A recombinant Escherichia coli for producing curcumin glucoside and its application

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to a recombinant Escherichia coli for producing curcumin glucoside and its application. The present invention uses Escherichia coli BL21(DE3) as the host cell, and transfers the double mutant of the polyphenol TDP-rhamnosyltransferase ppuG gene derived from Bacillus subtilis 168, the glucose-6-phosphate mutase Pgm gene and the UDP-glucose pyrophosphorylase GalU gene derived from Escherichia coli MG1655 into the Escherichia coli host cell. The double mutant of the polyphenol TDP-rhamnosyltransferase ppuG gene contains two mutation sites, G51A and Q345R. The recombinant Escherichia coli constructed by the present invention can efficiently produce curcumin diglucoside, and the yield can reach 7.10 g / L, which not only improves the water solubility and bioavailability of curcumin, but also provides strong support for the industrial production of curcumin glucoside.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a recombinant Escherichia coli for producing curcumin glucoside and its application. Background Art

[0002] The molecular formula of curcumin is C 21 H 20 O 6 , with a molecular weight of 368.38. It is a bioactive natural polyphenolic compound extracted from the rhizomes of some plants such as Zingiberaceae and Araceae, and its state is generally an orange crystalline powder. Curcumin has various effects and functions such as anti-inflammatory, antioxidant, anti-cancer, antibacterial, anti-malaria, liver and gallbladder protection, and lipid-lowering, and has wide applications in the fields of food additives, pharmaceuticals, cosmetics, etc. However, curcumin has characteristics such as high lipophilicity, insoluble in water, poor bioavailability, low cell uptake rate, and poor stability, which limit its pharmacological development and practical application. Therefore, finding a suitable method to synthesize new curcumin analogs to improve the pharmacological characteristics of natural compounds has extremely important scientific value and broad market application potential for the development of curcumin in various fields.

[0003] Modifying the structure of curcumin by microbial transformation method to improve its water solubility is one of the hot research contents at present. At present, the molecular structure modification of curcumin mainly includes glucosylation reaction, demethoxylation reaction and connection of hydrophilic groups on phenolic hydroxyl groups. These modifications can convert curcumin into curcumin analogs or derivatives, and can improve its solubility while retaining certain biological activities. Among them, glycosylation is a powerful method to produce compounds with higher water solubility and / or enhanced biological activity. Glycosyl Transferases (GTs) can catalyze the transfer of sugar groups from uridine diphosphate sugars (UDP sugars) to low molecular weight receptor substrates. They can strictly control regio- and stereoselectivity and high catalytic efficiency, making them potential biocatalysts in glycoside synthesis.

[0004] Patent document CN113322219A discloses a method for biocatalytic synthesis of curcumin glycoside compounds. In this method, the glycosyltransferase gene CaUGT2 and the sucrose synthase gene AtSUS1 are constructed onto an expression vector and introduced into Escherichia coli to obtain a recombinant strain. After induced expression of this recombinant strain, its soluble expression level is higher than that of other plant-derived glycosyltransferases, and it can efficiently catalyze the substrate curcumin to generate curcumin monoglycoside and curcumin diglycoside, whose water solubility is better than that of curcumin, solving the problem of poor water solubility of curcumin.

[0005] Patent document CN115975900A discloses the construction of a genetically engineered bacterium for synthesizing curcumin glucoside and its application. The construction method uses Escherichia coli BL21(DE3) as the engineering strain, overexpresses two key enzymes involved in UDP-glucose synthesis in Escherichia coli, namely glucose-6-phosphate mutase and UDP-glucose pyrophosphorylase, to increase the endogenous UDP-glucose level in the cells, effectively improving the synthesis efficiency of curcumin glucoside by whole-cell catalysis; at the same time, overexpresses Madagascar periwinkle the UDP-glucosyltransferase CaUGT2 derived from

[0006] This genetically engineered bacterium can efficiently synthesize curcumin monoglucoside and curcumin diglucoside, effectively solving the solubility problem of curcumin. Summary of the Invention

[0007] In order to overcome the deficiencies in the prior art, the present invention provides an Escherichia coli engineering bacterium for efficiently producing curcumin glucoside. The present invention uses Escherichia coli BL21(DE3) as the host cell, which can express a double mutant of the polyphenol TDP-rhamnosyltransferase ppuG gene derived from Bacillus subtilis 168 ( Bacillus subtilis 168), overexpresses the glucose-6-phosphate mutase Pgm gene and UDP-glucose pyrophosphorylase GalU gene derived from Escherichia coli MG1655 ( Escherichia coli MG1655). The double mutant of the polyphenol TDP-rhamnosyltransferase ppuG gene contains two mutation sites, G51A and Q345R. The constructed engineering strain can achieve the efficient production of curcumin glucoside by the microbial method, thereby improving the water solubility and bioavailability of curcumin.

[0008] In order to find a glycosyltransferase that can efficiently catalyze the reaction of curcumin with UDP-glucose to produce curcumin glucoside, the present invention first screened a variety of glycosyltransferases from different sources. These sources include Madagascar periwinkle roseus 、 Bacillus subtilis 168、 Lactobacillus zeae and other microorganisms. The specific steps are as follows:

[0009] Step S1: Obtain the glycosyltransferase gene sequences from the above-mentioned microbial sources in the NCBI database, including UDP-glucosyltransferases (CaUGT2, GT2, GH1), steroid glycosyltransferases (CaUGT74An3), and polyphenol TDP-rhamnosyltransferases (ppuG), etc.

