A method for synthesizing hydroxytyrosol by multi-enzyme cascade

Abstract

The present invention discloses a method for synthesizing hydroxytyrosol by multi-enzyme cascade, comprising: 1) preparing a hydroxytyrosol product by using 3,4-dihydroxybenzaldehyde, L-threonine, sodium formate, NAD + As raw material, L-threonine transaldolase, alcohol dehydrogenase, and formate dehydrogenase are used to catalyze the synthesis of L-threo-3-(3,4-dihydroxyphenyl)serine; 2) L-threo-3-(3,4-dihydroxyphenyl)serine is used as substrate, glucose, NAD + Hydroxytyrosol is synthesized through a cascade catalysis of phenylserine dehydratase, α-ketoacid decarboxylase, aldehyde reductase, and glucose dehydrogenase. The synthesis method provided by the present invention achieves a hydroxytyrosol yield of >99% and a space-time yield of 0.88 g / L / h, currently the highest, showing great potential for industrial application and suitable for further promotion and application.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalytic synthesis, and particularly relates to a method for synthesizing hydroxytyrosol by multi-enzyme cascade. Background Art

[0002] Hydroxytyrosol, chemically known as 3,4-dihydroxyphenylethanol, is a polyhydroxyphenolic compound with numerous biological activities, including antioxidant, anticancer, and antifungal properties. It is widely used in the food, cosmetics, and pharmaceutical industries. The market value of hydroxytyrosol is expected to reach $2.5 billion by 2030, growing at an annual rate exceeding 20%. Given its enormous market value, its synthesis methods have increasingly attracted the attention of researchers. Currently, industrial production of hydroxytyrosol primarily involves two methods: extraction from olive leaves and chemical synthesis. While both methods achieve efficient synthesis of hydroxytyrosol, they suffer from complex reaction steps, low product yields, and the generation of large amounts of acidic wastewater, violating the principles of sustainable development and green chemistry.

[0003] Biosynthesis of hydroxytyrosol, due to its mild reaction conditions and lack of organic wastewater, has become the most promising synthetic route for hydroxytyrosol. There are two different strategies for selecting starting materials. One is to use simple carbon sources such as glucose or glycerol as raw materials and, through metabolic engineering, introduce metabolic fluxes into the hydroxytyrosol synthesis pathway. While simple carbon sources are cheap and readily available, this strategy requires the use of living cells and involves the regulation of complex metabolic networks within numerous organisms, which can lead to the accumulation of numerous byproducts and a low yield of the desired product. The other is to select substances structurally similar to hydroxytyrosol (tyrosine and L-dopa) as substrates and screen for one or more enzymes that can efficiently catalyze the conversion of these compounds to hydroxytyrosol. This allows the construction of in vivo or in vitro enzymatic pathways. Whole-cell catalysts containing enzymes can be used for hydroxytyrosol biotransformation, offering advantages such as good catalytic activity and a single product, and are increasingly favored by researchers.

[0004] Currently reported enzymatic routes for the synthesis of hydroxytyrosol all use L-dopa and tyrosine as substrates. The high cost of these substrates has limited the large-scale synthesis of hydroxytyrosol using L-dopa as a substrate. Although tyrosine is cheaper and more readily available than L-dopa, the reaction requires the hydroxylation reaction mediated by 4-hydroxyphenylacetic acid-3-monooxygenase (HpaBC) to convert tyrosol to the substrate hydroxytyrosol. HpaBC has low activity and suffers from overoxidation, making it unsuitable for industrial production. While the synthesis of hydroxytyrosol using cheaper substrates is clearly more competitive, to date, no reports have been published on the synthesis of hydroxytyrosol using substrates other than tyrosine and L-dopa. Therefore, developing more substrates and more efficient reaction routes for the synthesis of hydroxytyrosol is of great significance. Summary of the Invention

[0005] In view of the above problems, the object of the present invention is to provide a method for synthesizing hydroxytyrosol by multi-enzyme cascade, using cheap 3,4-dihydroxybenzaldehyde and L-threonine as substrates, and efficiently synthesizing hydroxytyrosol through a one-pot two-step enzymatic process.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] A multi-enzyme cascade method for synthesizing hydroxytyrosol, comprising: 3,4-dihydroxybenzaldehyde, L-threonine, sodium formate, NAD + Glucose is converted into hydroxytyrosol through the cascade catalysis of L-threonine transaldolase, alcohol dehydrogenase, formate dehydrogenase, phenylserine dehydratase, α-ketoacid decarboxylase, aldehyde reductase, and glucose dehydrogenase.

