A genetically engineered bacterium for the whole-cell catalyzed synthesis of glucoside ferulate and its application

Abstract

The present invention discloses a genetically engineered bacterium for the whole-cell catalyzed synthesis of glucoside ferulate and its application, belonging to the technical field of bioengineering. In the present invention, by overexpressing the expression of two key enzymes involved in the synthesis of uridine diphosphate glucose (UDP-glucose) in the host cell, namely phosphoglucomutase and uridine diphosphate glucose pyrophosphorylase genes, the intracellular accumulation level of UDP-glucose is further increased; by transferring the UDP-glucosyltransferase gene into the host cell and achieving its high-efficiency expression, a genetically engineered bacterium capable of biocatalytic synthesis of glucoside ferulate is obtained. By adopting the technical scheme of the present invention, the problem of intracellular UDP-glucose supply can be solved, and glucoside ferulate is prepared by whole-cell catalysis using ferulic acid as a substrate. Finally, the yield of glucoside ferulate reaches 220.47 mg / L, and the molar conversion rate is 60.1%.

Description

Technical Field

[0001] The present invention relates to a genetically engineered bacterium for the whole-cell catalyzed synthesis of glucosyl ferulate and its application, belonging to the technical field of bioengineering. Background Art

[0002] Ferulic acid (FA) is widely present in various traditional Chinese medicines such as Angelica sinensis, Ligusticum chuanxiong, propolis, and Cimicifuga foetida, and has functions such as anti-inflammatory, analgesic, antithrombotic, antitumor, anti-free radical, and regulation of human immune function. Ferulic acid has stable properties and excellent antioxidant effects. However, due to its molecular structure, its liposolubility and water solubility are poor, and it cannot better play its value in many industrial fields. Therefore, in order to improve the practical application value of ferulic acid, the research and development of ferulic acid derivatives have increasingly become a hot topic.

[0003] There are four active groups in the molecular structure of ferulic acid, namely phenolic hydroxyl group, carboxyl group, alkene bond, and aromatic ring, which can be modified respectively to generate corresponding derivatives. Currently, five types of ferulic acid derivatives can be generated, namely amide derivatives, ester derivatives, ether derivatives, ketone derivatives, and aromatic ring substituent derivatives. These derivatives can not only maintain the original properties of ferulic acid, but also have some properties that ferulic acid itself does not possess.

[0004] Glycosylation is one of the most common and important modifications in nature. UDP-glucose in organisms can react with macromolecules such as proteins, lipids, and cell wall polysaccharides, as well as small molecules such as oligosaccharides, to generate one or more molecules of glycosylated products. Glycosylation usually greatly changes the solubility, stability, and bioavailability of the parent compound. Many secondary metabolites in plants and microorganisms (such as polyphenols, terpenoids, polyketides, etc.) exist in the form of glucosides in organisms and have bioactivities equivalent to or stronger than those of the parent compounds.

[0005] Research findings show that glucosyl ferulate has better water solubility, higher bioavailability than ferulic acid, exhibits more stable and significant efficacy than ferulic acid, and has lower toxicity. However, at present, the chemical synthesis methods for glucosylation of ferulic acid have complex reaction steps, low yields, and many by-products, making it difficult to scale up and produce on a large scale, and they do not have practical production value. Extracting from natural plants greatly limits the yield of glucosyl ferulate due to limited sources. Enzymatic synthesis requires the addition of expensive glycosyl donor UDP-glucose (uridinediphosphate glucose), making it difficult to scale up the reaction. By regulating the intracellular UDP-glucose biosynthesis pathway, increasing the level of UDP-glucose synthesis in cells, and simultaneously highly expressing UDP-glucosyltransferase, the synthesis level of glycosylated products can be significantly increased, which helps the industrial production of glucosyl ferulate by biocatalysis. Summary of the Invention

[0006] The main object of the present invention is to provide a genetically engineered bacterium with whole-cell catalytic synthesis of glucosyl ferulate. The preparation process mainly includes the following steps: (1) By overexpressing the expression of two key enzymes involved in the synthesis of uridine diphosphate glucose (UDP-glucose) in the host cell, namely phosphoglucomutase and uridine diphosphate glucose pyrophosphorylase genes, thereby increasing the intracellular accumulation level of UDP-glucose; (2) Transferring the UDP-glucosyltransferase gene into the host cell and achieving its high expression, that is, obtaining a genetically engineered bacterium with biocatalytic synthesis of glucosyl ferulate.