[0010] Step S2: Synthesize the obtained gene sequences into genes and construct them onto corresponding expression vectors, such as pgex-4t-1, pMAL-c5X, pET-28a(+), etc.

[0011] Step S3: Transform the constructed expression vector into Escherichia coli E. coli BL21(DE3), and induce expression through IPTG to obtain a genetically engineered bacterium expressing glycosyltransferase.

[0012] Step S4: React the above-mentioned genetically engineered bacterium with UDP-glucose and curcumin respectively, and detect and analyze the reaction products by HPLC and mass spectrometry to evaluate the catalytic performance of each glycosyltransferase.

[0013] Finally, it was determined that Bacillus subtilis the polyphenol TDP-rhamnosyltransferase (ppuG) from 168 has the best catalytic performance for UDP-glucose and curcumin, and can efficiently generate curcumin glucoside.

[0014] In order to further improve the catalytic performance of ppuG, the present invention conducted site-directed mutagenesis research on its gene. The specific steps are as follows:

[0015] Step S1: Use a consensus finder to search for 1000 homologous amino acid sequences of ppuG, and through multiple sequence alignment analysis, identify amino acid sites that are conserved in most sequences.

[0016] Step S2: Based on the results of sequence identity analysis, screen out amino acid sites with higher conservation as potential mutation sites, and a total of 17 mutation sites were determined.

[0017] Step S3: Use site-directed mutagenesis technology to design corresponding mutation primers for each screened mutation site, and perform PCR amplification and gene recombination to construct 17 ppuG single mutants.

[0018] Step S4: React each mutant with UDP-glucose and curcumin, and detect its catalytic performance. Through comparison, it was found that two mutants, ppuG / Q345R and ppuG / G51A, showed better catalytic performance.

[0019] Step S5: Based on the experimental results of the single mutants, further perform double mutations at positions 51 and 345 of ppuG to construct the ppuG / G51A / Q345R double mutant. The experimental results showed that this double mutant has the best catalytic performance and can significantly increase the yield of curcumin glucoside.

[0020] In order to further improve the ability of recombinant Escherichia coli to produce curcumin glucoside, the present invention also overexpresses phosphoglucomutase (Pgm) and UDP-glucose pyrophosphorylase (GalU). These two enzymes can increase the level of UDP-glucose in the cell, thereby providing more glycosyl donors for glycosyltransferases.

[0021] Specifically, Pgm catalyzes the interconversion between glucose-6-phosphate and glucose-1-phosphate and is a key enzyme in glycolysis and gluconeogenesis. By overexpressing Pgm, the conversion efficiency of glucose-6-phosphate in the cell can be increased, thus providing more raw materials for the synthesis of UDP-glucose. GalU catalyzes the reaction of uridine diphosphate (UDP) and glucose-1-phosphate to produce UDP-glucose and pyrophosphate. UDP-glucose is the glycosyl donor required for glycosyltransferases to carry out glycosylation reactions. By overexpressing GalU, the production amount of UDP-glucose in the cell can be directly increased, providing more substrates for glycosyltransferases.

[0022] The specific technical solution of the present invention is as follows:

[0023] The present invention provides a recombinant Escherichia coli for producing curcumin glucoside. The recombinant Escherichia coli BL21(DE3)CBG06 ( Escherichia coli BL21(DE3)CBG06) was deposited at the China Center for Type Culture Collection on December 20, 2024, and the deposit number is: CCTCC NO: M 20242869.

[0024] Furthermore, the recombinant Escherichia coli BL21(DE3)CBG06 is obtained by transferring the double mutant of the polyphenol TDP-rhamnosyltransferase ppuG gene shown in SEQ ID NO.1 derived from Bacillus subtilis 168 ( Bacillus subtilis 168), the phosphoglucomutase Pgm gene shown in SEQ ID NO.2 derived from Escherichia coli MG1655 ( Escherichia coli MG1655), and the UDP-glucose pyrophosphorylase GalU gene shown in SEQ ID NO.3 derived from Escherichia coli MG1655 ( Escherichia coli MG1655) into an Escherichia coli host cell.

[0025] Furthermore, the double mutant of the polyphenol TDP-rhamnosyltransferase ppuG gene contains two mutation sites, G51A and Q345R.

[0026] Further, the amino acid sequence of the protein encoded by the double mutant of the polyphenol TDP - rhamnosyltransferase ppuG gene is shown in SEQ ID NO.4; the amino acid sequence of the protein encoded by the glucose - 6 - phosphomutase Pgm gene is shown in SEQ ID NO.5; the amino acid sequence of the protein encoded by the UDP - glucose pyrophosphorylase GalU gene is shown in SEQ ID NO.6.

[0027] Further, the recombinant Escherichia coli BL21(DE3)CBG06 contains at least two recombinant vectors, and the recombinant vectors include but are not limited to pgex - 4t - 1, pMAL - c5X, pET - 28a(+), or pACYCDuet - 1.

[0028] Further, the recombinant Escherichia coli BL21(DE3)CBG06 contains two recombinant vectors, one of which contains the glucose - 6 - phosphomutase Pgm gene and the UDP - glucose pyrophosphorylase GalU gene, and the other recombinant vector contains the double mutant of the polyphenol TDP - rhamnosyltransferase ppuG gene.