[0008] As a possible embodiment, further, a method for synthesizing hydroxytyrosol by multi-enzyme cascade specifically comprises the following steps:

[0009] 1) 3,4-Dihydroxybenzaldehyde, L-threonine, sodium formate, NAD + L-threo-3-(3,4-dihydroxyphenyl)serine is synthesized by L-threonine transaldolase, alcohol dehydrogenase, and formate dehydrogenase;

[0010] 2) Using L-threo-3-(3,4-dihydroxyphenyl)serine as substrate, glucose and NAD + Hydroxytyrosol is synthesized by catalytic cascade of phenylserine dehydratase, α-ketoacid decarboxylase, aldehyde reductase and glucose dehydrogenase; the catalytic reaction synthesis route is as follows: Figure 1 shown.

[0011] As a preferred embodiment, preferably, the enzyme reaction is carried out in phosphate buffer.

[0012] As a preferred embodiment, preferably, PLP is also added during the enzyme reaction.

[0013] As a possible embodiment, further, the L-threonine transaldolase comprises the amino acid sequence shown in SEQ ID NO: 1 or a polypeptide protein having a homology greater than 80% to the amino acid sequence shown in SEQ ID NO.1; the alcohol dehydrogenase comprises the amino acid sequence shown in SEQ ID NO: 2 or a polypeptide protein having a homology greater than 80% to the amino acid sequence shown in SEQ ID NO.2; and the formate dehydrogenase comprises the amino acid sequence shown in SEQ ID NO: 3 or a polypeptide protein having a homology greater than 80% to the amino acid sequence shown in SEQ ID NO.3.

[0014] The phenylserine dehydratase comprises the amino acid sequence shown in SEQ ID NO: 4 or a polypeptide protein having a homology greater than 80% to the amino acid sequence shown in SEQ ID NO. 4; the α-ketoacid decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 5 or a polypeptide protein having a homology greater than 80% to the amino acid sequence shown in SEQ ID NO. 5; the aldehyde reductase comprises the amino acid sequence shown in SEQ ID NO: 6 or a polypeptide protein having a homology greater than 80% to the amino acid sequence shown in SEQ ID NO. 6; and the glucose dehydrogenase comprises the amino acid sequence shown in SEQ ID NO: 7 or a polypeptide protein having a homology greater than 80% to the amino acid sequence shown in SEQ ID NO. 7.

[0015] As a possible embodiment, further, the L-threonine transaldolase is PsLTTA, and the gene sequence encoding the PsLTTA is shown in SEQ ID NO.8; the alcohol dehydrogenase is ScADH, and the gene sequence encoding the ScADH is shown in SEQ ID NO.9; the formate dehydrogenase is CbFDH, and the gene sequence encoding the CbFDH is shown in SEQ ID NO.10.

[0016] The phenylserine dehydratase is PxPD, and the gene sequence encoding the PxPD is shown in SEQ ID NO.11; the α-ketoacid decarboxylase is ARO10, and the gene sequence encoding the ARO10 is shown in SEQ ID NO.12; the aldehyde reductase is YahK, and the gene sequence encoding the YahK is shown in SEQ ID NO.13; the glucose dehydrogenase is BtGDH, and the gene sequence encoding the BtGDH is shown in SEQ ID NO.14.