[0007] The present invention solves the problem of insufficient supply of UDP-glucose in the glucosylation reaction of ferulic acid. The content of glycosyl donor UDP-glucose in the host cell is very low, which is not conducive to the efficient synthesis of glucosyl ferulate. The present invention also solves the problem of low solubility of ferulic acid and provides a genetically engineered bacterium for biocatalytic synthesis of glucosyl ferulate. Using ferulic acid as a substrate, its water solubility is increased through glycosylation reaction, thereby improving its bioavailability. After glycosylation modification of the lead compound, it helps to modify ferulic acid and broaden the application scope of ferulic acid and its derivatives.

[0008] The present invention provides a genetically engineered bacterium that overexpresses phosphoglucomutase PGM and UDP-glucose pyrophosphorylase GalU derived from Escherichia coli, and simultaneously overexpresses UDP-glucosyltransferase.

[0009] In one embodiment of the present invention, the phosphoglucomutase PGM is derived from Escherichia coli, and its nucleotide sequence is as shown in SEQ ID NO.1, and its amino acid sequence is as shown in SEQ ID NO.2.

[0010] In one embodiment of the present invention, the UDP-glucose pyrophosphorylase GalU is derived from Escherichia coli, and its nucleotide sequence is as shown in SEQ ID NO.3, and its amino acid sequence is as shown in SEQ ID NO.4.

[0011] In one embodiment of the present invention, the UDP-glucosyltransferase is UDP-glucosyltransferase CsUGT84A22, which is derived from Camellia sinensis, and its nucleotide sequence is as shown in SEQ ID NO.5, and its amino acid sequence is as shown in SEQ ID NO.6.

[0012] In one embodiment of the present invention, the UDP-glucosyltransferase is UDP-glucosyltransferase AtUGT71C1, which is derived from Arabidopsis thaliana, and its nucleotide sequence is as shown in SEQ ID NO.7, and its amino acid sequence is as shown in SEQ ID NO.8.

[0013] In one embodiment of the present invention, the UDP-glucosyltransferase is UDP-glucosyltransferase NsCJ030-MR1G027745, which is derived from Nyssa sinensis, and its nucleotide sequence is as shown in SEQ ID NO.9, and its amino acid sequence is as shown in SEQ ID NO.10.

[0014] In one embodiment of the present invention, the UDP-glucosyltransferase is UDP-glucosyltransferase CmCMV-011720, which is derived from Castanea mollissima (Chinese chestnut), and its nucleotide sequence is as shown in SEQ ID NO.11, and its amino acid sequence is as shown in SEQ ID NO.12.

[0015] In one embodiment of the present invention, the UDP-glucosyltransferase is UDP-glucosyltransferase PgUGT84A23, which is derived from Punica granatum (Pomegranate), and its nucleotide sequence is as shown in SEQ ID NO.13, and its amino acid sequence is as shown in SEQ ID NO.14.

[0016] In one embodiment of the present invention, the genetically engineered bacterium uses pBAD(His A), pACYCDuet-1 or pGEX-2T as an expression vector.

[0017] In one embodiment of the present invention, the genetically engineered bacterium uses the pBAD plasmid as an expression vector, and is connected to the expression vector in the order of UDP-glucosyltransferase, phosphoglucomutase, and UDP-glucose pyrophosphorylase.

[0018] In one embodiment of the present invention, the genetically engineered bacterium uses Escherichia coli as an expression host.

