Production method of p-hydroxyacetophenone

Abstract

The invention provides a production method of p-hydroxyacetophenone, which comprises the following steps of: co-expressing phenylalanine amino mutase, L-aspartic acid-beta-decarboxylase, monoamine oxidase and catalase in escherichia coli, converting L-tyrosine into (R)-3-amino-3-(4-hydroxyphenyl) propionic acid through TAM (Transcriptional Amplification Molecule), and converting the (R)-3-amino-3-(4-hydroxyphenyl) propionic acid into (R)-3-amino-3-(4-hydroxyphenyl) propionic acid. According to the method, (R)-3-amino-3-(4-hydroxyphenyl) propionic acid is converted into (R)-4-(1-aminoethyl) phenol through ADC, (R)-4-(1-aminoethyl) phenol is converted into p-hydroxyacetophenone through AOD, and generated hydrogen peroxide is removed through CAT. According to the method, the reaction process is simple, no coenzyme needs to be added, the selected enzyme has the advantages of being high in activity, high in optical specificity and the like, and the p-hydroxyacetophenone is produced through conversion of the recombinant bacterium, so that the method is high in production efficiency, environmentally friendly, low in cost and good in industrial application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, relates to p-hydroxyacetophenone, and in particular to a production method of p-hydroxyacetophenone. Background Art

[0002] Parahydroxyacetophenone, also known as 4-acetylphenol, is a natural compound found in plants such as spruce, ginseng and Artemisia annua. It has antioxidant, antibacterial and preservative activities and is widely used in the pharmaceutical, chemical and cosmetic industries.

[0003] The main methods for preparing p-hydroxyacetophenone include plant extraction, chemical synthesis, and biotransformation. However, the low content of p-hydroxyacetophenone in plants and the high extraction cost limit its large-scale industrial application.

[0004] Chemical synthesis methods suffer from harsh reaction conditions, high separation and purification costs, and low safety, making them unsuitable for large-scale industrial production. Biotransformation methods have attracted widespread attention due to their advantages, such as high specificity, environmental friendliness, mild reaction conditions, and the absence of multiple separation and purification steps.

[0005] At present, scholars at home and abroad have reported a variety of biological preparation routes for p-hydroxyacetophenone.

[0006] For example: Chinese patent publication number CN115491368A, publication date December 20, 2022, is named a method for biosynthesis of p-hydroxyacetophenone and picolin. This patent uses Escherichia coli as a chassis strain to modify the synthesis pathway of p-hydroxyacetophenone. The obtained engineered strain uses glucose to ferment in a shake flask for more than 72 hours to obtain 710 mg / L of p-hydroxyacetophenone.

[0007] Chinese patent publication number CN118186027A, publication date June 14, 2024, is titled "A method for synthesizing p-hydroxyacetophenone". This patent prepares p-hydroxyacetophenone by a bioenzymatic method coupled with a chemical method. L-tyrosine is used as a substrate, and 4-hydroxystyrene is converted by tyrosine deaminase and phenolic acid decarboxylase, which is then catalyzed by a chemical method to obtain p-hydroxyacetophenone.

[0008] The above-reported biosynthetic routes of p-hydroxyacetophenone have the disadvantages of low yield, long reaction cycle, and complex reaction process.

[0009] In view of this, the present invention is proposed. Summary of the Invention

[0010] Based on the defects of various current methods, the present invention provides a method for producing p-hydroxyacetophenone.

[0011] In a first aspect, the present invention provides a recombinant Escherichia coli that co-expresses L-phenylalanine aminomutase, L-aspartate-β-decarboxylase, monoamine oxidase, and catalase.

[0012] The second aspect of the present invention provides a method for constructing the above-mentioned recombinant Escherichia coli.

[0013] The third aspect of the present invention provides a use of the recombinant E. coli, which is used to convert the inexpensive substrate L-tyrosine into p-hydroxyacetophenone.

[0014] Finally, the invention provides a novel production method of p-hydroxyacetophenone.

