A method for producing p-hydroxyphenylethanol

Abstract

This invention discloses a method for producing p-hydroxyphenylethanol. The method involves constructing genetically engineered bacteria capable of expressing L-amino acid deaminase, 4-hydroxymandelic acid synthase, glycolaldehyde dehydrogenase, lactaldehyde reductase, propylene glycol dehydratase, and alcohol dehydrogenase, followed by whole-cell transformation to synthesize p-hydroxyphenylethanol. L-tyrosine is widely available, has low toxicity, and is simple and inexpensive to prepare, making it an ideal substrate. The transformation process does not require the addition of expensive coenzymes, and the enzymes selected in this invention possess advantages such as high activity and strong optical specificity. Therefore, using the engineered bacteria of this invention to produce p-hydroxyphenylethanol is highly efficient, environmentally friendly, and low-cost, showing promising prospects for industrial application.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and more specifically, to a method for producing p-hydroxyphenylethanol. Background Art

[0002] p-Hydroxyphenylethanol, also known as tyrosol, is a natural phenolic compound with antioxidant and anti-inflammatory activities. Its derivatives, hydroxytyrosol and rhodioloside, are important antioxidant cardiovascular drugs. In addition, it can also be used as a food additive to enhance flavor.

[0003] The main methods for preparing p-hydroxyphenylethanol include extraction, chemical synthesis, and biotransformation. Extraction primarily uses olives as raw material, extracting p-hydroxyphenylethanol through hydrolysis and decolorization. However, the high cost and low yield of this method limit its industrial application. Chemical synthesis suffers from high raw material costs, cumbersome catalyst preparation, harsh reaction conditions, and low yields, and has been gradually phased out. Biotransformation, with its advantages of high specificity, environmental friendliness, mild reaction conditions, and the elimination of multi-step separation and purification, has attracted widespread attention.

[0004] Currently, scholars both domestically and internationally have reported various biological routes for the preparation of p-hydroxyphenylethanol. One such route uses L-tyrosine as a substrate, involving the conversion of L-amino acid deaminase, pyruvate decarboxylase, and alcohol dehydrogenase. Pyruvate decarboxylase is the rate-limiting enzyme, requiring thiamine pyrophosphate (TPP) as a coenzyme. However, TPP is expensive and has poor stability, limiting its industrial application. Another route uses L-tyrosine as a substrate, involving the conversion of aromatic aldehyde synthase (AAS) and alcohol dehydrogenase, but the final yield of p-hydroxyphenylethanol is only 1.4 g / L.

[0005] The above-reported routes for the preparation of p-hydroxyphenylethanol have drawbacks such as low production efficiency, high cost, and poor coenzyme stability. Therefore, it is of great significance to create a method for the preparation of p-hydroxyphenylethanol that is both efficient and low-cost.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a method for producing p-hydroxyphenylethanol. This method involves constructing genetically engineered bacteria capable of expressing L-amino acid deaminase, 4-hydroxymandelate synthase, glycolaldehyde dehydrogenase, lactaldehyde reductase, propylene glycol dehydratase, and alcohol dehydrogenase, followed by whole-cell transformation to synthesize p-hydroxyphenylethanol. This method is characterized by high production efficiency, environmental friendliness, and low cost, and has good prospects for industrial application. This invention uses L-tyrosine as a substrate to assemble a novel, efficient synthetic pathway that does not require TPP participation. Furthermore, the inventors screened the enzymes involved in the synthesis, increasing the yield of p-hydroxyphenylethanol, and optimized the whole-cell transformation system, thus obtaining a highly efficient whole-cell transformation system for the synthesis of p-hydroxyphenylethanol using L-tyrosine as a substrate.

[0008] Specifically, the present invention is implemented as follows:

[0009] In a first aspect, the present invention provides a catalytic system for synthesizing p-hydroxyphenylethanol from an L-tyrosine substrate, comprising L-amino acid deaminase (LAAD), 4-hydroxymandelate synthase (HmaS), glycolaldehyde dehydrogenase (GLDH), lactaldehyde reductase (LDR), propylene glycol dehydratase (PDH), and alcohol dehydrogenase (ADH).