[0019] The present invention provides a genetically engineered bacterium BadCsPmGu for whole-cell catalytic synthesis of glucoside ferulate, and its preparation process includes:

[0020] (1) Enhancing the expression of the endogenous or heterologous phosphoglucomutase gene in the host bacterium;

[0021] (2) Enhancing the expression of the endogenous or heterologous uridine diphosphate glucose pyrophosphorylase in the host bacterium;

[0022] (3) Transferring the UDP-glucosyltransferase gene into the host bacterium and enabling its high expression.

[0023] (4) After the operations of (1), (2), and (3) are completed in any order, the genetically engineered bacterium BadCsPmGu capable of catalytically synthesizing glucoside ferulate is obtained.

[0024] In one embodiment of the present invention, the genetically engineered bacterium uses Escherichia coli BL21(DE3) as an expression host.

[0025] In one embodiment of the present invention, the genetically engineered bacterium uses pBAD(His A) as an expression vector.

[0026] In one embodiment of the present invention, the phosphoglucomutase PGM gene is derived from Escherichia coli, and its nucleotide sequence is as shown in SEQ ID NO.1, and the amino acid sequence is as shown in SEQ ID NO.2; the UDP-glucose pyrophosphorylase GalU gene is derived from Escherichia coli, and its nucleotide sequence is as shown in SEQ ID NO.3, and the amino acid sequence is as shown in SEQ ID NO.4; the UDP-glucosyltransferase CsUGT84A22 gene is derived from Camellia sinensis, and its nucleotide sequence is as shown in SEQ ID NO.5, and the amino acid sequence is as shown in SEQ ID NO.6.

[0027] The present invention also provides a method for producing glucoside ferulate using ferulic acid as a substrate. The above-mentioned genetically engineered bacterium BadCsPmGu is used as a whole-cell catalyst and added to a whole-cell catalytic system containing the substrate ferulic acid, and glucoside ferulate is prepared by reaction.

[0028] In one embodiment of the present invention, the structural formula of the product glucosyl ferulate (I) is as follows:

[0029]

[0030] In one embodiment of the present invention, in the reaction system, the genetically engineered bacterium is recombinant Escherichia coli, and the added OD 600 reaches 5 to 15; the concentration of the substrate ferulic acid is: 0.2 to 0.5 g / L.

[0031] In one embodiment of the present invention, the reaction conditions in the reaction system are: the reaction temperature is 30 °C, the initial pH is 7.0, and the rotation speed is 200 rpm.

[0032] In one embodiment of the present invention, the whole-cell catalyst is obtained by culturing recombinant Escherichia coli in LB medium until the OD 600 is about 0.6 to 0.8, adding arabinose with a final concentration of 2 g / L, culturing overnight at 16 °C and 200 rpm to induce the expression of recombinant protein; after the induction ends, the bacterial cells are collected by centrifugation, washed twice with PBS buffer solution with pH 7.5 and 10 mmol / L, and the bacterial cells are collected to obtain the whole-cell catalyst.

[0033] In one embodiment of the present invention, the genetically engineered bacterium is BadCsPmGu, and whole-cell catalysis is carried out in a reaction flask with M9 synthetic medium. Substrate ferulic acid is added to the M9 synthetic medium for biocatalytic synthesis of the corresponding product glucosyl ferulate.

[0034] In one embodiment of the present invention, the whole-cell catalysis is carried out under the following reaction parameters: adding the substrate ferulic acid to the bacterial cells with an OD 600 of 10, the reaction temperature is 30 °C, the initial pH is 7.0, the final concentration of the substrate ferulic acid is 0.2 g / L, and the conversion is carried out for 50 h, and the content of each component in the conversion solution is detected by liquid phase.

[0035] The present invention also provides the application of the above-mentioned genetically engineered bacterium in the preparation of glucosyl ferulate or a product containing glucosyl ferulate.