[0015] The method for producing p-hydroxyacetophenone of the present invention has a simple reaction process and does not require the consideration of coenzyme addition. The enzyme selected in the present invention has advantages such as high activity and strong optical specificity. Therefore, the production of p-hydroxyacetophenone using the recombinant bacteria of the present invention has high production efficiency, is environmentally friendly, and has low cost, and has good prospects for industrial application.

[0016] In order to achieve the above object, the present invention adopts the following technical solutions:

[0017] The present invention first provides a recombinant bacterium, which co-expresses L-phenylalanine aminomutase, L-aspartate-β-decarboxylase, monoamine oxidase and catalase.

[0018] As a preferred embodiment of the present invention, the L-phenylalanine aminomutase is derived from Colletotrichumaenigma (Genbank No. KAF5502037.1) and Klebsiella pneumoniae (Genbank No. AVE25541.1), and is named CaTAM and KpTAM, respectively.

[0019] As a preferred embodiment of the present invention, the L-phenylalanine aminomutase includes CaTAM and KpTAM, and the amino acid sequence of the L-phenylalanine aminomutase is SEQ ID NO.2 and / or SEQ ID NO.4.

[0020] As a preferred embodiment of the present invention, the L-aspartate-β-decarboxylase is derived from Roseomonas mucosa (Genbank No. QDD94011.1) and Melissococcus plutonius (Genbank No. BBC61552.1), and is named RmADC and MpADC, respectively.

[0021] As a preferred embodiment of the present invention, the L-aspartate-β-decarboxylase includes RmADC and MpADC, and the amino acid sequence of the L-aspartate-β-decarboxylase is shown in SEQ ID NO. 6 and / or SEQ ID NO. 8.

[0022] As a preferred embodiment of the present invention, the monoamine oxidase is derived from Nocardia seriolae (Genbank No. APA96479.1) and Komagataeibacter europaeus (Genbank No. ARW15355.1), and is named NsAOD and KeAOD, respectively.

[0023] As a preferred embodiment of the present invention, the monoamine oxidase includes NsAOD and KeAOD, and the amino acid sequence of the monoamine oxidase is shown in SEQ ID NO.10 and / or SEQ ID NO.12.

[0024] As a preferred embodiment of the present invention, the catalase is derived from Pseudomonas koreensis (Genbank No. RVD76106.1) and Rhodopirellula baltica (Genbank No. CAD73747.1), and is named PkCAT and RbCAT, respectively.

[0025] As a preferred embodiment of the present invention, the catalase includes PkCAT and RbCAT, and the amino acid sequence of the catalase is shown in SEQ ID NO.14 and / or SEQ ID NO.16.

[0026] As a preferred embodiment of the present invention, the nucleotide sequence encoding the L-phenylalanine aminomutase is shown in SEQ ID NO.1 and / or SEQ ID NO.3;

[0027] The nucleotide sequence encoding the L-aspartate-β-decarboxylase is shown in SEQ ID NO.5 and / or SEQ ID NO.7;

[0028] The nucleotide sequence encoding the monoamine oxidase is shown in SEQ ID NO.9 and / or SEQ ID NO.11;

[0029] The nucleotide sequence encoding the catalase is shown in SEQ ID NO.13 and / or SEQ ID NO.15.

[0030] The present invention secondly provides a method for constructing the above-mentioned recombinant bacteria, which comprises: inserting the coding genes of L-phenylalanine aminomutase, L-aspartate-β-decarboxylase, monoamine oxidase and catalase into an expression vector to obtain a recombinant expression vector, and then introducing the obtained recombinant expression vector into a host bacterium to obtain the recombinant bacteria; preferably, the expression vector comprises pCDFDuet-1 and pACYCDuet-1.

[0031] In the present invention, one enzyme each of the above-mentioned L-phenylalanine aminomutase, L-aspartate-β-decarboxylase, monoamine oxidase and catalase is selected to perform co-expression of the four enzyme combination;

[0032] Any two enzyme encoding genes may be present on the same vector, or the four genes may be present on different vectors, which is not limited in the present invention.

