Construction and application of a genetically engineered bacterium for synthesizing curcumin glucoside

Abstract

The present invention discloses a genetically engineered bacterium construction and application for synthesizing curcumin glucoside, belonging to the field of genetic engineering. The present invention uses Escherichia coli BL21 (DE3) as an engineering strain, overexpresses two key enzymes involved in UDP-glucose synthesis in Escherichia coli, namely glucose-6-phosphomutase and UDP-glucose pyrophosphorylase, to increase the level of endogenous UDP-glucose in the cell, effectively improving the synthesis efficiency of whole-cell catalytic synthesis of curcumin glucoside; at the same time, overexpresses the UDP-glucosyltransferase CaUGT2 from Catharanthus roseus; the genetically engineered bacterium can efficiently synthesize curcumin monoglucoside and curcumin diglucoside, can effectively solve the solubility problem of curcumin, improve the biological absorption rate and biological availability of curcumin, and expand its application value in medicines, cosmetics and food.

Description

Technical Field

[0001] The invention relates to the construction of a genetically engineered bacterium for synthesizing curcumin glucoside and application thereof, belonging to the field of genetic engineering. Background Art

[0002] Curcumin is a diketone compound extracted from the rhizomes of some plants in the Zingiberaceae and Araceae families. Its chemical formula is C 21 H 20 O6. Turmeric contains approximately 3% to 6% curcumin, a rare diketone pigment in the plant kingdom. Curcumin is an orange-yellow crystalline powder with a slightly bitter taste and is insoluble in water. In food production, it is primarily used to color products such as sausages, canned foods, and pickled sauces. Curcumin was first isolated from turmeric in 1870 as a low-molecular-weight polyphenolic compound. Its chemical structure, diferuloylmethane, was elucidated in 1910, and subsequent research on its physiological and pharmacological effects has made significant progress. Curcumin exhibits a wide range of pharmacological activities, including anti-inflammatory, antioxidant, lipid-regulating, antiviral, anti-infective, anti-tumor, anticoagulant, anti-hepatic fibrosis, and anti-atherosclerotic properties, with low toxicity and minimal adverse reactions. Curcumin is currently one of the world's best-selling natural food pigments and is a food additive approved by the World Health Organization, the U.S. Food and Drug Administration, and many other countries. Curcumin is not only a nonsteroidal anti-inflammatory drug but also possesses a wide range of preventive properties. Given that modern medical research has found that the occurrence of many human diseases is related to the formation of free radicals and the involvement of inflammatory responses, the antioxidant and anti-inflammatory effects of curcumin have attracted widespread attention from scholars at home and abroad. However, due to its poor water solubility, curcumin has extremely poor biological absorption and bioavailability, which has limited its development in fields such as pharmaceuticals, cosmetics, and food. Although there are some solutions on the market, namely increasing its water solubility through the formulation of emulsifiers, this does not mean that it is truly water-soluble. To address the limited application of curcumin, many scholars are currently developing various curcumin derivatives to improve its water solubility, biological absorption rate, and bioavailability.

[0003] It is reported that improving the solubility of ketone compounds mainly relies on glycosylation modification, and the most studied glycosylation modification is glucosidation reaction. The use of chemical methods for glucosidation modification has complex steps, low yields, and many by-products. It is difficult to scale up and produce, and has no practical value for production. Microbial enzymatic glucosidation modification has mild reaction conditions, does not require protection and deprotection reactions, has high stereo and regioselectivity, and can glucosidate ketone compounds in a targeted and efficient manner. However, since the enzymatic reaction requires the addition of an activated glycosyl donor UDP-glucose, and this precursor is relatively expensive, it is difficult to scale up the reaction. Summary of the Invention

[0004] The present invention aims to provide a UDP-glucose synthesis pathway and a genetically engineered bacterium for synthesizing curcumin glucoside, thereby increasing the endogenous UDP-glucose level in cells and improving the solubility of curcumin, thereby expanding its scope of utilization.

