A method for producing hydroxytyrosol by using a dual-bacterial co-culture system

Abstract

The application discloses a method for producing hydroxytyrosol by using a double-bacterial co-culture system, and belongs to the technical field of microbial genetic engineering. The application constructs a double-bacterial co-culture system of a Saccharomyces cerevisiae engineering strain and an Escherichia coli engineering strain, optimizes and utilizes the double-bacterial co-culture system to synthesize hydroxytyrosol from scratch. The Saccharomyces cerevisiae engineering strain is obtained by eliminating the feedback inhibition of tyrosine in a host strain, introducing an exogenous path to improve the metabolic flux of a product and integrating a transformed GAL regulation system; the Escherichia coli engineering strain is obtained by overexpressing 4-hydroxyphenylacetic acid-3-hydroxylase and riboflavin oxidoreductase in a host strain, thereby realizing the preparation and efficient synthesis of hydroxytyrosol; the yield of hydroxytyrosol synthesized from scratch by using the double-bacterial co-culture system of the Saccharomyces cerevisiae engineering strain and the Escherichia coli engineering strain reaches 435.32 mg / L, the problem that a single wild strain cannot synthesize hydroxytyrosol from scratch is solved, and a new idea is provided for the efficient microbial fermentation production of hydroxytyrosol.

Description

Technical Field

[0001] This invention relates to a method for producing hydroxytyrosol using a dual-strain co-culture system, belonging to the field of microbial genetic engineering technology. Background Technology

[0002] Hydroxytyrosol (3,4-dihydroxyphenylethanol), as the main active substance in olive oil, has widely recognized nutritional and economic value. It has been shown to possess various pharmacological effects, such as antioxidant, antithrombotic, tumor-inhibiting, and osteoporosis-preventing effects. Natural extraction of hydroxytyrosol yields low amounts and purification is complex; chemical synthesis methods cause significant environmental pollution; and enzymatic catalysis is costly. In contrast, the synthesis of hydroxytyrosol using microbial cell factories offers advantages such as high efficiency, environmental friendliness, and low cost, attracting widespread attention from scholars both domestically and internationally. A comprehensive review of numerous studies on the synthesis of hydroxytyrosol reveals that tyrosol is an important precursor and can be synthesized de novo via the Ehrlich pathway in Saccharomyces cerevisiae. Research on tyrosol in Saccharomyces cerevisiae is relatively mature; however, Saccharomyces cerevisiae lacks suitable hydroxylases to oxidize tyrosol to hydroxytyrosol, thus hindering efficient de novo conversion. On the other hand, numerous studies have shown that the hydroxylase combination in Escherichia coli (4-hydroxyphenylacetic acid-3-hydroxylase HpaB and riboflavin oxidoreductase HpaC) has excellent tyrosol hydroxylation ability, but this strain lacks the ability to synthesize tyrosol de novo.

[0003] Therefore, how to achieve efficient conversion of glucose to hydroxytyrosol using microbial fermentation has become a key point in the green synthesis of hydroxytyrosol. Summary of the Invention

[0004] Natural extraction of hydroxytyrosol yields low efficiency and is complex to purify; chemical synthesis pollutes the environment; enzymatic synthesis is costly; microbial synthesis has advantages such as high efficiency, greenness, and low cost, but a single wild strain cannot achieve de novo synthesis of hydroxytyrosol.

[0005] This invention provides a method for producing hydroxytyrosol using a dual-strain co-culture system. Specifically, it involves constructing a dual-strain co-culture system of engineered *Saccharomyces cerevisiae* and engineered *Escherichia coli* strains, optimizing and utilizing this system for de novo synthesis of hydroxytyrosol. The dual-strain co-culture system constructed in this invention is environmentally friendly, low-cost, and effectively solves the problem that a single wild-type strain cannot synthesize hydroxytyrosol de novo. It provides a new approach and method for the efficient fermentation production of hydroxytyrosol by microorganisms, and is of great significance for promoting the green and efficient synthesis of hydroxytyrosol.

[0006] The first objective of this invention is to provide a recombinant brewer's yeast, characterized in that the recombinant brewer's yeast overexpresses the 3-deoxy-D-arabinohepetulose-7-phosphate synthase mutant aro4 derived from brewer's yeast.K229L A branching acid mutase mutant aro7 derived from Saccharomyces cerevisiae G141S tyrosine decarboxylase pcaas derived from parsley opt and phosphoketonease Bbxfpk derived from Bifidobacterium breve opt At the same time, the GAL80 gene and HO gene on the Saccharomyces cerevisiae genome were knocked out.

[0007] In one embodiment of the present invention, the 3-deoxy-D-arabinohepenolate-7-phosphate synthase mutant aro4 K229L The nucleotide sequence is shown in SEQ ID NO.1.