[0033] As a preferred embodiment of the present invention, the pCDFDuet-1 is loaded with genes for L-phenylalanine aminomutase and L-aspartate-β-decarboxylase, and the pACYCDuet-1 is loaded with genes for monoamine oxidase and catalase;

[0034] or ， pACYCDuet-1 carries genes for L-phenylalanine aminomutase and L-aspartate-β-decarboxylase, and pCDFDuet-1 carries genes for monoamine oxidase and catalase.

[0035] In some embodiments, the host bacteria is Escherichia coli. In other embodiments, the starting strain of the recombinant bacteria can be other strains, which are not specifically limited in the present invention.

[0036] As a preferred embodiment of the present invention, the host bacteria is Escherichia coli;

[0037] Preferably, the host bacteria is any one of Escherichia coli BL21 (DE3), Escherichia coli DH5α and / or Escherichia coli XL-Blue.

[0038] The present invention further provides the use of the recombinant bacteria in synthesizing p-hydroxyacetophenone and its downstream products.

[0039] Finally, the present invention provides a novel method for producing p-hydroxyacetophenone, wherein the recombinant bacteria are added to a reaction system containing L-tyrosine to carry out a catalytic reaction to obtain p-hydroxyacetophenone.

[0040] As a preferred embodiment of the present invention, p-hydroxyacetophenone is synthesized by whole-cell transformation, wherein the whole-cell transformation reaction system comprises: a recombinant bacterial cell amount of 1-20 g / L and an L-tyrosine concentration of 1-40 g / L.

[0041] As a preferred embodiment of the present invention, the pH of the reaction system for whole-cell transformation is 6.0-9.0, the temperature is 15-40° C., and the reaction time is 6-24 h.

[0042] The invention utilizes a recombinant Escherichia coli bacterium to prepare p-hydroxyacetophenone using L-tyrosine as a substrate. Four enzymes, namely L-phenylalanine aminomutase (TAM), L-aspartate-β-decarboxylase (ADC), monoamine oxidase (AOD) and catalase (CAT), are simultaneously expressed in the Escherichia coli. The reaction principle is as follows: L-tyrosine is converted into (R)-3-amino-3-(4-hydroxyphenyl)propionic acid by TAM, (R)-3-amino-3-(4-hydroxyphenyl)propionic acid is converted into (R)-4-(1-aminoethyl)phenol by ADC, (R)-4-(1-aminoethyl)phenol is converted into p-hydroxyacetophenone by AOD, and generated hydrogen peroxide is removed by CAT.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] The present invention provides a novel method for producing p-hydroxyacetophenone using a recombinant Escherichia coli bacterium and inexpensive L-tyrosine as a substrate. The reaction process is simple and eliminates the need for coenzyme addition. The enzyme selected in the present invention has advantages such as high activity and strong optical specificity. Therefore, the production of p-hydroxyacetophenone using the recombinant bacterium of the present invention is highly efficient, environmentally friendly, and low-cost, with promising prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0046] Figure 1 The invention discloses a synthetic route of p-hydroxyacetophenone. DETAILED DESCRIPTION

[0047] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0048] In a first aspect, the present invention provides a recombinant Escherichia coli that co-expresses L-phenylalanine aminomutase, L-aspartate-β-decarboxylase, monoamine oxidase, and catalase.

[0049] The second aspect of the present invention provides a method for constructing the above-mentioned recombinant Escherichia coli.

[0050] The third aspect of the present invention provides the use of the above-mentioned recombinant Escherichia coli, wherein the above-mentioned recombinant Escherichia coli is used as a biocatalyst to convert the cheap substrate L-tyrosine into p-hydroxyacetophenone.

[0051] Finally, the invention provides a novel production method of p-hydroxyacetophenone.

[0052] See also Figure 1 The present invention converts L-tyrosine into (R)-3-amino-3-(4-hydroxyphenyl)propionic acid through TAM, converts (R)-3-amino-3-(4-hydroxyphenyl)propionic acid into (R)-4-(1-aminoethyl)phenol through ADC, converts (R)-4-(1-aminoethyl)phenol into p-hydroxyacetophenone through AOD, and the generated hydrogen peroxide is removed by CAT.