[0017] As a possible embodiment, further, the L-threonine aldolase, alcohol dehydrogenase, and formate dehydrogenase are derived from whole cells or cell fragments of recombinant Escherichia coli that co-express L-threonine aldolase, alcohol dehydrogenase, and formate dehydrogenase using genetic engineering methods, or are whole cells or cell fragments of recombinant Escherichia coli that individually express L-threonine aldolase, alcohol dehydrogenase, and formate dehydrogenase.

[0018] The phenylserine dehydratase, α-ketoacid decarboxylase, aldehyde reductase, and glucose dehydrogenase are derived from whole cells or cell fragments of recombinant Escherichia coli that co-express phenylserine dehydratase, α-ketoacid decarboxylase, aldehyde reductase, and glucose dehydrogenase using genetic engineering methods, or are whole cells or cell fragments of recombinant Escherichia coli that individually express phenylserine dehydratase, α-ketoacid decarboxylase, aldehyde reductase, and glucose dehydrogenase.

[0019] Preferably, the L-threonine aldolase, alcohol dehydrogenase, and formate dehydrogenase in the present invention are derived from a recombinant Escherichia coli engineered bacterium BL21 (FM01) module that co-expresses L-threonine aldolase, alcohol dehydrogenase, and formate dehydrogenase using genetic engineering methods; the BL21 (FM01) engineered bacterium overexpresses L-threonine aldolase (PsLTTA), alcohol dehydrogenase (ScADH), and formate dehydrogenase (CbFDH), and the module can use 3,4-dihydroxybenzaldehyde and L-threonine as substrates to synthesize L-threo-3-(3,4-dihydroxyphenyl) serine. In the present invention, phenylserine dehydratase, α-ketoacid decarboxylase, aldehyde reductase, and glucose dehydrogenase are derived from a recombinant Escherichia coli engineered bacterium BL21 (SM13) module that co-expresses phenylserine dehydratase, α-ketoacid decarboxylase, aldehyde reductase, and glucose dehydrogenase by genetic engineering. The BL21 (SM13) engineered bacterium overexpresses phenylserine dehydratase (PxPD), α-ketoacid decarboxylase (ARO10), aldehyde reductase (YahK), and glucose dehydrogenase (BtGDH). The module can synthesize hydroxytyrosol using L-threo-3-(3,4-dihydroxyphenyl)serine as a substrate.

[0020] Specifically, the enzyme solution of the present invention is prepared by constructing recombinant expression vectors pRSFDuet-psltta, pCDFDuet-scadh / cbfdh, pCDFDuet-pxpd / aro10, and pETDuet-btgdh / yahk, respectively. The recombinant plasmids pRSFDuet-psltta and pCDFDuet-scadh / cbfdh are co-transformed into E. coli BL21 (DE3) competent cells to construct an E. coli engineered bacteria BL21 (FM01) module; and the recombinant plasmids pCDFDuet-pxpd / aro10, pETDuet-btgdh / yahk, and the commercial companion vector pACYC-groES / groEL are co-transformed into E. coli BL21 (DE3) competent cells to construct an E. coli engineered bacteria BL21 (SM13) module. BL21(FM01) and BL21(SM13) engineered bacteria were induced to express the corresponding proteins using IPTG, and protein solubility was determined by SDS-PAGE. Whole-cell reaction solutions of BL21(FM01) and BL21(SM13) engineered bacteria were prepared by resuspending the cells in phosphate buffer (100mM, pH 7.0) to a 250mg / mL stock solution. Alternatively, the cell suspensions were further ultrasonically disrupted to prepare crude enzyme solutions.

[0021] The reaction conditions for the multi-enzyme cascade synthesis of hydroxytyrosol are as follows: the reaction buffer is phosphate buffer at pH 7.0, 30 mM 3,4-dihydroxybenzaldehyde, 36 mM L-threonine, 36 mM sodium formate, 0.1 mM pyridoxal phosphate (PLP), NAD + 0.05mM, BL21 (FM01) whole cells or crushed crude enzyme solution 50g / L, react for 2h; continue to add glucose 36mM, NADP + 0.05mM, BL21 (SM13) whole cells or crushed crude enzyme solution 60g / L, continue the reaction at 30℃ for 3h, and detect the content of product and substrate by HPLC.

