CN116334011A - Hydroxylase mutant for preparing hydroxytyrosol by efficient conversion and application thereof - Google Patents
Hydroxylase mutant for preparing hydroxytyrosol by efficient conversion and application thereof Download PDFInfo
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- CN116334011A CN116334011A CN202210804481.6A CN202210804481A CN116334011A CN 116334011 A CN116334011 A CN 116334011A CN 202210804481 A CN202210804481 A CN 202210804481A CN 116334011 A CN116334011 A CN 116334011A
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Abstract
The invention discloses a hydroxylase mutant for preparing hydroxytyrosol by efficient conversion and application thereof, and belongs to the technical field of biology. The invention provides a hydroxylase mutant HpaB Y282H And HpaB S258C E.coli YMGR5A is used as host strain, pEtac plasmid is used as carrier, 4-hydroxyphenylacetic acid hydroxylase gene (GenBank accession number: CP 053601.1) and mutant HpaBC in colibacillus are expressed Y282H Or HpaB S258C The method comprises the steps of carrying out a first treatment on the surface of the The recombinationAfter the strain is fermented in 250mL shake flasks with 50mL LB culture medium, the yield of hydroxytyrosol is 1.82g/L and 1.81g/L respectively, and the yield of hydroxytyrosol produced by fermentation in the culture medium added with 50mM tyrosol is up to 49.98mM/L and 48.2mM/L respectively, and the conversion rate is over 99 percent and 96 percent respectively, which is higher than the results of most research teams at present.
Description
Technical Field
The invention relates to a hydroxylase mutant for preparing hydroxytyrosol by efficient conversion and application thereof, belonging to the technical field of biology.
Background
Hydroxytyrosol (hydroxytyrosol) is a natural polyphenol compound and is widely used in the food, feed, cosmetics, nutritional supplements and pharmaceutical industry. It is one of the strongest natural antioxidants known, e.g. anti-inflammatory and anti-cancer, and has the potential to act as a cardiac and neuroprotective agent. Accordingly, hydroxytyrosol has great commercial application value, and mass production of hydroxytyrosol is a necessary trend. The current production of hydroxytyrosol is still in the natural extraction and chemical synthesis stages. Natural extraction is mainly carried out from olive fruits, leaves and residues and wastewater generated in the preparation process of olive oil, and has the advantages of cheap and abundant raw materials, but has the disadvantages of using strong acid water vapor, low recovery rate and easy secondary pollution. The substrates used in chemical synthesis are relatively expensive, and these processes typically require protection and deprotection steps, reducing overall yields; high cost, serious pollution and low safety, and can not be applied to the addition of foods and medicines.
There are a number of currently known hydroxytyrosol effects: (1) synthesis of cardiovascular drugs: preventing and treating arteriosclerosis, hypertension, heart disease, cerebral hemorrhage, etc.; (2) safe and efficient antioxidant: the product is applied to a beauty product health care product; (3) anti-aging, beneficial to skeletal system: is helpful for the absorption of minerals by human body and reduces the osteoporosis; (4) Improving endocrine system function, promoting metabolism, promoting wound healing, scavenging free radicals, recovering health state of viscera and organs, and delaying aging; (5) anticancer and cancer preventing: promoting cancer later recovery and improving chemotherapy effect; (6) reducing and inhibiting the harm of smoking to human body: preventing and treating various diseases caused by smoking. Therefore, the mass production of hydroxytyrosol is a necessary trend.
In the prior report, although safe and efficient biological methods for synthesizing hydroxytyrosol exist, for example: production of hydroxytyrosol by expression of the gene HpaBC derived from E.coli BL21 hydroxylase (article "metabolic engineering E.coli Synthesis of hydroxytyrosol"), which is insufficient for complete conversion of the substrate into hydroxytyrosol.
Thus, researchers are currently working on a mutant of a highly active hydroxylase and a safe, efficient and inexpensive method for biologically synthesizing hydroxytyrosol.
Disclosure of Invention
Based on the prior art, the invention provides a high-activity hydroxylase mutant and a method for biosynthesizing hydroxytyrosol. By utilizing the technology of modern biological genetic engineering, a method for producing hydroxytyrosol in high yield is constructed on the basis of the existing strain producing tyrosol in high yield. The hydroxylase mutant obtained by the invention has higher conversion efficiency, and can utilize exogenously added substrate tyrosol besides completely converting tyrosol synthesized by the original metabolic pathway.