[0005] Escherichia coli, as a model strain, has the advantages of fast growth, easy cultivation, clear genetic background, and ease of genetic modification. It can also highly express UDP-glucose transferase for glucosidation modification, making it an excellent choice for whole-cell catalytic glucosidation modification. The present invention uses Escherichia coli BL21 (DE3) as an engineered strain to overexpress two key enzymes involved in UDP-glucose synthesis in E. coli, namely glucose-6-phosphate mutase (phosphoglucomutase, Pgm) and UDP-glucose pyrophosphorylase (UTP-glucose-1-phosphate uridylyltransferase, GalU), thereby increasing the endogenous UDP-glucose level in the cell and effectively improving the synthesis efficiency of curcumin glucoside catalyzed by whole-cell catalysis.

[0006] The invention provides a genetically engineered bacterium, which overexpresses glucose-6-phosphate mutase Pgm and UDP-glucose pyrophosphorylase GalU from Escherichia coli, and simultaneously overexpresses UDP-glucose transferase CaUGT2 from Catharanthus roseus.

[0007] In one embodiment of the present invention, the amino acid sequence of the glucose-6-phosphate mutase Pgm is shown as SEQ ID No. 1; the amino acid sequence of the UDP-glucose pyrophosphorylase GalU is shown as SEQ ID No. 2; and the amino acid sequence of the UDP-glucose transferase CaUGT2 is shown as SEQ ID No. 3.

[0008] In one embodiment of the present invention, the nucleotide sequence encoding the glucose-6-phosphate mutase Pgm is shown as SEQ ID No.4; the nucleotide sequence encoding the UDP-glucose pyrophosphorylase GalU is shown as SEQ ID No.5; and the nucleotide sequence encoding the UDP-glucose transferase CaUGT2 is shown as SEQ ID No.6.

[0009] In one embodiment of the present invention, the genetically engineered bacteria uses pGEX, pZSH, pBAD or pET as an expression vector.

[0010] In one embodiment of the present invention, the genetically engineered bacteria uses the pACYCDuet-1 plasmid to express glucose-6-phosphate mutase and UDP-glucose pyrophosphorylase; and uses pGEX to express UDP-glucosyltransferase.

[0011] In one embodiment of the present invention, the genetically engineered bacteria uses Escherichia coli, Bacillus subtilis or Pichia pastoris as the expression host.

[0012] In one embodiment of the present invention, the genetically engineered bacteria uses Escherichia coli as the expression host.

[0013] In one embodiment of the present invention, the Escherichia coli is Escherichia coli BL21 (DE3).

[0014] The present invention also provides a method for preparing curcumin glucoside by whole-cell conversion, wherein the recombinant Escherichia coli is added to a reaction system containing substrates curcumin and glucose, and the reaction is carried out to prepare curcumin glucoside.

[0015] In one embodiment of the present invention, the curcumin glucoside (I) and curcumin diglucoside (II) have the following structural formulas:

[0016]

[0017] In one embodiment of the present invention, the method is:

[0018] The recombinant E. coli was picked up and inoculated into liquid LB medium containing ampicillin and chloramphenicol resistance, and cultured until its OD 600 After reaching 0.6-1.0, 1M IPTG was added to induce expression, and curcumin substrate and glucose were added to react to prepare curcumin glucoside.

[0019] In one embodiment of the present invention, the recombinant E. coli was picked and inoculated into a liquid LB medium containing ampicillin and chloramphenicol resistance, and cultured at 37°C and 200 rpm in a shaking incubator until its OD 600 Reached 0.6.

[0020] In one embodiment of the present invention, the glucose concentration is between 0 g / L and 10 g / L.

[0021] In one embodiment of the present invention, the glucose concentration is 2 g / L.

[0022] In one embodiment of the present invention, the concentration of curcumin is between 0.1-2.5 g / L.

[0023] In one embodiment of the present invention, the concentration of curcumin is 1.0 g / L.

[0024] In one embodiment of the present invention, the curcumin substrate is added at a time point between 0 and 48 hours after induction.

[0025] In one embodiment of the present invention, the curcumin substrate is added 0 h after induction.

[0026] In one embodiment of the present invention, the induction temperature of the genetically engineered bacteria is 16-30°C.

[0027] In one embodiment of the present invention, the induction temperature of the genetically engineered bacteria is 30°C.

[0028] In one embodiment of the present invention, the reaction time is 24 to 72 hours.

[0029] In one embodiment of the present invention, the reaction time is 30 hours.

[0030] The present invention also provides the use of the recombinant Escherichia coli in preparing curcumin glucoside or a product containing curcumin glucoside.