[0008] In one embodiment of the present invention, the branched acid mutase mutant aro7 G141S The nucleotide sequence is shown in SEQ ID NO.2.

[0009] In one embodiment of the present invention, the tyrosine decarboxylase pcaas opt The nucleotide sequence is shown in SEQ ID NO.3.

[0010] In one embodiment of the present invention, the phosphate transketase Bbxfpk opt The nucleotide sequence is shown in SEQ ID NO.4.

[0011] In one embodiment of the present invention, the recombinant brewer's yeast is based on BY4741 as the starting strain.

[0012] In one embodiment of the present invention, the recombinant brewing yeast is aro4 K229L aro7 G141S pcaas opt Bbxfpk opt The GAL80, GAL80, HO, and HO sites were sequentially integrated into the genome of the starting strain BY4741, while the GAL80 and HO genes in the Saccharomyces cerevisiae genome were knocked out. The accession numbers of the GAL80 and HO sites on NCBI are 854954 and 851371, respectively.

[0013] The second objective of this invention is to provide a recombinant Escherichia coli strain, wherein the engineered Escherichia coli strain uses wild-type Escherichia coli BL21(DE3) as a host and overexpresses 4-hydroxyphenylacetic acid-3-hydroxylase HpaB (GenBank accession number: ARI00017.1) and riboflavin oxidoreductase HpaC (GenBank accession number: ARI00016.1) derived from Escherichia coli BL21(DE3).

[0014] In one embodiment of the present invention, the recombinant Escherichia coli expresses 4-hydroxyphenylacetic acid-3-hydroxylase HpaB using the PET30a plasmid and riboflavin oxidoreductase HpaC using the PET22b plasmid.

[0015] The third objective of this invention is to provide a method for improving the tyrosol conversion rate of Escherichia coli, wherein the method involves overexpressing 4-hydroxyphenylacetic acid-3-hydroxylase HpaB (GenBank accession number: ARI00017.1) and riboflavin oxidoreductase HpaC (GenBank accession number: ARI00016.1) derived from Escherichia coli BL21(DE3) in Escherichia coli.

[0016] In one embodiment of the present invention, the engineered strain of Escherichia coli uses wild-type Escherichia coli BL21(DE3) as the host.

[0017] In one embodiment of the present invention, the finally constructed Escherichia coli genetically engineered strain BL-EcBc has a high-efficiency tyrosol conversion capability.

[0018] A fourth objective of this invention is to provide a two-strain co-culture system for the de novo synthesis of hydroxytyrosol. This invention provides a method for the fermentation preparation of hydroxytyrosol, wherein the hydroxytyrosol is prepared using a two-strain fermentation system composed of the aforementioned recombinant Saccharomyces cerevisiae and recombinant Escherichia coli.

[0019] In one embodiment of the present invention, the substrate in the dual-strain fermentation system is one or more of glucose, sucrose, and glycerol.

[0020] In one embodiment of the present invention, the dual-strain co-culture system contains: tryptone (15 g / L), yeast extract (17.5 g / L), sodium chloride (5 g / L), and sucrose (10 g / L).

[0021] In one embodiment of the present invention, the dual-strain fermentation system is prepared by mixing recombinant Saccharomyces cerevisiae seed liquid and recombinant Escherichia coli seed liquid in a ratio of (1-10):(1-10) to obtain a mixed seed liquid, and then adding the mixed seed liquid to a reaction system containing substrate for fermentation.

[0022] In one embodiment of the present invention, the recombinant Saccharomyces cerevisiae seed liquid and the recombinant Escherichia coli seed liquid are mixed at a volume ratio of 5:1 to obtain a mixed seed liquid.

[0023] In one embodiment of the present invention, the fermentation conditions are as follows: the recombinant Saccharomyces cerevisiae seed liquid and the recombinant Escherichia coli seed liquid are inoculated in an optimal culture medium at a ratio of 5:1 and fermented for 24 to 96 hours; wherein, IPTG is added for induction during 2 to 10 hours of fermentation.

[0024] In one embodiment of the present invention, IPTG is added to induce fermentation after 8 hours.

[0025] In one embodiment of the present invention, during the co-culture of the two bacteria, the fermentation time of the co-culture system after adding the inducer IPTG is 72 hours.

[0026] The fifth object of the present invention is to provide a method for preparing hydroxytyrosol from a two-strain co-culture system.

[0027] In one embodiment of the present invention, the method for preparing hydroxytyrosol from a dual-culture system involves fermenting the co-culture system and then collecting the supernatant of the fermentation broth to separate hydroxytyrosol. Ascorbic acid is added to the fermentation system to achieve a final concentration of 1 g / L.

