A hydroxylase mutant for efficient conversion preparation of hydroxytyrosol and application thereof

Abstract

The application discloses a kind of efficient conversion preparation hydroxyl tyrosol hydroxylase mutant and its application, belong to biotechnology field.The application provides a kind of hydroxylase mutant HpaB Y282H And HpaB S258C , using E.coli YMGR5A as host strain, with pEtac plasmid as carrier, expressed 4- hydroxyphenylacetic acid hydroxylase gene (GenBank accession number: CP053601.1) in escherichia coli and mutant HpaBC Y282H Or HpaB S258C ;The recombinant strain is fermented in 250mL flask with 50mL LB medium, and the yield of hydroxytyrosol is 1.82g / L, 1.81g / L respectively, and the yield of hydroxytyrosol is 49.98mM / L, 48.2mM / L respectively in the medium added with 50mM tyrosol, and the conversion rate is more than 99%, 96% respectively, higher than the achievement of most research teams at present.

Description

TECHNICAL FIELD

[0001] The present application relates to a hydroxylase mutant for efficient conversion preparation of hydroxytyrosol and its application, belonging to the field of biotechnology. BACKGROUND

[0002] Hydroxytyrosol is a natural polyphenolic compound, which is widely used in food, feed, cosmetics, nutritional supplements and pharmaceutical industries. It is one of the strongest natural antioxidants known, for example, anti-inflammatory and anticancer, and has the potential to act as a heart and nerve protectant. Therefore, hydroxytyrosol has great commercial application value, and large-scale production of hydroxytyrosol has become an inevitable trend. At present, the production of hydroxytyrosol is still in the stage of natural extraction and chemical synthesis. Natural extraction is mainly from olive fruit, leaves, and residues and wastewater produced during olive oil production, which has the advantages of cheap and abundant raw materials, but the disadvantages of using strong acid steam, low recovery rate, and easy secondary pollution. The substrate used in chemical synthesis is relatively expensive, and these processes usually require protection and deprotection steps, which reduce the overall yield; high cost, serious pollution, low safety, and cannot be applied to food and drug additives.

[0003] At present, the known functions of hydroxytyrosol are as follows: (1) synthesis of cardiovascular drugs: prevention and treatment of arteriosclerosis, hypertension, heart disease, cerebral hemorrhage, etc.; (2) safe and efficient antioxidant: applied to beauty products and health products; (3) anti-aging and beneficial to the skeletal system: helpful to the absorption of minerals by the human body, reducing bone loss; (4) improving the function of the endocrine system, promoting metabolism, promoting wound healing, eliminating free radicals in the body, restoring the health of human organs, and delaying aging; (5) anticancer: promoting recovery after cancer and improving the effect of chemotherapy; (6) reducing and inhibiting the harm of smoking to the human body: preventing and treating various diseases caused by smoking. Therefore, large-scale production of hydroxytyrosol has become an inevitable trend.

[0004] In the existing reports, although there are safe and efficient biological methods for synthesizing hydroxytyrosol, for example: by expressing hydroxylase gene HpaBC derived from Escherichia coli BL21 to produce hydroxytyrosol (article "Metabolic engineering of Escherichia coli for synthesis of hydroxytyrosol"), which is not enough to convert the substrate to hydroxytyrosol.

[0005] Therefore, at present, researchers are committed to a high-activity hydroxylase mutant and a safe and efficient, low-cost biological method for synthesizing hydroxytyrosol. SUMMARY

[0006] On the basis of the prior art, the application provides a high-activity hydroxylase mutant and a method for biosynthesizing hydroxyl tyrosol. By using modern bioengineering technology, a method for constructing a high-yield hydroxyl tyrosol strain is provided on the basis of an existing tyrosol high-yield strain. The hydroxylase mutant obtained in the application has high conversion efficiency, and can convert not only tyrosol synthesized by the original metabolic pathway, but also exogenous added substrate tyrosol.

[0007] The application provides a HpaB hydroxylase mutant, which is obtained by mutating tyrosine at the 282th position into histidine on the basis of a parent enzyme HpaB with an amino acid sequence as shown in SEQ ID NO. 5, and is named as HpaB Y282H ; and has an amino acid sequence as shown in SEQ ID NO. 1.

[0008] Alternatively, the hydroxylase mutant is obtained by mutating serine at the 258th position into cysteine on the basis of the parent enzyme HpaB with an amino acid sequence as shown in SEQ ID NO. 5, and is named as HpaB S258C ; and has an amino acid sequence as shown in SEQ ID NO. 6.