[0029] Further, the present invention also provides a method for constructing the recombinant Escherichia coli for producing curcumin glucoside, including the following steps:

[0030] Step S1: Construct a recombinant vector containing the glucose - 6 - phosphomutase Pgm gene and the UDP - glucose pyrophosphorylase GalU gene;

[0031] Step S2: Construct a recombinant vector containing the double mutant of the polyphenol TDP - rhamnosyltransferase ppuG gene;

[0032] Step S3: Co - transform the recombinant vector obtained in Step S1 and the recombinant vector obtained in Step S2 into Escherichia coli to obtain the recombinant Escherichia coli.

[0033] In addition, the present invention also provides a method for producing curcumin glucoside. Using the above - mentioned recombinant Escherichia coli BL21(DE3)CBG06 for producing curcumin glucoside as the fermentation strain, curcumin as the substrate, and glucose as the glycosyl donor, culturing until the cell concentration OD600 = 5 - 10 at a temperature of 35 - 40 °C, adding an IPTG inducer, and catalytically synthesizing curcumin diglucoside to obtain curcumin glucoside.

[0034] Furthermore, the fermentation conditions are as follows: the fermentation temperature is 35 - 40 °C, the rotation speed is 120 - 650 rpm, the aeration rate is 0.7 - 1.5 VVM, the dissolved oxygen is controlled between 25 - 35%, the pH is controlled between 6 - 8, the final concentration of IPTG inducer is 0.5 - 1.5 mmol / L, the curcumin concentration is 0.2 - 4 g / L, and fermentation continues for 0 - 26 h after adding the inducer.

[0035] In addition, the present invention also claims the use of the recombinant Escherichia coli BL21(DE3)CBG06 for producing curcumin glucoside in the preparation of curcumin glucoside or products containing curcumin glucoside.

[0036] Furthermore, the product containing curcumin glucoside is a food additive, medicine or cosmetic containing curcumin glucoside.

[0037] The implementation steps of the specific overexpression strategy provided by the present invention are as follows:

[0038] Step S1: Clone the gene sequences of Pgm and GalU from the E. coli MG1655 strain. Then, construct these gene sequences onto an expression vector, such as pACYCDuet-1, for efficient expression in Escherichia coli.

[0039] Step S2: Co-transform the expression vector pACYCDuet-1-Pgm-GalU containing the Pgm and GalU genes and the expression vector pET28a(+)-ppuG / G51A / Q345R containing the ppuG gene into Escherichia coli E. coli BL21(DE3) competent cells. Through screening and verification, obtain the recombinant strain E. coli BL21(DE3)CBG06 that can simultaneously express these three enzymes.

[0040] Step S3: During the cultivation of the recombinant strain E. coli BL21(DE3)CBG06, optimize the growth and metabolic environment of the recombinant Escherichia coli by adjusting fermentation conditions such as medium composition, pH value, temperature, dissolved oxygen amount, etc., to improve the synthesis efficiency of UDP-glucose and the yield of curcumin glucoside.

[0041] Finally, the present invention successfully constructed a recombinant Escherichia coli BL21(DE3)CBG06 ( Escherichia coli BL21(DE3)CBG06) that can efficiently produce curcumin glucoside. Under suitable fermentation conditions, this strain can produce a high yield of curcumin diglucoside, with a yield reaching 7.10 g / L, which can fully meet the industrial demand.

[0042] In summary, compared with the prior art, the recombinant Escherichia coli for producing curcumin glucoside provided by the present invention has the following advantages:

[0043] (1) The recombinant Escherichia coli for producing curcumin glucoside provided by the present invention uses curcumin as a substrate and glucose as a glycosyl donor. After fermentation in a 5 L fermenter at 35 - 40 °C for 23 h, the yield of curcumin glucoside can reach 7.10 g / L, having the advantages of short fermentation time and high yield, meeting the requirements of industrial production.

[0044] (2) The curcumin glucoside produced by fermentation using the recombinant Escherichia coli provided by the present invention has good water solubility and bioavailability, providing the possibility for the wide application of curcumin in the fields of food additives, pharmaceuticals, cosmetics, etc., and helping to promote the research and development and market expansion of curcumin-related products.

[0045] Microbial information of the recombinant Escherichia coli BL21(DE3) CBG06 for producing curcumin glucoside provided by the present invention: The recombinant Escherichia coli BL21(DE3)CBG06 ( Escherichia coli BL21(DE3)CBG06) was deposited at the China Center for Type Culture Collection on December 20, 2024, with the deposit number: CCTCC NO: M 20242869, and the deposit address: Wuhan University, Wuhan, China, Zip Code: 430072. Description of the Drawings

[0046] Figure 1 It is a protein expression result diagram of different engineering bacteria CDG01, CDG02, CDG03, CDG04 and CDG05.

[0047] Figure 2 It is a high performance liquid chromatography detection result diagram of the engineering bacteria CDG03.

[0048] Figure 3 It is a detection result diagram of the catalytic performance of different engineering bacteria CDG01, CDG02, CDG03, CDG04 and CDG05 on UDP-glucose and curcumin.

[0049] Figure 4 It is a detection result diagram of the catalytic performance of the ppuG single mutant on UDP-glucose and curcumin.

[0050] Figure 5 It is a content detection result diagram of the production of curcumin diglucoside by the recombinant Escherichia coli BL21(DE3)CBG06. Detailed Embodiments

[0051] The present invention will be further described below through specific embodiments. However, this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of the present invention. As long as they do not depart from the basic idea of the present invention, they are within the scope of the present invention.

[0052] I. Strains and primers involved in the present invention

[0053] The strains and primers involved in Examples 1 to 5 of the present invention are shown in Tables 1 and 2.