[0028] Beneficial effects

[0029] (1) This invention successfully obtained engineered brewer's yeast strains LYJ-001, LYJ-002, LYJ-003 and LYJ-004 that synthesize tyrosol de novo by genetically modifying wild-type brewer's yeast BY4741. Compared with wild-type strains, LYJ-001 strain increased the yield of tyrosol from 17.60 mg / L to 106.05 mg / L; LYJ-002 strain further increased the yield of tyrosol to 116.83 mg / L; LYJ-003 strain further increased the yield of tyrosol to 371.09 mg / L; and finally LYJ-004 strain achieved a yield of 461.07 mg / L of tyrosol.

[0030] (2) This invention successfully obtained engineered Escherichia coli BL-Ecbc with high tyrosol conversion rate by genetically modifying wild-type Escherichia coli BL21(DE3). Compared with wild-type Escherichia coli, the conversion rate of tyrosol to hydroxytyrosol was increased from 0.03% to 86.02%.

[0031] (3) By optimizing the culture environment of the co-culture system, the present invention successfully obtained a dual-strain co-culture system for efficient de novo synthesis of hydroxytyrosol, which resulted in a de novo synthesis yield of hydroxytyrosol of 435.32 mg / L. Attached Figure Description

[0032] Figure 1 Gene maps (A) of engineered Saccharomyces cerevisiae strains LYJ-001, LYJ-002, LYJ-003 and LYJ-004, and the yield of tyrosol synthesized by the above engineered strains and wild-type strain BY4741 using sucrose and glycerol as substrates (B).

[0033] Figure 2The yield of hydroxytyrosol synthesized by engineered Escherichia coli BL-Ecbc and wild-type Escherichia coli BL21(DE3) using 300 mg / L tyrosol as a substrate.

[0034] Figure 3 Demonstration and optimization of a two-strain co-culture system for de novo synthesis of hydroxytyrosol, where A is a demonstration diagram of the two-strain co-culture system for de novo synthesis of hydroxytyrosol, B is the optimization of the culture medium for the two-strain co-culture system, C is the optimization of the initial inoculation ratio of the seed culture for the two-strain co-culture system, D is the optimization of the time for adding the inducer for the two-strain co-culture system, and E is the optimization of the fermentation time for the two-strain co-culture system. Detailed Implementation

[0035] The biochemical materials involved in the following embodiments are as follows:

[0036] Saccharomyces cerevisiae BY4741 and Escherichia coli BL21(DE3) (our laboratory); Escherichia coli Top10 competent cells (Novagen, USA); Tyrosol and hydroxytyrosol standards (Solarbio, China); restriction endonucleases, T4 DNA ligase, Prime STAR Max DNA polymerase, loading buffer, LE agarose gel and S4 nucleic acid electrophoresis dye (Takara); antibiotics, yeast whole genome extraction kit and SanPrep column-based plasmid DNA mini-extraction kit (Shanghai Sangon Biotech Co., Ltd.); DNA 5000-ladder and DNA 10000-ladder (Shanghai Takara Bio Co., Ltd.); tryptone and yeast extract (Oxoid, UK); tyrosine decarboxylase gene Pcaas from parsley. opt The phosphotransketase gene Bbxfpk in Bifidobacterium breve opt Codon optimization and synthesis (Shanghai Sangon Biotech Co., Ltd.)

[0037] The culture media involved in the following examples are as follows:

[0038] YPD liquid culture medium: glucose (20 g / L), tryptone (20 g / L), yeast extract (10 g / L), sterilized at 115°C for 30 min.

[0039] YPD agar medium: Add 15g of agar powder to 1L of YPD liquid medium, autoclave and pour into sterile plates to make antibiotic-free YPD solid medium; if antibiotics are added, it will be the corresponding antibiotic-selective YPD solid medium. After condensation, store in a 4℃ refrigerator for later use.

[0040] YPSG liquid medium: sucrose (10 g / L), glycerol (10 g / L), tryptone (20 g / L), yeast extract (10 g / L), sterilized at 115°C for 30 min.

[0041] YPSG agar medium: Add 15g of agar powder to 1L of YPSG liquid medium, autoclave and pour into sterile plates to make antibiotic-free YPSG solid medium; if antibiotics are added, it will be the corresponding antibiotic-selective YPSG solid medium. After condensation, store in a 4℃ refrigerator for later use.

[0042] LB liquid medium: NaCl (10 g / L), tryptone (10 g / L), yeast extract (5 g / L), sterilized at 120°C for 20 min.

[0043] LB agar medium: Add 15g of agar powder to 1L of LB liquid medium. The sterilization method is the same as that for YPD agar medium.