[0010] In this invention, the synthetic route for synthesizing p-hydroxyphenylethanol using L-tyrosine substrates is as follows: Figure 1 As shown, L-tyrosine is converted to p-hydroxyphenylpyruvic acid via LAAD, p-hydroxyphenylpyruvic acid is converted to L-p-hydroxymandelic acid via HmaS, L-p-hydroxymandelic acid is converted to 2-hydroxy-1-(4-hydroxyphenyl)ethyl ketone via GLDH, 2-hydroxy-1-(4-hydroxyphenyl)ethyl ketone is converted to 1-(4-hydroxyphenyl)ethane-1,2-diol via LDR, 1-(4-hydroxyphenyl)ethane-1,2-diol is converted to p-hydroxyphenylacetaldehyde via PDH, and p-hydroxyphenylethanol is generated under the action of ADH.

[0011] Based on the above-mentioned catalytic system, this invention has verified the effects of LAAD, HmaS, GLDH, LDR, PDH and ADH from different sources. The experiment found that the catalytic effects of enzymes from different sources are different when applied to the catalytic system of this invention. When the different catalytic systems obtained by the combination of six enzymes from different sources are used to produce p-hydroxyphenylethanol, the catalytic effects of each catalytic system are significantly different.

[0012] In some embodiments, the LAAD includes PvLAAD and PmLAAD. PvLAAD is derived from Proteus vulgaris, Genbank number BAA90864.1, and its nucleotide sequence is shown in SEQ ID NO.13, and its amino acid sequence is shown in SEQ ID NO.1. PmLAAD is derived from Proteus mirabilis, Genbank number AVK70349.1, and its nucleotide sequence is shown in SEQ ID NO.14, and its amino acid sequence is shown in SEQ ID NO.2.

[0013] In some embodiments, HmaS includes SaHmaS and StHmaS. SaHmaS is derived from Salinisporaarenicola, Genbank number TQL39149.1, and its nucleotide sequence is shown in SEQ ID NO.15, and its amino acid sequence is shown in SEQ ID NO.3. StHmaS is derived from Streptomyces turgidiscabies, Genbank number GAQ70618.1, and its nucleotide sequence is shown in SEQ ID NO.16, and its amino acid sequence is shown in SEQ ID NO.4.

[0014] In some embodiments, GLDH includes GsGLDH and KpGLDH. GsGLDH is derived from Geobacillus stearothermophilus, Genbank number ATA61514.1, and its nucleotide sequence is shown in SEQ ID NO.17, and its amino acid sequence is shown in SEQ ID NO.5. KpGLDH is derived from Klebsiella pneumoniae, Genbank number BAS35664.1, and its nucleotide sequence is shown in SEQ ID NO.18, and its amino acid sequence is shown in SEQ ID NO.6.

[0015] In some embodiments, the LDR includes AhLDR and BfLDR. AhLDR is derived from Aeromonas hydrophila, Genbank number CAD7522524.1, and its nucleotide sequence is shown in SEQ ID NO.19, and its amino acid sequence is shown in SEQ ID NO.7. BfLDR is derived from Bacteroides fragilis, Genbank number KXU39663.1, and its nucleotide sequence is shown in SEQ ID NO.20, and its amino acid sequence is shown in SEQ ID NO.8.

[0016] In some embodiments, the PDH includes SePDH and CpPDH. SePDH is derived from Salmonella enterica, Genbank number AVB05231.1, and its nucleotide sequence is shown in SEQ ID NO.21, and its amino acid sequence is shown in SEQ ID NO.9. CpPDH is derived from Citrobacter portucalensis, Genbank number OIY07269.1, and its nucleotide sequence is shown in SEQ ID NO.22, and its amino acid sequence is shown in SEQ ID NO.10.

[0017] In some embodiments, the ADH includes TtADH and AbADH. TtADH is derived from *Thermanaeromonastoyohensis*, Genbank number SMB99032.1, and its nucleotide sequence is shown in SEQ ID NO.23, and its amino acid sequence is shown in SEQ ID NO.11. PmLAAD is derived from *Actinobacteria bacterium*, Genbank number KPI21124.1, and its nucleotide sequence is shown in SEQ ID NO.24, and its amino acid sequence is shown in SEQ ID NO.12.

