Method for biologically synthesizing hydroxytyrosol from escherichia coli by using levodopa

Abstract

The invention provides a method for biologically synthesizing hydroxytyrosol from escherichia coli by using levodopa. The method comprises the following steps: by taking escherichia coli as a chassis host bacterium, constructing a way for biologically synthesizing hydroxytyrosol by taking levodopa as a substrate through overexpression of an alcohol reductase gene yqhD, a dopa decarboxylase gene dodc and a tyramine oxidase gene tyo; the method comprises the following steps: knocking out a phenylacetaldehyde oxidase gene feaB and a 4, 5-DOPA external diol dioxygenase gene ygiD, so as to reduce the generation of by-products; nusA tag protein is fused at the N end of tyramine oxidase gene tyo, and catalase gene KatG is overexpressed, so that a synthesis path of hydroxytyrosol is optimized; according to the present invention, the expression of the glucose-6-phosphate isomerase gene pgi is inhibited so as to adjust the carbon flux of the glycolysis pathway and the pentose phosphate pathway, such that the hydroxytyrosol biosynthesis pathway is reinforced, and the efficient hydroxytyrosol biosynthesis is finally achieved by using the levodopa; the invention provides a new scheme for biosynthesis of hydroxytyrosol.

Description

Technical Field

[0001] The present invention belongs to the field of metabolic engineering, and particularly relates to the use of genetic engineering techniques to transform Escherichia coli to obtain a recombinant Escherichia coli strain that can effectively utilize L-DOPA to synthesize hydroxytyrosol. Background Art

[0002] Hydroxytyrosol (3,4-dihydroxyphenylethanol) is a natural polyphenolic compound with a structural feature of a catechol group and an ethanol side chain, and is mainly present in olives and their derivatives. As a functional molecule with strong antioxidant activity, hydroxytyrosol exhibits significant anti-cancer, antibacterial and anti-inflammatory properties by scavenging free radicals, inhibiting lipid peroxidation and regulating oxidative stress pathways, and has important application values in the fields of medicine, food and cosmetics. At present, the industrial production of hydroxytyrosol mainly relies on plant extraction and chemical synthesis methods. However, the former is limited by the scarcity of olive resources, complex extraction processes and low yields, while the latter faces challenges such as harsh reaction conditions of high temperature and high pressure, environmental pollutant emissions and regulatory restrictions. In recent years, microbial synthesis technology has become a research hotspot for alternative strategies due to its green sustainability and controllable costs. As a model industrial microorganism, Escherichia coli has great development potential in theoretically establishing an Escherichia coli cell factory for effectively synthesizing hydroxytyrosol using L-DOPA as a substrate, relying on its clear genetic background, strong plasticity of metabolic network, fast growth rate and simple large-scale cultivation. Summary of the Invention

[0003] The present invention aims at the above problems and provides a method for Escherichia coli to biosynthesize hydroxytyrosol using L-DOPA.

[0004] To achieve the above object, the technical solution adopted by the present invention is as follows: A method for Escherichia coli to biosynthesize hydroxytyrosol using L-DOPA, which uses Escherichia coli as a chassis host bacterium, and constructs a pathway for biosynthesizing hydroxytyrosol using L-DOPA as a substrate by overexpressing alcohol reductase gene yqhD, dopa decarboxylase gene dodc and tyramine oxidase gene tyo; The method comprises the following steps: (1) Construct a recombinant plasmid carrying alcohol reductase gene yqhD, dopa decarboxylase gene dodc and tyramine oxidase gene tyo, and transform it into the chassis host bacterium E. coli JM109(DE3) to obtain a recombinant bacterium E. coli-yqhD-dodc-tyo; (2) Ferment and culture E. coli-yqhD-dodc-tyo in a fermentation medium containing the substrate L-DOPA to obtain a fermentation broth containing hydroxytyrosol.

