Method for producing p-hydroxycinnamic acid

Abstract

The present invention provides a recombinant bacterium and a method for producing p-hydroxycinnamic acid using the recombinant bacterium. Heterologous aldolase and fumarase genes are integrated into the genome of the recombinant bacterium.

Description

A method for producing p-hydroxycinnamic acid

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 202411187517.6, filed on August 28, 2024, entitled "A method for producing p-hydroxycinnamic acid", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the field of biotechnology, and more specifically, to a method for producing p-hydroxycinnamic acid. Background Technology

[0004] p-Hydroxycinnamic acid, also known as p-coumaric acid, is a natural phenolic compound that is widely found in plants. It has anti-mutagenic, anti-genotoxic, antimicrobial, antioxidant, and whitening effects, and is widely used in food, cosmetics, and pharmaceuticals.

[0005] The main methods for preparing p-hydroxycinnamic acid include extraction, chemical synthesis, and biotransformation. Extraction primarily uses natural plants as raw materials, involving acid or alkali hydrolysis followed by organic solvent extraction to obtain p-hydroxycinnamic acid. However, this method is complex and costly, limiting its industrial application. Chemical synthesis mainly involves condensation reactions to prepare p-hydroxycinnamic acid. However, this process easily generates toxic byproducts, resulting in low yields and high separation and purification costs, hindering its large-scale application. Biotransformation, with its advantages of high specificity, environmental friendliness, mild reaction conditions, and elimination of multi-step separation and purification, has attracted widespread attention.

[0006] Currently, scholars both domestically and internationally have reported several bio-based preparation routes for p-hydroxycinnamic acid. For example, Yuqi Liu et al. expressed phenylalanine ammonia-lyase (RgPAL) in *E. coli* and prepared p-hydroxycinnamic acid via whole-cell catalysis of L-tyrosine. Under optimal conditions, the yield of p-hydroxycinnamic acid was only 525 mg / L, with a conversion rate of 57.9%. For instance, Chinese patent CN117965514A molecularly modified phenylalanine ammonia-lyase (ZmPAL2) from *Zea mays* to obtain the optimal ZmPAL2 mutant. The highest yield of p-hydroxycinnamic acid reached 3.12 g / L when converted from L-tyrosine using this mutant.

[0007] The existing biosynthetic methods for preparing p-hydroxycinnamic acid have drawbacks such as high substrate cost, low yield, low conversion rate, and low production efficiency. Therefore, how to develop a low-cost, widely available substrate, high-yield, high-conversion-rate, and high-efficiency method for preparing p-hydroxycinnamic acid is an urgent problem to be solved by researchers in this field.

[0008] Therefore, this application is hereby submitted. Summary of the Invention

[0009] The purpose of this application is to provide a method for producing p-hydroxycinnamic acid, which involves constructing a recombinant bacterium expressing heterologous aldolase and fumarate, and then using it as a biocatalyst to convert inexpensive substrates p-hydroxybenzaldehyde and acetic acid to obtain p-hydroxycinnamic acid.

[0010] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0011] As shown in Figure 1, the p-hydroxycinnamic acid synthesis pathway used in this application is as follows: p-hydroxybenzaldehyde and acetic acid are converted to 3-hydroxy-3-(4-hydroxyphenyl)propionic acid via aldolase (ALD), and 3-hydroxy-3-(4-hydroxyphenyl)propionic acid is converted to p-hydroxycinnamic acid via fumarate enzyme (FR). Based on the p-hydroxycinnamic acid synthesis pathway shown in Figure 1, this application designs a novel recombinant bacterium capable of expressing the key enzymes of this synthesis pathway, namely ALD and FR. The inventors screened three enzymes with high p-hydroxycinnamic acid production efficiency from various sources of ALD and FR.

[0012] Among them, ALD includes SsALD derived from Sulfurihydrogenibium sp., LhALD derived from Lachnellula hyalina, and GsALD derived from Geobacter sp.

[0013] FRs include HpFR from Halanaebium praevalens, OpFR from Oceanithermus profundus, and AaFR from Acetohalobium arabaticum.

[0014] The GenBank accession number for SsALD is ACD65676.1, and its amino acid sequence is shown in SEQ ID NO.1, while its nucleotide sequence is shown in SEQ ID NO.4. The GenBank accession number for LhALD is TVY29750.1, and its amino acid sequence is shown in SEQ ID NO.2, while its nucleotide sequence is shown in SEQ ID NO.5. The GenBank accession number for GsALD is GFE61120.1, and its amino acid sequence is shown in SEQ ID NO.3, while its nucleotide sequence is shown in SEQ ID NO.6.

[0015] The GenBank accession number for HpFR is ADO76634.1, and its amino acid sequence is shown in SEQ ID NO.7, while its nucleotide sequence is shown in SEQ ID NO.10. The GenBank accession number for OpFR is ADR36409.1, and its amino acid sequence is shown in SEQ ID NO.8, while its nucleotide sequence is shown in SEQ ID NO.11. The GenBank accession number for AaFR is ADL12064.1, and its amino acid sequence is shown in SEQ ID NO.9, while its nucleotide sequence is shown in SEQ ID NO.12.