[0031] Beneficial effects

[0032] (1) The present invention increases the concentration of UDP-glucose in the cell metabolism pool by overexpressing glucose-6-phosphate mutase (phosphoglucomu-tase, Pgm) and UDP-glucose pyrophosphorylase (UTP-glucose-1-phosphate pyrophosphorylase, GalU) in the host cell. Under normal conditions, the concentration of UDP-glucose in the cell metabolism pool is very low. Even if the cell overexpresses UDP-glucosyltransferase with the activity of catalyzing curcumin to form curcumin glucoside, it is difficult to obtain curcumin glucoside efficiently and in large quantities. The present invention provides UDP-glucose through engineered bacteria, uses curcumin as a substrate, and catalyzes the efficient synthesis of curcumin monoglucoside and curcumin diglucoside in the whole cell, which can effectively solve the solubility problem of curcumin, improve the biological absorption rate and biological availability of curcumin, and expand its application value in medicines, cosmetics and food.

[0033] (2) Compared with the prior art, the present invention constructs a genetically engineered bacterium that provides UDP-glucose, and uses curcumin as a substrate to efficiently synthesize curcumin glucoside and curcumin diglucoside. This effectively solves the solubility problem of curcumin, greatly improves its biological absorption rate and bioavailability, and expands its application value in pharmaceuticals, cosmetics, and food. After 30 hours of reaction, the conversion rate of curcumin to curcumin glucoside is 62%, and the conversion rate to curcumin diglucoside is 28%. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 : Synthesis pathway of curcumin glucoside.

[0035] Figure 2 : Curcumin, curcumin glucoside and curcumin glucoside contents change with time.

[0036] Figure 3 : Liquid phase detection results.

[0037] Figure 4 : Mass spectrometry detection results; wherein, A is the mass spectrometry detection result of curcumin glucose monosaccharide, and B is the mass spectrometry detection result of curcumin glucose disaccharide.

[0038] Figure 5: Conversion effects of UDP-glucosyltransferases from different sources; Figure 5A To use the genetic engineering bacteria BL21-AhUGT83A1 transformation effect, Figure 5B For the transformation effect of genetically engineered bacteria BL21-UGT75B1, Figure 5C To use the genetic engineering bacteria BL21-UGT74F2 transformation effect, Figure 5D This is the transformation effect of the genetically engineered bacteria BL21-Ugt3. DETAILED DESCRIPTION

[0039] The present invention will be further described below with reference to specific embodiments. However, it should be understood that the embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements shall fall within the scope of protection of the present invention.

[0040] The culture medium involved in the following examples is as follows:

[0041] LB medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L.

[0042] The detection methods involved in the following embodiments are as follows:

[0043] Detection of curcumin monoglucoside and curcumin diglucoside:

[0044] HPLC detection:

[0045] The fermentation broth was collected and diluted with methanol in a 1:3 volume ratio. The broth was then filtered through a 0.22 μm filter. Chromatographic conditions: Column: InfinityLab Poroshell 120EC-C18, 4.6 x 150 mm, 2.7 μm; Mobile phase A: Water (0.1% trifluoroacetic acid), Mobile phase B: Methanol (0.1% trifluoroacetic acid). Elution program: 40-79% B (0-15 min); 100% B (15-20 min). Flow rate: 1.0 mL / min, Column temperature: 30°C, Injection volume: 15 μL. Detector: UV Detector, Detection wavelength: 423 nm.

[0046] Mass spectrometry conditions:

[0047] Ion source: ESI, simultaneous determination in positive and negative modes; heating gas flow rate: 10.0 L / min; nebulizing gas flow rate: 3.0 L / min; drying gas flow rate: 10.0 L / min; heating module temperature: 400°C; DL temperature: 250°C; interface temperature: 300°C.

[0048] Example 1: Construction of genetically engineered bacteria

[0049] The specific steps are as follows:

[0050] (1) Glucose-6-phosphate mutase Pgm (pgm, GenBank no. NP_415214) and UDP-glucose pyrophosphorylase GalU (galU, Genbank no. NP_415752) were both from Escherichia coli, and UDP-glucosyltransferase CaUGT2 (caUGT2 Genbank no. AB159213) was from Catharanthus roseus. The DNA sequences of the above enzymes were codon-optimized and synthesized in vitro by Beijing Qingke Biotechnology Co., Ltd.

