Recombinant escherichia coli for producing curcumin glucoside and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to recombinant escherichia coli for producing curcumin glucoside and application of the recombinant escherichia coli. According to the invention, escherichia coli BL21 (DE3) is taken as a host cell, and a double mutant of a polyphenol TDP-rhamnosyltransferase ppuG gene derived from bacillus subtilis 168, a glucose-6-phosphate mutase Pgm gene derived from escherichia coli MG1655 and a UDP-glucose pyrophosphorylase GalU gene are transferred into the escherichia coli host cell, so that the recombinant escherichia coli is obtained. The double mutants of the polyphenol TDP-rhamnosyltransferase ppuG gene comprise two mutation sites, namely, G51A and Q345R. The invention further discloses a preparation method of the polyphenol TDP-rhamnosyltransferase ppuG gene. The recombinant escherichia coli constructed by the invention can efficiently produce curcumin diglucoside, and the yield can reach 7.10 g / L, so that not only are the water solubility and bioavailability of curcumin improved, but also powerful support can be provided for industrial production of curcumin glucoside.

Description

Technical Field

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

[0002] The molecular formula of curcumin is C 21 H 20 O6, with a molecular weight of 368.38, is a natural polyphenol compound with biological activity extracted from the rhizomes of some plants such as the Zingiberaceae and Araceae. It is generally in the form of an orange-yellow crystalline powder. Curcumin has a variety of functions and effects such as anti-inflammatory, antioxidant, anti-cancer, antibacterial, anti-malarial, liver-protecting and choleretic, and lipid-lowering, and is widely used in the fields of food additives, medicines, and cosmetics. However, curcumin is highly lipophilic, insoluble in water, has poor bioavailability, low cellular uptake rate, and poor stability, which limits its pharmacological development and practical application. Therefore, finding a suitable method to synthesize new curcumin analogs to improve the pharmacological characteristics of natural compounds is of extremely important scientific value and broad market application potential for the development of curcumin applications in various fields.

[0003] Modifying the structure of curcumin by microbial transformation to improve its water solubility is one of the hot topics of current research. At present, the molecular structure modification of curcumin mainly includes glucosylation reaction, demethoxylation reaction and connection of hydrophilic groups on the phenolic hydroxyl group. These modifications can convert curcumin into curcumin analogs or derivatives, and 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 (GT) can catalyze the transfer of sugar groups from uridine diphosphate sugar (UDP sugar) to low molecular weight receptor substrates. They can strictly control the regional 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. The method constructs the glycosyltransferase gene CaUGT2 and the sucrose synthase gene AtSUS1 into an expression vector, and introduces them into Escherichia coli to obtain a recombinant strain. After the recombinant strain is induced to express, its soluble expression amount is higher than that of other plant-derived glycosyltransferases, and the substrate curcumin can be efficiently catalyzed to generate curcumin monosaccharide and curcumin disaccharide, which have better water solubility than curcumin, solving the problem of poor water solubility of curcumin.

[0005] Patent document CN115975900A discloses a genetically engineered bacterium for synthesizing curcumin glucoside and its application. The construction method uses Escherichia coli BL21 (DE3) as an 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 cell, effectively improving the synthesis efficiency of curcumin glucoside catalyzed by the whole cell; at the same time, overexpresses Catharanthus roseus The UDP-glucosyltransferase CaUGT2 from the source; the genetically engineered bacteria can efficiently synthesize curcumin monoglucoside and curcumin diglucoside, which can effectively solve the solubility problem of curcumin.

[0006] However, the efficiency and yield of curcumin glucoside synthesized by genetically engineered bacteria are generally low and cannot meet the production requirements of industrialization. Summary of the invention

[0007] In order to overcome the shortcomings of the prior art, the present invention provides an engineered Escherichia coli strain that efficiently produces curcumin glucoside. The present invention uses Escherichia coli BL21 (DE3) as a host cell and is capable of expressing a gene derived from Bacillus subtilis 168 ( Bacillus subtilits 168) double mutant of the polyphenol TDP-rhamnosyltransferase ppuG gene, overexpressed from Escherichia coli MG1655 ( Escherichia coli The double mutant of the polyphenol TDP-rhamnosyltransferase ppuG gene contains two mutation sites, G51A and Q345R. The constructed engineered strain can realize the efficient production of curcumin glucoside by 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 and UDP-glucose to generate curcumin glucoside, the present invention first screened a variety of glycosyltransferases from different sources. These sources include Catharanthus roseus , Bacillus subtilis 168. Lactobacillus zeae And other microorganisms. The specific steps are as follows: Step S1, obtaining the glycosyltransferase gene sequences of the above-mentioned microorganisms from the NCBI database, including UDP-glucosyltransferase (CaUGT2, GT2, GH1), steroid glycosyltransferase (CaUGT74An3) and polyphenol TDP-rhamnosyltransferase (ppuG), etc.