[0016] The above-mentioned method for constructing recombinant bacteria includes: ligating the genes of ALD and FR into an expression vector, and then introducing the obtained recombinant expression vector into the starting strain to obtain recombinant bacteria.

[0017] In some embodiments, the starting strain of the recombinant bacteria is *Escherichia coli*. In other embodiments, the starting strain of the recombinant bacteria can be other strains, and this application does not specifically limit them.

[0018] In some embodiments, Escherichia coli is selected from any one of Escherichia coli BL21(DE3), Escherichia coli DH5α, and Escherichia coli XL-Blue.

[0019] In some embodiments, the expression vector includes pCDFDuet-1 plasmid and pACYCDuet-1 plasmid.

[0020] The following recombinant vectors can be constructed using the above expression vectors: pCDFDuet-SsALD, pCDFDuet-LhALD, pCDFDuet-GsALD, pACYCDuet-HpFR, pACYCDuet-OpFR, and pACYCDuet-AaFR.

[0021] Preferably, the combination of pCDFDuet-LhALD and pACYCDuet-OpFR can increase the yield of p-hydroxycinnamic acid when constructing recombinant Escherichia coli.

[0022] Furthermore, this application also provides a whole-cell catalyst containing the aforementioned recombinant bacteria.

[0023] Using the above recombinant bacteria as whole-cell catalysts, the ALD and FR expressed by them can efficiently convert p-hydroxybenzaldehyde and acetic acid into p-hydroxycinnamic acid.

[0024] This application also provides the application of the above-mentioned recombinant bacteria in the synthesis of p-hydroxycinnamic acid and its downstream products. Based on this, this application can use the recombinant bacteria to produce p-hydroxycinnamic acid, as follows:

[0025] The recombinant bacteria were added to a reaction system containing p-hydroxybenzaldehyde and acetic acid to perform whole-cell catalysis and obtain p-hydroxycinnamic acid.

[0026] In some embodiments, the recombinant bacteria are induced and cultured before whole-cell transformation. The induction and culture process is as follows: the recombinant bacteria are inoculated into LB medium containing 30-60 mg / L chloramphenicol and 30-60 mg / L streptomycin, and seed culture is obtained after culture. The seed culture is then inoculated into fresh LB medium until the bacterial concentration OD600nm reaches 0.6-0.8. An inducer is added, and after induction, the bacteria are separated and washed to obtain wet cells.

[0027] In some embodiments, the culture conditions for the recombinant bacteria are: temperature of 32-38℃, rotation speed of 150-250 rpm, and culture time of 10-16 h.

[0028] In some embodiments, the culture conditions for the above seed solution are: temperature of 32-38℃ and rotation speed of 150-250 rpm.

[0029] 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.

[0030] In some embodiments, the separation and cleaning conditions are as follows: rotation speed of 7500-8500 rpm, centrifugation time of 5-15 min; and washing of wet bacterial cells with saline solution at a concentration of 0.9%.

[0031] In some embodiments, the whole-cell catalytic reaction system comprises: 1-10 g / L of recombinant bacteria wet weight, 1-60 g / L of p-hydroxybenzaldehyde, 1-35 g / L of acetic acid, and 0.01-0.05 g / L of pyridoxal phosphate (PLP).

[0032] In some embodiments, the whole-cell catalytic reaction conditions are: pH = 6.0-9.0, temperature = 15-40℃, rotation speed = 150-250 rpm, and reaction time = 8-24h.

[0033] This application has the following beneficial effects:

[0034] This application describes the construction of a recombinant strain of *Escherichia coli* capable of efficiently synthesizing p-hydroxycinnamic acid. The genome of this recombinant strain integrates heterologous aldolase and fumarate enzyme genes, enabling it to efficiently synthesize p-hydroxycinnamic acid using p-hydroxybenzaldehyde and acetic acid as substrates. p-hydroxybenzaldehyde and acetic acid are widely available, simple to prepare, and inexpensive, making them ideal substrates. The enzymes selected in this application possess advantages such as high activity and strong optical specificity. Therefore, the production of p-hydroxycinnamic acid using this recombinant strain offers high yield, high conversion rate, high production efficiency, is environmentally friendly, and low-cost, demonstrating promising prospects for industrial application. Attached Figure Description

[0035] To more clearly illustrate the technical solutions of the embodiments of this application, 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 this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 shows the synthetic route of p-hydroxycinnamic acid in this application. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application 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.

[0038] The features and performance of this application will be further described in detail below with reference to the embodiments.

[0039] 1. Selection of bacteria and plasmids

[0040] pCDFDuet-1 plasmid, pACYCDuet-1 plasmid, Escherichia coli BL21(DE3), Escherichia coli DH5α and Escherichia coli XL-Blue were purchased from Novagen.

[0041] 2. Enzyme selection

[0042] (1) Selection of aldolase

[0043] The amino acid sequences of aldolases SsALD, LhALD, and GsALD 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 techniques for total synthesis, as shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively. EcoRI and HindIII restriction enzyme sites were added to both ends of the nucleotide sequences.