[0054] Table 1 Strains involved in Examples 1 to 5

[0055]

[0056] Table 2 Primer sequences used in Examples 1 to 5

[0057] Primer name Sequence (5′-3′) Pgm-F CTTTAATAAGGAGATATAATGGCAATCCACAATCGTGCAGG Pgm-R GACTTAAGCATTATTTACGCGTTTTTCAGAACTTCGC GalU-F GGAGATATAATGGCTGCCATTAATACGAAAGTC GalU-R GCGGTTTCTTTACCAGATTACTTCTTAATGCCCATCTCTTCTTCAAG

[0058] II. The culture media involved in Examples 1 to 5 of the present invention are as follows:

[0059] (1) LB culture medium formula: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0. For solid culture medium, add 1.5% - 2% (mass ratio) of agar powder.

[0060] (2) Fermentation medium:

[0061] Fermentation medium formula: 10 g / L glucose, 13.4 g / L potassium dihydrogen phosphate, 4 g / L ammonium sulfate, 1.8 g / L citric acid, 0.6 g / L magnesium sulfate heptahydrate, 300 μL / L antifoaming agent, 10 mL / L trace element solution.

[0062] The formula of the trace metal solution: 0.84 g / L ethylenediaminetetraacetic acid (EDTA), 0.25 g / L cobalt chloride hexahydrate (CoCl 2 -6H 2 O), 1.50 g / L manganese chloride tetrahydrate (MnCl 2 -4H 2 O), 0.15 g / L copper chloride dihydrate (CuCl 2 -2H 2 O), 0.30 g / L boric acid (H 3 BO 3 ), 0.25 g / L sodium molybdate dihydrate (Na 2 MoO 4 -2H 2 O), 1.30 g / L zinc acetate dihydrate [Zn(CH 3 COO)2 -2H 2 O], 10.0 g / L ammonium ferric citrate.

[0063] III. The nucleotide sequences and amino acid sequences involved in Examples 1-5 of the present invention are as follows:

[0064] (1) The nucleotide sequence of the double mutant of the polyphenol TDP - rhamnosyltransferase ppuG gene derived from Bacillus subtilis 168 ( Bacillus subtilis 168) is (SEQ ID NO.1):

[0065]

[0066] (2) The nucleotide sequence of the phosphoglucomutase Pgm gene derived from Escherichia coli MG1655 ( Escherichia coli MG1655) is (SEQ ID NO.2):

[0067]

[0068] (3) The nucleotide sequence of the UDP-glucose pyrophosphorylase GalU gene derived from Escherichia coli MG1655 ( Escherichia coli MG1655) is (SEQ ID NO.3):

[0069] ATGGCTGCCATTAATACGAAAGTCAAAAAAGCCGTTATCCCCGTTGCGGGATTAGGAACCAGGATGTTGCCGGCGACGAAAGCCATCCCGAAAGAGATGCTGCCACTTGTCGATAAGCCATTAATTCAATACGTCGTGAATGAATGTATTGCGGCTGGCATTACTGAAATTGTGCTGGTTACACACTCATCTAAAAACTCTATTGAAAACCACTTTGATACCAGTTTTGAACTGGAAGCAATGCTGGAAAAACGTGTAAAACGTCAACTGCTTGATGAAGTGCAGTCTATTTGTCCACCGCACGTGACTATTATGCAAGTTCGTCAGGGTCTGGCGAAAGGCCTGGGACACGCGGTATTGTGTGCTCACCCGGTAGTGGGTGATGAACCGGTAGCTGTTATTTTGCCTGATGTTATTCTGGATGAATATGAATCCGATTTGTCACAGGATAACCTGGCAGAGATGATCCGCCGCTTTGATGAAACGGGTCATAGCCAGATCATGGTTGAACCGGTTGCTGATGTGACCGCATATGGCGTTGTGGATTGCAAAGGCGTTGAATTAGCGCCGGGTGAAAGCGTACCGATGGTTGGTGTGGTAGAAAAACCGAAAGCGGATGTTGCGCCGTCTAATCTCGCTATTGTGGGTCGTTACGTACTTAGCGCGGATATTTGGCCGTTGCTGGCAAAAACCCCTCCGGGAGCTGGTGATGAAATTCAGCTCACCGACGCAATTGATATGCTGATCGAAAAAGAAACGGTGGAAGCCTATCATATGAAAGGGAAGAGCCATGACTGCGGTAATAAATTAGGTTACATGCAGGCCTTCGTTGAATACGGTATTCGTCATAACACCCTTGGCACGGAATTTAAAGCCTGGCTTGAAGAAGAGATGGGCATTAAGAAGTAA。

[0070] (4)The amino acid sequence of the protein encoded by the double mutant of the polyphenol TDP - rhamnosyltransferase ppuG gene is (SEQ ID NO.4):

[0071] MKKYHISMINIPAYGHVNPTLALVEKLCEKGHRVTYATTEEFAPAVQQAGAEALIYHTSLNIDPKQIREMMEKNDAPLSLLKESLSILPQLEELYKDDQPDLIIYDFVALAGKLFAEKLNVPVIKLCSSYAQNESFQLGNEDMLKKIREAEAEFKAYLEQEKLPAVSFEQLAVPEALNIVFMPKSFQIQHETFDDRFCFVGPSLGERKEKESLLIDKDDRPLMLISLGTAFNAWPEFYKMCIKAFRDSSWQVIMSVGKTIDPESLEDIPANFTIRQSVPQLEVLEKADLFISHGGMNSTMEAMNAGVPLVVIPQMYEQELTANRVDELGLGVYLPKEEVTVSSLREAVQAVSSDQELLSRVKNMQKDVKEAGGAERAAAEIEAFMKKSAVPQ*。