[0009] In an embodiment of the application, the parent enzyme hydroxylase HpaB is derived from Escherichia coli BL21 (DE3) and is optimized, and has a nucleotide sequence as shown in SEQ ID NO. 3.

[0010] The application further provides a gene encoding the above-mentioned hydroxylase mutant HpaB Y282H or HpaB S258C .

[0011] The application further provides a recombinant vector carrying the above-mentioned gene.

[0012] The application further provides a recombinant cell expressing the above-mentioned hydroxylase mutant HpaB Y282H or HpaB S258C , or containing the above-mentioned gene or containing the above-mentioned recombinant vector.

[0013] In an embodiment of the application, the recombinant cell takes bacteria or fungi as a host cell.

[0014] The application further provides a recombinant Escherichia coli, which expresses the above-mentioned hydroxylase mutant HpaB Y282H or HpaB S258C and a hydroxylase HpaC.

[0015] In an embodiment of the application, the hydroxylase HpaC has an amino acid sequence as shown in SEQ ID NO. 2.

[0016] In an embodiment of the present application, the nucleotide sequence encoding the hydroxylase HpaC is shown as SEQ ID NO. 4.

[0017] In an embodiment of the present application, the hydroxylase HpaC is derived from Escherichia coli.

[0018] In an embodiment of the present application, the recombinant Escherichia coli takes Escherichia coli BL21 (DE3), Escherichia coli MG1655 or Escherichia coli CCTCC NO: M2019390 as a host cell.

[0019] In an embodiment of the present application, the Escherichia coli CCTCC NO: M2019390 is recorded in the Chinese patent application text with the publication number CN110452865A, and is named as E. coli YMGR5A.

[0020] In an embodiment of the present application, the recombinant Escherichia coli takes pEtac, pkk223-3 as an expression vector.

[0021] The present application also provides a method for constructing the above-mentioned recombinant Escherichia coli, which comprises the following steps:

[0022] (1) chemically synthesizing a hydroxylase HpaB mutant.

[0023] (2) preparing a recombinant plasmid pEtac-HpaC: chemically synthesizing a hydroxylase HpaC fragment, and then connecting HpaC with the plasmid pEtac after double enzyme digestion to obtain the recombinant plasmid pEtac-HpaC.

[0024] (3) inserting the obtained hydroxylase HpaB mutant into the plasmid pEtac-HpaC to obtain a recombinant plasmid pEtac-HpaB mutant-HpaC, and then introducing the recombinant plasmid pEtac-HpaB mutant-HpaC into the tyrosol high-yield strain E. coli YMGR5A to obtain a recombinant hydroxytyrosol high-yield strain E. coli YMGR5A / pEtac-HpaB mutant-HpaC.

[0025] The present application also provides a method for producing hydroxytyrosol, which uses the above-mentioned recombinant Escherichia coli E. coli YMGR5A / pEtac-HpaB mutant-HpaC to produce hydroxytyrosol.

[0026] In an embodiment of the present application, the method is to inoculate the recombinant Escherichia coli E. coli YMGR5A / pEtac-HpaB mutant-HpaC into a seed culture medium for cultivation to prepare a seed liquid; and then inoculate the seed liquid into a reaction system containing glucose to ferment and prepare hydroxytyrosol.

[0027] In an embodiment of the present application, the reaction system containing glucose is a fermentation medium, and the fermentation medium is as follows: 50 mL of M9Y liquid medium in a 250 mL conical flask.

[0028] In an embodiment of the present application, the reaction system further comprises tyrosol, and the amount of tyrosol added is at least 30 mM.

[0029] In an embodiment of the present application, the recombinant E. coli YMGR5A / pEtac-HpaB mutant-HpaC is inoculated on LB solid medium to obtain single colonies, and the single colonies are inoculated in LB liquid medium to prepare seed liquid under the conditions of 35-39°C and 180-220 r / min for 10-14 h.

[0030] In an embodiment of the present application, the recombinant E. coli YMGR5A / pEtac-HpaB mutant-HpaC is inoculated on LB solid medium to obtain single colonies, and the single colonies are inoculated in LB liquid medium to prepare seed liquid under the conditions of 35-39°C and 180-220 r / min for 10-14 h.

[0031] In an embodiment of the present application, the seed liquid is inoculated in LB liquid medium, and the bacteria are collected after being cultured under the conditions of 35-39°C and 180-220 r / min for 10-14 h; and the collected bacteria are inoculated in M9Y liquid medium for fermentation culture to prepare hydroxytyrosol.