[0022] The beneficial effects of the present invention are:

[0023] The present invention uses cheap 3,4-dihydroxybenzaldehyde and L-threonine as substrates and utilizes a multi-enzyme cascade method to efficiently synthesize hydroxytyrosol. HPLC detection shows that the yield of hydroxytyrosol is greater than 99%, and the space-time yield reaches 0.88 g / L / h, which is the highest level currently. This fully demonstrates the huge industrial application prospects of the multi-enzyme cascade synthesis route proposed by the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A multi-enzyme cascade synthesis roadmap for hydroxytyrosol.

[0025] Figure 2The SDS-PAGE electrophoresis diagram of BL21 (FM01) and BL21 (SM13) engineered Escherichia coli bacteria; in the figure, A: engineered bacteria BL21 (FM01), B: engineered bacteria BL21 (SM13); P: cell disruption precipitate, S: cell disruption supernatant.

[0026] Figure 3 HPLC detection diagram (A) and mass spectrum (B) of the synthesis of L-threo-3-(3,4-dihydroxyphenyl)serine catalyzed by engineered bacteria BL21 (FM01); in the figure, peak 1: 3,4-dihydroxybenzaldehyde (3.5 min), peak 2: L-threo-3-(3,4-dihydroxyphenyl)serine (2.7 min).

[0027] Figure 4 The HPLC detection diagram (A) and mass spectrum (B) of the synthesis of hydroxytyrosol catalyzed by the engineered bacteria BL21 (SM13); in the figure, peak 2: L-threo-3-(3,4-dihydroxyphenyl)serine (2.7 min), peak 3: 3,4-dihydroxyphenylpyruvate (7.7 min), peak 4: 3.4-dihydroxyphenylacetaldehyde (6.3 min), peak 5: hydroxytyrosol (6.7 min), peak 6: 3,4-dihydroxybenzyl alcohol (5.8 min).

[0028] Figure 5 This is a time curve diagram of the one-pot two-step multi-enzyme cascade synthesis of hydroxytyrosol; in the figure, curve 1: 3,4-dihydroxybenzaldehyde change curve; curve 2: L-threo-3-(3,4-dihydroxyphenyl)serine change curve; curve 3: 3,4-dihydroxyphenylpyruvate change curve; curve 4: 3.4-dihydroxyphenylacetaldehyde change curve; curve 5: hydroxytyrosol synthesis curve. DETAILED DESCRIPTION

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0030] The following describes the main experimental process and main implementation plan of this application in accordance with the research ideas of this application. For the sake of simplicity and space saving, the following examples are only representative experiments after repeated verification by the applicant. Some experiments not listed are general techniques in this technical field. The reagents and experimental instruments used in this application are all existing experimental supplies in the applicant's laboratory, and the specific information is not repeated here.

[0031] Example 1: Fermentation preparation of BL21 (FM01) engineered bacteria whole cell reaction solution

[0032] L-threonine transaldolase (PsLTTA, whose amino acid sequence is shown in SEQ ID NO: 1) is encoded by the gene psltta (base sequence is shown in SEQ ID NO: 8), and a recombinant expression plasmid pRSFDuet-psltta was constructed.

[0033] Alcohol dehydrogenase (ScADH, amino acid sequence shown in SEQ ID NO: 2) and formate dehydrogenase (CbFDH, amino acid sequence shown in SEQ ID NO: 3) are encoded by genes scadh (base sequence shown in SEQ ID NO: 9) and cbadh (base sequence shown in SEQ ID NO: 10), respectively, and recombinant expression plasmid pCDFDuet-scadh / cbfdh was constructed.