[0072] (5)The amino acid sequence of the protein encoded by the glucose-6-phosphate mutase Pgm gene is (SEQ ID NO.5):

[0073] MAIHNRAGQPAQQSDLINVAQLTAQYYVLKPEAGNAEHAVKFGTSGHRGSAARHSFNEPHILAIAQAIAEERAKNGITGPCYVGKDTHALSEPAFISVLEVLAANGVDVIVQENNGFTPTPAVSNAILVHNKKGGPLADGIVITPSHNPPEDGGIKYNPPNGGPADTNVTKVVEDRANALLADGLKGVKRISLDEAMASGHVKEQDLVQPFVEGLADIVDMAAIQKAGLTLGVDPLGGSGIEYWKRIGEYYNLNLTIVNDQVDQTFRFMHLDKDGAIRMDCSSECAMAGLLALRDKFDLAFANDPDYDRHGIVTPAGLMNPNHYLAVAINYLFQHRPQWGKDVAVGKTLVSSAMIDRVVNDLGRKLVEVPVGFKWFVDGLFDGSFGFGGEESAGASFLRFDGTPWSTDKDGIIMCLLAAEITAVTGKNPQEHYNELAKRFGAPSYNRLQAAATSAQKAALSKLSPEMVSASTLAGDPITARLTAAPGNGASIGGLKVMTDNGWFAARPSGTEDAYKIYCESFLGEEHRKQIEKEAVEIVSEVLKNA*。

[0074] (6)The amino acid sequence of the protein encoded by the UDP - glucose pyrophosphorylase GalU gene is (SEQ ID NO.6):

[0075] MAAINTKVKKAVIPVAGLGTRMLPATKAIPKEMLPLVDKPLIQYVVNECIAAGITEIVLVTHSSKNSIENHFDTSFELEAMLEKRVKRQLLDEVQSICPPHVTIMQVRQGLAKGLGHAVLCAHPVVGDEPVAVILPDVILDEYESDLSQDNLAEMIRRFDETGHSQIMVEPVADVTAYGVVDCKGVELAPGESVPMVGVVEKPKADVAPSNLAIVGRYVLSADIWPLLAKTPPGAGDEIQLTDAIDMLIEKETVEAYHMKGKSHDCGNKLGYMQAFVEYGIRHNTLGTEFKAWLEEEMGIKK*。

[0076] Example 1: Screening of glycosyltransferase strains from different sources

[0077] 1. Recombinant plasmid construction:

[0078] Obtained from the NCBI database Madagascar periwinkle UDP - glucosyltransferase (CaUGT2, GenBank: BAD29722.1), steroid glucosyltransferase (CaUGT74An3, GenBank: AYC35244.1) from Bacillus subtilis Polyphenol TDP - rhamnosyltransferase (ppuG, GenBank: NP_389104.1) from 168 Bacillus subtilis UDP - glucosyltransferase (GT2, GenBank: QAT94473.1) from ATCC 6633 and Lactobacillus zeae The gene sequences of GH1 (GenBank: KRK12807.1). These gene sequences were sent to Beijing Tsingke Biotechnology Co., Ltd. for gene synthesis and were respectively constructed into pgex - 4t - 1, pMAL - c5X, pET - 28a(+), pET - 28a(+) and pET - 28a(+) vectors to obtain recombinant plasmids pgex - 4t - 1 - CaUGT2, recombinant plasmid pMAL - c5X - CaUGT74An3, recombinant plasmid pET - 28a(+) - ppuG, recombinant plasmid pET - 28a(+) - GT2, recombinant plasmid pET - 28a(+) - GH1.

[0079] 2. Engineering strain construction:

[0080] The recombinant plasmids pgex - 4t - 1 - CaUGT2, pMAL - c5X - CaUGT74An3, pET - 28a(+) - ppuG, pET - 28a(+) - GT2, pET - 28a(+) - GH1 were respectively transferred into Escherichia coli DH5α by chemical transformation method. Then the plasmids were extracted from the transformed Escherichia coli DH5α and transformed into E. coli BL21(DE3). Finally, the genetically engineered bacteria CDG01, CDG02, CDG03, CDG04, CDG05 expressing different glycosyltransferases (GTs) were obtained. The specific engineering bacteria are: CDG01 ( E. coli BL21(DE3) pgex - 4t - 1 - CaUGT2), CDG02 ( E. coli BL21(DE3) pMAL - c5X - CaUGT74An3), CDG03 ( E. coli BL21(DE3)pET - 28a(+) - ppuG), CDG04 (E. coli BL21(DE3) pET-28a(+)-GT2), CDG05 ( E. coli BL21(DE3) pET-28a(+)-GH1).

[0081] 3. Engineering strain fermentation:

[0082] Pick single colonies from the transformed E. coli BL21(DE3) plate, inoculate them into 5 mL of LB liquid medium containing Amp (ampicillin, 100 μg / mL) or Kan (kanamycin, 50 μg / mL), and culture overnight at 37°C and 180 rpm. Inoculate the seed liquid into 200 mL of LB liquid medium containing Amp (100 μg / mL) or Kan (50 μg / mL) at an inoculation amount of 1% (V / V), and culture at 37°C and 180 rpm. Culture for about 3 - 4 h until the OD 600 value reaches 0.6 - 0.8, then add IPTG with a final concentration of 1 mmol / L and induce overnight at 25°C and 150 rpm.