[0018] Secondly, the present invention provides a genetically engineered bacterium capable of expressing the aforementioned LAAD, HmaS, GLDH, LDR, PDH and ADH.

[0019] In this invention, after obtaining the amino acid sequence of the above-mentioned enzyme, the inventors optimized the codons based on the E. coli preference and synthesized two optimized nucleotide sequences by total synthesis.

[0020] In some embodiments, the method for constructing the recombinant bacteria includes: ligating the genes of LAAD, HmaS, GLDH, LDR, PDH and ADH into a dual promoter expression vector, and then introducing the obtained recombinant expression vector into a host bacterium to obtain a genetically engineered bacterium.

[0021] In some embodiments, the host of the genetically engineered bacteria is *Escherichia coli*. In other embodiments, the host of the genetically engineered bacteria can be other bacteria, and the present invention does not specifically limit them.

[0022] In some embodiments, the *Escherichia coli* is selected from any one of *Escherichia coli* BL21, *Escherichia coli* DH5α, and *Escherichia coli* XL-Blue.

[0023] In some embodiments, the dual promoter expression vector includes pETDuet-1, pCDFDuet-1 plasmid, and pACYCDuet-1 plasmid.

[0024] In this invention, one enzyme is selected from LAAD, HmaS, GLDH, LDR, PDH and ADH to perform co-expression of the six enzymes. As for the method of introducing plasmids carrying the coding genes of the above enzymes, it can be that any two coding genes exist on the same plasmid, or that the six genes exist on different plasmids, or other introduction methods. This invention does not limit this.

[0025] Preferably, the pETDuet-1, pCDFDuet-1 and pACYCDuet-1 plasmids are used to co-express the encoding genes of 6 enzymes.

[0026] More preferably, pETDuet-1 is loaded with LAAD and HmaS, pCDFDuet-1 is loaded with GLDH and LDR, and pACYCDuet-1 is loaded with PDH and ADH.

[0027] Thirdly, the present invention provides the application of the above-mentioned catalytic system or genetically engineered bacteria in the synthesis of p-hydroxyphenylethanol and its downstream products.

[0028] Fourthly, the present invention also provides a method for synthesizing p-hydroxyphenylethanol, which includes adding the above-mentioned catalytic system or genetically engineered bacteria to a solution containing L-tyrosine for catalysis to obtain p-hydroxyphenylethanol.

[0029] Specifically, the synthesis method of p-hydroxyphenylethanol is as follows: Figure 1 As shown: L-tyrosine is converted to p-hydroxyphenylpyruvate via LAAD, p-hydroxyphenylpyruvate is converted to L-p-hydroxymandelic acid via HmaS, L-p-hydroxymandelic acid is converted to 2-hydroxy-1-(4-hydroxyphenyl)ethyl ketone via GLDH, 2-hydroxy-1-(4-hydroxyphenyl)ethyl ketone is converted to 1-(4-hydroxyphenyl)ethane-1,2-diol via LDR, 1-(4-hydroxyphenyl)ethane-1,2-diol is converted to p-hydroxyphenylacetaldehyde via PDH, and p-hydroxyphenylethanol is generated under the action of ADH. The coenzymes required in the reaction process are provided by glucose metabolized by the bacteria. In addition, there is a complementary relationship between the corresponding coenzymes of GLDH, LDR and ADH, which can realize the cyclic regeneration of coenzymes.

[0030] In some embodiments, the genetically engineered bacteria synthesize p-hydroxyphenylethanol through whole-cell transformation. Before whole-cell transformation, the genetically engineered bacteria undergo induction culture. This induction culture process involves inoculating the genetically engineered bacteria into LB medium containing 30-60 mg / L kanamycin, 30-60 mg / L chloramphenicol, and 30-60 mg / L streptomycin. After cultivation, a seed culture is obtained, which is then inoculated into fresh LB medium until the bacterial concentration reaches OD0.05. 600nm When the concentration reaches 0.6-0.8, add an inducer, and after induction, separate and wash to obtain wet bacterial cells.