[0032] In an embodiment of the present application, the seed liquid is inoculated in LB liquid medium at a ratio of 1% (v / v).

[0033] In an embodiment of the present application, the shake flask fermentation conditions are as follows: the seed liquid is inoculated in a 250 mL conical flask containing 50 mL of LB liquid medium at a ratio of 1% (v / v), the bacteria are collected after being cultured at 37°C and 200 r / min for 10 h, the bacteria are washed once with normal saline, and then the bacteria are transferred into a 250 mL conical flask containing 50 mL of M9Y liquid medium, and the bacteria are cultured at 30°C and 200 r / min for 48 h.

[0034] In an embodiment of the present application, the shake flask fermentation conditions are as follows: inoculating into a 250 mL conical flask containing 50 mL LB liquid medium at an inoculation amount of 1% (v / v), collecting the bacteria after culturing at 37℃, 200 r / min for 10 h, and washing the bacteria once with normal saline. Then, transferring into a 250 mL conical flask containing 50 mL M9Y liquid medium, and culturing at 30℃, 200 r / min for 48 h.

[0035] In an embodiment of the present application, the reaction system further comprises a substrate tyrosol.

[0036] In an embodiment of the present application, the concentration of the substrate tyrosol is at least 30 mM.

[0037] In an embodiment of the present application, the concentration of the substrate tyrosol is 50 mM.

[0038] In an embodiment of the present application, the reaction condition in the reaction system is 30℃, 200 r / min.

[0039] In an embodiment of the present application, the HpaB mutant is HpaB Y282H or HpaB S258C .

[0040] The present application also provides the use of the recombinant E. coli YMGR5A / pEtac-HpaB mutant-HpaC described above in the preparation of hydroxytyrosol and products containing hydroxytyrosol.

[0041] Advantages

[0042] (1) The recombinant E. coli YMGR5A / pEtac-HpaB mutant-HpaC provided by the present application can realize the conversion of glucose into hydroxytyrosol, and the E. coli YMGR5A / pEtac-HpaB Y282H mutant-HpaC, and the E. coli YMGR5A / pEtac-HpaB S258C mutant-HpaC can produce hydroxytyrosol up to 1.82 g / L and 1.81 g / L, respectively, which is higher than the results of most research teams at present.

[0043] (2) The recombinant E. coli YMGR5A / pEtac-HpaB mutant-HpaC provided by the present application can produce hydroxytyrosol in a culture medium added with 50 mM tyrosol, and the E. coli YMGR5A / pEtac-HpaB Y282H mutant-HpaC, and the E. coli YMGR5A / pEtac-HpaB S258CThe yield of HpaC can be up to 49.98 mM / L (7.705 g / L) and 48.2 mM / L (7.431 g / L) respectively, and the conversion rate is more than 99% and 96% respectively, which is higher than the achievements of most research teams at present. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 The strain E. coli YMGR5A / pEtac-HpaB-HpaC, E. coli YMGR5A / pEtac-HpaB Y282H HpaC, E. coli YMGR5A / pEtac-HpaB S258C HpaC, E. coli YMGR5A / pEtac-HpaB Q212E HpaC, E. coli YMGR5A / pEtac-HpaB G340A The yield of hydroxytyrosol and the residual amount of tyrosol of HpaC under the condition of adding 30 mM tyrosol.

[0045] Figure 2 The strain E. coli YMGR5A / pEtac-HpaB Y282H HpaC, E. coli YMGR5A / pEtac-HpaB S258C The yield of hydroxytyrosol of HpaC fermentation.

[0046] Figure 3 The strain E. coli YMGR5A / pEtac-HpaB Y282H The yield of hydroxytyrosol of HpaC fermentation under the condition of adding tyrosol.

[0047] Figure 4 The strain E. coli YMGR5A / pEtac-HpaB S258C The yield of hydroxytyrosol of HpaC fermentation under the condition of adding tyrosol. DETAILED DESCRIPTION

[0048] The construction method of the recombinant plasmid pEtac involved in the following examples is described in "Secretory expression of archaeal Pyrococcus furiosus high-thermostable alpha amylase gene in E. coli" by Shen Wei et al. in the literature of Jiangnan University:

[0049] The construction method is as follows: the fragment containing tac promoter in the E. coli expression vector pKK223-3 is cut by BamHI and EcoRI enzymes and then connected to the expression vector pET28a to construct a new expression vector pEtac.