The invention provides a HpaB hydroxylase mutant, which is obtained by mutating 282 rd tyrosine into histidine on the basis of a parent enzyme HpaB with an amino acid sequence shown as SEQ ID NO.5, and is named HpaB Y282H The method comprises the steps of carrying out a first treatment on the surface of the The amino acid sequence is shown as SEQ ID NO. 1.
Or, the hydroxylase mutant is obtained by mutating serine at 258 th site into cysteine based on a parent enzyme HpaB with an amino acid sequence shown as SEQ ID NO.5, and is named HpaB S258C The method comprises the steps of carrying out a first treatment on the surface of the The amino acid sequence is shown as SEQ ID NO. 6.
In one embodiment of the invention, the parent enzyme hydroxylase HpaB is derived from Escherichia coli BL21 (DE 3) and is optimized and has a nucleotide sequence shown in SEQ ID NO. 3.
The invention also provides a method for coding the hydroxylase mutant HpaB Y282H Or HpaB S258C Is a gene of (a).
The invention also provides a recombinant vector carrying the gene.
The invention also provides a method for expressing the hydroxylase mutant HpaB Y282H Or HpaB S258C Or a recombinant cell containing the above gene or the above recombinant vector.
In one embodiment of the invention, the recombinant cell is a bacterial or fungal host cell.
The invention also provides a recombinant escherichia coli which expresses the hydroxylase mutant HpaB Y282H Or HpaB S258C And hydroxylase HpaC.
In one embodiment of the present invention, the amino acid sequence of the hydroxylase HpaC is shown in SEQ ID NO. 2.
In one embodiment of the invention, the nucleotide sequence encoding the hydroxylase HpaC is shown in SEQ ID NO. 4.
In one embodiment of the invention, the hydroxylase HpaC is derived from E.coli.
In one embodiment of the invention, the recombinant E.coli is selected from E.coli BL21 (DE 3), E.coli MG1655 or E.coli CCTCC NO: m2019390 is a host cell.
In one embodiment of the invention, the escherichia coli cctccc NO: m2019390 is described in the Chinese patent application publication No. CN110452865A and is named E.coli YMGR5A.
In one embodiment of the invention, the recombinant escherichia coli takes pEtac and pkk223-3 as expression vectors.
The invention also provides a method for constructing the recombinant escherichia coli, which comprises the following steps:
(1) Chemically synthesizing hydroxylase HpaB mutant.
(2) Preparing a recombinant plasmid pEtac-HpaC: and (3) chemically synthesizing a hydroxylase HpaC fragment, and connecting the HpaC fragment and the plasmid pEtac after double enzyme digestion to obtain a recombinant plasmid pEtac-HpaC.
(3) Inserting the obtained hydroxylase HpaB mutant into a plasmid pEtac-HpaC to obtain a recombinant plasmid pEtac-HpaB mutant-HpaC, and introducing the recombinant plasmid pEtac-HpaB mutant-HpaC into a tyrosol high-yield strain E.coli YMGR5A to obtain a recombinant hydroxytyrosol high-yield strain E.coli YMGR5A/pEtac-HpaB mutant-HpaC.
The invention also provides a method for producing hydroxytyrosol by adopting the recombinant E.coli YMGR5A/pEtac-HpaB mutant-HpaC.
In one embodiment of the invention, the method comprises inoculating the recombinant E.coli YMGR5A/pEtac-HpaB mutant-HpaC into a seed culture medium for culture to prepare seed solution; inoculating the seed liquid into a reaction system containing glucose, and fermenting to prepare the hydroxytyrosol.
In one embodiment of the present invention, the glucose-containing reaction system is a fermentation medium, which is: 50mL M9Y broth in a 250mL Erlenmeyer flask.
In one embodiment of the invention, the reaction system further comprises tyrosol, and the adding amount of the tyrosol is at least 30mM.
In one embodiment of the invention, the recombinant E.coli YMGR5A/pEtac-HpaB mutant-HpaC is inoculated on an LB solid medium for culture to obtain a single colony, the single colony is picked and inoculated on an LB liquid medium, and the seed liquid is prepared by culturing for 10-14 h under the conditions of 35-39 ℃ and 180-220 r/min.