[0044] SD agar medium: yeast free of amino acid nitrogen (1.7 g / L), anhydrous ammonium sulfate (5 g / L), yeast total culture amino acid premix (1.31 g / L), glucose (2 g / L) and (15 g / L) agar powder, sterilized at 115℃ for 30 min.

[0045] SD-FOA agar medium: First, prepare a 100 mg / mL stock solution of 5-fluoroorotic acid (FOA). Weigh 1 g of 5-FOA powder and dissolve it in 10 mL of dimethyl sulfoxide. Filter the solution through a sterile 0.22 μm organic syringe filter for sterilization and store at -20°C. When preparing SD-FOA plates, cool 100 mL of hyperthermically sterilized SD agar medium to approximately 60°C and add 1 mL of the FOA stock solution. This results in a working concentration of 1 mg / mL of 5-FOA.

[0046] Preparation of mixed liquid culture medium for co-culture of two bacteria:

[0047] Culture medium 1: tryptone (15 g / L), yeast extract (17.5 g / L), NaCl (5 g / L) and glucose (10 g / L);

[0048] Culture medium 2: tryptone (15 g / L), yeast extract (17.5 g / L), NaCl (5 g / L) and sucrose (10 g / L);

[0049] Culture medium 3: tryptone (15 g / L), yeast extract (17.5 g / L), NaCl (5 g / L), sucrose (5 g / L) and glycerol (5 g / L);

[0050] Culture medium 4: yeast extract (5 g / L), glucose (40 g / L), KH2PO4 (13.3 g / L), (NH4)2HPO4 (4 g / L), citric acid (1.7 g / L), EDTA (0.0084 g / L), CoCl2 (0.0025 g / L), MnCl2 (0.015 g / L), CuCl2 (0.0015 g / L), H3BO3 (0.003 g / L), Na2MoO4 (0.0025 g / L), Zn(CH3COO)2 (0.008 g / L), Fe(III)citrate (0.06 g / L) and MgSO4 (1.3 g / L);

[0051] Culture medium 5: yeast extract (5 g / L), sucrose (20 g / L), glycerol (20 g / L), KH2PO4 (13.3 g / L), (NH4)2HPO4 (4 g / L), citric acid (1.7 g / L), EDTA (0.0084 g / L), CoCl2 (0.0025 g / L), MnCl2 (0.015 g / L), CuCl2 (0.0015 g / L), H3BO3 (0.003 g / L), Na2MoO4 (0.0025 g / L), Zn(CH3COO)2 (0.008 g / L), Fe(III)citrate (0.06 g / L) and MgSO4 (1.3 g / L), sterilized at 115℃ for 30 min.

[0052] All the above culture media were treated with ascorbic acid, ampicillin and kanamycin after filtration sterilization to achieve final concentrations of 1 g / L, 50 μg / mL and 50 μg / mL, respectively.

[0053] The strains, plasmids, and primers involved in the following examples are shown in Tables 1-3:

[0054] Table 1: Strains and their genotypes

[0055]

[0056] Table 2: Plasmids and their genotypes

[0057]

[0058] Table 3: Primer sequences

[0059]

[0060]

[0061] Basic operations in molecular biology:

[0062] (1) Unless otherwise specified, the procedures for E. coli plasmid extraction, genome extraction, PCR product purification, and gel recovery shall be performed in accordance with the instructions of the corresponding kit.

[0063] (2) DNA fragment amplification and site-directed mutagenesis were performed using PCR and overlap extension PCR techniques, respectively, with different reaction systems and procedures adopted according to different experimental requirements. The reaction systems are shown in Table 4, and the reaction procedures are shown in Table 5.

[0064] Table 4: PCR reaction system

[0065]

[0066] Table 5: PCR reaction procedure

[0067]

[0068] (3) Enzyme digestion and ligation: Unless otherwise specified, the double DNA digestion and T4 ligase reaction system and reaction conditions shall be performed according to the Takara company website (https: / / www.takarabiomed.com.cn / ).

[0069] (4) Transformation of competent Escherichia coli by heat shock method: First, take 5 μL of ligation product and add it to 50 μL of freeze-thawed competent cells and incubate on ice for 20 min; then place the competent cells in a 42℃ metal bath for heat shock for 90 s, and quickly place them on ice for 5 min; then add 1 mL of fresh LB medium and incubate at 37℃ with shaking for 60 min; finally, take 200 μL of bacterial culture and spread it on solid LB plates with the corresponding resistance and incubate at 37℃ overnight.