[0036] Beneficial effects

[0037] The present invention constructs a co-expression engineering bacterium of phosphoglucomutase, uridine diphosphate glucose pyrophosphorylase, and UDP-glucose transferase genes. By adopting the technical solution of the present invention, the problem of intracellular UDP-glucose supply can be solved, and ferulic acid glucoside is prepared by whole-cell catalysis using ferulic acid as a substrate. Finally, the yield of ferulic acid glucoside reaches 220.47 mg / L, and the molar conversion rate is 60.1%. The genetically engineered bacterium and the whole-cell catalysis method constructed in this study lay a theoretical and practical foundation for the industrial production and application of ferulic acid glucoside. Description of the Drawings

[0038] Figure 1 : The pathway for the synthesis of ferulic acid glucoside by whole-cell catalysis of recombinant Escherichia coli.

[0039] Figure 2 : HPLC chromatograms of ferulic acid and ferulic acid glucoside; among them, peak 1 is ferulic acid, and peak 2 is ferulic acid glucoside. Detailed Embodiments

[0040] The present invention discloses a genetically engineered bacterium for the whole-cell catalysis synthesis of ferulic acid glucoside and its application. The concept and technical effects of the present invention are clearly and completely described. The described embodiments are only part of the embodiments of the present invention, not all embodiments. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve. All similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as the scope protected by the present invention.

[0041] In the following examples, the phosphoglucomutase PGM is derived from Escherichia coli (Uniprot: P36938), and its protein sequence is shown in SEQ ID NO.2; the UDP-glucose pyrophosphorylase GalU is derived from Escherichia coli (Uniprot: P0AEP3), and its protein sequence is as shown in SEQ ID NO.4; the UDP-glucosyltransferase CsUGT84A22 is derived from Camellia sinensis (Uniprot: A0A125QVX1), and its protein sequence is as shown in SEQ ID NO.6; the UDP-glucosyltransferase AtUGT71C1 is derived from Arabidopsis thaliana (Uniprot: O82381), and its protein sequence is as shown in SEQ ID NO.8; the UDP-glucosyltransferase NsCJ030-MR1G027745 is derived from Nyssasinensis (Uniprot: A0A6A1WR66), and its protein sequence is as shown in SEQ ID NO.10; the UDP-glucosyltransferase CmCMV-011720 is derived from Castanea mollissima (Chinese chestnut) (Uniprot: A0A8J4VNJ2), and its protein sequence is as shown in SEQ ID NO.12; the gene of the UDP-glucosyltransferase PgUGT84A23 is derived from Punica granatum (Pomegranate) (Uniprot: A0A193AUF6), and its protein sequence is as shown in SEQ ID NO.14.

[0042] The culture media involved in the following examples are as follows:

[0043] LB medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride (2% agar powder is added to the solid medium).

[0044] M9 medium: 4 g / L glucose, 2 mM MgSO4, 0.1 mM CaCl2, 12.8 g / L Na2HPO4·7H2O, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl.

[0045] The detection methods involved in the following examples are as follows:

[0046] Detection of the content of each component in the catalytic system

[0047] Collect the whole-cell catalytic reaction solution, centrifuge it at 12,000 rpm for 1 min, take 300 μL of the supernatant, add 3 volumes of methanol for dilution, and filter it using a 0.22-μm filter membrane. Chromatographic conditions: Chromatographic column: InfinityLab Poroshell 120 EC-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: 0 - 10 min, 40% B; 10 - 15 min, 40 - 70% B; 15 - 20 min, 100% B. Flow rate: 1.0 mL / min, Column temperature: 30 °C, Injection volume: 2 μL. Detector: UV Detector, Detection wavelength: 320 nm.