In one embodiment of the invention, the recombinant E.coli YMGR5A/pEtac-HpaB mutant-HpaC is inoculated on LB solid medium for streak culture, single colony is selected and inoculated in a 100mL conical flask filled with 20mL LB liquid medium, and the culture is carried out for 10-14 h under the conditions of 35-39 ℃ and 180-220 r/min, thus obtaining seed liquid.
In one embodiment of the invention, the prepared seed liquid is inoculated into LB liquid culture medium, and the thallus is collected after culturing for 10-14 h under the conditions of 35-39 ℃ and 180-220 r/min; inoculating the collected thalli into an M9Y liquid culture medium for fermentation culture, and preparing the hydroxytyrosol.
In one embodiment of the present invention, the seed solution is inoculated into LB liquid medium in a ratio of 1% (v/v) of the inoculum size.
In one embodiment of the invention, shake flask fermentation conditions: inoculating 1% (v/v) into a 250mL conical flask containing 50mL LB liquid medium, culturing at 37deg.C for 10h at 200r/min, collecting the cells, washing the cells with physiological saline once, transferring into a 250mL conical flask containing 50mL M9Y liquid medium, and culturing at 30deg.C for 48h at 200r/min.
In one embodiment of the invention, shake flask fermentation conditions: 1% (v/v) of the cells were inoculated into a 250mL Erlenmeyer flask containing 50mL of LB liquid medium, cultured at 37℃for 10 hours at 200r/min, and then the cells were collected and washed with physiological saline once. Transfer into a 250mL Erlenmeyer flask containing 50mL M9Y liquid medium, and culture at 30deg.C and 200r/min for 48h.
In one embodiment of the invention, the reaction system further comprises a substrate tyrosol.
In one embodiment of the invention, the substrate tyrosol has a concentration of at least: 30mM.
In one embodiment of the invention, the substrate tyrosol has a concentration of 50mM.
In one embodiment of the present invention, the reaction conditions in the reaction system are: 30 ℃ and 200r/min.
In one embodiment of the invention, the HpaB mutant HpaB Y282H Or HpaB S258C 。
The invention also provides application of the recombinant E.coli YMGR5A/pEtac-HpaB mutant-HpaC in preparing hydroxytyrosol and products containing the same.
Advantageous effects
(1) The recombinant escherichia coli E.coli YMGR5A/pEtac-HpaB mutant-HpaC provided by the invention can convert glucose into hydroxytyrosol, and the hydroxytyrosol is produced by fermentation in a glucose culture medium, and E.coli YMGR5A/pEtac-HpaB is obtained Y282H -HpaC、E.coli YMGR5A/pEtac-HpaB S258C The yields of HpaC can be up to 1.82g/L, 1.81g +.L is higher than the results of most research teams currently.
(2) The recombinant escherichia coli E.coli YMGR5A/pEtac-HpaB mutant-HpaC provided by the invention is adopted to ferment and produce hydroxytyrosol in a culture medium added with 50mM tyrosol, and E.coli YMGR5A/pEtac-HpaB Y282H -HpaC、E.coli YMGR5A/pEtac-HpaB S258C The HpaC yield can be up to 49.98mM/L (7.705 g/L) and 48.2mM/L (7.431 g/L), and the conversion rate is over 99% and 96% respectively, which is higher than the current achievement of most research teams.
Drawings
Fig. 1: the strain E.coli YMGR5A/pEtac-HpaB-HpaC and E.coli YMGR5A/pEtac-HpaB constructed by the invention 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 fermented by HpaC with the addition of 30mM tyrosol.
Fig. 2: the strain E.coli YMGR5A/pEtac-HpaB constructed by the invention Y282H -HpaC、E.coli YMGR5A/pEtac-HpaB S258C Results of HpaC fermentation of hydroxytyrosol production.
Fig. 3: the strain E.coli YMGR5A/pEtac-HpaB constructed by the invention Y282H Results of hydroxytyrosol production by fermentation of HpaC with exogenous tyrosol addition.
Fig. 4: the strain E.coli YMGR5A/pEtac-HpaB constructed by the invention S258C Results of hydroxytyrosol production by fermentation of HpaC with exogenous tyrosol addition.