[0083] Centrifuge the induced bacterial liquid at 4°C and 12,000 rpm for 10 min to collect the bacterial cells. After washing the bacterial cells once with physiological saline (physiological saline: 9 g NaCl, made up to 1 L with deionized water), centrifuge at 4°C and 12,000 rpm for 10 min, discard the supernatant, and collect the bacterial cell precipitate.

[0084] 4. Detection of protein expression in engineering strains:

[0085] 4.1 Detection method:

[0086] Detect the protein expression in glycosyltransferase strains from different sources of CDG01, CDG02, CDG03, CDG04, and CDG05. The detection method uses SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The protein electrophoresis buffer: 1 g SDS, 14.4 g glycine, 3.03 g tris(hydroxymethyl)aminomethane, made up to 1 L with deionized water; the gel decolorizing solution: 100 mL glacial acetic acid, 50 mL ethanol, made up to a total volume of 1 L with deionized water.

[0087] 4.2 Detection results:

[0088] The protein expression detection results of different engineering bacteria of CDG01, CDG02, CDG03, CDG04, and CDG05 are as Figure 1 shown. Figure 1Protein expression detection results of different engineering bacteria CDG01, CDG02, CDG03, CDG04, and CDG05. Here, M: Maeker; 1 - 4: whole cells before induction, whole cells after induction, cell supernatant after disruption, and precipitate after disruption. CaUGT2, 81.1 KD; CaUGT74An3, 96.3 KD; ppuG, 46.2 KD; GH1, 52.9 KD; GT2, 46.7 KDa.

[0089] 5. Catalytic performance detection of engineering strains for UDP - glucose and curcumin:

[0090] 5.1 Detection method:

[0091] Detection of the catalytic performance of glycosyltransferases from different sources of CDG01, CDG02, CDG03, CDG04, and CDG05 for UDP - glucose and curcumin. After high - performance liquid chromatography (HPLC) analysis of the engineering strains, purification is carried out, followed by mass spectrometry detection and structural analysis.

[0092] 5.1.1 High - performance liquid chromatography (HPLC) analysis:

[0093] Taking the CDG03 engineering strain as an example, in a 3 - mL detection system containing 1.6 mmol / L (0.9 g / L) UDP - glucose, 0.54 mmol / L (0.2 g / L) curcumin (dissolved in DMSO), and 15 g / L cells suspended in Tris - HCl (50 mmol / L Tris - HCl buffer: 6.06 g Tris, add 800 mL of ionized water, adjust the solution pH to 8.0 with hydrochloric acid and sodium hydroxide, and make up to 1 L with ionized water), the catalytic reaction is carried out at 37 °C and 180 rpm for 0 h to 26 h. At the end of fermentation, methanol is used to terminate the reaction, fermentation broth: methanol (v:v) = 1:3, mix well, centrifuge at 10000 rpm for 10 min, take the supernatant, filter the sample through a 0.22 - μm microporous membrane, and perform high - performance liquid chromatography (HPLC) analysis using a C18 column (250×4.6 mm, 5 μm).

[0094] The HPLC column was C18 (150 mm×4.6 mm, 5 μm), the detector was an ultraviolet (UV) detector with a wavelength of 425 nm, the column temperature was 30 °C, the flow rate was 1.0 mL / min, the injection volume was 5 μL, the mobile phase was pure water (A) and acetonitrile (B), and the gradient elution program (v / v) was 90% A: 10% B for 0 - 5 min; 70% A: 30% B for 5 - 10 min; 60% A: 40% B for 10 - 15 min; 40% A: 60% B for 15 - 20 min; 60% A: 40% B for 20 - 25 min; 90% A: 10% B for 25 - 30 min.

[0095] 5.1.2, Mass spectrometry detection:

[0096] Taking the CDG03 engineered strain as an example, the detectors used were Agilent 1290 UPLC and Agilent qtof6550, the chromatographic column was waters BEH C18 (2.1×100 mm ×1.7 μm), the ion source was ESI, the voltage was 4000 V, the mass spectrometry scanning range was 50 - 1800 m / z for the first stage, the atomization temperature was 350 °C; the atomization gas flow rate was 12 L / min, the mobile phase was 0.1% formic acid aqueous solution and acetonitrile, the flow rate was 0.3 mL / min, and the injection volume was 5 μL.

[0097] 5.1.3, Structure analysis:

[0098] Taking the CDG03 engineered strain as an example, in order to determine the structures of curcumin monoglucoside and curcumin diglucoside, the purified pure products were sent to Hangzhou Yanqu Information Technology Co., Ltd. for structure analysis. Nuclear magnetic resonance spectrometer (NMR) and high-resolution mass spectrometry were used in combination to determine their structures.

[0099] 5.2, Detection results:

[0100] 5.2.1, HPLC detection results of CDG03 engineered bacteria:

[0101] The HPLC detection results of CDG03 engineered bacteria are as Figure 2 shown. Figure 2 It is the HPLC detection result diagram of CDG03 engineered bacteria. From Figure 2 it can be seen that in addition to the substrate curcumin (15.138 min), two obvious peaks appeared at 11.026 min and 8.667 min.

[0102] 5.2.2, Mass spectrometry detection and structure analysis results of CDG03 engineered bacteria:

[0103] Analysis Figure 2 The results of the analysis showed that curcumin monoglucoside was at 11.026 min and curcumin diglucoside was at 8.667 min. Among them, the structural formula of curcumin monoglucoside is: and the structural formula of curcumin diglucoside is: .