[0009] Step S2: synthesize the obtained gene sequence into a gene and construct it into a corresponding expression vector, such as pgex-4t-1, pMAL-c5X, pET-28a(+), etc.

[0010] Step S3: Transform the constructed expression vector into Escherichia coli E. coli In BL21(DE3), the expression was induced by IPTG to obtain a genetically engineered bacterium expressing glycosyltransferase.

[0011] Step S4, reacting the genetically engineered bacteria with UDP-glucose and curcumin respectively, analyzing the reaction products by HPLC and mass spectrometry, and evaluating the catalytic performance of each glycosyltransferase.

[0012] Finally confirmed Bacillus subtilis The polyphenol TDP-rhamnosyltransferase (ppuG) from 168 has the best catalytic performance for UDP-glucose and curcumin, and can efficiently produce curcumin glucoside.

[0013] 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: Step S1: Use a consensus finder to search for 1000 homologous amino acid sequences of ppuG, and identify the amino acid sites conserved in most sequences through multiple sequence alignment analysis.

[0014] Step S2: Based on the sequence consistency analysis results, amino acid sites with higher conservatism were screened as potential mutation sites, and a total of 17 mutation sites were identified.

[0015] Step S3: using site-directed mutagenesis technology, designing corresponding mutation primers for each screened mutation site, and performing PCR amplification and gene recombination to construct 17 ppuG single mutants.

[0016] Step S4, reacting each mutant with UDP-glucose and curcumin to detect its catalytic performance. By comparison, it was found that two mutants, ppuG / Q345R and ppuG / G51A, showed better catalytic performance.

[0017] Step S5: Based on the experimental results of the single mutant, double mutations were further performed at sites 51 and 345 of ppuG to construct a ppuG / G51A / Q345R double mutant. The experimental results showed that the double mutant had the best catalytic performance and could significantly increase the yield of curcumin glucoside.

[0018] In order to further improve the ability of recombinant Escherichia coli to produce curcumin glucoside, the present invention also overexpressed glucose-6-phosphate mutase (Pgm) and UDP-glucose pyrophosphorylase (GalU). These two enzymes can increase the level of UDP-glucose in cells, thereby providing more glycosyl donors for glycosyltransferases.

[0019] Specifically, Pgm catalyzes the mutual conversion 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, thereby 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 of UDP-glucose in the cell can be directly increased, providing more substrates for glycosyltransferases.

[0020] The specific technical solutions of the present invention are as follows: The present invention provides a recombinant Escherichia coli for producing curcumin glucoside, wherein the recombinant Escherichia coli BL21(DE3)CBG06 ( 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.

[0021] Furthermore, the recombinant Escherichia coli BL21 (DE3) CBG06 is derived from Bacillus subtilis 168 ( Bacillus subtilits 168) double mutant of polyphenol TDP-rhamnosyltransferase ppuG gene, derived from Escherichia coli MG1655 ( Escherichia coli MG1655) of glucose-6-phosphate mutase Pgm gene, SEQ ID NO.3 is from Escherichia coli MG1655 ( Escherichia coli The UDP-glucose pyrophosphorylase GalU gene of MG1655 was transformed into Escherichia coli host cells.

[0022] Furthermore, the double mutant of the polyphenol TDP-rhamnosyltransferase ppuG gene contains two mutation sites, G51A and Q345R.

[0023] Furthermore, the amino acid sequence of the protein encoded by the double mutant of the polyphenol TDP-rhamnosyltransferase ppuG gene is shown as SEQ ID NO.4; the amino acid sequence of the protein encoded by the glucose-6-phosphate mutase Pgm gene is shown as SEQ ID NO.5; and the amino acid sequence of the protein encoded by the UDP-glucose pyrophosphorylase GalU gene is shown as SEQ ID NO.6.

[0024] Furthermore, the recombinant Escherichia coli BL21 (DE3) CBG06 contains at least two recombinant vectors, including but not limited to pgex-4t-1, pMAL-c5X, pET-28a (+) or pACYCDuet-1.

[0025] Furthermore, the recombinant Escherichia coli BL21 (DE3) CBG06 comprises two recombinant vectors, one of which comprises the glucose-6-phosphate mutase Pgm gene and the UDP-glucose pyrophosphorylase GalU gene, and the other comprises a double mutant of the polyphenol TDP-rhamnosyltransferase ppuG gene.