[0048] Identification of glucoside ferulate

[0049] Collect the whole-cell catalytic product for mass spectrometry and nuclear magnetic resonance spectroscopy (NMR) analysis. The nuclear magnetic resonance data are as follows: ESIMS: m / z 379 [M+Na] + , 1H NMR (400 MHz, CD3OD): δ 3.89 (3H, s, OCH3), 5.58 (1H, d, J = 7.6 Hz, H-1’), 6.40 (1H, d, J = 15.6 Hz, H-8), 6.81 (1H, d, J = 8.0 Hz, H-5), 7.09 (1H, dd, J = 8.4, 2.0 Hz, H-6), 7.20 (1H, d, J = 2.0 Hz, H-2), 7.72 (1H, d, J = 15.6 Hz, H-7);

[0050] 13 13C NMR (100 MHz, CD3OD): δ 56.4 (OCH3), 62.3 (C-6’), 71.0 - 78.7 (C-2’, 3’, 4’, 5’), 95.7 (C-1’), 111.6 (C-2), 114.6 (C-8), 116.4 (C-5), 124.2 (C-6), 127.4 (C-1), 148.1 (C-7), 149.2 (C-3), 150.7 (C-4), 167.5 (C-9).

[0051] Example 1: Construction of genetically engineered bacteria

[0052] (1) Synthesize related genes

[0053] According to the Escherichia coli phosphoglucomutase PGM protein sequence published by Uniprot, after codon optimization, the gene pgm encoding phosphoglucomutase Pgm with the sequence shown in SEQ ID NO.1 is obtained.

[0054] According to the protein sequence of Escherichia coli UDP-glucose pyrophosphorylase GalU published by Uniprot, after codon optimization, the gene galU encoding UDP-glucose pyrophosphorylase GalU with the sequence shown in SEQ ID NO.3 was obtained.

[0055] According to the protein sequence of Camellia sinensis UDP-glucosyltransferase CsUGT84A22 published by Uniprot, after codon optimization, the gene csUGT84A22 encoding UDP-glucosyltransferase CsUGT84A22 with the sequence shown in SEQ ID NO.5 was obtained.

[0056] (2) Obtaining of recombinant vector

[0057] Using a multi-fragment homologous recombination kit (Nanjing Novoprotein Biotechnology Co., Ltd.), the three genes obtained in step (1) were ligated with the expression plasmid pBAD(His A) (the three were placed after the araBAD promoter and ligated in the order of csUGT84A22, pgm, galU, that is, ligated in the order of UDP-glucosyltransferase, phosphoglucomutase, UDP-glucose pyrophosphorylase). The ligation product was transformed into competent cells of E.coli Trans1-T1 (TransGen Biotech Co., Ltd., Beijing) and spread on an LB plate containing ampicillin.

[0058] Positive transformants were screened and verified by colony PCR, and the recombinant plasmid pBAD-csUGT84A22-pgm-galU was extracted.

[0059] (3) Construction of genetically engineered bacteria

[0060] The recombinant plasmid pBAD-csUGT84A22-pgm-galU was transformed into E.coli BL21(DE3). Colonies grown on an LB plate containing ampicillin were picked and cultured in a shake flask, and thus the genetically engineered bacteria BadCsPmGu were obtained.

[0061] (4) Construction of genetically engineered bacteria: BadAtPmGu, BadNsPmGu, BadCmPmGu, BadPg-PmGu

[0062] The specific implementation is the same as steps (1) to (3), except that the adjusted Camellia sinensis UDP-glucosyltransferase CsUGT84A22 protein sequences are respectively: UDP-glucosyltransferase from Arabidopsis thaliana, UDP-glucosyltransferase from Nyssa sinensis, UDP-glucosyltransferase from Castanea mollissima (Chinese chestnut), UDP-glucosyltransferase from Punica granatum (Pomegranate), specifically as follows:

[0063] According to the Arabidopsis thaliana UDP-glucosyltransferase AtUGT71C1 protein sequence published by Uniprot, after codon optimization, the gene atUGT71C1 encoding UDP-glucosyltransferase AtUGT71C1 with the sequence shown in SEQ ID NO.7 is obtained.