[0104] 5.2.3 Detection results of the catalytic performance of different engineered bacteria CDG01, CDG02, CDG03, CDG04, and CDG05 on UDP-glucose and curcumin:

[0105] The detection results of the catalytic performance of different engineered bacteria CDG01, CDG02, CDG03, CDG04, and CDG05 on UDP-glucose and curcumin are as Figure 3 shown. Figure 3 It is a detection result diagram of the catalytic performance of different engineered bacteria CDG01, CDG02, CDG03, CDG04, and CDG05 on UDP-glucose and curcumin. From Figure 3 it can be seen that through comparison, it is found that compared with the other 4 enzymes, B. subtilis the ppuG from the 168 source has the best catalytic ability for UDP-glucose and curcumin, and finally produces 116.36 mg / L of curcumin monoglucoside and 100.98 mg / L of curcumin diglucoside.

[0106] Example 2 Construction and screening of double mutants of polyphenol TDP-rhamnosyltransferase ppuG gene

[0107] 1. Design and construct mutants:

[0108] Using the method of sequence identity, based on the GT2 amino acid sequence obtained from the NCBI database, 1000 homologous amino acid sequences of ppuG (B. subtilits 168) were searched using the Consensus Finder (http: / / cbs-kazlab.oit.umn.edu), and the overexpressed sequences (redundancy of 0.9) were deleted using the CD-Hit Suite software. According to the results of sequence identity analysis, residues with a proportion of amino acids at a site in the multiple sequence alignment reaching more than 60% (conservative threshold of 0.6) were mutated. A total of 17 mutation sites (S7A, M8F, K26E, C28V, K30R, G51A, K169G, H190G, K210Q, M223L, I253V, E266G, M303L, Q345R, K369R, M385L, and A379D) were screened out. A total of 17 ppuG single mutants were designed and constructed using the site-directed mutagenesis method. The sequence identity analysis is shown in Table 3.

[0109] Table 3 Sequence identity analysis table

[0110] Original amino acid Amino acid site Mutated amino acid Conservation threshold S 7 A 72 M 8 F 65 K 26 E 91 C 28 V 76 K 30 R 92 G 51 A 94 K 169 G 73 H 190 G 65 K 210 Q 62 M 223 L 83 I 253 V 63 E 266 G 87 M 303 L 81 Q 345 R 83 K 369 R 61 M 385 L 72 A 379 D 73

[0111] 2. Detection of the catalytic performance of the mutant strain on UDP-glucose and curcumin:

[0112] Detect the catalytic performance of UDP-glucose and curcumin of the 17 ppuG single mutants designed and constructed above. The detection results of the catalytic performance of the ppuG single mutants on UDP-glucose and curcumin are as Figure 4 shown. Figure 4 It is a graph showing the detection results of the catalytic performance of the ppuG single mutants on UDP-glucose and curcumin. From Figure 4 it can be seen that a total of 8 beneficial mutants were obtained by sequence identity mutation. Compared with the conversion rate of 67.30% of the wild type, the two better strains are ppuG / G51A and ppuG / Q345R, and the conversion rates are 77.75% and 82.34% respectively.

[0113] Subsequently, double mutations were carried out at positions 51 and 345 of ppuG to obtain the optimal double mutant ppuG / G51A / Q345R (conversion rate is 87.00%).

[0114] Compared with the wild type, ppuG / G51A, ppuG / Q345R and ppuG / G51A / Q345R have better catalytic ability on UDP-glucose and curcumin, and produce 142.21 mg / L, 153.06 mg / L and 164.17 mg / L of curcumin monoglucoside, and 106.78 mg / L, 109.64 mg / L and 112.78 mg / L of curcumin diglucoside respectively after catalysis.

[0115] Based on the detection results of the catalytic performance, the mutant ppuG / G51A / Q345R was selected for subsequent experiments.

[0116] Example 3. Construction of recombinant Escherichia coli for producing curcumin glucoside

[0117] Step S1. Construction of recombinant plasmid pACYCDuet-1-Pgm-GalU:

[0118] To increase E. coli the UDP-glucose level of the cells themselves, overexpress glucose-6-phosphate mutase (Pgm) and UDP-glucose pyrophosphorylase (GalU). Using E. coliUsing the MG1655 strain as a template, PCR was performed with Pgm-F / R and GalU-F / R to obtain the Pgm and GalU gene sequences, and the recombinant plasmid pACYCDuet-1-Pgm-GalU was obtained through homologous recombination.

[0119] Step S2: Construction of the recombinant plasmid pET28a(+)-ppuG / G51A / Q345R:

[0120] The optimized glycosyltransferase gene (ppuG / G51A / Q345R) was constructed into the pET28a(+) vector to obtain the recombinant plasmid pET28a(+)-ppuG / G51A / Q345R.

[0121] Step S3: Construction of the CDG06 engineering strain:

[0122] The recombinant plasmid pACYCDuet-1-Pgm-GalU prepared in Step S1 and the recombinant plasmid pET28a(+)-ppuG / G51A / Q345R prepared in Step S2 were co-transformed into E. coli BL21(DE3) competent cells, spread on an LB solid medium containing Cm (chloramphenicol, 30 μg / mL) and Kan (50 μg / mL), and cultured overnight in a 37°C incubator. Single colonies were picked and inoculated into an LB liquid medium containing Cm (30 μg / mL) and Kan (50 μg / mL), cultured overnight at 37°C and 180 rpm, and the overnight cultured bacterial solution was stored in glycerol (final concentration 25%) and stored at -80°C to obtain the recombinant strain

[0123] E. coli BL21(DE3) pACYCDuet-1-Pgm-GalU pET28a(+)-ppuG / G51A / Q345R, namely the CDG06 strain (recombinant Escherichia coli BL21(DE3)CBG06).