[0026] Furthermore, the present invention also provides a method for constructing the recombinant Escherichia coli for producing curcumin glucoside, comprising the following steps: Step S1, constructing a recombinant vector containing the glucose-6-phosphate mutase Pgm gene and the UDP-glucose pyrophosphorylase GalU gene; Step S2, constructing a recombinant vector containing a double mutant of the polyphenol TDP-rhamnosyltransferase ppuG gene; Step S3, transforming the recombinant vector prepared in step S1 and the recombinant vector prepared in step S2 into Escherichia coli to obtain.

[0027] In addition, the present invention also provides a method for producing curcumin glucoside, which comprises taking the recombinant Escherichia coli BL21 (DE3) CBG06 for producing curcumin glucoside as a fermentation strain, taking curcumin as a substrate, taking glucose as a glycosyl donor, culturing at a temperature of 35-40° C. until the bacterial concentration OD600 is 5-10, adding an IPTG inducer, and catalyzing the synthesis of curcumin diglucoside to obtain the product.

[0028] Furthermore, 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 0-26 h after adding the inducer.

[0029] In addition, the present invention also claims to protect the use of the recombinant Escherichia coli BL21 (DE3) CBG06 producing curcumin glucoside in the preparation of curcumin glucoside or products containing curcumin glucoside.

[0030] Furthermore, the product containing curcumin glucoside is a food additive, medicine or cosmetic containing curcumin glucoside.

[0031] The implementation steps of the specific overexpression strategy provided by the present invention are: Step S1: E. coli The gene sequences of Pgm and GalU were cloned from the MG1655 strain and then constructed into an expression vector, such as pACYCDuet-1, to achieve efficient expression in Escherichia coli.

[0032] 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, a recombinant strain capable of simultaneously expressing the three enzymes was obtained. E. coli BL21(DE3)CBG06.

[0033] Step S3: in the recombinant strain E. coli During the cultivation of BL21(DE3)CBG06, the growth and metabolic environment of the recombinant Escherichia coli were optimized by adjusting the fermentation conditions such as medium composition, pH value, temperature, and dissolved oxygen content to improve the synthesis efficiency of UDP-glucose and the yield of curcumin glucoside.

[0034] Finally, the present invention successfully constructed a recombinant Escherichia coli BL21 (DE3) CBG06 ( Escherichia coli BL21(DE3)CBG06). Under suitable fermentation conditions, this strain can produce a high yield of curcumin diglucoside, with a yield of 7.10 g / L, which can fully meet the needs of industrialization.

[0035] In summary, compared with the prior art, the recombinant Escherichia coli for producing curcumin glucoside provided by the present invention has the following advantages: (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 at 35-40°C in a 5 L fermenter for 23 h, the yield of curcumin glucoside can reach 7.10 g / L. This has the advantages of short fermentation time and high yield, thus meeting the needs of industrial production.

[0036] (2) Curcumin glucoside produced by fermentation with the recombinant Escherichia coli provided by the present invention has good water solubility and bioavailability, which provides the possibility for the wide application of curcumin in the fields of food additives, medicines, cosmetics, etc., and helps to promote the research and development and market expansion of curcumin-related products.

[0037] The microbial information of the recombinant Escherichia coli BL21 (DE3) CBG06 for producing curcumin glucoside provided by the present invention is as follows: 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, and the deposit address: Wuhan University, Wuhan, China, Postal Code: 430072. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a graph showing the protein expression results of different engineered bacteria: CDG01, CDG02, CDG03, CDG04 and CDG05.

[0039] Figure 2 This is a graph showing the results of high performance liquid chromatography testing of CDG03 engineered bacteria.

[0040] Figure 3 This is a graph showing the test results of the catalytic performance of different engineered bacteria, CDG01, CDG02, CDG03, CDG04 and CDG05, on UDP-glucose and curcumin.

[0041] Figure 4 This is a graph showing the catalytic performance test results of the ppuG single mutant towards UDP-glucose and curcumin.

[0042] Figure 5 This is a graph showing the results of detecting the content of curcumin diglucoside produced by recombinant Escherichia coli BL21 (DE3) CBG06. DETAILED DESCRIPTION

[0043] The present invention is further illustrated below through the description of specific implementation modes, but 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, but as long as they do not deviate from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0044] 1. Strains and primers involved in the present invention The strains and primers involved in Examples 1 to 5 of the present invention are shown in Tables 1 and 2.