[0005] A method for the biosynthesis of hydroxytyrosol from L - dopa by Escherichia coli, which uses Escherichia coli as the chassis host bacterium, constructs a pathway for the biosynthesis of hydroxytyrosol using L - dopa as the substrate by overexpressing the alcohol dehydrogenase gene yqhD, the dopa decarboxylase gene dodc, and the tyramine oxidase gene tyo; reduces the generation of by - products by knocking out the phenylacetaldehyde oxidase gene feaB and the 4,5 - DOPA extradiol dioxygenase gene ygiD; The method comprises the following steps: (1) Using E. coli JM109(DE3) as the chassis host bacterium, sequentially knocking out the phenylacetaldehyde oxidase gene feaB and the 4,5 - DOPA extradiol dioxygenase gene ygiD by the λ - Red homologous recombination method to obtain the recombinant bacterium E. coli - ΔfeaB - ΔygiD; (2) Constructing a recombinant plasmid carrying the alcohol dehydrogenase gene yqhD, the dopa decarboxylase gene dodc, and the tyramine oxidase gene tyo, and transforming it into E. coli - ΔfeaB - ΔygiD to obtain the recombinant bacterium E. coli - ΔfeaB - ΔygiD - yqhD - dodc - tyo; (3) Fermenting and culturing E. coli - ΔfeaB - ΔygiD - yqhD - dodc - tyo in a fermentation medium containing the substrate L - dopa to obtain a fermentation broth containing hydroxytyrosol.

[0006] A method for the biosynthesis of hydroxytyrosol from L - dopa by Escherichia coli, which uses Escherichia coli as the chassis host bacterium, constructs a pathway for the biosynthesis of hydroxytyrosol using L - dopa as the substrate by overexpressing the alcohol dehydrogenase gene yqhD, the dopa decarboxylase gene dodc, and the tyramine oxidase gene tyo; reduces the generation of by - products by knocking out the phenylacetaldehyde oxidase gene feaB and the 4,5 - DOPA extradiol dioxygenase gene ygiD; optimizes the synthesis pathway of hydroxytyrosol by fusing the NusA tag protein to the N - terminus of the tyramine oxidase gene tyo and overexpressing the catalase gene KatG; The method comprises the following steps: (1) Using E. coli JM109(DE3) as the chassis host bacterium, sequentially knocking out the phenylacetaldehyde oxidase gene feaB and the 4,5 - DOPA extradiol dioxygenase gene ygiD by the λ - Red homologous recombination method to obtain the recombinant bacterium E. coli - ΔfeaB - ΔygiD; (2) Construct a recombinant plasmid carrying the alcohol dehydrogenase gene yqhD, the dopa decarboxylase gene dodc, the fusion gene nusA tyo, and the catalase gene KatG, and transform it into E. coli-ΔfeaB-ΔygiD to obtain the recombinant strain E. coli-ΔfeaB-ΔygiD-yqhD-dodc-nusA tyo-KatG; (3) Ferment and culture E. coli-ΔfeaB-ΔygiD-yqhD-dodc-nusA tyo-KatG in a fermentation medium containing the substrate L-dopa to obtain a fermentation broth containing hydroxytyrosol.