[0031] In some embodiments, the culture conditions for the above-mentioned genetically engineered bacteria are: temperature of 32-38℃, rotation speed of 150-250rpm, and culture time of 10-16h.

[0032] In some embodiments, the culture conditions for the above seed solution are: temperature of 32-38℃ and rotation speed of 150-250 rpm.

[0033] In some embodiments, the induction conditions are: 0.2-0.6 mM IPTG inducer, temperature of 25-30°C, and time of 10-16 h.

[0034] In some embodiments, the whole-cell transformation production system includes: L-tyrosine concentration of 1-80 g / L, glucose concentration of 10-100 g / L, cobalt chloride concentration of 0.02-0.1 g / L, and the amount of genetically engineered bacteria of 1-20 g / L.

[0035] In some embodiments, the production system for whole-cell transformation has a pH of 6.0-9.0, a temperature of 15-40°C, and a reaction time of 6-24 hours.

[0036] The present invention has the following beneficial effects:

[0037] This invention provides a novel method for producing p-hydroxyphenylethanol using an engineered strain of *Escherichia coli* with L-tyrosine as a substrate. L-tyrosine is widely available, has low toxicity, and its preparation process is simple and inexpensive, making it an ideal substrate. The conversion process does not require the addition of expensive coenzymes. The enzyme selected in this invention has advantages such as high activity and strong optical specificity. Therefore, the production of p-hydroxyphenylethanol using the engineered strain of this invention is highly efficient, environmentally friendly, and low-cost, showing promising prospects for industrial application. Attached Figure Description

[0038] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 This is the synthetic route for p-hydroxyphenylethanol of the present invention. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. The features and performance of the present invention will be further described in detail below with reference to the embodiments.

[0041] 1. Selection of bacteria and plasmids

[0042] The following plasmids were purchased from Novagen: pETDuet-1, pCDFDuet-1, pACYCDuet-1, Escherichia coli BL21, Escherichia coli DH5α, and Escherichia coli XL-Blue.

[0043] 2. Enzyme selection

[0044] (1) Selection of L-amino acid deaminase

[0045] The amino acid sequences of L-amino acid deaminases PvLAAD and PmLAAD were obtained from the NCBI database. Codon optimization was performed based on E. coli preferences, and two nucleotide sequences were synthesized using standard genetic engineering methods, as shown in SEQ ID NO.13 and SEQ ID NO.14, respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. EcoRI and HindIII restriction enzyme sites were added to both ends of the nucleotide sequences.

[0046] (2) Selection of 4-hydroxymandelic acid synthase

[0047] The amino acid sequences of 4-hydroxymandelic acid synthases SaHmaS and StHmaS were obtained from the NCBI database. Codon optimization was performed based on E. coli preferences, and two nucleotide sequences were synthesized using standard genetic engineering methods, as shown in SEQ ID NO.15 and SEQ ID NO.16, respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. Enzyme cleavage sites NdeI and XhoI were added to both ends of the nucleotide sequences.

[0048] (3) Selection of alcohol aldehyde dehydrogenase

[0049] The amino acid sequences of glycolaldehyde dehydrogenases GsGLDH and KpGLDH were obtained from the NCBI database. Codon optimization was performed based on E. coli preferences, and two nucleotide sequences were synthesized using standard genetic engineering methods, as shown in SEQ ID NO.17 and SEQ ID NO.18, respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively. EcoRI and HindIII restriction enzyme sites were added to both ends of the nucleotide sequences.

[0050] (4) Selection of lactalaldehyde reductase

[0051] The amino acid sequences of lactaldehyde reductases AhLDR and BfLDR were obtained from the NCBI database. Codon optimization was performed based on E. coli preferences, and a nucleotide sequence was synthesized using standard genetic engineering methods, as shown in SEQ ID NO. 19 and SEQ ID NO. 20. The amino acid sequence encoding the enzyme is shown in SEQ ID NO. 7 and SEQ ID NO. 8. NdeI and XhoI restriction enzyme sites were added to both ends of the nucleotide sequence.