[0034] Construction of BL21 (FM01) engineering bacteria: co-transform the recombinant expression plasmids pRSFDuet-psltta and pCDFDuet-scadh / cbfdh into E. coli BL21 (DE3) cells; pick a single clone containing the recombinant plasmid and inoculate it into 20 mL LB liquid medium (containing 50 μg / mL kanamycin and 50 μg / mL streptomycin) and culture it at 37°C and 180 rpm overnight. Pipette 10 mL of culture solution and transfer it to 1 L LB liquid medium (containing 50 μg / mL kanamycin and 50 μg / mL streptomycin) and culture it at 37°C and 200 rpm. When OD 600 When the concentration of IPTG was 0.5, the final concentration of IPTG was 0.1 mM. After induction at 28 ° C for 16 h, the culture was stopped and the cells were harvested by centrifugation at 6000 rpm for 10 min at 4 ° C. The cells were detected by SDS-PAGE (such as Figure 2 (As shown in A), specific bands for PsLTTA, ScADH, and CbFDH appeared near 45 kDa, 42 kDa, and 38 kDa, respectively, indicating successful expression of all three proteins. The cells were resuspended in an appropriate amount of phosphate buffer (100 mM, pH 7.0) to prepare a 250 mg / mL whole-cell suspension.

[0035] Example 2: Fermentation preparation of BL21 (SM13) engineered bacteria whole cell reaction solution

[0036] Phenylserine dehydratase (PxPD, whose amino acid sequence is shown in SEQ ID NO: 4) and α-ketoacid decarboxylase (ARO10, whose amino acid sequence is shown in SEQ ID NO: 5) are encoded by genes pxpd (base sequence shown in SEQ ID NO: 11) and aro10 (base sequence shown in SEQ ID NO: 12), respectively, and recombinant expression plasmid pCDFDuet-pxpd / aro10 was constructed.

[0037] Aldehyde reductase (YahK, whose amino acid sequence is shown in SEQ ID NO: 6) and glucose dehydrogenase (BtGDH, whose amino acid sequence is shown in SEQ ID NO: 7) are encoded by genes yahk (base sequence shown in SEQ ID NO: 13) and btgdh (base sequence shown in SEQ ID NO: 14), respectively, and recombinant expression plasmid pETDuet-btgdh / yahk was constructed.

[0038] The commercial chaperone vector pACYC-groES / groEL was purchased from Takara Biotechnology (Dalian) Co., Ltd.

[0039] Construction of BL21 (SM13) engineering bacteria: The recombinant expression plasmids pCDFDuet-pxpd / aro10, pETDuet-btgdh / yahk and pACYC-groES / groEL were co-transformed into Escherichia coli BL21 (DE3) cells; a single clone containing the recombinant plasmid was picked and inoculated into 20 mL LB liquid medium (containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, 50 μg / mL streptomycin), and cultured at 37°C, 180 rpm overnight. 10 mL of culture solution was transferred to 1 L LB liquid medium (containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, 50 μg / mL streptomycin), and cultured at 37°C, 200 rpm. When OD 600 When the concentration of IPTG was 0.5, the final concentration of IPTG was 0.1 mM. After induction at 28 ° C for 16 h, the culture was stopped and the cells were harvested by centrifugation at 6000 rpm for 10 min at 4 ° C. The cells were detected by SDS-PAGE (such as Figure 2 (As shown in Figure 2B), specific bands for Aro10, the molecular chaperone proteins GroES-GroEL, Yahk, PxPD, and BtGDH appeared near 70 kDa, 65 kDa, 45 kDa, 40 kDa, and 26 kDa, respectively, indicating successful expression of these five proteins. The cells were resuspended in an appropriate amount of phosphate buffer (100 mM, pH 7.0) to prepare a 250 mg / mL whole-cell suspension.

[0040] Example 3: Synthesis of L-threo-3-(3,4-dihydroxyphenyl)serine catalyzed by BL21 (FM01)

[0041] The reaction conditions were as follows: 1 mL of reaction system, reaction temperature 30°C, reaction buffer pH 7.0 phosphate buffer (100 mM), 3,4-dihydroxybenzaldehyde 30 mM, L-threonine 36 mM, sodium formate 36 mM, pyridoxal phosphate (PLP) 0.1 mM, NAD + 0.05mM, BL21 whole cell catalyst or crude enzyme solution 50g / L, reaction for 2h.

