the microbial synthesis of cyanidin-3-O-rutin. 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-rutin glycoside, and a method for producing cyanidin 3-O-rutin glycoside.

[0009] The specific technical solution of the present invention is as follows: This invention provides a genetically engineered bacterium for producing cyanidin-3-O-rutin, using *Escherichia coli* as the starting strain, and obtaining the genetically engineered bacterium through gene introduction. The introduced genes include any one of the following groups: (1) Anthocyanin synthase encoding gene, UDP glucose transferase encoding gene, UDP rhamnosyltransferase encoding gene, UDP rhamnosyl synthase encoding gene and membrane transport protein encoding gene MdtH; (2) Anthocyanin synthase encoding gene, UDP glucose transferase encoding gene, UDP rhamnosyltransferase encoding gene, UDP rhamnosyl synthase 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 ) and Dutch iris ( Iris hollandica One of them, the UDP rhamnosyltransferase, is derived from petunia ( Petunia hybrida Arabidopsis thaliana ( ) Arabidopsis thaliana ), sweet orange ( Citrus sinensis ) and Lobelia chinensis ( Lobelia erinus One of them, the UDP rhamnose synthase is derived from grape ( Vitis vinifera The UTP glucose-1-phosphate uridine transferase, phosphoglucose mutase, cytidine kinase, nucleoside diphosphate kinase, and membrane transport protein MdtH are derived from *Escherichia coli* (E. coli). Escherichia coli DH5α.

[0011] Furthermore, the anthocyanin synthase encoding gene, UDP glucosyltransferase encoding gene, UDP rhamnosyltransferase encoding gene, and UDP rhamnosyltransferase 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, UDP glucosyltransferase, UDP rhamnosyltransferase, and UDP rhamnosyltransferase based on their amino acid sequences. Based on codon rules, the nucleotide sequences of the genes encoding the above proteins are not unique, but all nucleotide sequences encoding the above anthocyanin synthase, UDP glucosyltransferase, UDP rhamnosyltransferase, and UDP rhamnosyltransferase are within the scope of protection of this invention.

[0012] Preferably, the GenBank number of the UDP glucosyltransferase is either AYC35394.1 or BAD83701.1, the GenBank number of the anthocyanin synthase is NP_001268147.1, the GenBank number of the UDP rhamnosyltransferase is one of ABA18631.1, AEE85357.1, BAU68119.1, or CAA50376.1, and the GenBank number of the UDP rhamnosyltransferase is X. P_002285634.1, the GenBank number of the UTP glucose-1 phosphate uridine transferase is NP_415752.1, the GenBank number of the phosphoglucose mutase is NP_415214.1, the GenBank number of the cytidine kinase is NP_415430.1, the GenBank number of the nucleoside diphosphate kinase is NP_417013.1, and the GenBank number of the membrane transport protein MdtH is NP_415583.4.

[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 bacteria that produce cyanidin 3-O-rutin, wherein the introduced genes include anthocyanin synthase encoding genes, UDP glucose transferase encoding genes, UDP rhamnosyltransferase encoding genes, UDP rhamnosyl synthase encoding genes, and membrane transport protein encoding gene MdtH. 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 UDP rhamnosyltransferase encoding gene and the UDP rhamnosyl synthase encoding gene were cloned into the expression vector to obtain recombinant vector 2; (3) The sequence of the membrane transport protein encoding gene MdtH was cloned into the expression vector to obtain recombinant expression vector 3; (4) The recombinant expression vector 1 in step (1), the recombinant expression vector 2 in step (2) and the recombinant expression vector 3 in step (3) are transformed into Escherichia coli to obtain the genetically engineered bacteria that produce cyanidin 3-O-rutin.

[0015] Specifically, pET-28a(+) was used as the expression plasmid for the genes encoding anthocyanin synthase and UDP glucosyltransferase, pCDFDuet was used as the expression plasmid for the genes encoding UDP rhamnosyltransferase and UDP rhamnosyl synthase, and pACYCDuet-1 was used as the expression plasmid for the gene encoding the membrane transport protein MdtH.

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

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

[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 18 h of induction at 20°C, the bacterial cells were collected, resuspended in the catalytic reaction solution, and catalyzed at 18–30°C for 24 h. The catalytic reaction solution used included 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 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, UDP rhamnosyltransferase, UDP rhamnosyltransferase and membrane transport protein MdtH with an inducer, collecting bacterial cells and using the collected bacterial cells to produce cyanidin 3-O-rutin glycosides by whole-cell catalysis with (+)-catechin as a substrate.