[0052] (5) Selection of propylene glycol dehydrating enzyme

[0053] The amino acid sequences of propylene glycol dehydratases SePDH and CpPDH were obtained from the NCBI database. Codon optimization was performed based on E. coli preferences, and two nucleotide sequences were synthesized using standard genetic engineering methods, as shown in SEQ ID NO. 21 and SEQ ID NO. 22, respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NO. 9 and SEQ ID NO. 10, respectively. EcoRI and HindIII restriction sites were added to both ends of the nucleotide sequences.

[0054] (6) Selection of alcohol dehydrogenase

[0055] The amino acid sequences of alcohol dehydrogenases TtADH and AbADH were obtained from the NCBI database. Codon optimization was performed based on E. coli preferences, and a nucleotide sequence was synthesized using standard genetic engineering methods, as shown in SEQ ID NO. 21 and SEQ ID NO. 22. The amino acid sequence encoding the enzyme is shown in SEQ ID NO. 11 and SEQ ID NO. 12. Enzyme cleavage sites NdeI and XhoI were added to both ends of the nucleotide sequence.

[0056] 3. Construction of a six-enzyme co-expression system and cell culture

[0057] One enzyme from each of the selected L-amino acid deaminase, 4-hydroxymandelic acid synthase, glycolaldehyde dehydrogenase, lactaldehyde reductase, propylene glycol dehydratase, and alcohol dehydrogenase was chosen for co-expression of six enzymes. The encoding genes of all six enzymes were co-expressed using three plasmids: pETDuet-1, pCDFDuet-1, and pACYCDuet-1. pETDuet-1 contained L-amino acid deaminase and 4-hydroxymandelic acid synthase; pCDFDuet-1 contained glycolaldehyde dehydrogenase and lactaldehyde reductase; and pACYCDuet-1 contained propylene glycol dehydratase and alcohol dehydrogenase. After obtaining the co-expression recombinant plasmids, the three recombinant plasmids were simultaneously transformed into Escherichia coli BL21 competent cells. Positive transformants were obtained by screening with plates containing ampicillin, streptomycin, and chloramphenicol, thus obtaining recombinant E. coli. The obtained recombinant bacteria were inoculated into fresh liquid culture medium, induced, centrifuged, and the wet bacterial cells were obtained.

[0058] 4. Whole-cell conversion of L-tyrosine to prepare p-hydroxyphenylethanol

[0059] Transformation system: L-tyrosine concentration 1-80 g / L, glucose concentration 10-100 g / L, pH adjusted between 6.0 and 9.0, fresh cell mass 1-20 g / L, then transformed at 15-40℃ and 200 rpm for 6-24 h. After transformation, the yield of p-hydroxyphenylethanol was determined by liquid chromatography.

[0060] 5. Sample detection and analysis

[0061] The conversion solution was analyzed using a Shimadzu 2030C high-performance liquid chromatograph (HPLC). The chromatographic conditions were as follows: mobile phase: methanol:water (v / v = 1:1), Inertsustain C18 column (4.6 × 250 mm, 5 μm), flow rate: 1 mL / min, column temperature: 30 °C, injection volume: 20 μL, and detection wavelength: 275 nm.

[0062] Example 1

[0063] This example describes the construction of engineered Escherichia coli, as detailed below:

[0064] The fully synthesized LAAD recombinant plasmid and pETDuet-1 vector were double-digested with restriction endonucleases EcoRI and HindIII, respectively. The fully synthesized HmaS recombinant plasmid and pETDuet-1 vector were double-digested with restriction endonucleases NdeI and XhoI, respectively. LAAD and HmaS from different sources were then ligated into the pETDuet-1 vector in pairs using T4 DNA ligase to obtain recombinant plasmid 1. The fully synthesized GLDH recombinant plasmid and pCDFDuet-1 vector were double-digested with restriction endonucleases EcoRI and HindIII, respectively. The fully synthesized LDR recombinant plasmid and pCDFDuet-1 vector were double-digested with restriction endonucleases NdeI and XhoI, respectively. The t-1 vector was double-digested, and GLDH and LDR from different sources were ligated into the pCDFDuet-1 vector in pairs using T4 DNA ligase to obtain recombinant plasmid 2. The fully synthesized PDH recombinant plasmid and pACYCDuet-1 vector were double-digested with restriction endonucleases EcoRI and HindIII, and the fully synthesized ADH recombinant plasmid and pACYCDuet-1 vector were double-digested with restriction endonucleases NdeI and XhoI. The PDH and ADH from different sources were ligated into the pACYCDuet-1 vector in pairs using T4 DNA ligase to obtain recombinant plasmid 3. The different recombinant plasmids 1, 2, and 3 were combined and transformed into E. coli BL21(DE3) competent cells to obtain recombinant Escherichia coli.