[0064] According to the Nyssa sinensis UDP-glucosyltransferase NsCJ030-MR1G027745 protein sequence published by Uniprot, after codon optimization, the gene nsCJ030-MR1G027745 encoding UDP-glucosyltransferase NsCJ030-MR1G027745 with the sequence shown in SEQ ID NO.9 is obtained.

[0065] According to the Castanea mollissima (Chinese chestnut) UDP-glucosyltransferase CmCMV-011720 protein sequence published by Uniprot, after codon optimization, the gene cmCMV-011720 encoding UDP-glucosyltransferase CmCMV-011720 with the sequence shown in SEQ ID NO.11 is obtained.

[0066] According to the Punica granatum (Pomegranate) UDP-glucosyltransferase PgUGT84A23 protein sequence published by Uniprot, after codon optimization, the gene pgUGT84A23 encoding UDP-glucosyltransferase PgUGT84A23 with the sequence shown in SEQ ID NO.13 is obtained.

[0067] Recombinant plasmids were separately prepared: pBAD-atUGT71C1-pgm-galU, pBAD-nsCJ030-MR1G027745-pgm-galU, pBAD-cmCMV-011720-pgm-galU, pBAD-pgUGT84A23-pgm-galU;

[0068] The recombinant plasmids were transformed into E. coli BL21(DE3), and the colonies grown on the LB plate containing ampicillin were picked and cultured in a shaking flask to obtain the genetically engineered bacteria BadAtPmGu, BadNsPmGu, BadCmPmGu, BadPg-PmGu.

[0069] Example 2: Preparation of whole-cell catalyst

[0070] The specific steps are as follows:

[0071] (1) The genetically engineered strains BadCsPmGu, BadAtPmGu, BadNsPmGu, BadCmPmGu, BadPg-PmGu prepared in Example 1 were respectively placed in 5 mL of LB medium containing 100 mg / L ampicillin and cultured overnight at 37 °C to obtain seed solutions;

[0072] (2) The seed solutions were transferred into 50 mL of LB medium containing 100 μg / L ampicillin at an inoculation amount of 1% (v / v) and cultured at 37 °C. When the OD 600 was approximately 0.6 - 0.8, arabinose with a final concentration of 2 g / L was added for induction, and induction culture was carried out overnight at 16 °C and 200 rpm; centrifuged at 4000 rpm to collect the cells, washed twice with PBS buffer (pH 7.5, 10 mM) and resuspended the cells to obtain the whole-cell catalyst.

[0073] Example 3: Whole-cell catalytic synthesis of glucoside ferulate

[0074] The whole-cell catalytic synthesis of glucoside ferulate by recombinant Escherichia coli is as Figure 1 shown, and the specific steps are as follows:

[0075] (1) The separately collected whole-cell catalysts BadCsPmGu, BadAtPmGu, BadNsPmGu, BadCmPmGu, BadPg-PmGu were added to the M9 medium, and the final OD of the cells 600 was approximately 10.

[0076] (2) Add ferulic acid with a final concentration of 0.2 g / L as a substrate to the reaction system obtained in step (1), and add 100 μg / mL of ampicillin and arabinose with a final concentration of 2 g / L. Conduct whole-cell catalytic reaction on a constant-temperature shaker at 30 °C and 200 rpm. Reaction time: 24 h.

[0077] (3) Use E. coli BL21(DE3) containing the empty plasmid pBAD(His A) as a blank control. Take samples after fermentation for 24 h and detect the content of each component in the catalytic system by liquid phase ( Figure 2 ), and the results are shown in Table 1.