[0020] Preferably, the induction is at OD 600 When the pH is 0.6–0.8, the expression of anthocyanin synthase, UDP-glucosyltransferase, UDP-rhamnosyltransferase, UDP-rhamnosyltransferase, and the membrane transport protein MdtH 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 18 h of induction, resuspended in the catalytic reaction solution, and subjected to whole-cell catalysis at 18–30°C for 24 h. Most preferably, whole-cell catalysis is performed at 25°C for 24 h, at which the highest yield is achieved, reaching 529.01 mg / L, which is 1.25 times the yield before catalytic temperature optimization.

[0021] The beneficial effects of this invention are as follows: This invention provides the use of *Escherichia coli* in the production of cyanidin-3-O-rutin glycoside. The modified recombinant *Escherichia coli* can be used for the efficient whole-cell catalytic biosynthesis of cyanidin-3-O-rutin glycoside. Compared with plant extraction, the production conditions of this invention are milder, improving product yield and achieving product-specific synthesis, thus providing an efficient, environmentally friendly method with broad development prospects for the production and application of cyanidin-3-O-rutin glycoside. Attached Figure Description

[0022] Figure 1 The spectrum of the recombinant expression vector pET28a-T7-VvANS-Vc3GT-Ph3RT-VvRHM.

[0023] Figure 2 The spectrum of the recombinant expression vector pET28a-T7-VvANS-Vc3GT-CsRhat-VvRHM.

[0024] Figure 3 The map of the recombinant expression vector pET28a-T7-VvANS-Vc3GT-LeRhat-VvRHM.

[0025] Figure 4 The map of the recombinant expression vector pET28a-T7-VvANS-Vc3GT-AtRhat-VvRHM.

[0026] Figure 5The map of the recombinant expression vector pACYCDuet-T7-EcgalU-Ecpgm-Eccmk-Ecndk.

[0027] Figure 6 The spectrum of the recombinant expression vector pET28a-T7-VvANS-Ih3GT.

[0028] Figure 7 The spectrum of the recombinant expression vector pACYCDuet-T7-EcMdtH.

[0029] Figure 8 The map shows the recombinant expression vector pCDF-T7-LeRhat-VvRHM.

[0030] Figure 9 Qualitative analysis of recombinant Escherichia coli fermentation products was performed using liquid chromatography and liquid chromatography-mass spectrometry; where A is cyanidin-3-O-rutin glycoside standard; B is recombinant Escherichia coli fermentation broth; C is cyanidin-3-O-rutin glycoside standard m / z 595; and D is recombinant Escherichia coli fermentation broth m / z 595.

[0031] Figure 10 Analysis of cyanidin 3-O-rutin glycoside yield in recombinant Escherichia coli containing UDP-rhamnosyltransferases from different sources.

[0032] Figure 11 To compare the yield of cyanidin 3-O-rutin glycoside in recombinant Escherichia coli under different amounts of catechin and glucose.

[0033] Figure 12 Comparison of cyanidin-3-O-rutin glycoside yield in recombinant Escherichia coli at different catalytic temperatures. Detailed Implementation

[0034] The primers used in the embodiments of this invention are shown in Table 1.

[0035] Table 1

[0036] Example 1: Construction of Recombinant Escherichia coli 1. Obtain the target gene nucleic acid sequence from the NCBI database. The GenBank accession number for anthocyanin synthase (ANS) is NP_001268147.1; for UDP-glucosyltransferase (3GT), one of the GenBank accession numbers is AYC35394.1 or BAD83701.1; and for UDP-rhamnosyltransferase (Rhat), one of the GenBank accession numbers is ABA18631.1, AEE85357.1, BAU68119.1, or CAA50376. UDP-rhamnosyltransferase... The GenBank accession numbers for anthocyanin synthase (RHM), UDP-glucosyltransferase, UDP-rhamnosyltransferase, and UDP-rhamnosyltransferase were XP_002285634.1, NP_415752.1, phosphoglucose mutase, NP_415214.1, cytidine kinase, NP_415430.1, nucleoside diphosphate kinase, and MdtH were NP_415583.4. Codon optimization was performed on anthocyanin synthase, UDP-glucosyltransferase, UDP-rhamnosyltransferase, and UDP-rhamnosyltransferase based on E. coli codon bias. Their gene sequences were then optimized according to this bias, and related gene fragments were synthesized. The remaining genes were directly amplified from the E. coli genome.