[0065] Example 2

[0066] This example describes the induction culture of engineered Escherichia coli, as detailed below:

[0067] Recombinant *E. coli* was inoculated into LB medium containing 50 mg / L ampicillin, 50 mg / L streptomycin, and 50 mg / L chloramphenicol, and cultured at 37°C and 200 rpm for 12 h to obtain a seed culture. The seed culture was then inoculated into fresh LB medium at a 2% inoculation rate and cultured at 37°C and 200 rpm until the bacterial concentration reached OD500. 600nm When the concentration reaches 0.7, add 0.5 mM IPTG, induce at 28℃ for 15 h, centrifuge at 8000 rpm for 10 min, discard the supernatant, wash the wet bacterial cells twice with 0.9% physiological saline, centrifuge, and set aside.

[0068] Example 3

[0069] This example compares the transformation capabilities of various recombinant Escherichia coli strains:

[0070] The collected recombinant E. coli were resuspended in a 50 mL system with a final cell concentration of 20 g / L, L-tyrosine concentration of 80 g / L, glucose concentration of 100 g / L, and pH of 8.0. The reaction was carried out at 30 °C with a shaking speed of 200 rpm for 24 h. After transformation, the yield of p-hydroxyphenylethanol was determined by HPLC.

[0071] Table 1 Comparison of the yield of various recombinant bacteria against hydroxyphenylethanol

[0072]

[0073] Example 4

[0074] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 1 g / L, L-tyrosine 1 g / L, glucose 10 g / L, cobalt chloride 0.02 g / L, pH 8.0, temperature 30 °C, shaking speed 200 rpm, and transformation time 24 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 0.74 g / L.

[0075] Example 5

[0076] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 3 g / L, L-tyrosine 10 g / L, glucose 20 g / L, cobalt chloride 0.02 g / L, pH 8.0, temperature 35℃, shaker speed 200 rpm, and transformation time 24 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 7.5 g / L.

[0077] Example 6

[0078] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 5 g / L, L-tyrosine 18 g / L, glucose 30 g / L, cobalt chloride 0.04 g / L, pH 8.0, temperature 30 °C, shaking speed 200 rpm, and transformation time 24 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 13.3 g / L.

[0079] Example 7

[0080] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 8 g / L, L-tyrosine 30 g / L, glucose 40 g / L, cobalt chloride 0.04 g / L, pH 8.0, temperature 30 °C, shaking speed 200 rpm, and transformation time 24 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 22.1 g / L.

[0081] Example 8

[0082] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 10 g / L, L-tyrosine 40 g / L, glucose 50 g / L, cobalt chloride 0.05 g / L, pH 8.0, temperature 30 °C, shaker speed 200 rpm, and transformation time 24 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 30.2 g / L.

[0083] Example 9

[0084] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 15 g / L, L-tyrosine 60 g / L, glucose 80 g / L, cobalt chloride 0.08 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, and transformation time 24 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 44.7 g / L.

[0085] Example 10

[0086] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 18 g / L, L-tyrosine 70 g / L, glucose 100 g / L, cobalt chloride 0.1 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, and transformation time 24 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 52.5 g / L.

[0087] Example 11

[0088] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 20 g / L, L-tyrosine 45 g / L, glucose 60 g / L, cobalt chloride 0.1 g / L, pH 8.0, temperature 30 °C, shaking speed 200 rpm, and transformation time 12 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 33.9 g / L.