[0070] (5) Preparation of competent cells of Saccharomyces cerevisiae and electroporation transformation: ① Pick a single colony and inoculate it into 5 mL of YPD liquid medium. Incubate overnight in a constant temperature shaker at 30°C and a rotation speed of 200 rpm; ② Transfer 1 mL of bacterial culture to 50 mL of YPD medium and incubate under the same conditions until OD. 600① The concentration of the precipitate is approximately 1.3–2; ② Transfer the precipitate to a 50 mL sterile centrifuge tube, incubate on ice for 10 min, centrifuge at 2500 rpm, and recover the cells; ③ Resuspend the bacterial culture in 30 mL of pre-cooled sterile water, centrifuge at 4 °C and 2500 rpm for 10 min, and discard the supernatant; ④ Resuspend the cells in 30 mL of pre-cooled 1 M sorbitol solution, centrifuge at 4 °C and 2500 rpm for 10 min, and discard the supernatant; ⑤ Dissolve the precipitate in 200 μL of sorbitol solution, transfer it to a pre-cooled 1.5 mL centrifuge tube, and the competent cells of Saccharomyces cerevisiae are obtained. ⑦ Take 5 μL of plasmid or 10 μL of concentrated linear fragment, add it to 50 μL of competent cells, incubate on ice for 5 min, and transfer to a pre-cooled 1 mm sterile electroporation cuvette; ⑧ Electroporate the cuvette at 750 V / mm, then immediately add 1 mL of LYPD medium, suspend the cells, and transfer them to a 1.5 mL centrifuge tube, and incubate at 30 °C for 2 h; ⑨ Wash the revived cells with sterile water, take 200 μL and spread it on solid YPD / SD agar plates with corresponding resistance or defective selection markers, and incubate at 30 °C.

[0071] (6) Strain Cultivation and Fermentation Methods: Single colonies were picked from agar plates and inoculated into 5 mL of YPD / LB liquid medium. Yeast was cultured overnight in a constant-temperature shaker at 30°C and 200 rpm, while Escherichia coli was cultured overnight in a constant-temperature shaker at 37°C and 200 rpm. For single-strain cultivation, 1 mL of bacterial culture was transferred to 50 mL of YPSG / LB medium for expansion. Appropriate inducers and antibiotics were added according to the strain requirements. For co-culture, 1 mL of mixed bacterial culture with different strain ratios was transferred to 50 mL of different mixed media for expansion to screen for optimal fermentation conditions. Appropriate inducers, antibiotics, and ascorbic acid were added according to the strain requirements.

[0072] Methods for detecting tyrosol and hydroxytyrosol:

[0073] Tyrosol and hydroxytyrosol in the fermentation broth supernatant were detected using an HPLC-UV system (Waters 2695). After fermentation, 2 mL of fermentation broth was centrifuged at 10000 g for 5 min. The supernatant was filtered through a 0.22 μm aqueous membrane and then detected using a C18 column (250 × 4.6 mm id, 5 μm; Agilent). The injection volume was 10 μL, the detection wavelength was 280 nm, the column temperature was 30 °C, and the flow rate was 0.3 mL / min. The mobile phase consisted of 5% formic acid (A) and 100% acetonitrile (B). A gradient elution program was used, as shown in Table 6.

[0074] Table 6: Liquid Phase Elution Procedure

[0075]

[0076] The pUMRI-B-△GAL80 and pUMRI-A-△HO involved in the following embodiments are disclosed in the following papers: Lv,XM; Wang,F.; Zhou,PP; Ye,LD; Xie,WP; Xu,HM; Yu,HWD. *Dual regulation of cytoplasmic and mitochondrial acetyl-CoA utilization for improved isoprene production in Saccharomyces cerevisiae*. Nat Commun. 2016, 7, No. 12851.

[0077] Example 1: Genetically engineered wild-type Saccharomyces cerevisiae BY4741 and detection of its de novo tyrosol synthesis yield.

[0078] The specific steps are as follows:

[0079] 1. Preparation of engineered yeast strain YLYJ-001

[0080] (1) The aro4 enzyme with restriction sites was amplified from the whole genome of wild-type yeast BY4741 using overlap extension PCR. K229L Gene fragment, primer is aro4 K229L -F1, aro4 K229L -R1(SalI), aro4 K229L -F2 (BamHI) and aro4 K229L -R2(SalI), primer nucleotide sequences are referenced in Table 3, PCR reaction system is shown in Table 4, and PCR reaction procedure is shown in Table 5.

[0081] (2) For aro4 with restriction sites K229L The gene fragment and plasmid pUMRI-B-△GAL80 were double-digested with enzymes. After gel recovery of the double-digested gene fragment and vector fragment, the gene fragment and vector fragment were cloned to obtain the recombinant plasmid pB-△GAL80-aro4. K229L .