[0050] The culture medium involved in the following examples is as follows:

[0051] The formula of LB culture medium (g / L): yeast powder 5, peptone 10, NaCl 10, and 1.5%-2.0% agar powder is additionally added to solid culture medium.

[0052] The formula of M9Y culture medium (g / L): Na2HPO4·12H2O 17.1, KH2PO4 3, NaCl 0.5, NH4Cl 1, glucose 20, yeast powder 0.25, and MgSO4 5 mmol·L is additionally added to a final concentration. -1 .

[0053] The detection method involved in the following examples is as follows:

[0054] Detection method of tyrosol and hydroxytyrosol

[0055] High performance liquid chromatography (HPLC) is used for detection. The chromatographic detection conditions are as follows: Agela Innoval C18 chromatographic column (4.6*250 mm, pore size 5 μm); mobile phase is 80% 0.1% formic acid aqueous solution and 20% methanol; flow rate is 1 mL / min; injection volume is 10 μL; ultraviolet detector, detection wavelength is 280 nm; column temperature is 28°C.

[0056] Detection method of hydroxylase enzyme activity: hydroxytyrosol sodium periodate detection method is used.

[0057] Example 1: Construction of recombinant plasmid pEtac-HpaBC

[0058] (1) Construction of recombinant plasmid pEtac-HpaC:

[0059] According to the sequence disclosed in NCBI, HpaC gene (nucleotide sequence is shown as SEQ ID NO. 4) is chemically synthesized, and Pst I and Hind III enzyme cutting sites are designed in primers p-HpaC-L and p-HpaC-R (as shown in Table 1). The target fragment is subjected to two-site enzyme cutting and purification, and then is connected by Solution I ligase. After chemical transformation, it is transformed into E. coli JM109, and is coated on LB solid culture medium plate containing kanamycin resistance and is cultured in a 37°C incubator for 10-12 h. The single colony grown is subjected to enzyme cutting verification, and the correct strain is cultured to extract the recombinant plasmid pEtac HpaC.

[0060] (2) Construction of recombinant plasmid pEtac-HpaB-HpaC

[0061] According to the sequence disclosed in NCBI, the HpaB gene (nucleotide sequence as shown in SEQ ID NO. 3) is chemically synthesized, and EcoR I and Pst I enzyme cutting sites are designed in primers p-HpaB-L and p-HpaB-R (as shown in Table 1). The target fragment is subjected to two-site enzyme cutting and purification, and then connected by Solution I ligase, and then cultured and extracted after verification by the above method to obtain the recombinant plasmid pEtac-HpaB-HpaC.

[0062] Example 2: Construction of recombinant plasmid containing hydroxylase mutant

[0063] The specific steps are as follows:

[0064] The site-directed mutation primer is designed, and the recombinant plasmid pEtac-HpaB-HpaC is used as a template to construct a mutant HpaB Y282H , HpaB S258C , HpaB Q212E , HpaB G340A containing recombinant plasmid pEtac-HpaB Y282H -HpaC, pEtac-HpaB S258C -HpaC, pEtac-HpaB Q212E -HpaC, pEtac-HpaB G340A -HpaC.

[0065] After the PCR mutation is verified by sequencing to be successful, the subsequent operation can be carried out.

[0066] The primer sequences involved are shown in Table 1:

[0067] Table 1: Primer sequences

[0068]

[0069]

[0070] The PCR reaction system is shown in Table 2:

[0071] Table 2: Preparation of PCR system

[0072]

[0073] Example 3: Construction of recombinant strain containing hydroxylase mutant

[0074] The recombinant plasmids prepared in Example 2 were verified by sequencing and then transformed into E. coli JM109 by chemical transformation method, and then coated on LB solid medium containing kanamycin and cultured in a 37°C incubator for 10-12 h. The verified strains were cultured to extract recombinant plasmids pEtac-HpaB-HpaC, pEtac-HpaB Y282H -HpaC, pEtac-HpaB S258C -HpaC, pEtac-HpaB Q212E -HpaC, pEtac-HpaB G340A -HpaC, pEtac-HpaB Y282H -HpaC, pEtac-HpaB S258C -HpaC, pEtac-HpaB Q212E -HpaC, pEtac-HpaB G340A -HpaC.