[0007] A method for Escherichia coli to biosynthesize hydroxytyrosol using L-dopa, which uses Escherichia coli as a chassis host bacterium, constructs a pathway for biosynthesizing hydroxytyrosol using L-dopa as a substrate by overexpressing the alcohol dehydrogenase gene yqhD, the dopa decarboxylase gene dodc, and the tyramine oxidase gene tyo; reduces the generation of by-products by knocking out the phenylacetaldehyde oxidase gene feaB and the 4,5-DOPA extradiol dioxygenase gene ygiD; optimizes the synthesis pathway of hydroxytyrosol by fusing the NusA tag protein to the N-terminus of the tyramine oxidase gene tyo and overexpressing the catalase gene KatG; regulates the carbon flux of the glycolysis pathway and the pentose phosphate pathway by inhibiting the expression of the glucose-6-phosphate isomerase gene pgi, thereby strengthening the biosynthetic pathway of hydroxytyrosol; The method includes the following steps: (1) Using E. coli JM109(DE3) as a chassis host bacterium, sequentially knock out the phenylacetaldehyde oxidase gene feaB and the 4,5-DOPA extradiol dioxygenase gene ygiD by the λ-Red homologous recombination method to obtain the recombinant strain E. coli-ΔfeaB-ΔygiD; (2) Construct a recombinant plasmid carrying the alcohol dehydrogenase gene yqhD, the dopa decarboxylase gene dodc, the fusion gene nusA tyo, and the catalase gene KatG, and transform it into E. coli-ΔfeaB-ΔygiD to obtain the recombinant strain E. coli-ΔfeaB-ΔygiD-yqhD-dodc-nusA tyo-KatG; (3) Construct a recombinant plasmid carrying an antisense RNA fragment of the glucose-6-phosphate isomerase gene pgi, and transform it into E. coli-ΔfeaB-ΔygiD-yqhD-dodc-nusA tyo-KatG to obtain the recombinant strain E. coli-ΔfeaB-ΔygiD-yqhD-dodc-nusA tyo-KatG-aspgi; (4) Ferment and culture E.coli-ΔfeaB-ΔygiD-yqhD-dodc-nusA tyo-KatG-aspgi in a fermentation medium containing the substrate L-dopa to obtain a fermentation broth containing hydroxytyrosol. Description of the Drawings

[0008] Figure 1 : Diagram of the cell factory.

[0009] Figure 2 : Recombinant plasmid map.

[0010] Figure 3 : Fermentation diagram of E.coli-yqhD-dodc-tyo product.

[0011] Figure 4 : LC-MS analysis of the fermentation broth of E.coli-yqhD-dodc-tyo.

[0012] Figure 5 : Fermentation diagram of E.coli-ΔfeaB-ΔygiD-yqhD-dodc-tyo product.

[0013] Figure 6 : Fermentation diagram of E.coli-ΔfeaB-ΔygiD-yqhD-dodc-nusA tyo-KatG product.

[0014] Figure 7 : Fermentation diagram of E.coli-ΔfeaB-ΔygiD-yqhD-dodc-nusA tyo-KatG-aspgi product. Detailed Embodiments

[0015] To make the content of the present invention easier to understand, the following further describes the technical solutions of the present invention in conjunction with specific embodiments, but the present invention is not limited thereto.

[0016] The formula of the LB liquid medium in the following examples is: containing 5 g of yeast extract, 10 g of tryptone, 10 g of NaCl per 1 L volume, and the rest is water.

[0017] The formula of the M9 medium in the following examples is: containing MgSO 4 ·7H 2 O 0.2465 g, CaCl 2 0.033 g, biotin 0.001 g, thiamine 0.001 g, glucose 10 g, 100×trace element solution 10 mL, M9 low-salt medium (5-fold concentrated) 20 mL, and the rest is water. The formula of the 100×trace element solution is: containing 5 g of EDTA, FeCl 3·6H 2 O 0.83 g, ZnCl 2 84 mg, CuCl 2 ·2H 2 O 13 mg, CoCl 2 ·2H 2 O 10 mg, H 3 BO 3 10 mg and MnCl 2 ·4H 2 O 1.6 mg, and the rest is water. The formula of M9 low-salt medium (5-fold concentrated) is: containing Na 2 HPO 4 33.9 g, KH 2 PO 4 15 g, NaCl 2.5 g, NH 4 Cl 5 g, and the rest is water.

[0018] Example 1: The dopa decarboxylase gene dodc is derived from Pseudomonas putida, and the UniProt serial number is Q88JU5; the tyramine oxidase gene tyo is derived from Micrococcus luteus, and the UniProt serial number is O82865; the alcohol reductase gene yqhD is derived from Escherichia coli, and the UniProt serial number is Q46856.