[0037] Genes of UDP-rhamnosyltransferase, anthocyanin synthase, UDP-glucosyltransferase, and rhamnosyl synthase from different sources were amplified and inserted into vector pET28a using the Gibson assembly method. The specific construction is as follows: CsRhat, AtRhat, Ph3RT, and LeRhat were amplified using primers P1 / P2, P3 / P4, P5 / P6, and P7 / P8, respectively; VvRHM was amplified using primers P11 / P12; VvANS was amplified using primers P13 / P14; and Vc3GT was amplified using primers P15 / 16. Fragments CsRhat and VvRHM were amplified using primers P1 / P11 to obtain CsRhat-VvRHM; fragments AtRhat and VvRHM were amplified using primers P3 / P11 to obtain AtRhat-VvRHM; fragments Ph3RT and VvRHM were amplified using primers P5 / P11 to obtain Ph3RT-VvRHM; and fragments LeRhat and VvRHM were amplified using primers P7 / P11 to obtain LeRhat-VvRHM. The pET28a backbone was amplified using primers P9 / P19 and recombinated with fragment CsRhat-VvRHM to obtain the recombinant vector pET28a-T7-CsRhat-VvRHM. The pET28a backbone was amplified using primers P9 / P21 and recombinated with fragment AtRhat-VvRHM to obtain the recombinant vector pET28a-T7-AtRhat-VvRHM. The pET28a backbone was amplified using primers P9 / P20 and recombinated with the Ph3RT-VvRHM fragment to obtain the recombinant vector pET28a-T7-Ph3RT-VvRHM. The pET28a backbone was also amplified using primers P9 / P18 and recombinated with the LeRhat-VvRHM fragment to obtain the recombinant vector pET28a-T7-LeRhat-VvRHM. Using primers P11 / P39, fragments T7-CsRhat-VvRHM, T7-AtRhat-VvRHM, T7-Ph3RT-VvRHM, and T7-LeRhat-VvRHM were amplified using pET28a-T7-CsRhat-VvRHM, pET28a-T7-AtRhat-VvRHM, T7-Ph3RT-VvRHM, and T7-LeRhat-VvRHM as templates. The pET28a backbone was then amplified using primers P9 / P17 and recombined with VvANS and Vc3GT to obtain the recombinant vector pET28a-T7-VvANS-Vc3GT.Using primers P9 / P10, pET28a-T7-VvANS-Vc3GT was amplified as a template to obtain the linearized vector pET28a-T7-VvANS-Vc3GT backbone. Subsequently, it was recombinated with fragments CsRhat-VvRHM, AtRhat-VvRHM, Ph3RT-VvRHM, and LeRhat-VvRHM to obtain the desired results. Figure 1-4 The recombinant vectors shown are pET28a-T7-VvANS-Vc3GT-Ph3RT-VvRHM, pET28a-T7-VvANS-Vc3GT-CsRhat-VvRHM, pET28a-T7-VvANS-Vc3GT-LeRhat-VvRHM, and pET28a-T7-VvANS-Vc3GT-AtRhat-VvRHM.

[0038] The genes for UTP glucose-1-phosphouridine transferase (Gene ID: 945730), phosphoglucose mutase (Gene ID: 945271), cytidine kinase (Gene ID: 945535), and nucleoside diphosphate kinase (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 P22 / P23, Ecpgm using primers P24 / P25, Eccmk using primers P26 / P27, and Ecndk using primers P28 / P29. The EcgalU and Ecpgm fragments were then extended using primers P22 / P25 to obtain the EcgalU-Ecpgm fragment, and the Eccmk and Ecndk fragments were extended using primers P26 / P29 to obtain the Eccmk-Ecndk fragment. The pACYCDuet-1 backbone was amplified with primers P30 / 31, and the recombination of the Eccmk-Ecndk fragment was amplified to obtain the recombinant expression vector pACYCDuet-T7-Eccmk-Ecndk. Using primers P29 / 32, pACYCDuet-T7-Eccmk-Ecndk was amplified as a template to obtain the T7-Eccmk-Ecndk fragment. The pACYCDuet-1 backbone was amplified with primers P30 / P37, and the recombination of the EcgalU-Ecpgm and T7-Eccmk-Ecndk fragments was amplified to obtain the following results: Figure 5The 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-AtRhat-VvRHM, pET28a-T7-VvANS-Vc3GT-LeRhat-VvRHM, pET28a-T7-VvANS-Vc3GT-CsRhat-VvRHM, and pET28a-T7-VvANS-Vc3GT-Ph3RT-VvRHM, respectively, and transformed into *E. coli* BL21(DE3) using a CaCl2-treated *E. coli* chemical transformation method to obtain recombinant expression strains C3R-F1, C3R-F2, C3R-F3, and C3R-F4.