[0053] 1. Selection of bacteria and plasmids

[0054] pCDFDuet-1 plasmid, pACYCDuet-1 plasmid, Escherichia coli BL21 (DE3), Escherichia coli DH5α, and Escherichia coli XL-Blue were purchased from Novagen.

[0055] 2. Enzyme selection

[0056] (1) Selection of L-phenylalanine aminomutase

[0057] The amino acid sequences of L-phenylalanine aminomutases CaTAM and KpTAM were obtained from the NCBI database. Codon optimization was performed based on the preference of Escherichia coli. Two nucleotide sequences were synthesized by total synthesis using conventional genetic engineering techniques, as shown in SEQ ID NOs. 1 and 3, respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NOs. 2 and 4, respectively. EcoRI and HindIII restriction sites were added to both ends of the nucleotide sequences.

[0058] (2) Selection of L-aspartate-β-decarboxylase

[0059] The amino acid sequences of L-aspartate-β-decarboxylases RmADC and MpADC were obtained from the NCBI database. Codon optimization was performed based on E. coli preferences. Two nucleotide sequences were synthesized by total synthesis using conventional genetic engineering techniques, as shown in SEQ ID NOs. 5 and 7, respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NOs. 6 and 8, respectively. Restriction sites NdeI and XhoI were added to both ends of the nucleotide sequences.

[0060] (3) Selection of monoamine oxidase

[0061] The amino acid sequences of monoamine oxidases NsAOD and KeAOD were obtained from the NCBI database. Codon optimization was performed based on E. coli preferences, and the nucleotide sequences were synthesized by total synthesis using conventional genetic engineering techniques, as shown in SEQ ID NOs. 9 and 11. The amino acid sequences encoding the enzymes are shown in SEQ ID NOs. 10 and 12. EcoRI and HindIII restriction sites were added to both ends of the nucleotide sequences.

[0062] (4) Selection of catalase

[0063] The amino acid sequences of catalase PkCAT and RbCAT were obtained from the NCBI database. Codon optimization was performed based on E. coli preferences, and the nucleotide sequences were synthesized by total synthesis using conventional genetic engineering techniques as shown in SEQ ID NOs. 13 and 15. The amino acid sequences encoding the enzymes are shown in SEQ ID NOs. 14 and 16. Restriction sites NdeI and XhoI were added to both ends of the nucleotide sequences.

[0064] 3. Construction of the Four-enzyme Co-expression System and Bacterial Culture

[0065] One enzyme from each of the selected enzyme categories, L-phenylalanine aminomutase, L-aspartate β-decarboxylase, monoamine oxidase, and catalase, was co-expressed in a four-enzyme combination. The genes encoding these four enzymes were co-expressed using a dual plasmid, pCDFDuet-1 and pACYCDuet-1; pCDFDuet-1 was loaded with L-phenylalanine aminomutase and L-aspartate β-decarboxylase, while pACYCDuet-1 was loaded with monoamine oxidase and catalase. After obtaining the co-expression recombinant plasmids, both recombinant plasmids were simultaneously transformed into competent Escherichia coli BL21(DE3) cells. Positive transformants were screened using plates containing streptomycin and chloramphenicol to obtain recombinant E. coli. The resulting recombinant bacteria were inoculated into fresh liquid culture medium, induced, and centrifuged to obtain wet cells.

[0066] 4. Whole Cell Conversion of L-Tyrosine to Prepare p-Hydroxyacetophenone

[0067] Transformation system: L-tyrosine concentration of 1-40 g / L, pH adjusted between 6.0-9.0, fresh bacterial cell volume of 1-20 g / L, then incubate at 15-40°C, 200 rpm, for 6-24 hours. After conversion, determine the p-hydroxyacetophenone yield by liquid chromatography.

[0068] 5. Sample Detection and Analysis

[0069] The conversion solution was analyzed by Shimadzu 2030C high performance liquid chromatography (HPLC) under the following chromatographic conditions: the mobile phase was methanol: water (V / V = 1:1), an Inertsustain C18 column (4.6 × 250 mm, 5 μm), a flow rate of 1 mL / min, a column temperature of 30 °C, an injection volume of 20 μL, and a detection wavelength of 275 nm.