[0089] Example 12

[0090] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 20 g / L, L-tyrosine 20 g / L, glucose 30 g / L, cobalt chloride 0.1 g / L, pH 8.0, temperature 30 °C, shaking speed 200 rpm, and transformation time 6 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 15.1 g / L.

[0091] Example 13

[0092] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 15 g / L, L-tyrosine 17 g / L, glucose 30 g / L, cobalt chloride 0.05 g / L, pH 8.0, temperature 30 °C, shaking speed 200 rpm, and transformation time 8 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 12.8 g / L.

[0093] Example 14

[0094] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 12 g / L, L-tyrosine 55 g / L, glucose 70 g / L, cobalt chloride 0.05 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, and transformation time 15 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 40.4 g / L.

[0095] Example 15

[0096] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 10 g / L, L-tyrosine 22 g / L, glucose 30 g / L, cobalt chloride 0.05 g / L, pH 6.0, temperature 35℃, shaker speed 200 rpm, and transformation time 12 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 16.3 g / L.

[0097] Example 16

[0098] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 15 g / L, L-tyrosine 28 g / L, glucose 40 g / L, cobalt chloride 0.08 g / L, pH 7.0, temperature 35℃, shaker speed 200 rpm, and transformation time 12 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 20.6 g / L.

[0099] Example 17

[0100] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 13 g / L, L-tyrosine 25 g / L, glucose 40 g / L, cobalt chloride 0.08 g / L, pH 7.5, temperature 35℃, shaker speed 200 rpm, and transformation time 12 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 18.5 g / L.

[0101] Example 18

[0102] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 7 g / L, L-tyrosine 15 g / L, glucose 30 g / L, cobalt chloride 0.05 g / L, pH 8.5, temperature 35℃, shaker speed 200 rpm, and transformation time 12 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 11.2 g / L.

[0103] Example 19

[0104] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 4 g / L, L-tyrosine 9 g / L, glucose 20 g / L, cobalt chloride 0.05 g / L, pH 9.0, temperature 35℃, shaker speed 200 rpm, and transformation time 12 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 6.7 g / L.

[0105] Example 20

[0106] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 16 g / L, L-tyrosine 65 g / L, glucose 80 g / L, cobalt chloride 0.1 g / L, pH 7.5, temperature 15℃, shaker speed 200 rpm, and transformation time 24 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 48.9 g / L.

[0107] Example 21

[0108] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 10 g / L, L-tyrosine 38 g / L, glucose 50 g / L, cobalt chloride 0.08 g / L, pH 7.5, temperature 25℃, shaker speed 200 rpm, and transformation time 24 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 28.4 g / L.

[0109] Example 22

[0110] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 11 g / L, L-tyrosine 42 g / L, glucose 55 g / L, cobalt chloride 0.08 g / L, pH 7.5, temperature 40℃, shaking speed 200 rpm, and transformation time 24 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 31.2 g / L.

[0111] Comparative Example 1

[0112] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 30 g / L, L-tyrosine 100 g / L, glucose 120 g / L, cobalt chloride 0.1 g / L, pH 7.5, temperature 35℃, shaker speed 200 rpm, and transformation time 36 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 11.8 g / L.

[0113] Comparative Example 2

[0114] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 25 g / L, L-tyrosine 70 g / L, glucose 100 g / L, cobalt chloride 0.12 g / L, pH 5.5, temperature 35℃, shaking speed 200 rpm, and transformation time 36 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 9.4 g / L.

[0115] Comparative Example 3

[0116] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 15 g / L, L-tyrosine 55 g / L, glucose 80 g / L, cobalt chloride 0.1 g / L, pH 9.5, temperature 35℃, shaker speed 200 rpm, and transformation time 36 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 7.8 g / L.

[0117] Comparative Example 4

[0118] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 20 g / L, L-tyrosine 75 g / L, glucose 100 g / L, cobalt chloride 0.1 g / L, pH 7.5, temperature 10℃, shaker speed 200 rpm, and transformation time 36 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 11.3 g / L.