[0039] Optimized codon sequences of UDP-glucosyltransferases from different sources were amplified along with the anthocyanin synthase, UDP-rhamnosyltransferase, and UDP-rhamnosyltransferase genes, and inserted into the vector pET28a using the Gibson assembly method. The specific construction is as follows: Ih3GT was amplified using primers P33 / P34. Using primers P9 / P14, pET28a-T7-VvANS-Vc3GT was amplified as a template to obtain the linearized vector pET28a-T7-VvANS backbone, which was then recombinated with IhGT to obtain the desired result. Figure 6 The recombinant expression vector shown is pET28a-T7-VvANS-Ih3GT.

[0040] The membrane transport protein MdtH (Gene ID: 946920) was amplified and used Gi... b The pACYCDuet-1 vector was inserted using the Son assembly method. The specific construction is as follows: The pACYCDuet backbone was amplified using primers P35 / P38 and recombined with MdtH to obtain the desired result. Figure 7 The recombinant vector pACYCDuet-T7-EcMdtH is shown.

[0041] The UDP rhamnosyltransferase and rhamnosyl synthase genes were amplified and inserted into the vector pCDFDuet-1 using the Gibson assembly method. The specific construction is as follows: Using primers P11 / P39, the fragment T7-LeRhat-VvRHM was amplified using pET28a-T7-VvANS-Vc3GT-LeRhat-VvRHM as a template. The pCDFDuet backbone was then amplified using primers P9 / P36 to recombine with the T7-LeRhat-VvRHM fragment, resulting in the following... Figure 8The recombinant vector pCDFDuet-T7-LeRhat-VvRHM is shown. The above recombinant vector, along with pET28a-T7-VvANS-Ih3GT and pACYCDuet-T7-EcMdtH, were co-transformed into E. coli BL21(DE3) using a CaCl2-treated E. coli chemical transformation method to obtain recombinant E. coli C3R-F5.

[0042] Example 2: Analysis of the Synthetic Ability of Recombinant Escherichia coli Cyanide 3-O-Rutinoside 1. This embodiment uses the recombinant Escherichia coli C3R-F1, C3R-F2, C3R-F3, and C3R-F4 prepared in Example 1 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. 600 When 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 was 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.

[0043] The catalytic reaction solution used 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 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.

[0044] 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.

[0045] Qualitative analysis of the synthesized product was performed using liquid chromatography and liquid chromatography-mass spectrometry, and the results are as follows: Figure 9As shown, recombinant Escherichia coli can synthesize cyanidin 3-O-rutin using (+)-catechin as a substrate. Figure 10 As shown, the catalytic activities of UDP rhamnosyltransferases from different sources are different. Among them, the UDP rhamnosyltransferase from Arabidopsis thaliana has no ability to synthesize cyanidin-3-O-rutin, while the UDP rhamnosyltransferase from Lobelia chinensis significantly increased the yield of cyanidin-3-O-rutin, reaching 97.15 mg / L.

[0046] Example 3: Analysis of cyanidin 3-O-rutin yield in recombinant Escherichia coli under different amounts of catechin and glucose addition. 1. In this embodiment, the recombinant Escherichia coli C3R-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. The 18-h 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 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 11 As shown, the yield of cyanidin 3-O-rutinoside varied significantly with different amounts of catechin and glucose added. With increasing catechin addition, the yield of cyanidin 3-O-rutinoside initially increased and then decreased, reaching a maximum of 348.93 mg / L at a concentration of 3 mM, representing a 9% increase compared to before catechin addition optimization. Similarly, with increasing glucose addition, the yield of cyanidin 3-O-rutinoside also showed an initial increase followed by a decrease, reaching a maximum of 424.11 mg / L at a glucose addition of 3%, representing a 22% increase compared to before glucose addition optimization. Therefore, optimizing the amounts of catechin and glucose significantly improved the yield of cyanidin 3-O-rutinoside.