[0051] (2) Preparation of recombinant vectors pACYCDuet-pgm-galU and pGEX-caUGT2

[0052] 1) The genes pgm and galU obtained in step (1) were seamlessly ligated with the vector pACYCDuet-1 in the order of pgm and galU according to the method described in the ClonExpress MultiS One Step Cloning Kit (Vazyme, China). The ligation products were transformed into E. coli Trans1-T1 (Beijing Quanshijin Biotechnology Co., Ltd.) competent cells and spread on LB plates containing chloramphenicol. Positive transformants were verified by colony PCR screening to obtain the recombinant plasmid pACYCDuet-pgm-galU.

[0053] 2) The caUGT2 gene obtained in step (1) was seamlessly ligated with the vector pGEX according to the sequence described in the ClonExpress MultiS One Step Cloning Kit (Vazyme, China). The ligation product was transformed into E. coli Trans1-T1 competent cells (Beijing Quanshijin Biotechnology Co., Ltd.), plated on LB plates containing ampicillin, and positive transformants were screened by colony PCR to obtain the recombinant plasmid pGEX-caUGT2. Positive transformants were screened by colony PCR and the recombinant plasmid was extracted.

[0054] 3) The recombinant plasmids pACYCDuet-pgm-galU and pGEX-caUGT2 were transformed into E. coli BL21(DE3). Colonies that grew on LB plates containing ampicillin and chloramphenicol were selected and cultured in shake flasks to obtain the genetically engineered strain E. coli BL21(DE3) / pACYCDuet-pgm-galU / pGEX-caUGT2, designated BL21-I.

[0055] Example 2: Whole-cell transformation of curcumin by genetically engineered bacteria BL21-I

[0056] The synthetic pathway of curcumin glucoside is as follows Figure 1 The specific steps are as follows:

[0057] (1) A single clone of the recombinant strain BL21-I obtained in Example 1 was picked and inoculated into a liquid LB medium containing a final concentration of 100 μg / ml ampicillin and 17 μg / ml chloramphenicol, and cultured at 37°C and 200 rpm in a shaking incubator until its OD 600 The pH value reached 0.6, and then 1 M IPTG was added to a final concentration of 1 mM. At the same time, curcumin substrate was added to a final concentration of 0.5 g / L, and glucose was added to a final concentration of 2.0 g / L. The culture conditions were changed to 30 ° C, 200 rpm, and cultured in the dark for 72 h to complete the biotransformation of curcumin.

[0058] (2) Samples were taken on time, the fermentation broth was collected, and the contents of curcumin monoglucoside and curcumin diglucoside were detected by HPLC.

[0059] The results show that ( Figures 2-3 ): After culturing in the dark for 72 hours, most of the curcumin and curcumin monoglucoside were converted into curcumin diglucoside. The content of the curcumin diglucoside reached 682 mg / L, and the molar conversion rate was 72.6%.

[0060] (3) Meanwhile, the mass spectrometry results of curcumin monoglucoside and curcumin diglucoside are shown in Table 1 and Figure 4 As shown in A and 4B.

[0061] Table 1: Mass spectrometry results of curcumin monoglucoside and curcumin diglucoside

[0062]

[0063] The results showed that in the constructed genetically engineered bacteria BL21-I biotransformation curcumin system, the content of curcumin was reduced, and two curcumin glycoside derivatives, curcumin monoglucoside and curcumin diglucoside, were produced.

[0064] The results verified that the constructed genetically engineered bacteria BL21-I recombinant strain can produce a large amount of UDP-glucose inside the cell, and catalyze the synthesis of curcumin monoglucoside and curcumin diglucoside from curcumin in vitro.

[0065] Quantitative analysis of curcumin monoglucoside and curcumin diglucoside revealed that after 72 h of reaction, most of the curcumin and curcumin monoglucoside were converted into curcumin diglucoside. The content of curcumin diglucoside reached 682 mg / L, and the molar conversion rate was 72.6%.