[0070] Example 1

[0071] This example provides the construction of recombinant Escherichia coli, as follows:

[0072] The fully synthetic TAM recombinant plasmid and the pCDFDuet-1 vector were double-digested with restriction endonucleases EcoRI and HindIII, respectively, and TAMs from different sources were ligated to the pCDFDuet-1 vector using T4 DNA ligase. The fully synthetic ADC recombinant plasmid and the pCDFDuet-1 vector were double-digested with restriction endonucleases NdeI and XhoI, respectively, and TAMs and ADCs from different sources were ligated in pairs to the pCDFDuet-1 vector using T4 DNA ligase to obtain recombinant plasmid 1;

[0073] The fully synthetic AOD recombinant plasmid and the pACYCDuet-1 vector were double-digested with restriction endonucleases EcoRI and HindIII, and the fully synthetic CAT recombinant plasmid and the pACYCDuet-1 vector were double-digested with restriction endonucleases NdeI and XhoI, respectively. AOD and CAT from different sources were ligated in pairs to the pACYCDuet-1 vector using T4 DNA ligase to obtain recombinant plasmid 2.

[0074] Different recombinant plasmids 1 and 2 were combined and transformed into competent E. coli BL21 (DE3) to obtain recombinant E. coli.

[0075] Example 2

[0076] This example provides the induction culture of recombinant Escherichia coli, which is as follows:

[0077] The recombinant E. coli was inoculated into LB medium containing 50 mg / L streptomycin and 50 mg / L chloramphenicol, and cultured at 37°C and 200 rpm for 12 h to obtain seed solution. The seed solution was inoculated into fresh LB medium at a 2% inoculum volume, and cultured at 37°C and 200 rpm until the bacterial concentration OD 600nm When the pH reaches 0.7, 0.5 mM IPTG was added and induced at 28 °C for 15 h. The cells were centrifuged at 8000 rpm for 10 min, and the supernatant was discarded. The wet cells were washed twice with 0.9% saline, centrifuged, and set aside.

[0078] Example 3

[0079] This example provides a comparison of the transformation abilities of various recombinant E. coli:

[0080] The harvested recombinant E. coli was resuspended in 50 mL of a reaction system with a final cell concentration of 20 g / L and an L-tyrosine concentration of 40 g / L at pH 8.0. The reaction was incubated at 30°C, shaken at 200 rpm, and the conversion time was 24 hours. After the conversion, the yield of p-hydroxyacetophenone was determined by HPLC. The results are shown in Table 1.

[0081] Table 1 Comparison of the production of hydroxyacetophenone by various recombinant bacteria

[0082]

[0083]

[0084] Example 4

[0085] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. The final cell concentration was 1 g / L in a 50 mL system, the L-tyrosine concentration was 1 g / L, the pH was 8.0, the reaction was carried out at 30°C, the shaking speed was 200 rpm, and the conversion time was 24 h. The results of HPLC determination showed that the yield of p-hydroxyacetophenone was 0.73 g / L.

[0086] Example 5

[0087] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. In a 50 mL system, the final cell concentration was 3 g / L, the L-tyrosine concentration was 6 g / L, the pH was 8.0, and the reaction was carried out at 35°C, the shaking speed was 200 rpm, and the conversion time was 24 h. The results of HPLC determination showed that the yield of p-hydroxyacetophenone was 4.4 g / L.

[0088] Example 6

[0089] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. The final cell concentration was 5 g / L in a 50 mL system, the L-tyrosine concentration was 10 g / L, the pH was 8.0, the reaction was carried out at 30°C, the shaking speed was 200 rpm, and the conversion time was 24 h. The results of HPLC determination showed that the yield of p-hydroxyacetophenone was 7.3 g / L.