[0082] (3) The obtained recombinant plasmid pB-△GAL80-aro4 K229L SfiI single enzyme digestion was performed, and homologous recombination was used to integrate it into the GAL80 site of the BY4741 chromosome. The GAL80 gene of BY4741 was then knocked out, resulting in the yeast engineered strain YLYJ-001. The genotype of YLYJ-001 is as follows: Figure 1 As shown in Figure A.

[0083] 2. Preparation of engineered yeast strain YLYJ-002

[0084] (1) The aro7 enzyme with restriction sites was amplified from the whole genome of wild-type yeast BY4741 using overlap extension PCR. G141S Gene fragment, primer is aro7 G141S -F1(EcoRI), aro7 G141S -R1、aro7 G141S -F2 and aro7 G141S -R2(NotI), the primer nucleotide sequences are shown in Table 3, the PCR reaction system is shown in Table 4, and the PCR reaction procedure is shown in Table 5.

[0085] (2) Preparation of aro4 with restriction sites from YLYJ-001 bacteria K229L and the aforementioned aro7 G141S The gene fragment and plasmid pUMRI-B-△GAL80 were double-digested with enzymes. After gel recovery of the double-digested gene fragment and vector fragment, the gene fragment and vector fragment were cloned to obtain the recombinant plasmid pB-△GAL80-aro4. K229L -aro7 G141S .

[0086] (3) The obtained recombinant plasmid pB-△GAL80-aro4 K229L -aro7 G141S SfiI single enzyme digestion was performed, and homologous recombination was used to integrate it into the GAL80 site of the BY4741 chromosomal cell line. The GAL80 gene of BY4741 was then knocked out, resulting in the yeast engineered strain YLYJ-002. The genotype of YLYJ-002 is as follows: Figure 1 As shown in Figure A.

[0087] 3. Preparation of engineered yeast strain YLYJ-003

[0088] (1) Following the above method, optimize the Pcaas codon. opt The gene fragment (nucleotide sequence shown in SEQ ID NO.3) was cloned with plasmid pUMRI-A-△HO to obtain the recombinant plasmid pA-△HO-Pcaas. opt .

[0089] (2) The obtained recombinant plasmid pA-△HO-Pcaas opt SfiI single enzyme digestion was performed, and homologous recombination was used to integrate it into the HO site on the chromosome of YLYJ-002 bacteria while simultaneously knocking out the HO gene, resulting in the yeast engineered strain YLYJ-003. The genotype of YLYJ-003 is as follows: Figure 1 As shown in Figure A.

[0090] 4. Preparation of engineered yeast strain YLYJ-004

[0091] (1) Following the above method, optimize the Pcaas codon. opt and Bbxfpk opt Gene fragments (nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4, respectively) were cloned with plasmid pUMRI-A-△HO to obtain recombinant plasmid pA-△HO-Pcaas. opt -Bbxfpk opt .

[0092] (2) The obtained recombinant plasmid pA-△HO-Pcaas opt -Bbxfpk opt SfiI single enzyme digestion was performed, and homologous recombination was used to integrate it into the HO site on the chromosome of YLYJ-002 bacteria while simultaneously knocking out the HO gene, resulting in the yeast engineered strain YLYJ-004. The genotype of YLYJ-004 is as follows: Figure 1 As shown in Figure A.

[0093] 5. Production of Tyrosol

[0094] Wild-type Saccharomyces cerevisiae BY4741, YLYJ-001, YLYJ-002, YLYJ-003 and YLYJ-004 were inoculated into YPSG medium and fermented at 30℃ and 200rpm for 72 hours to obtain fermentation broth.

[0095] The yield of tyrosol in the fermentation broth supernatant was determined using an HPLC-UV system. The results are as follows: Figure 1 As shown in B.

[0096] The results showed that, compared with wild-type fungus BY4741, YLYJ-001 increased the production of tyrosol from 17.60 mg / L to 106.05 mg / L; YLYJ-002 further increased the production of tyrosol to 116.83 mg / L; YLYJ-003 further increased the production of tyrosol to 371.09 mg / L; and finally, YLYJ-004 increased the production of tyrosol to 461.07 mg / L.

[0097] The results showed that simultaneous overexpression of aro4 K229L aro7 G141S Pcaas opt and Bbxfpk opt The recombinant strain YLYJ-004 had the highest tyrosol production, therefore, YLYJ-004 was used to continue the experiment.

[0098] Example 2: Genetically engineered wild-type Escherichia coli BL21(DE3) and its tyrosol conversion rate was tested.

[0099] (1) Using PCR technology, Echpab and Echpac fragments with restriction sites were amplified from the whole genome of wild-type Escherichia coli BL 21(DE3) (GenBank accession numbers: ARI00017.1 and ARI00016.1, respectively). The primers are shown in Table 3.