Detailed Description
The method for constructing the recombinant plasmid pEtac according to the following examples is described in: shen Wei et al, "secretory expression of the archaebacteria Pyrococcus furiosus hyperthermophilic alpha amylase gene in E.coli", literature from university of Jiangnan:
the construction method comprises the following steps: the fragment containing the tac promoter in the escherichia coli expression vector pKK223-3 is cut by BamHI and EcoRI and then is connected into the expression vector pET28a to construct a new expression vector pEtac.
The following examples relate to the following media:
LB medium formulation (g/L): yeast powder 5, peptone 10, naCl 10, and 1.5% -2.0% agar powder.
M9Y Medium formulation (g/L): na (Na) 2 HPO 4 ·12H 2 O 17.1，KH 2 PO 4 3，NaCl 0.5，NH 4 Cl 1, glucose 20, yeast powder 0.25, and MgSO with final concentration 4 5mmol·L -1 。
The detection method involved in the following examples is as follows:
tyrosol and hydroxytyrosol detection method
High Performance Liquid Chromatography (HPLC) is used for detection. The chromatographic detection conditions are specifically as follows: agela Innoval C18 column (4.6X1250 mm, pore size 5 μm); a mobile phase of 80% aqueous solution of 0.1% formic acid, 20% methanol; the flow rate is 1mL/min; the sample injection amount is 10 mu L; an ultraviolet detector for detecting a wavelength of 280nm; the column temperature was 28 ℃.
The method for detecting the hydroxylase enzyme activity comprises the following steps: the method adopts hydroxytyrosol sodium periodate detection method.
Example 1: construction of recombinant plasmid pEtac-HpaBC
(1) Construction of recombinant plasmid pEtac-HpaC:
according to the sequence disclosed in NCBI, hpaC gene (nucleotide sequence is shown as SEQ ID NO. 4) is chemically synthesized, pst I and HindIII cleavage sites are designed in primers p-HpaC-L and p-HpaC-R (shown in table 1), a target fragment and pEtac plasmid are subjected to cleavage purification at two sites, then are connected by using Solution I ligase, are transferred into E.coli JM109 by a chemical conversion method, are coated on LB solid medium plates containing kanamycin resistance, are cultured for 10-12 hours in a 37 ℃ incubator, the single colony which grows out is subjected to cleavage verification, and the recombinant plasmid pEtac HpaC is extracted after the correct strain is cultured.
(2) Construction of recombinant plasmid pEtac-HpaB-HpaC
According to the sequence disclosed in NCBI, hpaB gene (nucleotide sequence is shown as SEQ ID NO. 3) is chemically synthesized, ecoR I and PstI cleavage sites are designed in primers p-HpaB-L and p-HpaB-R (shown in table 1), a target fragment and pEtac plasmid are subjected to two-site cleavage purification, then are connected by using Solution I ligase, and then are subjected to verification by using the method and then are cultured to extract recombinant plasmid pEtac-HpaB-HpaC.
Example 2: construction of recombinant plasmid containing hydroxylase mutant
The method comprises the following specific steps:
designing a site-directed mutagenesis primer, constructing and obtaining a mutant HpaB by taking a recombinant plasmid pEtac-HpaB-HpaC as a template and carrying out single-point mutagenesis Y282H 、HpaB S258C 、HpaB Q212E 、HpaB G340A Recombinant plasmid pEtac-HpaB of (E) Y282H -HpaC、pEtac-HpaB S258C -HpaC、pEtac-HpaB Q212E -HpaC、pEtac-HpaB G340A -HpaC。
The PCR mutation can enter the subsequent operation after the successful mutation is verified by sequencing.