[0078] Table 1 Comparison of fermentation conditions of different engineered bacteria

[0079]

[0080]

[0081] (4) Identification of glucoside ferulate

[0082] Collect the whole-cell catalytic product for mass spectrometry and nuclear magnetic resonance spectroscopy (NMR) analysis. The nuclear magnetic resonance data are as follows: ESIMS: m / z 379 [M+Na] + , 1H NMR (400 MHz, CD3OD): δ 3.89 (3H, s, OCH3), 5.58 (1H, d, J = 7.6 Hz, H-1’), 6.40 (1H, d, J = 15.6 Hz, H-8), 6.81 (1H, d, J = 8.0 Hz, H-5), 7.09 (1H, dd, J = 8.4, 2.0 Hz, H-6), 7.20 (1H, d, J = 2.0 Hz, H-2), 7.72 (1H, d, J = 15.6 Hz, H-7);

[0083] 13 C NMR (100 MHz, CD3OD): δ 56.4 (OCH3), 62.3 (C-6’), 71.0 - 78.7 (C-2’, 3’, 4’, 5’), 95.7 (C-1’), 111.6 (C-2), 114.6 (C-8), 116.4 (C-5), 124.2 (C-6), 127.4 (C-1), 148.1 (C-7), 149.2 (C-3), 150.7 (C-4), 167.5 (C-9).

[0084] The collected catalytic product is identified as glucoside ferulate, and its structural formula is shown in (Ⅰ).

[0085]

[0086] In summary, the present invention first provides a genetically engineered bacterium BadCsPmGu for the whole-cell catalytic synthesis of glucoside ferulate. Finally, using ferulic acid as the substrate, fermentation was carried out in a shake flask for 24 hours, and the yield of the glycosylation product glucoside ferulate reached 220.47 mg / L, with a molar conversion rate of 60.1%.

[0087] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A genetically engineered bacterium, characterized in that, The genetically engineered bacterium uses Escherichia coli as the expression host, overexpresses phosphoglucomutase PGM and UDP-glucose pyrophosphorylase GalU derived from Escherichia coli, and simultaneously overexpresses UDP-glucosyltransferase CsUGT84A22 derived from Camellia sinensis. The amino acid sequence of the phosphoglucomutase PGM is shown in SEQ ID NO.2; the amino acid sequence of the UDP-glucose pyrophosphorylase GalU is shown in SEQ ID NO.4; the amino acid sequence of the UDP-glucosyltransferase CsUGT84A22 is shown in SEQ ID NO.

6.

2. The genetically engineered bacterium according to claim 1, characterized in that, The nucleotide sequence encoding the phosphoglucomutase PGM is shown in SEQ ID NO.1; the nucleotide sequence encoding the UDP-glucose pyrophosphorylase GalU is shown in SEQ ID NO.3; the nucleotide sequence encoding the UDP-glucosyltransferase CsUGT84A22 is shown in SEQ ID NO.

5.

3. The genetically engineered bacterium according to claim 2, characterized in that, The genetically engineered bacterium uses pBAD(His A), pACYCDuet-1 or pGEX-2T as the expression vector.

4. The genetically engineered bacterium according to claim 3, wherein The genetically engineered bacterium uses the pBAD(HisA) plasmid as the expression vector, and is ligated into the expression vector in the order of UDP-glucosyltransferase, phosphoglucomutase, and UDP-glucose pyrophosphorylase.

5. A method for preparing glucoside ferulate by whole-cell transformation, characterized in that, The method is to add the genetically engineered bacterium according to any one of claims 1 to 4 to a reaction system containing the substrate ferulic acid, carry out the reaction, and prepare ferulic acid glucoside.

6. The method according to claim 5, characterized in that, In the reaction system, the genetically engineered bacterium is recombinant Escherichia coli, and the added OD 600 reaches 5 to 15; the concentration of the substrate ferulic acid is 0.2 to 0.5 g / L.

7. The method according to claim 6, wherein The reaction conditions in the reaction system are: the reaction temperature is 30 °C, the initial pH is 7.0, and the rotation speed is 200 rpm.

8. Use of the genetically engineered bacterium according to any one of claims 1 to 4 in the preparation of ferulic acid glucoside or a product containing ferulic acid glucoside.

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