[0124] Example 4: Flask fermentation process of recombinant Escherichia coli BL21(DE3)CBG06

[0125] Step S1: Preparation of the seed solution:

[0126] The recombinant Escherichia coli BL21(DE3)CBG06 prepared in Example 3 was streaked on an LB solid medium containing Cm (30 μg / mL) and Kan (50 μg / mL) to obtain single colonies. Single colonies were picked and inoculated into 5 mL of an LB liquid medium containing Cm (30 μg / mL) and Kan (50 μg / mL), and cultured overnight at 37°C and 180 rpm to prepare the seed solution.

[0127] Step S2, Fermentation culture:

[0128] The obtained seed liquid was inoculated into 200 mL of LB liquid medium containing Cm (30 μg / mL) and Kan (50 μg / mL) at an inoculation amount of 1% (v / v), and cultured at 37 °C and 180 rpm. After culturing for about 3 - 4 h until the OD600 value reached 0.6 - 0.8, IPTG with a final concentration of 1 mmol / L, glucose with a concentration of 10 g / L, and curcumin with a concentration of 0.2 g / L were added, and the catalytic reaction was carried out at 37 °C and 180 rpm for 26 h. Recombinant Escherichia coli BL21(DE3)CBG06 produced 24.39 mg / L of curcumin monoglucoside and 315.90 mg / L of curcumin diglucoside, and the utilization rate of the substrate curcumin reached 92.56%.

[0129] Example 5, 5L fermentation process of recombinant Escherichia coli BL21(DE3)CBG06

[0130] Step S1, Pick the single colony obtained in Step S1 of Example 4 and inoculate it into 5 mL of LB liquid medium containing Cm (30 μg / mL) and Kan (50 μg / mL), and culture overnight at 37 °C and 180 rpm to prepare the primary seed liquid.

[0131] Step S2, Inoculate the primary seed liquid obtained in Step S1 into 200 mL of LB liquid medium containing Cm (30 μg / mL) and Kan (50 μg / mL) at an inoculation amount of 1% (v / v), and culture at 37 °C and 180 rpm for 6 - 8 h to prepare the secondary seed liquid.

[0132] Step S3, Inoculate the secondary seed liquid obtained in Step S2 into a 5 L fermenter containing 3 L of fermentation medium containing Cm (30 μg / mL) and Kan (50 μg / mL) at an inoculation amount of 2% (v / v), control the dissolved oxygen at about 30%, the ventilation rate is 1VVM, and start culturing at 37 °C.

[0133] Step S4, When the cell concentration OD600 value reaches 5 - 10, add IPTG with a final concentration of 1 mmol / L for induction, and at the same time add 1 g / L of curcumin. Induction and catalysis are carried out simultaneously at 40 °C, and 1 g / L of curcumin is added once at 4 h, 7 h, and 12 h during the catalytic reaction, and ferment for 26 h to obtain the product.

[0134] After culturing for 26 h, the yield of curcumin diglucoside produced by recombinant Escherichia coli BL21(DE3)CBG06 was detected. The detection results of the yield of curcumin diglucoside produced by recombinant Escherichia coli BL21(DE3)CBG06 are asFigure 5 as shown

[0135] Figure 5 It is a graph showing the yield detection results of curcumin diglucoside produced by recombinant Escherichia coli BL21(DE3)CBG06. From Figure 5 it can be seen that the highest yield of curcumin diglucoside produced by the recombinant Escherichia coli BL21(DE3)CBG06 constructed in the present invention reaches 7.10 g / L.

[0136] The above embodiments are only illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A recombinant Escherichia coli BL21(DE3)CBG06 for producing curcumin glucoside ( Escherichia coli BL21(DE3)CBG06), characterized in that, The recombinant Escherichia coli BL21 (DE3) CBG06 was deposited in the China Center for Type Culture Collection on December 20, 2024, with the deposit number: CCTCC NO: M 20242869.

2. A method for producing curcumin glucoside, characterized in that: The recombinant Escherichia coli BL21 (DE3) CBG06 for producing curcumin glucoside according to claim 1 is used as a fermentation strain, curcumin is used as a substrate, glucose is used as a glycosyl donor, and the culture is carried out at a temperature of 35-40° C. until the bacterial concentration OD600 is 5-10, and an IPTG inducer is added to catalyze the synthesis of curcumin diglucoside.

3. The method for producing curcumin glucoside as claimed in claim 2, wherein The fermentation conditions are as follows: fermentation temperature is 35-40°C, rotation speed is 120-650 rpm, ventilation volume is 0.7-1.5 VVM, dissolved oxygen is controlled between 25-35%, pH is controlled between 6-8, final concentration of IPTG inducer is 0.5-1.5 mmol / L, curcumin concentration is 0.2-4 g / L, and fermentation is continued for 26 h after adding the inducer.

4. Use of the recombinant Escherichia coli BL21 (DE3) CBG06 for producing curcumin glucoside as claimed in claim 1 in preparing curcumin glucoside or a product containing curcumin glucoside.

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