The primer sequences involved are shown in Table 1:
table 1: primer sequences
The PCR reaction system is shown in Table 2:
table 2: preparation of PCR System
Example 3: construction of recombinant strains containing hydroxylase mutants
The recombinant plasmids with correct sequencing verification, which are prepared in the example 2, are respectively transferred into E.coli JM109 by a chemical transformation method, coated on LB solid culture medium plates with kanamycin resistance, cultured for 10-12 hours in a 37 ℃ incubator, and the recombinant plasmids pEtac-HpaB-HpaC and pEtac-HpaB are extracted after the correct strains are cultured Y282H -HpaC、pEtac-HpaB S258C -HpaC、pEtac-HpaB Q212E -HpaC、pEtac-HpaB G340A HpaC; transferring the recombinant plasmids into E.coli YMGR5A respectively by an electrotransformation method, coating on LB solid culture medium plates containing kanamycin resistance, culturing for 10-12 h in a 37 ℃ incubator, picking single colony for culture to obtain recombinant escherichia coli E.coli YMGR5A/pEtac-HpaB-HpaC and E.coli YMGR5A/pEtac-HpaB respectively Y282H -HpaC、E.coli YMGR5A/pEtac-HpaB S258C -HpaC、E.coli YMGR5A/pEtac-HpaB Q212E -HpaC、E.coli YMGR5A/pEtac-HpaB G340A -HpaC。
Example 4: verification of the conversion of tyrosol by the mutant Strain
The method comprises the following specific steps:
(1) The recombinant E.coli YMGR5A/pEtac-HpaB-HpaC obtained in example 3, E.coli YMGR5A/pEtac-HpaB, respectively Y282H -HpaC、E.coli YMGR5A/pEtac-HpaB S258C -HpaC、E.coli YMGR5A/pEtac-HpaB Q212E -HpaC、E.coli YMGR5A/pEtac-HpaB G340A Streaking the HpaC on the LB solid medium to obtain single colonies, picking the single colonies, inoculating the single colonies into a 100mL conical flask filled with 20mL LB liquid medium, and culturing for 12h at 37 ℃ and 200 r/min; seed solutions are prepared respectively.
(2) Respectively inoculating the seed solution prepared in the step (1) into a 250mL conical flask filled with 50mL LB liquid medium according to the inoculum size of 1% (v/v), culturing at 37 ℃ for 10 hours at 200r/min, and collecting thalli;
(3) After washing the thalli obtained in the step (2) once by using normal saline, transferring the thalli into a 250mL conical flask filled with 50mL M9Y liquid culture medium (30 mM tyrosol is added into the liquid culture medium), culturing for 12 hours at 30 ℃ and 200r/min to prepare a fermentation broth, and detecting the yield and the residual tyrosol of hydroxytyrosol in the fermentation broth. The hydroxytyrosol production and the tyrosol residual amounts of each recombinant strain are shown in table 3 and fig. 1.
Table 3: hydroxytyrosol yield and tyrosol residual amount of recombinant strain under exogenous addition of 30mM tyrosol
| Strain | Tyrosol residual quantity (g/L) | Yield of hydroxytyrosol (g/L) |
| E.coli YMGR5A/pEtac-HpaB-HpaC | 9.75 | 19.82 |
| E.coli YMGR5A/pEtac-HpaB Y282H - |
0 | 27.51 |
| E.coli YMGR5A/pEtac-HpaB S258C - |
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 |
Example 5: shake flask fermentation to verify high-yield hydroxytyrosol of mutant strain
The method comprises the following specific steps:
(1) Recombinant E.coli YMGR5A/pEtac-HpaB from example 4 was isolated Y282H -HpaC、E.coli YMGR5A/pEtac-HpaB S258C Streaking the HpaC on the LB solid medium to obtain single colonies, picking the single colonies, inoculating the single colonies into a 100mL conical flask filled with 20mL LB liquid medium, and culturing for 12h at 37 ℃ and 200 r/min; seed solutions are prepared respectively.
(2) Respectively inoculating the seed solution prepared in the step (1) into a 250mL conical flask filled with 50mL LB liquid medium according to the inoculum size of 1% (v/v), culturing at 37 ℃ for 10 hours at 200r/min, and collecting thalli;
(3) And (2) respectively washing the thalli obtained in the step (2) by using normal saline once, transferring the thalli into a 250mL conical flask filled with 50mL M9Y liquid culture medium, culturing at 30 ℃ for 48 hours at 200r/min, sampling every 12 hours, and detecting the content of hydroxytyrosol in the fermentation broth, wherein the yield results of the hydroxytyrosol are shown in Table 4 and figure 2.
Table 4: hydroxytyrosol production by different recombinant E.coli
The result shows that the recombinant escherichia coli E.coli YMGR5A/pEtac-HpaBC provided by the invention Y282H -HpaC、E.coli YMGR5A/pEtac-HpaBC S258C HpaC can realize the conversion of glucose into hydroxytyrosol, and the yield of the hydroxytyrosol produced by fermentation in a glucose culture medium can be up to 1.82g/L and 1.81g/L respectively.