[0045] Table 1 Strains involved in Examples 1 to 5

[0046] Table 2 Primer sequences used in Examples 1 to 5 Primer name Sequence (5′-3′) Pgm-F CTTTAATAAGGAGATATAATGGCAATCCACAATCGTGCAGG Pgm-R GACTTAAGCATTATTTACGCGTTTTTCAGAACTTCGC GalU-F GGAGATATAATGGCTGCCATTAATACGAAAGTC GalU-R GCGGTTTCTTTACCAGATTACTTCTTAATGCCCATCTCTTCTTCAAG 2. The culture medium involved in Examples 1 to 5 of the present invention is as follows: (1) LB medium formula: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0. For solid medium, add 1.5%~2% (mass ratio) agar powder.

[0047] (2) Fermentation medium: 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 defoamer, 10 mL / L trace element solution.

[0048] The trace metal solution formula is: 0.84 g / L ethylenediaminetetraacetic acid (EDTA), 0.25 g / L cobalt chloride hexahydrate (CoCl2-6H2O), 1.50 g / L manganese chloride tetrahydrate (MnCl2-4H2O), 0.15 g / L copper chloride dihydrate (CuCl2-2H2O), 0.30 g / L boric acid (H3BO3), 0.25 g / L sodium molybdate dihydrate (Na2MoO4-2H2O), 1.30 g / L zinc acetate dihydrate [Zn(CH3COO)2-2H2O], and 10.0 g / L ammonium ferric citrate.

[0049] 3. The nucleotide sequences and amino acid sequences involved in Examples 1 to 5 of the present invention are as follows: (1) Derived from Bacillus subtilis 168 ( Bacillus subtilits The nucleotide sequence of the double mutant of the polyphenol TDP-rhamnosyltransferase ppuG gene of 168 is (SEQ ID NO.1):

[0050] (2) Derived from Escherichia coli MG1655 ( Escherichia coli The nucleotide sequence of the glucose-6-phosphate mutase Pgm gene of MG1655 is (SEQ ID NO.2):

[0051] (3) Derived from Escherichia coli MG1655 ( Escherichia coli The nucleotide sequence of the UDP-glucose pyrophosphorylase GalU gene of MG1655 is (SEQ ID NO.3): ATGGCTGCCATTAATACGAAAGTCAAAAAAGCCGTTATCCCCGTTGCGGGATTAGGAACCAGGATGTTGCCGGCGACGAAAGCCATCCCGAAAGAGATGCTGCCACTTGTCGATAAGCCATTAATTCAATACGTCGTGAATGAATGTATTGCGGCTGGCATTACTGAAATTGTGCTGGTTACACACTCATCTAAAAACTCTATTGAAAACCACTTTGATACCAGTTTTGAACTGGAAGCAATGCTGGAAAAACGTGTAAAACGTCAACTGCTTGATGAAGTGCAGTCTATTTGTCCACCGCACGTGACTATTATGCAAGTTCGTCAGGGTCTGGCGAAAGGCCTGGGACACGCGGTATTGTGTGCTCACCCGGTAGTGGGTGATGAACCGGTAGCTGTTATTTTGCCTGATGTTATTCTGGATGAATATGAATCCGATTTGTCACAGGATAACCTGGCAGAGATGATCCGCCGCTTTGATGAAACGGGTCATAGCCAGATCATGGTTGAACCGGTTGCTGATGTGACCGCATATGGCGTTGTGGATTGCAAAGGCGTTGAATTAGCGCCGGGTGAAAGCGTACCGATGGTTGGTGTGGTAGAAAAACCGAAAGCGGATGTTGCGCCGTCTAATCTCGCTATTGTGGGTCGTTACGTACTTAGCGCGGATATTTGGCCGTTGCTGGCAAAAACCCCTCCGGGAGCTGGTGATGAAATTCAGCTCACCGACGCAATTGATATGCTGATCGAAAAAGAAACGGTGGAAGCCTATCATATGAAAGGGAAGAGCCATGACTGCGGTAATAAATTAGGTTACATGCAGGCCTTCGTTGAATACGGTATTCGTCATAACACCCTTGGCACGGAATTTAAAGCCTGGCTTGAAGAAGAGATGGGCATTAAGAAGTAA。

[0052] (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): MKKYHISMINIPAYGHVNPTLALVEKLCEKGHRVTYATTEEFAPAVQQAGAEALIYHTSLNIDPKQIREMMEKNDAPLSLLKESLSILPQLEELYKDD QPDLIIYDFVALAGKLFAEKLNVPVIKLCSSYAQNESFQLGNEDMLKKIREAEAEFKAYLEQEKLPAVSFEQLAVPEALNIVFMPKSFQIQHETFDDRF CFVGPSLGERKEKESLLIDKDDRPLMLISLGTAFNAWPEFYKMCIKAFRDSSWQVIMSVGKTIDPESLEDIPANFTIRQSVPQLEVLEKADLFISHGGMNSTMEAMNAGVPLVVIPQMYEQELTANRVDELGLGVYLPKEEVTVSSLREAVQAVSSDQELLSRVKNMQKDVKEAGGAERAAAEIEAFMKKSAVPQ*.