[0100] Then, the Echpab and Echpac gene fragments containing restriction enzyme sites were double-digested with plasmids PET 30a and PET 22b, respectively. After gel recovery of the double-digested gene fragments and vector fragments, the gene fragments were cloned with a linear vector to obtain recombinant plasmids PET 30a-Echpab and PET 22b-Echpac. The recombinant plasmids PET 30a-Echpab and PET 22b-Echpac were then introduced together into competent E. coli BL 21(DE3) cells using a heat shock method to obtain engineered E. coli BL21(DE3) / PET 30a-Echpab / PET 22b-Echpac, named BL-EcBc.

[0101] (2) Wild-type Escherichia coli BL 21(DE3) and BL-EcBc were inoculated into LB medium and fermented for 24 hours at 37℃ and 200 rpm with exogenous addition of tyrosol to make the final concentration of tyrosol 300 mg / L to prepare fermentation broth.

[0102] The yields of tyrosol and hydroxytyrosol in the fermentation broth supernatant were determined using an HPLC-UV system. The results are as follows: Figure 2 As shown, overexpression of the Echpab and Echpac genes increased the conversion rate of tyrosol to hydroxytyrosol from 0.03% to 86.02%.

[0103] Example 3: Establishment and optimization of a dual-strain co-culture system for efficient de novo synthesis of hydroxytyrosol

[0104] 1. Preparation of Hydroxytyrosol

[0105] The engineered strains YLYJ-004 and BL-EcBc obtained in Example 1 and Example 2 were co-cultured to synthesize hydroxytyrosol de novo, demonstrating the process as follows: Figure 3 As shown in Figure A.

[0106] (1) Preparation of mixed seed liquid

[0107] Preparation of YLYJ-004 seed solution:

[0108] Pick a single colony from an agar plate and inoculate it into 5 mL of YPD liquid medium containing 200 mg / L G418 resistance. Place the medium in a constant temperature shaker at 200 rpm and 30°C and incubate for 15 h.

[0109] Preparation of BL-EcBc seed culture:

[0110] Pick a single colony from an agar plate and inoculate it into 5 mL of LB liquid medium containing 50 μg / mL ampicillin and 50 μg / mL kanamycin resistance. Place the medium in a constant temperature shaker at 37°C and incubate for 15 h.

[0111] Preparation of mixed seed solution:

[0112] A mixed seed solution was prepared by mixing YLYJ-004 seed solution and BL-EcBc seed solution in a 1:1 ratio.

[0113] (2) Preparation of hydroxytyrosol

[0114] Take 1 mL (i.e., mix 0.5 mL of YLYJ-004 seed solution and 0.5 mL of BL-EcBc seed solution separately) of the mixed seed solution prepared in step (1) and inoculate it into 50 mL of different culture media (culture media 1-5). Ferment at 30℃ and 200 rpm for 4 h. Then, add 1 mM isopropyl-β-D-thiogalactoside (IPTG) for induction, and continue fermentation at 27℃ and 200 rpm for 72 h. Take the supernatant to measure the yield of hydroxytyrosol. The results are as follows: Figure 3 As shown in B.

[0115] The results showed that the most suitable culture medium for co-culture of bacteria was medium 2, at which the yield of hydroxytyrosol synthesized de novo by the co-culture system was 254.31 mg / L.

[0116] 2. Optimization of preparation conditions for hydroxytyrosol

[0117] (1) Optimize the bacterial strain ratio in the dual-strain co-culture system.

[0118] YLYJ-004 seed solution and BL-EcBc seed solution were prepared into mixed seed solutions at volume ratios of 10:1, 5:1, 1:1, 1:5, and 1:10, respectively.

[0119] 1 ml of the mixed seed culture was inoculated into 50 mL of medium 2 and fermented at 30 °C and 200 rpm for 4 h. Then, 1 mM IPTG was added for induction, followed by fermentation at 27 °C and 200 rpm for 72 h. The supernatant was used to determine the yield of hydroxytyrosol. The results are as follows: Figure 3 As shown in C.

[0120] The results showed that the optimal initial bacterial ratio for co-culture was YLYJ-004 / BL-Ecbc = 5:1. Under this ratio, the yield of hydroxytyrosol synthesized de novo in the co-culture system was 375.02 mg / L, which was 0.47 times higher than the yield synthesized under the condition of initial bacterial ratio YLYJ-004 / BL-Ecbc = 1:1.

[0121] (2) Optimization of IPTG addition time in the dual-strain co-culture system

[0122] YLYJ-004 seed solution and BL-EcBc seed solution were respectively prepared into mixed seed solutions at a volume ratio of 5:1.