Example 6: shake flask fermentation demonstrated high conversion of tyrosol by mutant strains
The method comprises the following specific steps:
(1) The recombinant E.coli YMGR5A/pEtac-HpaB of example 5 Y282H -HpaC、E.coli YMGR5A/pEtac-HpaB S258C Streaking the HpaC on the LB solid medium to obtain single colonies, picking the single colonies, inoculating the single colonies into a 100mL conical flask filled with 20mL LB liquid medium, and culturing for 12h at 37 ℃ and 200 r/min; preparing seed liquid.
(2) Inoculating the seed solution prepared in the step (1) into a 250mL conical flask filled with 50mL LB liquid medium according to the inoculum size of 1% (v/v), culturing at 37 ℃ for 10 hours at 200r/min, and collecting thalli;
(3) After washing the thalli obtained in the step (2) by using normal saline for one time, transferring the thalli into a 250mL conical flask filled with 50mL M9Y (containing different concentrations of substrate tyrosol: 30mM, 40mM, 50mM, 60mM and 70mM, specifically shown in table 5), culturing at 30 ℃ for 12 hours at 200r/min to respectively prepare fermentation solutions, detecting the content of hydroxytyrosol in the fermentation solutions, and carrying out hydroxytyrosol under the conditions of different concentrations of substrate tyrosol.
Wherein E.coli YMGR5A/pEtac-HpaB Y282H The HpaC yield and the unit cell concentration yield are shown in Table 5 and FIG. 3.
Table 5: hydroxytyrosol yield and unit bacterial concentration yield of recombinant escherichia coli under different concentrations of substrate tyrosol
| Substrate concentration mM | Hydroxytyrosol yield mM | Yield of concentrated hydroxytyrosol per |
| 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 |
Wherein E.coli YMGR5A/pEtac-HpaB S258C The HpaC yield and the unit cell concentration yield are shown in Table 6 and FIG. 4.
Table 6: hydroxytyrosol yield and unit bacterial concentration yield of recombinant escherichia coli under different concentrations of substrate tyrosol
| Substrate concentration mM | Hydroxytyrosol yield mM | Yield of concentrated hydroxytyrosol per |
| 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 |
The results show that:
recombinant E.coli YRG 5A/pEtac-HpaB provided by the invention Y282H The production of hydroxytyrosol by HpaC fermentation in a medium supplemented with 50mM tyrosol can be as high as 49.98mM (7.705 g/L), with a conversion of more than 99%;
recombinant E.coli YMGR5A/pEtac-HpaB provided by the invention S258C The production of hydroxytyrosol by HpaC fermentation in a medium supplemented with 50mM tyrosol can be up to 48.2mM (7.431 g/L), with a conversion of over 96%;
thus, the recombinant E.coli YMRG5A/pEtac-HpaB of the present invention Y282H -HpaC、E.coli YMGR5A/pEtac-HpaB S258C HpaC has a wide range of industrial applications.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A hydroxylase HpaB mutant is characterized in that the hydroxylase HpaB mutant is obtained by mutating tyrosine at 282 rd site into histidine on the basis of a parent enzyme HpaB with an amino acid sequence shown as SEQ ID NO.5, and the amino acid sequence of the hydroxylase HpaB mutant is shown as SEQ ID NO. 1;
or, the hydroxylase mutant is obtained by mutating serine at 258 th site into cysteine on the basis of parent enzyme HpaB with an amino acid sequence shown as SEQ ID NO.5, and the amino acid sequence of the hydroxylase mutant is shown as 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 comprising the gene of claim 2 or comprising 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 which expresses the hydroxylase HpaB mutant and the hydroxylase HpaC of claim 1, wherein the amino acid sequence of the hydroxylase HpaC is shown as SEQ ID NO. 2.
7. A method for producing hydroxytyrosol, which is characterized in that the seed solution of the recombinant escherichia coli according to claim 6 is added into a reaction system containing glucose for fermentation.
8. The method of claim 7, further comprising a substrate tyrosol in the reaction system.
9. The method according to claim 7 or 8, wherein in the reaction system, the reaction conditions are: inoculating the prepared seed liquid into an LB liquid culture medium, culturing for 10-14 h at 35-40 ℃ and 180-220 r/min, and collecting thalli; inoculating the collected thalli into an M9Y liquid culture medium for fermentation culture, and preparing the hydroxytyrosol.
10. Use of the recombinant escherichia coli of claim 6 for preparing hydroxytyrosol and products containing hydroxytyrosol.
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