[0042] After the reaction, the mixture was centrifuged at 8000 rpm for 5 minutes using a high-speed centrifuge, and the supernatant of the reaction solution was collected and diluted with methanol a certain multiple and then detected by HPLC. The HPLC detection conditions were as follows: detection wavelength: 280 nm; chromatographic column: Agilent C18 column (250×4.6 mm, 5 μm); mobile phase: 5.7 g / L sodium octane sulfonate (1 g / L, dissolved in sodium acetate buffer at pH 3.5): methanol = 5:6; flow rate: 1 mL / min, temperature: 35°C; sample loading: 10 μL. A chromatographic peak appeared in the reaction solution at 3.5 minutes, which was consistent with the retention time of the L-threo-3-(3,4-dihydroxyphenyl)serine standard (such as Figure 3 A), indicating that BL21 (FM01) can catalyze the synthesis of L-threo-3-(3,4-dihydroxyphenyl)serine from 3,4-dihydroxybenzaldehyde and L-threonine. The mass spectrum shows that in the positive ion mode, a signal with an m / z ratio of 214.0707 can be detected (as shown in FIG. Figure 3 B), which is consistent with the theoretical molecular weight of L-threo-3-(3,4-dihydroxyphenyl) (214.0710), further demonstrating that the synthesized product is L-threo-3-(3,4-dihydroxyphenyl)serine.

[0043] Example 4: Synthesis of Hydroxytyrosol Catalyzed by BL21 (SM13)

[0044] The reaction conditions were as follows: 1 mL reaction system, reaction temperature 30°C, reaction buffer pH 7.0 phosphate buffer (100 mM), L-threo-3-(3,4-dihydroxyphenyl)serine 30 mM, glucose 36 mM, NADP + 0.05mM, BL21 whole cell catalyst or crude enzyme solution 60g / L, reaction for 3h.

[0045] After the reaction, the mixture was centrifuged at 8000 rpm for 5 minutes using a high-speed centrifuge, and the supernatant of the reaction solution was collected and diluted with methanol for a certain multiple before being detected by HPLC. The HPLC detection conditions were as follows: detection wavelength: 210 nm; chromatographic column: Agilent C18 column (250×4.6 mm, 5 μm); mobile phase: solution A (100% methanol), solution B (0.1% formic acid aqueous solution); elution conditions: 0-10 min, 10-100% solution A; 10-15 min, 100% solution A; 15-20 min, 100-10% solution A; and 20-25 min, 10% solution A; flow rate: 1 mL / min, temperature: 30°C; sample loading: 10 μL. A chromatographic peak appeared at 6.7 min in the reaction solution, which was consistent with the retention time of the hydroxytyrosol standard (e.g. Figure 4 A), indicating that BL21 (SM13) can catalyze the production of hydroxytyrosol from L-threo-3-(3,4-dihydroxyphenyl)serine. The mass spectrum shows that in negative ion mode, a signal with an m / z ratio of 153.0574 can be detected (as shown in Figure 4 B), which is consistent with the theoretical molecular weight of hydroxytyrosol (153.0557), further demonstrating that the synthesized product is hydroxytyrosol.

[0046] Example 5: One-pot two-step synthesis of hydroxytyrosol

[0047] The reaction conditions for the multi-enzyme cascade synthesis of hydroxytyrosol were as follows: 100 mL of the reaction system, the reaction buffer was a pH 7.0 phosphate buffer, 30 mM 3,4-dihydroxybenzaldehyde, 36 mM L-threonine, 36 mM sodium formate, 0.1 mM pyridoxal phosphate (PLP), NAD + 0.05mM, BL21 (FM01) whole cells 50g / L, reaction for 2h, HPLC detection substrate 3,4-dihydroxybenzaldehyde conversion rate>99%; continue to add glucose 36mM, NADP + 0.05mM, BL21 (SM13) whole cells 60g / L, continue the reaction at 30 ℃ for 3h. The yield of the product hydroxytyrosol was >99% by HPLC detection, and the space-time yield reached 0.88g / L / h, which is the highest level at present (such as Figure 5 shown).