[0053] (5) The amino acid sequence of the protein encoded by the glucose-6-phosphate mutase Pgm gene is (SEQ ID NO.5): *.

[0054] (6) The amino acid sequence of the protein encoded by the UDP-glucose pyrophosphorylase GalU gene is (SEQ ID NO.6): MAAINTKVKKAVIPVAGLGTRMLPATKAIPKEMLPLVDKPLIQYVVNECIAAGITEIVLVTHSSKNSIENHFDTSFELEAMLEKRVKRQLLDEVQSICPPHVTIMQVRQGLAKGLGHAVLCAHPVVGDEPVAVILPDVILDEYESDLSQDNL AEMIRRFDETGHSQIMVEPVADVTAYGVVDCKGVELAPGESVPMVGVVEKPKADVAPSNLAIVGRYVLSADIWPLLAKTPPGAGDEIQLTDAIDMLIEKETVEAYHMKGKSHDCGNKLGYMQAFVEYGIRHNTLGTEFKAWLEEEMGIKK*.

[0055] Example 1. Screening of glycosyltransferase strains from different sources 1. Construction of recombinant plasmid: Obtained from NCBI database Catharanthus roseus UDP-glucosyltransferase (CaUGT2, GenBank: BAD29722.1), steroid glycosyltransferase (CaUGT74An3, GenBank: AYC35244.1), Bacillus subtilits Polyphenol TDP-rhamnosyltransferase (ppuG, GenBank: NP_389104.1) from 168 Bacillus subtilis UDP-glucosyltransferase from ATCC 6633 (GT2, GenBank: QAT94473.1) and Lactobacillus zeae The gene sequences of GH1 (GenBank: KRK12807.1) were submitted to Beijing Qingke Biotechnology Co., Ltd. for gene synthesis and constructed into pgex-4t-1, pMAL-c5X, pET-28a(+), pET-28a(+) and pET-28a(+) vectors, respectively, to obtain recombinant plasmids pgex-4t-1-CaUGT2, pMAL-c5X-CaUGT74An3, pET-28a(+)-ppuG, pET-28a(+)-GT2 and pET-28a(+)-GH1, respectively.

[0056] 2. Construction of engineered strains: The recombinant plasmids pgex-4t-1-CaUGT2, pMAL-c5X-CaUGT74An3, pET-28a(+)-ppuG, pET-28a(+)-GT2 and pET-28a(+)-GH1 were respectively transferred into Escherichia coli DH5α. Then, from the transformed Escherichia coli The plasmid was extracted from DH5α and transformed into E. coli BL21(DE3), finally obtained genetically engineered bacteria CDG01, CDG02, CDG03, CDG04, CDG05 expressing different glycosyltransferases (GTs), 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. coliBL21(DE3) pET-28a(+)-GH1).

[0057] 3. Fermentation of engineered strains: From the converted E. coli Pick a single colony from the BL21(DE3) plate and inoculate it 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 a 1% (V / V) inoculation rate, and culture at 37°C and 180 rpm. Culture for about 3-4 hours until the bacterial OD reaches 0. 600 When the value reached 0.6-0.8, IPTG was added at a final concentration of 1 mmol / L and induced overnight at 25°C and 150 rpm.

[0058] The induced bacterial solution was centrifuged at 4°C and 12,000 rpm for 10 min to collect the bacteria. The bacterial cells were washed once with physiological saline (physiological saline: 9 g NaCl, dilute to 1 L with deionized water), centrifuged at 4°C and 12,000 rpm for 10 min, the supernatant was discarded, and the bacterial precipitate was collected.

[0059] 4. Detection of protein expression of engineering strains: 4.1. Detection method: The protein expression of glycosyltransferase strains from different sources, CDG01, CDG02, CDG03, CDG04 and CDG05, was detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The protein electrophoresis buffer included 1 g SDS, 14.4 g glycine, 3.03 g tris(hydroxymethyl)aminomethane, and the volume was adjusted to 1 L with deionized water. The gel destaining solution included 100 mL glacial acetic acid, 50 mL ethanol, and the volume was adjusted to 1 L with deionized water.

[0060] 4.2 Test results: The protein expression test results of different engineered bacteria CDG01, CDG02, CDG03, CDG04 and CDG05 are shown in Figure 1 shown. Figure 1The results of protein expression detection of different engineered bacteria CDG01, CDG02, CDG03, CDG04 and CDG05 are shown in Figure 1, where M: Maeker; 1-4: whole cells before induction, whole cells after induction, supernatant of cells 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.