[0090] Example 7

[0091] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. In a 50 mL system, the final cell concentration was 8 g / L, the L-tyrosine concentration was 17 g / L, the pH was 8.0, the reaction was carried out at 30°C, the shaking speed was 200 rpm, and the conversion time was 24 h. The results of HPLC determination showed that the yield of p-hydroxyacetophenone was 12.6 g / L.

[0092] Example 8

[0093] According to the induction expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. In a 50 mL system, the final cell concentration was 10 g / L, the L-tyrosine concentration was 19 g / L, the pH was 8.0, and the reaction was carried out at 30°C, the shaking speed was 200 rpm, and the conversion time was 24 h. HPLC determination results showed that the yield of p-hydroxyacetophenone was 14.1 g / L.

[0094] Example 9

[0095] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. In a 50 mL system, the final cell concentration was 15 g / L, the L-tyrosine concentration was 28 g / L, the pH was 8.0, and the reaction was carried out at 30°C, the shaking speed was 200 rpm, and the conversion time was 24 h. HPLC determination results showed that the yield of p-hydroxyacetophenone was 20.6 g / L.

[0096] Example 10

[0097] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. In a 50 mL system, the final cell concentration was 18 g / L, the L-tyrosine concentration was 35 g / L, the pH was 8.0, and the reaction was carried out at 30°C, the shaking speed was 200 rpm, and the conversion time was 24 h. HPLC determination results showed that the yield of p-hydroxyacetophenone was 26.1 g / L.

[0098] Example 11

[0099] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. The final cell concentration was 20 g / L in a 50 mL system, the L-tyrosine concentration was 20 g / L, the pH was 8.0, the reaction was carried out at 30°C, the shaking speed was 200 rpm, and the conversion time was 12 h. The results of HPLC determination showed that the yield of p-hydroxyacetophenone was 14.7 g / L.

[0100] Example 12

[0101] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. In a 50 mL system, the final cell concentration was 20 g / L, the L-tyrosine concentration was 11 g / L, the pH was 8.0, the reaction was carried out at 30°C, the shaking speed was 200 rpm, and the conversion time was 6 h. HPLC determination results showed that the yield of p-hydroxyacetophenone was 8.1 g / L.

[0102] Example 13

[0103] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. The final cell concentration was 15 g / L in a 50 mL system, the L-tyrosine concentration was 10 g / L, the pH was 8.0, the reaction was carried out at 30°C, the shaking speed was 200 rpm, and the conversion time was 8 h. The results of HPLC determination showed that the yield of p-hydroxyacetophenone was 7.4 g / L.

[0104] Example 14

[0105] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. In a 50 mL system, the final cell concentration was 12 g / L, the L-tyrosine concentration was 13 g / L, the pH was 8.0, and the reaction was carried out at 30°C, the shaking speed was 200 rpm, and the conversion time was 15 h. HPLC analysis showed that the yield of p-hydroxyacetophenone was 9.7 g / L.

[0106] Example 15

[0107] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. In a 50 mL system, the final cell concentration was 10 g / L, the L-tyrosine concentration was 9 g / L, the pH was 6.0, and the reaction was carried out at 35°C, the shaking speed was 200 rpm, and the conversion time was 12 h. The results of HPLC determination showed that the yield of p-hydroxyacetophenone was 6.7 g / L.

[0108] Example 16

[0109] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. In a 50 mL system, the final cell concentration was 15 g / L, the L-tyrosine concentration was 14 g / L, the pH was 7.0, and the reaction was carried out at 35 ° C. The shaking speed was 200 rpm and the conversion time was 12 h. The results of HPLC determination showed that the yield of p-hydroxyacetophenone was 10.3 g / L.

[0110] Example 17

[0111] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. In a 50 mL system, the final cell concentration was 13 g / L, the L-tyrosine concentration was 12 g / L, the pH was 7.5, and the reaction was carried out at 35°C, the shaking speed was 200 rpm, and the conversion time was 12 h. The results of HPLC determination showed that the yield of p-hydroxyacetophenone was 8.9 g / L.