[0066] Example 3: Effects of UDP-glucosyltransferases from different sources on whole-cell conversion of curcumin

[0067] The present invention evaluated UDP-glucosyltransferases from various sources. In addition to the UDP-glucosyltransferase CaUGT2 (GenBank no. AB159213) from Catharanthus roseus described in Example 1, other enzymes evaluated included AhUGT83A1-like (GenBank no. KF411463) from Arachis hypogaea L., UGT75B1 (GenBank no. NC_003070.9) from Arabidopsis thaliana, UGT74F2 (GenBank no. NC_003071.7) from Arabidopsis thaliana, and Ugt3 (GenBank no. NC_051364.1) from Bombyx mori.

[0068] The specific embodiment is the same as Examples 1-2, except that the UDP-glucosyltransferase CaUGT2 (caUGT2 Genbank no. AB159213) from Catharanthus roseus in Example 1 is replaced with: AhUGT83A1-like (GenBank no. KF411463) from Arachis hypogaea L., UGT75B1 (GenBank no. NC_003070.9) from Arabidopsis thaliana, UGT74F2 (GenBank no. NC_003071.7) from Arabidopsis thaliana, and Ugt3 (GenBank no. NC_051364.1) from Bombyx mori, respectively, to prepare genetically engineered bacteria: BL21-AhUGT83A1, BL21-UGT75B1, BL21-UGT74F2, and BL21-Ugt3;

[0069] According to the method of Example 2, whole cell transformation was performed to prepare curcumin diglucoside, and the cells were cultured in the dark for 72 h. The content and molar conversion rate of curcumin diglucoside were detected. The results are shown in Table 2 and Figure 5A ~D shown.

[0070] Table 2: Content and molar conversion of curcumin diglucoside prepared by different genetically engineered bacteria

[0071] Different sources Content (mg / L) Molar conversion rate (%) AhUGT83A1-like 146 29.43 UGT75B1 230 36.00 UGT74F2 241 36.29 Ugt3 91 15.23

[0072] The results showed that the transferases from other sources were not as efficient as the UDP-glucosyltransferase CaUGT2 (Genbank no. AB159213) from Catharanthus roseus in Example 1.

[0073] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria uses Escherichia coli as an expression host, overexpresses glucose-6-phosphate mutase Pgm and UDP-glucose pyrophosphorylase GalU from Escherichia coli, and overexpresses Catharanthus roseus The amino acid sequence of the glucose-6-phosphate mutase Pgm is shown in SEQ ID No. 1; the amino acid sequence of the UDP-glucose pyrophosphorylase GalU is shown in SEQ ID No. 2; the amino acid sequence of the UDP-glucose pyrophosphorylase CaUGT2 is shown in SEQ ID No.

3. The genetically engineered bacteria uses the pACYCDuet plasmid to express the glucose-6-phosphate mutase and the UDP-glucose pyrophosphorylase; and the pGEX plasmid is used to express the UDP-glucose syltransferase.

2. The genetically engineered bacterium according to claim 1, characterized in that The nucleotide sequence encoding the glucose-6-phosphate mutase Pgm is shown in SEQ ID No. 4; the nucleotide sequence encoding the UDP-glucose pyrophosphorylase GalU is shown in SEQ ID No. 5; and the nucleotide sequence encoding the UDP-glucose syltransferase CaUGT2 is shown in SEQ ID No.

6.

3. A method for preparing curcumin glucoside by whole cell transformation, characterized in that: The method comprises adding the genetically engineered bacteria according to claim 1 or 2 to a reaction system containing substrates curcumin and glucose, and carrying out a reaction to prepare curcumin glucoside.

4. The method according to claim 3, characterized in that In the reaction system, the added OD of the genetically engineered bacteria 600 Reach 0.6-1.0; the concentration of the substrate curcumin is: 0.1-2.5 g / L; the concentration of glucose is 2 g / L.

5. The method according to claim 3, characterized in that The method comprises the following steps: culturing the culture in a shaking platform at 37°C and 200 rpm until the OD600 reaches 0.6, then adding 1M IPTG to a final concentration of 1 mM, adding a curcumin substrate to a final concentration of 0.5 g / L, and adding glucose to a final concentration of 2.0 g / L. The culture conditions are changed to 30°C and 200 rpm, and the culture is carried out in the dark for 72 hours to complete the biotransformation of curcumin.

6. Use of the genetically engineered bacteria according to claim 1 or 2 in the preparation of curcumin glucoside or products containing curcumin glucoside.

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