[0061] 5. Test of the catalytic performance of engineered strains on UDP-glucose and curcumin: 5.1. Detection method: The catalytic performance of glycosyltransferases from different sources, CDG01, CDG02, CDG03, CDG04 and CDG05, on UDP-glucose and curcumin was tested. The engineered strains were analyzed by high performance liquid chromatography (HPLC), purified, and then detected by mass spectrometry and structural elucidation.

[0062] 5.1.1 High Performance Liquid Chromatography (HPLC) Analysis: Taking the CDG03 engineered strain as an example, the 3 mL test system contained 1.6 mmol / L (0.9 g / L) UDP-glucose, 0.54 mmol / L (0.2 g / L) curcumin (DMSO dissolved) and 15 g / L cells suspended in Tris-HCl (50 mmol / L Tris-HCl buffer: 6.06 g Tris, 800 mL of ionized water, hydrochloric acid and sodium hydroxide were added to adjust the solution pH to 8.0, and ionized water was fixed to 1L). The catalytic reaction was carried out at 37°C and 180 rpm for 0 h to 26 h. At the end of the fermentation, methanol was used to terminate the reaction, and the fermentation broth: methanol (v:v) = 1:3. After mixing, the supernatant was taken and the sample was filtered through a 0. 22 μm microporous membrane and analyzed by high performance liquid chromatography (HPLC) using a C18 column (250×4.6 mm, 5 μm).

[0063] 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 0-5 min, 90% A: 10% B; 5-10 min, 70% A: 30% B; 10-15 min, 60%A: 40% B; 15-20 min, 40% A: 60% B; 20-25 min, 60% A: 40% B; 25-30 min, 90% A: 10% B.

[0064] 5.1.2 Mass spectrometry detection: Taking the CDG03 engineered strain as an example, the detectors selected 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 primary 50~1800 m / z, the nebulization temperature was 350℃; the nebulization 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.

[0065] 5.1.3 Structural analysis: Taking the CDG03 engineered strain as an example, in order to determine the structure of curcumin monoglucoside and curcumin diglucoside, the extracted and purified pure products were sent to Hangzhou Yanqu Information Technology Co., Ltd. for structural analysis. Nuclear magnetic resonance spectrometer (NMR) and high-resolution mass spectrometry were used to determine its structure.

[0066] 5.2. Test results: 5.2.1. High performance liquid chromatography test results of CDG03 engineered bacteria: The results of HPLC detection of CDG03 engineered bacteria are as follows Figure 2 shown. Figure 2 The figure is the result of high performance liquid chromatography test of CDG03 engineering bacteria. 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.

[0067] 5.2.2. Mass spectrometry and structural analysis results of CDG03 engineered bacteria: analyze Figure 2The results show that 11.026 min is curcumin monoglucoside and 8.667 min is curcumin diglucoside. Among them: The structural formula of curcumin monoglucoside is: , the structural formula of curcumin diglucoside is: .

[0068] 5.2.3. Test results of the catalytic performance of different engineered bacteria CDG01, CDG02, CDG03, CDG04 and CDG05 on UDP-glucose and curcumin: The catalytic performance test results of different engineered bacteria CDG01, CDG02, CDG03, CDG04 and CDG05 on UDP-glucose and curcumin are shown in Figure 3 shown. Figure 3 The figure shows the results of the catalytic performance test of different engineered bacteria CDG01, CDG02, CDG03, CDG04 and CDG05 on UDP-glucose and curcumin. Figure 3 It can be seen that, by comparison, compared with the other four enzymes, B. subtilits The ppuG from 168 had the best catalytic ability for UDP-glucose and curcumin, ultimately producing 116.36 mg / L of curcumin monoglucoside and 100.98 mg / L of curcumin diglucoside.

[0069] Example 2: Construction and screening of double mutants of polyphenol TDP-rhamnosyltransferase ppuG gene 1. Design and construction of mutants: Using the sequence consistency method, based on the amino acid sequence of GT2 obtained in the NCBI database, the consensus finder (http: / / cbs-kazlab.oit.umn.edu) was used to search for 1000 homologous amino acid sequences of ppuG (B. subtilits 168), and the over-represented sequences (redundancy of 0.9) were deleted using CD-Hit Suite software. According to the results of sequence consistency analysis, the residues with a proportion of amino acids at the site of more than 60% (conservative threshold of 0.6) in the multiple sequence alignment were mutated, and 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. A total of 17 ppuG single mutants were designed and constructed using site-directed mutagenesis, and the sequence consistency analysis is shown in Table 3.