[0019] Using the pRSFDuet-1 plasmid as the backbone plasmid, the yqhD gene was inserted between the NcoⅠ and BamHⅠ sites of the backbone plasmid, the dodc gene was inserted between the BamHⅠ and HindⅢ sites of the backbone plasmid, and the tyo gene was inserted between the XhoⅠ and AvrⅡ sites of the backbone plasmid to obtain the recombinant plasmid pRSF-yqhD-dodc-tyo. The recombinant plasmid pRSF-yqhD-dodc-tyo was electrotransformed into E. coli JM109(DE3) to obtain the recombinant bacterium E. coli-yqhD-dodc-tyo.

[0020] Fermentation verification: Inoculate a single colony of E. coli-yqhD-dodc-tyo into 4 mL of LB liquid medium containing 50 μg / mL kanamycin, culture overnight at 37 °C and 220 rpm to obtain a seed solution; take 1 mL of the seed solution and inoculate it into 50 mL of M9 medium containing 0.3 g / L L-dopa, culture at 37 °C and 220 rpm until OD 600When the value was 0.6, IPTG with a final concentration of 0.4 mM was added to induce protein expression, and then fermentation culture was carried out at 30 °C and 220 rpm for 72 h. Sampling was performed every 12 h during this period. The samples were detected by high-performance liquid chromatography, and a Chromaster system (Hitachi, Japan) equipped with an ultraviolet detector (UV) and a Waters SunFire C18 column (5 μm, 4.6 × 250 mm) was used for high-performance liquid chromatography analysis. The column temperature was 35 °C, the detection wavelength was 280 nm, the injection volume was 10 μL; the flow rate was 1 mL / min. Mobile phase A was water containing 0.1% formic acid, mobile phase B was 100% acetonitrile, A:B = 94:6, and isocratic elution was carried out for 20 min. The fermentation broth samples were detected for hydroxytyrosol by liquid chromatography-mass spectrometry (LC-MS) in the negative ion mode using electrospray ionization (ESI).

[0021] The results showed that the hydroxytyrosol production of E. coli-yqhD-dodc-tyo reached a maximum of 0.015 g / L at 72 h of fermentation, and the corresponding yield was 5.7%; the product hydroxytyrosol was also identified by LC-MS analysis. It was shown that the present invention successfully constructed a pathway for the biosynthesis of hydroxytyrosol using L-dopa as a substrate in Escherichia coli.

[0022] Example 2: The UniProt serial number of the phenylacetaldehyde oxidase gene feaB is P80668; the UniProt serial number of the 4,5-DOPA extradiol dioxygenase gene ygiD is P24197.

[0023] Based on E. coli JM109(DE3), the feaB gene was knocked out using the λ-Red homologous recombination method to obtain the recombinant strain E. coli-ΔfeaB; then, based on E. coli-ΔfeaB, the ygiD gene was knocked out using the λ-Red homologous recombination method to obtain the recombinant strain E. coli-ΔfeaB-ΔygiD. The recombinant plasmid pRSF-yqhD-dodc-tyo was electrotransformed into E. coli-ΔfeaB-ΔygiD to obtain the recombinant strain E. coli-ΔfeaB-ΔygiD-yqhD-dodc-tyo.

[0024] Fermentation verification: A single colony of E. coli-ΔfeaB-ΔygiD-yqhD-dodc-tyo was inoculated into 4 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37 °C and 220 rpm to obtain a seed solution; 1 mL of the seed solution was inoculated into 50 mL of M9 medium containing 0.3 g / L L-dopa and cultured at 37 °C and 220 rpm until OD 600When the value was 0.6, IPTG with a final concentration of 0.4 mM was added to induce protein expression, and then fermentation culture was carried out at 30 °C and 220 rpm for 72 h. Sampling was performed every 12 h during this period. The samples were detected by high performance liquid chromatography, and high performance liquid chromatography analysis was carried out using a Chromaster system (Hitachi, Japan) equipped with an ultraviolet detector (UV) and a Waters SunFire C18 column (5 μm, 4.6×250 mm). The column temperature was 35 °C, the detection wavelength was 280 nm, the injection volume was 10 μL; the flow rate was 1 mL / min. Mobile phase A was water containing 0.1% formic acid, mobile phase B was 100% acetonitrile, A:B = 94:6, and isocratic elution was carried out for 20 min. The fermentation broth samples were detected for hydroxytyrosol by liquid chromatography-mass spectrometry (LC-MS) in the negative ion mode by electrospray ionization (ESI).