[0075] Example 4: Verification of the conversion rate of tyrosol by the mutant strain

[0076] The specific steps are as follows:

[0077] (1) The recombinant E. coli strains E. coli YMGR5A / pEtac-HpaB-HpaC, E. coli YMGR5A / pEtac-HpaB Y282H -HpaC, pEtac-HpaB S258C -HpaC, pEtac-HpaB Q212E -HpaC, pEtac-HpaB G340A -HpaC were streaked on LB solid medium to obtain single colonies, and the single colonies were inoculated into 100 mL conical flasks containing 20 mL of LB liquid medium and cultured at 37°C and 200 r / min for 12 h. Seed liquids were prepared.

[0078] (2) The seed culture prepared in step (1) was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of LB liquid medium at an inoculation rate of 1% (v / v). After incubation at 37 °C and 200 r / min for 10 h, the bacterial cells were collected.

[0079] (3) After washing the bacterial cells obtained in step (2) once with physiological saline, the cells were transferred to a 250 mL Erlenmeyer flask containing 50 mL of M9Y liquid culture medium (with 30 mM tyrosol added to the liquid culture medium) and cultured at 30 °C and 200 r / min for 12 h to prepare the fermentation broth. The yield of hydroxytyrosol and the amount of tyrosol remaining in the fermentation broth were then measured. The yield of hydroxytyrosol and the amount of tyrosol remaining for each recombinant strain are shown in Table 3 and [Table 4]. Figure 1 As shown.

[0080] Table 3: Hydroxytyrosol production and residual tyrosol in recombinant strains under exogenous 30mM tyrosol supplementation.

[0081] Strains Residual tyrosol (g / L) Hydroxytyrosol production (g / L) E. coli YMGR5A / pEtac-HpaB-HpaC 9.75 19.82 E. coli YMGR5A / pEtac-HpaB Y282H -HpaC]]> 0 27.51 E. coli YMGR5A / pEtac-HpaB S258C -HpaC]]> 0 27.29 E. coli YMGR5A / pEtac-HpaB Q212E -HpaC]]> 9.35 19.98 E. coli YMGR5A / pEtac-HpaB G340A -HpaC]]> 8.25 20.47

[0082] Example 5: Shake-flask fermentation to verify high hydroxytyrosol production by mutant strain

[0083] The specific steps are as follows:

[0084] (1) The recombinant E. coli YMGR5A / pEtac-HpaB from Example 4 were respectively... Y282H -HpaC, E.coli YMGR5A / pEtac-HpaB S258C -HpaC was streaked on LB solid medium to obtain single colonies. A single colony was picked and inoculated into a 100mL Erlenmeyer flask containing 20mL of LB liquid medium and cultured at 37℃ and 200r / min for 12h; seed liquids were prepared accordingly.

[0085] (2) The seed culture prepared in step (1) was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of LB liquid medium at an inoculation rate of 1% (v / v), and the cells were collected after being cultured at 37 °C and 200 r / min for 10 h.

[0086] (3) After washing the bacterial cells obtained in step (2) once with physiological saline, they were transferred into 250mL Erlenmeyer flasks containing 50mL of M9Y liquid culture medium and cultured at 30℃ and 200r / min for 48h. Samples were taken every 12h to detect the hydroxytyrosol content in the fermentation broth. The hydroxytyrosol yield results are shown in Table 4 and Figure 2 As shown.

[0087] Table 4: Hydroxytyrosol production of different recombinant Escherichia coli strains

[0088]

[0089] The results show that the recombinant E. coli E. coli YMGR5A / pEtac-HpaBC provided by the present application can convert glucose into hydroxytyrosol, and the yield of hydroxytyrosol produced by fermentation in the glucose culture medium can be as high as 1.82 g / L and 1.81 g / L, respectively. Y282H -HpaC, E. coli YMGR5A / pEtac-HpaBC S258C -HpaC, E. coli YMGR5A / pEtac-HpaBC

[0090] Example 6: Shake flask fermentation verifies high conversion rate of mutant strain to tyrosol

[0091] The specific steps are as follows:

[0092] (1) The recombinant E. coli E. coli YMGR5A / pEtac-HpaB in Example 5 was inoculated into a 100 mL conical flask containing 20 mL of LB liquid medium at a inoculation amount of 1% (v / v), and cultured at 37°C and 200 r / min for 12 h to prepare a seed liquid. Y282H -HpaC, E. coli YMGR5A / pEtac-HpaB S258C -HpaC was inoculated into a 100 mL conical flask containing 20 mL of LB liquid medium at a inoculation amount of 1% (v / v), and cultured at 37°C and 200 r / min for 12 h to prepare a seed liquid.