[0070] Table 3 Sequence consistency analysis table Original amino acids Amino acid position Amino acid after mutation Conservative 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 2. Catalytic performance test of mutant strains on UDP-glucose and curcumin: The catalytic performance of the 17 ppuG single mutants designed and constructed above on UDP-glucose and curcumin was tested. The results of the catalytic performance test of the ppuG single mutants on UDP-glucose and curcumin are shown in Figure 4 shown. Figure 4 The figure shows the results of the catalytic performance test of the ppuG single mutant on UDP-glucose and curcumin. Figure 4 It can be seen that a total of 8 beneficial mutants were obtained by sequence consistency mutation. Compared with the conversion rate of 67.30% of the wild type, the two better strains were ppuG / G51A and ppuG / Q345R, with conversion rates of 77.75% and 82.34%, respectively.

[0071] Subsequently, double mutations were performed at sites 51 and 345 of ppuG to obtain the optimal double mutant ppuG / G51A / Q345R (conversion rate was 87.00%).

[0072] Compared with the wild type, ppuG / G51A, ppuG / Q345R and ppuG / G51A / Q345R had better catalytic ability for UDP-glucose and curcumin, producing 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.

[0073] Based on the catalytic performance test results, the mutant ppuG / G51A / Q345R was selected for subsequent experiments.

[0074] Example 3: Construction of recombinant Escherichia coli producing curcumin glucoside Step S1, construction of recombinant plasmid pACYCDuet-1-Pgm-GalU: To improve E. coli The UDP-glucose level of the cell itself was increased by overexpressing glucose-6-phosphate mutase (Pgm) and UDP-glucose pyrophosphorylating enzyme (GalU). E. coli MG1655 strain was used as a template, Pgm-F / R and GalU-F / R were used for PCR to obtain the Pgm and GalU gene sequences, and the recombinant plasmid pACYCDuet-1-Pgm-GalU was obtained by homologous recombination.

[0075] Step S2, construction of recombinant plasmid pET28a(+)-ppuG / G51A / Q345R: The optimized glycosyltransferase gene (ppuG / G51A / Q345R) was constructed into the pET28a(+) vector to obtain the recombinant plasmid pET28a(+)-ppuG / G51A / Q345R.

[0076] Step S3: Construction of CDG06 engineering strain: 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 In BL21(DE3) competent cells, spread onto LB solid medium containing Cm (chloramphenicol, 30 μg / mL) and Kan (50 μg / mL), and culture in a 37°C incubator overnight. Pick a single clone and transfer it to LB liquid medium containing Cm (30 μg / mL) and Kan (50 μg / mL), culture it at 37°C 180 rpm overnight, and store the overnight cultured bacterial solution in glycerol (final concentration 25%) and store it at -80°C to obtain the recombinant strain. E. coli BL21(DE3) pACYCDuet-1-Pgm-GalU pET28a(+)-ppuG / G51A / Q345R, i.e., CDG06 strain (recombinant Escherichia coli BL21(DE3)CBG06).

[0077] Example 4 Shake flask fermentation process of recombinant Escherichia coli BL21 (DE3) CBG06 Step S1, seed solution preparation: 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 a single colony, and a single colony was picked and inoculated into 5 mL of LB liquid medium containing Cm (30 μg / mL) and Kan (50 μg / mL), and cultured overnight at 37°C and 180 rpm to prepare a seed solution.

[0078] Step S2, fermentation culture: The prepared seed solution was inoculated into 200 mL LB liquid medium containing Cm (30 μg / mL) and Kan (50 μg / mL) at an inoculum size of 1% (v / v), and cultured at 37°C and 180 rpm. After culturing for about 3-4 h, when the OD600 value reached 0.6-0.8, IPTG with a final concentration of 1 mmol / L, 10 g / L glucose and 0.2 g / L curcumin were added, and the reaction was catalyzed at 37°C and 180 rpm for 26 h. The recombinant Escherichia coli BL21(DE3)CBG06 produced 24.39 mg / L curcumin monoglucoside and 315.90 mg / L curcumin diglucoside, and the utilization rate of the substrate curcumin reached 92.56%.

[0079] Example 5: 5 L fermentation process of recombinant Escherichia coli BL21 (DE3) CBG06 Step S1. Pick a single colony obtained in step S1 of Example 4 and inoculate it into 5 mL LB liquid culture medium containing Cm (30 μg / mL) and Kan (50 μg / mL), and culture it overnight at 37° C. and 180 rpm to prepare a primary seed solution.

[0080] Step S2: Inoculate the primary seed solution prepared in step S1 into 200 mL LB liquid culture medium containing Cm (30 μg / mL) and Kan (50 μg / mL) at an inoculum amount of 1% (v / v), and culture at 37°C and 180 rpm for 6-8 h to prepare a secondary seed solution.