[0112] Example 18

[0113] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. In a 50 mL system, the final cell concentration was 7 g / L, the L-tyrosine concentration was 7 g / L, the pH was 8.5, and the reaction was carried out at 35°C, the shaking speed was 200 rpm, and the conversion time was 12 h. The results of HPLC determination showed that the yield of p-hydroxyacetophenone was 5.2 g / L.

[0114] Example 19

[0115] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. In a 50 mL system, the final cell concentration was 4 g / L, the L-tyrosine concentration was 4 g / L, the pH was 9.0, and the reaction was carried out at 35°C, the shaking speed was 200 rpm, and the conversion time was 12 h. The results of HPLC determination showed that the yield of p-hydroxyacetophenone was 3 g / L.

[0116] Example 20

[0117] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. In a 50 mL system, the final cell concentration was 16 g / L, the L-tyrosine concentration was 31 g / L, the pH was 7.5, the reaction was carried out at 15 ° C, the shaking speed was 200 rpm, and the conversion time was 24 h. The results of HPLC determination showed that the yield of p-hydroxyacetophenone was 23.1 g / L.

[0118] Example 21

[0119] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. In a 50 mL system, the final cell concentration was 10 g / L, the L-tyrosine concentration was 18 g / L, the pH was 7.5, the reaction was carried out at 25 ° C, the shaking speed was 200 rpm, and the conversion time was 24 h. The results of HPLC determination showed that the yield of p-hydroxyacetophenone was 13.3 g / L.

[0120] Example 22

[0121] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. In a 50 mL system, the final cell concentration was 11 g / L, the L-tyrosine concentration was 22 g / L, the pH was 7.5, and the reaction was carried out at 40°C, the shaking speed was 200 rpm, and the conversion time was 24 h. HPLC determination results showed that the yield of p-hydroxyacetophenone was 16.2 g / L.

[0122] Example 23

[0123] According to the induction expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. In a 50 mL system, the final cell concentration was 30 g / L, the L-tyrosine concentration was 45 g / L, the pH was 7.5, and the reaction was carried out at 35°C, the shaking speed was 200 rpm, and the conversion time was 36 h. The results of HPLC determination showed that the yield of p-hydroxyacetophenone was 4.1 g / L.

[0124] Example 24

[0125] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. In a 50 mL system, the final cell concentration was 25 g / L, the L-tyrosine concentration was 40 g / L, the pH was 5.5, and the reaction was carried out at 35°C, the shaking speed was 200 rpm, and the conversion time was 36 h. The results of HPLC determination showed that the yield of p-hydroxyacetophenone was 4.8 g / L.

[0126] Example 25

[0127] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. In a 50 mL system, the final cell concentration was 15 g / L, the L-tyrosine concentration was 27 g / L, the pH was 9.5, and the reaction was carried out at 35°C, the shaking speed was 200 rpm, and the conversion time was 36 h. HPLC determination results showed that the yield of p-hydroxyacetophenone was 3.7 g / L.

[0128] Example 26

[0129] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. In a 50 mL system, the final cell concentration was 20 g / L, the L-tyrosine concentration was 37 g / L, the pH was 7.5, the reaction was carried out at 10 ° C, the shaking speed was 200 rpm, and the conversion time was 36 h. The results of HPLC determination showed that the yield of p-hydroxyacetophenone was 4.7 g / L.

[0130] Example 27

[0131] According to the inducible expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. In a 50 mL system, the final cell concentration was 30 g / L, the L-tyrosine concentration was 33 g / L, the pH was 8.0, and the reaction was carried out at 45°C, the shaking speed was 200 rpm, and the conversion time was 36 h. The results of HPLC determination showed that the yield of p-hydroxyacetophenone was 3.7 g / L.

[0132] Example 28

[0133] According to the induction expression method described in Example 2, E. coli BL21 (DE3) / pCDFDuet-CaTAM-RmADC + pACYCDuet-KeAOD-PkCAT was induced and the cells were collected after expression was completed. In a 50 mL system, the final cell concentration was 0.5 g / L, the L-tyrosine concentration was 0.5 g / L, the pH was 8.0, and the reaction was carried out at 30°C, the shaking speed was 200 rpm, and the conversion time was 36 h. The results of HPLC determination showed that the yield of p-hydroxyacetophenone was 0.1 g / L.