[0025] The results showed that the hydroxytyrosol production of E.coli-ΔfeaB-ΔygiD-yqhD-dodc-tyo reached the maximum value of 0.069 g / L at 72 h of fermentation, and the corresponding yield was 29.1%.

[0026] Example 3: The UniProt serial number of the NusA-tagged protein is P0AFF6; the catalase gene KatG is derived from Escherichia coli, and the UniProt serial number is P13029. The NusA-tagged protein was fused to the N-terminus of the tyo gene to obtain the fusion gene nusA tyo.

[0027] Using the pRSFDuet-1 plasmid as the backbone plasmid, the yqhD gene was inserted between the NcoⅠ and BamHⅠ sites of the backbone plasmid, the dodc gene was inserted between the BamHⅠ and HindⅢ sites of the backbone plasmid, the nusA tyo gene was inserted between the MfeⅠ and KpnⅠ sites of the backbone plasmid, and the KatG was inserted between the KpnⅠ and AvrⅡ sites of the backbone plasmid to obtain the recombinant plasmid pRSF-yqhD-dodc-nusA tyo-KatG. The recombinant plasmid pRSF-yqhD-dodc-nusA tyo-KatG was electrotransformed into E.coli-ΔfeaB-ΔygiD to obtain the recombinant strain E.coli-ΔfeaB-ΔygiD-yqhD-dodc-nusA tyo-KatG.

[0028] Fermentation verification: Inoculate a single colony of E.coli-ΔfeaB-ΔygiD-yqhD-dodc-nusA tyo-KatG into 4 mL of LB liquid medium containing 50 μg / mL kanamycin, and culture overnight at 37 °C and 220 rpm to obtain a seed solution; Take 1 mL of the seed solution and inoculate it into 50 mL of M9 medium containing 0.3 g / L L-dopa, and culture at 37 °C and 220 rpm until the OD 600 value reaches 0.6, then add IPTG with a final concentration of 0.4 mM to induce protein expression, and then perform fermentation culture at 30 °C and 220 rpm for 72 h. Samples are taken every 12 h during this period. The samples are detected by high performance liquid chromatography. High performance liquid chromatography analysis is carried out using a Chromaster system (Hitachi, Japan) equipped with an ultraviolet detector (UV) and a Waters SunFire C18 column (5 μm, 4.6×250 mm). The column temperature is 35 °C, the detection wavelength is 280 nm, and the injection volume is 10 μL; The flow rate is 1 mL / min. Mobile phase A is water containing 0.1% formic acid, mobile phase B is 100% acetonitrile, A:B = 94:6, and isocratic elution is carried out for 20 min. The fermentation broth sample is detected for hydroxytyrosol by liquid chromatography-mass spectrometry (LC-MS) in the negative ion mode using electrospray ionization (ESI).

[0029] The results show that the hydroxytyrosol production of E.coli-ΔfeaB-ΔygiD-yqhD-dodc-nusA tyo-KatG reaches a maximum value of 0.113 g / L at 72 h of fermentation, and the corresponding yield is 37.8%.

[0030] Example 4: The UniProt serial number of the glucose-6-phosphate isomerase gene pgi is P0A6T1; The nucleotide sequence of the antisense RNA fragment targeting the pgi gene (i.e., the aspgi gene) is shown in SEQ ID NO.1.

[0031] Using the pACYCDuet-1 plasmid as the backbone plasmid, insert the aspgi gene between the AflⅡ and NcoⅠ sites of the backbone plasmid to obtain the recombinant plasmid pACYC-aspgi (also known as pACYC-asRNA). Electrotransform the recombinant plasmid pACYC-aspgi into E.coli-ΔfeaB-ΔygiD-yqhD-dodc-nusA tyo-KatG to obtain the recombinant bacterium E.coli-ΔfeaB-ΔygiD-yqhD-dodc-nusA tyo-KatG-aspgi.