[0123] 1 mL of the mixed seed culture was inoculated into 50 mL of medium 2 and fermented at 30 °C and 200 rpm. After induction with 1 mM IPTG at 2 h, 4 h, 6 h, 8 h, and 10 h of fermentation, fermentation was continued at 27 °C and 200 rpm for 72 h. The yield of hydroxytyrosol was measured from the supernatant. The results are as follows: Figure 3 As shown in D.

[0124] The results showed that the optimal time for IPTG addition in the dual-strain co-culture system was 8 hours after fermentation, at which point the yield of hydroxytyrosol synthesized de novo in the dual-strain co-culture system was 435.32 mg / L.

[0125] (3) Optimize the fermentation time of the dual-strain co-culture system

[0126] YLYJ-004 seed solution and BL-EcBc seed solution were respectively prepared into mixed seed solutions at a volume ratio of 5:1.

[0127] 1 mL of the mixed seed culture was inoculated into 50 mL of medium 2 and fermented at 30 °C and 200 rpm for 8 h. Then, 1 mM IPTG was added for induction, and fermentation was continued at 27 °C and 200 rpm for 24 h, 36 h, 48 h, 72 h, and 96 h. The yield of hydroxytyrosol was measured from the supernatant. The results are as follows: Figure 3 As shown in E.

[0128] The results showed that the optimal fermentation time for co-culture was 72 hours, at which point the yield of hydroxytyrosol synthesized de novo by the co-culture system was 435.32 mg / L.

[0129] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A recombinant brewing yeast, characterized in that, The recombinant Saccharomyces cerevisiae was overexpressed with a mutant of 3-deoxy-D-arabinohepenolate-7-phosphate synthase derived from Saccharomyces cerevisiae. aro4 K229L A branching acid mutase mutant derived from Saccharomyces cerevisiae aro7 G141S Tyrosine decarboxylase derived from parsley pcaas opt and phosphotransketase derived from Bifidobacterium breve Bbxfpk opt Simultaneously, the GAL80 and HO genes on the Saccharomyces cerevisiae genome were knocked out; the 3-deoxy-D-arabinohepantrolonic acid-7-phosphate synthase mutant aro4 K229L The nucleotide sequence is shown in SEQ ID NO.1; the branched acid mutase mutant aro7 G141S The nucleotide sequence is shown in SEQ ID NO.2; the tyrosine decarboxylase pcaas opt The nucleotide sequence is shown in SEQ ID NO.3; the phosphotransketase Bbxfpk opt The nucleotide sequence is shown in SEQ ID NO.

4.

2. The recombinant brewing yeast as described in claim 1, characterized in that, The recombinant brewer's yeast was based on strain BY4741.

3. A method for preparing hydroxytyrosol, characterized in that, The recombinant Saccharomyces cerevisiae and recombinant Escherichia coli, as described in claim 1 or 2, were prepared using a dual-strain fermentation system; the recombinant Escherichia coli expressed 4-hydroxyphenylacetic acid-3-hydroxylase. HpaB and riboflavin oxidoreductase HpaC The 4-hydroxyphenylacetic acid-3-hydroxylase HpaB The riboflavin oxidoreductase described is accessed on the NCBI database with the number ARI00017.

1. HpaC The login number on NCBI is ARI00016.

1.

4. The method as described in claim 3, characterized in that, The recombinant *E. coli* strain used *E. coli* BL21(DE3) as the expression host and expressed 4-hydroxyphenylacetic acid-3-hydroxylase using the PET30a plasmid. HpaB Riboflavin oxidoreductase was expressed using the PET22b plasmid. HpaC .

5. The method as described in claim 4, characterized in that, The substrate in the dual-strain fermentation system is one or more of glucose, sucrose, and glycerol.

6. The method as described in claim 4, characterized in that, Recombinant Saccharomyces cerevisiae seed liquid and recombinant Escherichia coli seed liquid were mixed in a ratio of (1~10):(1~10) to obtain a mixed seed liquid, which was then inoculated into the reaction system for fermentation.

7. The method as described in claim 6, characterized in that, The recombinant Saccharomyces cerevisiae seed liquid and the recombinant Escherichia coli seed liquid were mixed at a volume ratio of 5:1 to obtain a mixed seed liquid; the mixed seed liquid was inoculated into the reaction system for fermentation, and IPTG was added for induction after 2-10 h of fermentation.

8. The method as described in claim 7, characterized in that, After 8 hours of fermentation, IPTG was added for induction. The fermentation time of the dual-strain co-culture system after adding IPTG was 24-96 hours.

9. The method as described in claim 8, characterized in that, The fermentation time of the dual-strain co-culture system after adding the inducer IPTG was 72 h.

Images