[0048] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for synthesizing hydroxytyrosol by multi-enzyme cascade, characterized in that: The specific steps include: 1) Using 3,4-dihydroxybenzaldehyde and L-threonine as substrates, sodium formate and NAD were added + , L-threo-3-(3,4-dihydroxyphenyl)serine is synthesized by L-threonine transaldolase, alcohol dehydrogenase and formate dehydrogenase; 2) Using L-threo-3-(3,4-dihydroxyphenyl)serine as substrate, adding glucose and NAD + Hydroxytyrosol is synthesized through a cascade of phenylserine dehydratase, α-ketoacid decarboxylase, aldehyde reductase, and glucose dehydrogenase.

2. The method for synthesizing hydroxytyrosol by multi-enzyme cascade according to claim 1, wherein The enzyme reaction was carried out in phosphate buffered saline.

3. The method for synthesizing hydroxytyrosol by multi-enzyme cascade according to claim 1, wherein PLP was also added during the enzyme reaction.

4. The method for synthesizing hydroxytyrosol by multi-enzyme cascade according to claim 1, wherein The L-threonine aldolase, alcohol dehydrogenase, and formate dehydrogenase are derived from whole cells or cell fragments of recombinant Escherichia coli that co-express L-threonine aldolase, alcohol dehydrogenase, and formate dehydrogenase using genetic engineering methods, or are whole cells or cell fragments of recombinant Escherichia coli that individually express L-threonine aldolase, alcohol dehydrogenase, and formate dehydrogenase.

5. The method for synthesizing hydroxytyrosol by multi-enzyme cascade according to claim 1, wherein The phenylserine dehydratase, α-ketoacid decarboxylase, aldehyde reductase, and glucose dehydrogenase are derived from whole cells or cell fragments of recombinant Escherichia coli that co-express phenylserine dehydratase, α-ketoacid decarboxylase, aldehyde reductase, and glucose dehydrogenase using genetic engineering methods, or are whole cells or cell fragments of recombinant Escherichia coli that individually express phenylserine dehydratase, α-ketoacid decarboxylase, aldehyde reductase, and glucose dehydrogenase.

6. The method for synthesizing hydroxytyrosol by multi-enzyme cascade according to claim 1 or 4, wherein: The amino acid sequence of the L-threonine transaldolase is shown in SEQ ID NO: 1; The amino acid sequence of the alcohol dehydrogenase is shown in SEQ ID NO: 2; The amino acid sequence of the formate dehydrogenase is shown in SEQ ID NO:

3.

7. The method for synthesizing hydroxytyrosol by multi-enzyme cascade according to claim 1 or 5, wherein: The amino acid sequence of the phenylserine dehydratase is shown in SEQ ID NO: 4; The amino acid sequence of the α-ketoacid decarboxylase is shown in SEQ ID NO: 5; The amino acid sequence of the aldehyde reductase is shown in SEQ ID NO: 6; The amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO:

7.

8. The method for synthesizing hydroxytyrosol by multi-enzyme cascade according to claim 6, wherein The L-threonine transaldolase is PsLTTA, and the gene sequence encoding the PsLTTA is shown in SEQ ID NO.8; The alcohol dehydrogenase is ScADH, and the gene sequence encoding the ScADH is shown in SEQ ID NO.9; The formate dehydrogenase is CbFDH, and the gene sequence encoding the CbFDH is shown in SEQ ID NO.

10.

9. The method for synthesizing hydroxytyrosol by multi-enzyme cascade according to claim 7, wherein The phenylserine dehydratase is PxPD, and the gene sequence encoding the PxPD is shown in SEQ ID NO.11; The α-ketoacid decarboxylase is ARO10, and the gene sequence encoding the ARO10 is shown in SEQ ID NO.12; The aldehyde reductase is YahK, and the gene sequence encoding the YahK is shown in SEQ ID NO.13; The glucose dehydrogenase is BtGDH, and the gene sequence encoding the BtGDH is shown in SEQ ID NO.14.

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