[0032] Fermentation verification: Inoculate a single colony of E. coli-ΔfeaB-ΔygiD-yqhD-dodc-nusA tyo-KatG-aspgi into 4 mL of LB liquid medium containing 50 μg / mL kanamycin, and culture overnight at 37 °C and 220 rpm to obtain a seed solution; Take 1 mL of the seed solution and inoculate it into 50 mL of M9 medium containing 0.3 g / L L-dopa, and culture at 37 °C and 220 rpm until the OD 600 value reaches 0.6, then add IPTG with a final concentration of 0.4 mM to induce protein expression, and then ferment and culture at 30 °C and 220 rpm for 72 h. Samples are taken every 12 h during this period. The samples are detected by high performance liquid chromatography, and a Chromaster system (Hitachi, Japan) equipped with an ultraviolet detector (UV) and a Waters SunFire C18 column (5 μm, 4.6×250 mm) is used for high performance liquid chromatography analysis. The column temperature is 35 °C, the detection wavelength is 280 nm, the injection volume is 10 μL; the flow rate is 1 mL / min. Mobile phase A is water containing 0.1% formic acid, mobile phase B is 100% acetonitrile, A:B = 94:6, and isocratic elution is carried out for 20 min. The fermentation broth sample is detected for hydroxytyrosol by liquid chromatography-mass spectrometry (LC-MS) in the negative ion mode using electrospray ionization (ESI).

[0033] The results show that the hydroxytyrosol production of E. coli-ΔfeaB-ΔygiD-yqhD-dodc-nusA tyo-KatG-aspgi reaches a maximum value of 0.153 g / L at 72 h of fermentation, and the corresponding yield is 58.4%.

Claims

1. A method for biosynthesizing hydroxytyrosol using L-DOPA in Escherichia coli, characterized in that: Using Escherichia coli as a chassis host bacterium, a pathway for biosynthesizing hydroxytyrosol using L-DOPA as a substrate is constructed by overexpressing an alcohol reductase gene yqhD, a dopa decarboxylase gene dodc and a tyramine oxidase gene tyo; wherein the UniProt sequence number of the alcohol reductase gene yqhD is Q46856, the UniProt sequence number of the dopa decarboxylase gene dodc is Q88JU5, and the UniProt sequence number of the tyramine oxidase gene tyo is O82865.

2. The method according to claim 1, characterized in that: The following steps are involved: (1) constructing a recombinant plasmid carrying the alcohol reductase gene yqhD, the dopa decarboxylase gene dodc and the tyramine oxidase gene tyo, and transforming it into the chassis host bacteria E. coli JM109 (DE3) to obtain the recombinant bacteria E. coli-yqhD-dodc-tyo; (2) E. coli-yqhD-dodc-tyo is fermented and cultured in a fermentation medium containing a substrate, L-dopa, to obtain a fermentation liquid containing hydroxytyrosol.

3. The method according to claim 1, characterized in that: The method also includes: knocking out the phenylacetaldehyde oxidase gene feaB and the 4,5-DOPA extra-diol dioxygenase gene ygiD to reduce the generation of by-products; wherein the UniProt sequence number of the phenylacetaldehyde oxidase gene feaB is P80668, and the UniProt sequence number of the 4,5-DOPA extra-diol dioxygenase gene ygiD is P24197.