[0134] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form or substance. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention. Any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A recombinant bacterium, characterized in that The recombinant bacteria co-express L-phenylalanine aminomutase, L-aspartate-β-decarboxylase, monoamine oxidase and catalase.

2. A recombinant bacterium according to claim 1, characterized in that The L-phenylalanine aminomutase includes CaTAM and KpTAM, and the amino acid sequence of the L-phenylalanine aminomutase is shown in SEQ ID NO.2 and / or SEQ ID NO.4; The L-aspartate-β-decarboxylase includes RmADC and MpADC, and the amino acid sequence of the L-aspartate-β-decarboxylase is shown in SEQ ID NO.6 and / or SEQ ID NO.8; The monoamine oxidase includes NsAOD and KeAOD, and the amino acid sequence of the monoamine oxidase is shown in SEQ ID NO.10 and / or SEQ ID NO.12; The catalase includes PkCAT and RbCAT, and the amino acid sequence of the catalase is shown in SEQ ID NO.14 and / or SEQ ID NO.

16.

3. A recombinant bacterium according to claim 1, characterized in that The nucleotide sequence encoding the L-phenylalanine aminomutase is shown in SEQ ID NO.1 and / or SEQ ID NO.3; The nucleotide sequence encoding the L-aspartate-β-decarboxylase is shown in SEQ ID NO.5 and / or SEQ ID NO.7; The nucleotide sequence encoding the monoamine oxidase is shown in SEQ ID NO.9 and / or SEQ ID NO.11; The nucleotide sequence encoding the catalase is shown in SEQ ID NO.13 and / or SEQ ID NO.

15.

4. A method for constructing a recombinant bacterium according to any one of claims 1 to 3, characterized in that: The construction method of the recombinant bacteria includes: inserting the coding genes of L-phenylalanine aminomutase, L-aspartate-β-decarboxylase, monoamine oxidase and catalase into an expression vector to obtain a recombinant expression vector, and then introducing the obtained recombinant expression vector into a host bacteria to obtain the recombinant bacteria; preferably, the expression vector includes pCDFDuet-1 and pACYCDuet-1.

5. The method for constructing a recombinant bacterium according to claim 4, characterized in that: The pCDFDuet-1 is loaded with genes for L-phenylalanine aminomutase and L-aspartate-β-decarboxylase, and the pACYCDuet-1 is loaded with genes for monoamine oxidase and catalase; or, pACYCDuet-1 is loaded with genes for L-phenylalanine aminomutase and L-aspartate-β-decarboxylase, and the pCDFDuet-1 is loaded with genes for monoamine oxidase and catalase.

6. The method for constructing a recombinant bacterium according to claim 4, characterized in that: The host bacteria is Escherichia coli; Preferably, the host bacteria is any one of Escherichia coli BL21 (DE3), Escherichia coli DH5α and / or Escherichia coli XL-Blue.

7. A use of the recombinant bacterium according to any one of claims 1 to 3, characterized in that: The recombinant bacteria is used in the synthesis of p-hydroxyacetophenone and its downstream products.

8. A method for producing p-hydroxyacetophenone, characterized in that: The method comprises adding the recombinant bacteria according to any one of claims 1 to 3 or the recombinant bacteria obtained by the construction method according to any one of claims 4 to 6 into a reaction system containing L-tyrosine to carry out a catalytic reaction to obtain p-hydroxyacetophenone.

9. The method for producing p-hydroxyacetophenone according to claim 8, wherein Parahydroxyacetophenone is synthesized by whole-cell transformation, wherein the whole-cell transformation reaction system comprises: a cell mass of the recombinant bacteria of 1-20 g / L and an L-tyrosine concentration of 1-40 g / L.

10. The method for producing p-hydroxyacetophenone according to claim 9, wherein: The pH of the reaction system for whole-cell transformation is 6.0-9.0, the temperature is 15-40° C., and the reaction time is 6-24 hours.

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