[0081] Step S3, inoculate the secondary seed solution prepared in step S2 into a 5 L fermentation tank containing 3 L fermentation medium containing Cm (30 μg / mL) and Kan (50 μg / mL) at an inoculum amount of 2% (v / v), control the dissolved oxygen at about 30%, the ventilation volume at 1VVM, and start culturing at 37°C.

[0082] Step S4, when the OD600 value of the bacterial concentration reaches 5-10, add IPTG with a final concentration of 1 mmol / L for induction, and add 1 g / L curcumin at the same time, and carry out induction and catalysis at the same time at 40°C, and add 1 g / L curcumin once at 4 h, 7 h and 12 h of the catalytic reaction, and ferment for 26 h.

[0083] After 26 h of cultivation, the yield of curcumin diglucoside produced by recombinant E. coli BL21 (DE3) CBG06 was detected. The yield detection results of curcumin diglucoside produced by recombinant E. coli BL21 (DE3) CBG06 are shown in Figure 2. Figure 5 shown.

[0084] Figure 5This is a graph showing the production test results of curcumin diglucoside produced by recombinant Escherichia coli BL21 (DE3) CBG06. 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 is 7.10 g / L.

[0085] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A recombinant Escherichia coli for producing curcumin glucoside, characterized in that: The recombinant Escherichia coli BL21 (DE3) CBG06 ( 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. The recombinant Escherichia coli for producing curcumin glucoside according to claim 1, characterized in that The recombinant Escherichia coli BL21 (DE3) CBG06 is derived from Bacillus subtilis 168 ( Bacillus subtilits 168) double mutant of polyphenol TDP-rhamnosyltransferase ppuG gene, derived from Escherichia coli MG1655 ( Escherichia coli MG1655) of glucose-6-phosphate mutase Pgm gene, SEQ ID NO.3 is from Escherichia coli MG1655 ( Escherichia coli The UDP-glucose pyrophosphorylase GalU gene of MG1655 was transformed into Escherichia coli host cells.

3. The recombinant Escherichia coli for producing curcumin glucoside according to claim 2, characterized in that The double mutant of the polyphenol TDP-rhamnosyltransferase ppuG gene comprises two mutation sites, G51A and Q345R.

4. The recombinant Escherichia coli for producing curcumin glucoside according to claim 2, characterized in that 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-phosphate mutase Pgm gene is shown in SEQ ID NO.5; and the amino acid sequence of the protein encoded by the UDP-glucose pyrophosphorylase GalU gene is shown in SEQ ID NO.

6.

5. The recombinant Escherichia coli for producing curcumin glucoside according to claim 1, wherein The recombinant Escherichia coli BL21 (DE3) CBG06 comprises at least two recombinant vectors, including but not limited to pgex-4t-1, pMAL-c5X, pET-28a (+) or pACYCDuet-1.

6. The recombinant Escherichia coli for producing curcumin glucoside according to claim 5, characterized in that The recombinant Escherichia coli BL21 (DE3) CBG06 comprises two recombinant vectors, one of which comprises the glucose-6-phosphate mutase Pgm gene and the UDP-glucose pyrophosphorylase GalU gene, and the other comprises the double mutant of the polyphenol TDP-rhamnosyltransferase ppuG gene.

7. A method for constructing a recombinant Escherichia coli for producing curcumin glucoside as claimed in any one of claims 1 to 6, characterized in that: The following steps are involved: Step S1, constructing a recombinant vector containing the glucose-6-phosphate mutase Pgm gene and the UDP-glucose pyrophosphorylase GalU gene; Step S2, constructing a recombinant vector containing a double mutant of the polyphenol TDP-rhamnosyltransferase ppuG gene; Step S3, transforming the recombinant vector prepared in step S1 and the recombinant vector prepared in step S2 into Escherichia coli to obtain.

8. A method for producing curcumin glucoside, characterized in that: The recombinant Escherichia coli BL21 (DE3) CBG06 for producing curcumin glucoside according to any one of claims 1 to 6 is used as a fermentation strain, curcumin is used as a substrate, glucose is used as a glycosyl donor, and the mixture is cultured at a temperature of 35 to 40° C. until the bacterial concentration OD600 is 5 to 10, and an IPTG inducer is added to catalyze the synthesis of curcumin diglucoside.

9. The method for producing curcumin glucoside as claimed in claim 8, 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 0-26 h after adding the inducer.

10. Use of the recombinant Escherichia coli BL21 (DE3) CBG06 for producing curcumin glucoside according to any one of claims 1 to 6 in the preparation of curcumin glucoside or a product containing curcumin glucoside.

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