[0049] Example 4: Analysis of the yield of cyanidin 3-O-rutin from recombinant Escherichia coli at different catalytic temperatures. 1. In this embodiment, the recombinant Escherichia coli C3R-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 performed by adding 1 mM IPTG at a final concentration when the concentration was 0.6–0.8. The culture was incubated at 20°C and 200 rpm for 18 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 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 culture was incubated at 18°C, 20°C, 25°C, and 30°C at 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 12 As shown, catalytic temperature has a significant impact on the yield of cyanidin 3-O-rutin from recombinant Escherichia coli. Optimizing the catalytic temperature can further increase the yield. With increasing catalytic temperature, the yield of cyanidin 3-O-rutin first increases and then decreases, reaching its highest value of 529.01 mg / L at 25℃, which is 1.25 times that before catalytic temperature optimization.

Claims

1. A genetically engineered bacterium for producing cornflower-3-O-rutin, 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, UDP rhamnosyltransferase encoding gene, UDP rhamnosyl synthase encoding gene and membrane transport protein encoding gene MdtH; (2) Anthocyanin synthase encoding gene, UDP glucose transferase encoding gene, UDP rhamnosyltransferase encoding gene, UDP rhamnosyl synthase 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-rutin 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 ) or Dutch iris ( Iris hollandica ), The UDP-rhamnosyltransferase was derived from petunia ( Petunia hybrida Arabidopsis thaliana ( ) Arabidopsis thaliana ), sweet orange ( Citrus sinensis ) and Lobelia chinensis ( Lobelia erinus )one, The UDP-rhamnosyl synthase is derived from grapes ( Vitis vinifera ), The UTP glucose-1-phosphourydyltransferase, phosphoglucose mutase, cytidine kinase, nucleoside diphosphate kinase, and membrane transport protein MdtH are derived from *Escherichia coli*. Escherichia coli ).

3. The genetically engineered bacterium for producing cyanidin-3-O-rutin according to claim 2, characterized in that, The anthocyanin synthase encoding gene, UDP glucosyltransferase encoding gene, UDP rhamnosyltransferase encoding gene, and UDP rhamnosyltransferase encoding gene were optimized according to the codon preference of Escherichia coli.

4. The genetically engineered bacterium for producing cyanidin-3-O-rutin according to claim 2, characterized in that, The UDP glucosyltransferase has either the GenBank number AYC35394.1 or BAD83701.

1. The anthocyanin synthase has the GenBank accession number NP_001268147.

1. The GenBank accession number for the UDP rhamnosyltransferase is one of ABA18631.1, AEE85357.1, BAU68119.1, or CAA50376.

1. The GenBank accession number for the UDP rhamnosyl synthase is XP_002285634.

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 transport protein MdtH is NP_415583.

4.

5. The genetically engineered bacterium for producing cyanidin-3-O-rutin 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-rutin as described in any one of claims 1 to 5, characterized in that, The imported genes include anthocyanin synthase encoding gene, UDP glucose transferase encoding gene, UDP rhamnosyltransferase encoding gene, UDP rhamnosyl synthase encoding gene, and membrane transport protein encoding gene MdtH. The construction method includes the following steps: The sequences of the anthocyanin synthase encoding gene and the UDP glucosyltransferase encoding gene were cloned into an expression vector to obtain recombinant expression vector 1; The sequences of the UDP rhamnosyltransferase encoding gene and the UDP rhamnosyl synthase encoding gene were cloned into an expression vector to obtain recombinant vector 2; The sequence of the membrane transport protein encoding gene MdtH was cloned into an expression vector to obtain recombinant expression vector 3; The recombinant expression vector 1 in step (1), the recombinant expression vector 2 in step (2), and the recombinant expression vector 3 in step (3) are all transformed into Escherichia coli to obtain the genetically engineered bacteria that produce cyanidin 3-O-rutin.

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

8. A method for producing cornflower 3-O-rutin, 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-rutin glycoside is extracted.

9. The method for producing cornflower 3-O-rutin 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 value was 0.6 or 0.

8.

10. The method for producing cornflower 3-O-rutin according to claim 9, characterized in that, The bacterial cells were collected after induction at 20℃ for 18 hours, and the collected bacterial cells were resuspended in the catalytic reaction solution and catalyzed at 18-30℃ for 24 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 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.

Images