[0119] Comparative Example 5

[0120] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 30 g / L, L-tyrosine 60 g / L, glucose 90 g / L, cobalt chloride 0.1 g / L, pH 8.0, temperature 45℃, shaker speed 200 rpm, and transformation time 36 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 5.5 g / L.

[0121] Comparative Example 6

[0122] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pETDuet-PmLAAD-SaHmaS+pCDFDuet-KpGLDH-AhLDR+pACYCDuet-CpPDH-TtADH cells were collected after induction expression. The cells were then placed in a 50 mL system with the following parameters: cell wet weight 0.5 g / L, L-tyrosine 0.5 g / L, glucose 5 g / L, cobalt chloride 0.01 g / L, pH 8.0, temperature 30 °C, shaker speed 200 rpm, and transformation time 36 h. HPLC analysis showed a p-hydroxyphenylethanol yield of 0.05 g / L.

[0123] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A genetically engineered bacterium for synthesizing p-hydroxyphenylethanol, characterized in that, The genetically engineered bacteria express L-amino acid deaminase, 4-hydroxymandelic acid synthase, glycolaldehyde dehydrogenase, lactaldehyde reductase, propylene glycol dehydratase, and alcohol dehydrogenase. The L-amino acid deaminase is PvLAAD or PmLAAD, the amino acid sequence of PvLAAD is shown in SEQ ID NO. 1, and the amino acid sequence of PmLAAD is shown in SEQ ID NO. 2; The 4-hydroxymandelic acid synthase is SaHmaS or StHmaS, the amino acid sequence of SaHmaS is shown in SEQ ID NO. 3, and the amino acid sequence of StHmaS is shown in SEQ ID NO. 4; The glycolaldehyde dehydrogenase is GsGLDH or KpGLDH, the amino acid sequence of GsGLDH is shown in SEQ ID NO. 5, and the amino acid sequence of KpGLDH is shown in SEQ ID NO.

6. The lactaldehyde reductase is AhLDR or BfLDR, the amino acid sequence of AhLDR is shown in SEQ ID NO.7, and the amino acid sequence of BfLDR is shown in SEQ ID NO.8; The propylene glycol dehydratase is SePDH or CpPDH, the amino acid sequence of SePDH is shown in SEQ ID NO. 9, and the amino acid sequence of CpPDH is shown in SEQ ID NO. 10; The alcohol dehydrogenase is TtADH or AbADH, the amino acid sequence of TtADH is shown in SEQ ID NO. 11, and the amino acid sequence of AbADH is shown in SEQ ID NO. 12; The host bacterium of the genetically engineered bacteria is Escherichia coli.

2. The genetically engineered bacterium according to claim 1, characterized in that, The host bacteria of the genetically engineered bacteria are Escherichia coli BL21, Escherichia coli DH5α, or Escherichia coli XL-Blue.

3. The genetically engineered bacteria according to claim 2, characterized in that, The method for constructing the genetically engineered bacteria includes: ligating the genes of L-amino acid deaminase, 4-hydroxymandelic acid synthase, glycolaldehyde dehydrogenase, lactaldehyde reductase, propylene glycol dehydratase, and alcohol dehydrogenase into an expression vector, and then introducing the obtained recombinant expression vector into a host bacterium to obtain the genetically engineered bacteria.

4. The use of the genetically engineered bacteria as described in any one of claims 1-3 in the synthesis of p-hydroxyphenylethanol.

5. A method for synthesizing p-hydroxyphenylethanol, characterized in that, It includes adding the genetically engineered bacteria according to any one of claims 1-3 to a solution containing L-tyrosine for a catalytic reaction to obtain p-hydroxyphenylethanol.

6. The method according to claim 5, characterized in that, The genetically engineered bacteria synthesize p-hydroxyphenylethanol through whole-cell transformation. The whole-cell transformation production system includes: L-tyrosine concentration of 1-80 g / L, glucose concentration of 10-100 g / L, cobalt chloride concentration of 0.02-0.1 g / L, and the bacterial cell mass of the genetically engineered bacteria of 1-20 g / L.

7. The method according to claim 6, characterized in that, The production system for whole-cell transformation has a pH of 6.0-9.0, a temperature of 15-40℃, and a reaction time of 6-24h.

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