4. The method according to claim 3, characterized in that: The following steps are involved: (1) Using E. coli JM109 (DE3) as the host bacteria, the phenylacetaldehyde oxidase gene feaB and the 4,5-DOPA exodiol dioxygenase gene ygiD were knocked out in sequence using the λ-Red homologous recombination method to obtain the recombinant bacteria E. coli-ΔfeaB-ΔygiD; (2) constructing a recombinant plasmid carrying the alcohol reductase gene yqhD, the dopa decarboxylase gene dodc, and the tyramine oxidase gene tyo, and transforming it into E. coli-ΔfeaB-ΔygiD to obtain the recombinant bacteria E. coli-ΔfeaB-ΔygiD-yqhD-dodc-tyo; (3) Fermenting E. coli-ΔfeaB-ΔygiD-yqhD-dodc-tyo in a fermentation medium containing a substrate, L-DOPA, to obtain a fermentation broth containing hydroxytyrosol.

5. The method according to claim 3, characterized in that: The method further comprises: optimizing the synthesis pathway of hydroxytyrosol by fusing NusA tag protein to the N-terminus of tyramine oxidase gene tyo and overexpressing catalase gene KatG; wherein the UniProt sequence number of the NusA tag protein is P0AFF6, and the UniProt sequence number of the catalase gene KatG is P13029.

6. The method according to claim 5, characterized in that: The following steps are involved: (1) Using E. coli JM109 (DE3) as the host bacteria, the phenylacetaldehyde oxidase gene feaB and the 4,5-DOPA exodiol dioxygenase gene ygiD were knocked out in sequence using the λ-Red homologous recombination method to obtain the recombinant bacteria E. coli-ΔfeaB-ΔygiD; (2) constructing a recombinant plasmid carrying the alcohol reductase gene yqhD, the dopa decarboxylase gene dodc, the fusion gene nusA tyo, and the catalase gene KatG, and transforming it into E. coli-ΔfeaB-ΔygiD to obtain the recombinant bacteria E. coli-ΔfeaB-ΔygiD-yqhD-dodc-nusA tyo-KatG; (3) E. coli-ΔfeaB-ΔygiD-yqhD-dodc-nusA tyo-KatG is fermented in a fermentation medium containing a substrate L-dopa to obtain a fermentation broth containing hydroxytyrosol.

7. The method according to claim 5, characterized in that: The method also includes: regulating the carbon flux of the glycolysis pathway and the pentose phosphate pathway by inhibiting the expression of the glucose-6-phosphate isomerase gene pgi, thereby strengthening the biosynthesis pathway of hydroxytyrosol; wherein the UniProt sequence number of the glucose-6-phosphate isomerase gene pgi is P0A6T1.

8. The method according to claim 7, characterized in that: The following steps are involved: (1) Using E. coli JM109 (DE3) as the host bacteria, the phenylacetaldehyde oxidase gene feaB and the 4,5-DOPA exodiol dioxygenase gene ygiD were knocked out in sequence using the λ-Red homologous recombination method to obtain the recombinant bacteria E. coli-ΔfeaB-ΔygiD; (2) constructing a recombinant plasmid carrying the alcohol reductase gene yqhD, the dopa decarboxylase gene dodc, the fusion gene nusA tyo, and the catalase gene KatG, and transforming it into E. coli-ΔfeaB-ΔygiD to obtain the recombinant bacteria E. coli-ΔfeaB-ΔygiD-yqhD-dodc-nusA tyo-KatG; (3) constructing a recombinant plasmid carrying the antisense RNA fragment of the glucose-6-phosphate isomerase gene pgi, and transforming it into E. coli-ΔfeaB-ΔygiD-yqhD-dodc-nusA tyo-KatG to obtain the recombinant bacteria E. coli-ΔfeaB-ΔygiD-yqhD-dodc-nusA tyo-KatG-aspgi; (4) Fermenting E. coli-ΔfeaB-ΔygiD-yqhD-dodc-nusA tyo-KatG-aspgi in a fermentation medium containing a substrate L-dopa to obtain a fermentation broth containing hydroxytyrosol.

9. The method according to claim 8, characterized in that: The nucleotide sequence of the antisense RNA fragment of the glucose-6-phosphate isomerase gene pgi is shown in SEQ ID NO.

1.

10. Use of the method according to any one of claims 1 to 9 in the production of hydroxytyrosol.

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