[0093] (2) The seed liquid prepared in step (1) was inoculated into a 250 mL conical flask containing 50 mL of LB liquid medium at a inoculation amount of 1% (v / v), and cultured at 37°C and 200 r / min for 10 h, and then the bacterial cells were collected.

[0094] (3) The bacterial cells obtained in step (2) were washed once with physiological saline, and then transferred into a 250 mL conical flask containing 50 mL of M9Y liquid medium containing different concentrations of substrate tyrosol (30 mM, 40 mM, 50 mM, 60 mM, and 70 mM, as shown in Table 5), and cultured at 30°C and 200 r / min for 12 h to prepare fermentation liquids, and the content of hydroxytyrosol in the fermentation liquids was detected.

[0095] E. coli YMGR5A / pEtac-HpaB Y282H -HpaC, E. coli YMGR5A / pEtac-HpaB Figure 3

[0096] Table 5: Hydroxytyrosol yield and unit bacterial concentration yield of recombinant E. coli under different concentrations of substrate tyrosol

[0097] Substrate concentration mM Hydroxytyrosol production mM Hydroxytyrosol production per cell concentration mM 30 29.89 7.12 40 39.92 8.58 50 49.98 14.19 60 21.81 9.49 70 17.38 8.39

[0098] ​The E. coli YMGR5A / pEtac-HpaB S258C The HpaC production and the production per unit of bacteria concentration are shown in Table 6 and Figure 4

[0099] Table 6: The hydroxytyrosol production and the production per unit of bacteria concentration of the recombinant E. coli under different concentrations of substrate tyrosol

[0100] Substrate concentration mM Hydroxytyrosol production mM Hydroxytyrosol production per cell concentration mM 30 29.10 7.11 40 38.24 8.51 50 48.22 13.98 60 20.07 10.23 70 18.56 8.05

[0101] The results show that:

[0102] The recombinant E. coli E. coli YMRG5A / pEtac-HpaB Y282H The HpaC production of the recombinant E. coli E. coli YMRG5A / pEtac-HpaB

[0103] The recombinant E. coli E. coli YMRG5A / pEtac-HpaB S258C The HpaC production of the recombinant E. coli E. coli YMRG5A / pEtac-HpaB

[0104] The recombinant E. coli E. coli YMRG5A / pEtac-HpaB Y282H The HpaC production of the recombinant E. coli E. coli YMRG5A / pEtac-HpaB S258C The HpaC production of the recombinant E. coli E. coli YMRG5A / pEtac-HpaB

[0105] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.​

Claims

1. A hydroxylase HpaB mutant, characterized in that, the hydroxylase HpaB mutant is obtained by mutating the tyrosine at position 282 to histidine based on the parent enzyme HpaB with the amino acid sequence as shown in SEQ ID NO. 5, and has the amino acid sequence as shown in SEQ ID NO. 1; or, the hydroxylase mutant is obtained by mutating the serine at position 258 to cysteine based on the parent enzyme HpaB with the amino acid sequence as shown in SEQ ID NO. 5, and has the amino acid sequence as shown in SEQ ID NO.

6.

2. A gene encoding the hydroxylase mutant of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. A recombinant cell expressing the hydroxylase HpaB mutant of claim 1 or containing the gene of claim 2 or containing the recombinant vector of claim 3.

5. The recombinant cell of claim 4, wherein The recombinant cell is a bacterial or fungal host cell.

6. A recombinant Escherichia coli, characterized in that, The recombinant Escherichia coli expresses the hydroxylase HpaB mutant of claim 1 and a hydroxylase HpaC with the amino acid sequence as shown in SEQ ID NO.

2.

7. A method of producing hydroxytyrosol, characterized by, The seed liquid of the recombinant Escherichia coli of claim 6 is added to a reaction system containing glucose to prepare by fermentation.

8. The method of claim 7, wherein, The reaction system further contains a substrate tyrosol.

9. The method of claim 7 or 8, wherein, In the reaction system, the reaction conditions are as follows: the prepared seed liquid is inoculated into LB liquid medium, and the bacterial cells are collected after being cultured at 35-40°C and 180-220 r / min for 10-14 h; the collected bacterial cells are inoculated into M9Y liquid medium for fermentation culture to prepare hydroxytyrosol.

10. Use of the recombinant Escherichia coli of claim 6 in the preparation of hydroxytyrosol and products containing hydroxytyrosol.

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