[0044] (2) Selection of fumarate

[0045] The amino acid sequences of fumarate enzymes HpFR, OpFR, and AaFR 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 techniques for total synthesis, as shown in SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively. The amino acid sequences encoding the enzymes are shown in SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively. EcoRI and HindIII restriction enzyme sites were added to both ends of the nucleotide sequences.

[0046] 3. Construction of the dual-enzyme co-expression system and cell culture

[0047] One enzyme from each of the selected aldolases and fumarates was chosen for co-expression. The encoding genes of both enzymes were co-expressed using the pCDFDuet-1 and pACYCDuet-1 plasmids; pCDFDuet-1 contained aldolase, and pACYCDuet-1 contained fumarate. After obtaining the co-expressed recombinant plasmids, both plasmids were simultaneously transformed into *Escherichia coli* BL21(DE3) competent cells. Positive transformants were obtained by screening with streptomycin and chloramphenicol plates, thus obtaining recombinant *E. coli*. The obtained recombinant bacteria were inoculated into fresh liquid culture medium, induced, centrifuged, and wet cells were obtained.

[0048] 4. Preparation of p-hydroxycinnamic acid by whole-cell transformation of p-hydroxybenzaldehyde and acetic acid

[0049] Transformation system: p-hydroxybenzaldehyde concentration 1-60 g / L, acetic acid concentration 1-35 g / L, pyridoxal phosphate (PLP) 0.01-0.05 g / L, pH adjusted between 6.0-9.0, fresh cell count 1-10 g / L, then transformed at 15-40℃ and 200 rpm for 8-24 h. After transformation, the yield of p-hydroxycinnamic acid was determined by liquid chromatography.

[0050] 5. Sample detection and analysis

[0051] 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: 309 nm.

[0052] Example 1

[0053] Construction of recombinant Escherichia coli

[0054] The fully synthesized ALD recombinant plasmid and pCDFDuet-1 vector were double-digested with restriction endonucleases EcoRI and HindIII, respectively. ALDs from different sources were ligated into the pCDFDuet-1 vector using T4 DNA ligase to obtain recombinant plasmid 1. The fully synthesized FR recombinant plasmid and pACYCDuet-1 vector were double-digested with restriction endonucleases EcoRI and HindIII, respectively. FRs from different sources were ligated into the pACYCDuet-1 vector using T4 DNA ligase to obtain recombinant plasmid 2. Recombinant plasmids 1 and 2 were transformed into E. coli BL21(DE3) competent cells to obtain recombinant Escherichia coli.

[0055] Example 2

[0056] Induction culture of recombinant Escherichia coli

[0057] 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 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.

[0058] Example 3

[0059] Comparison of transformation capabilities of various recombinant Escherichia coli strains

[0060] The collected recombinant E. coli were resuspended in a 50 mL system with a final cell concentration of 10 g / L, p-hydroxybenzaldehyde 60 g / L, acetic acid 35 g / L, PLP 0.05 g / L, and pH 7.0. The reaction was carried out at 30 °C with a shaking speed of 200 rpm for 24 h. After transformation, the yield of p-hydroxycinnamic acid was determined by HPLC.

[0061] Table 1 Comparison of the yield of hydroxycinnamic acid by various recombinant strains

[0062] Example 4

[0063] Following the induction expression method described in Example 2, after induction expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 1 g / L, p-hydroxybenzaldehyde 1 g / L, acetic acid 1 g / L, PLP 0.01 g / L, pH 8.0, temperature 30℃, shaking speed 200 rpm, and transformation time 24 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 1.31 g / L, the conversion rate was 97.5%, and the production efficiency was 0.05 g / L / h.

[0064] Example 5

[0065] Following the induction expression method described in Example 2, after induction expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 1 g / L, p-hydroxybenzaldehyde 6 g / L, acetic acid 4 g / L, PLP 0.01 g / L, pH 8.0, temperature 35℃, shaking speed 200 rpm, and transformation time 24 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 7.82 g / L, the conversion rate was 97.0%, and the production efficiency was 0.33 g / L / h.

[0066] Example 6

[0067] Following the induction expression method described in Example 2, after induction expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 3 g / L, p-hydroxybenzaldehyde 19 g / L, acetic acid 11 g / L, PLP 0.01 g / L, pH 8.0, temperature 30℃, shaking speed 200 rpm, and transformation time 24 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 24.6 g / L, the conversion rate was 96.3%, and the production efficiency was 1.03 g / L / h.

[0068] Example 7

[0069] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 4 g / L, p-hydroxybenzaldehyde 23 g / L, acetic acid 13 g / L, PLP 0.03 g / L, pH 8.0, temperature 30℃, shaking speed 200 rpm, and transformation time 24 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 29.7 g / L, the conversion rate was 96.1%, and the production efficiency was 1.24 g / L / h.

[0070] Example 8

[0071] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 6 g / L, p-hydroxybenzaldehyde 34 g / L, acetic acid 19 g / L, PLP 0.03 g / L, pH 8.0, temperature 30℃, shaking speed 200 rpm, and transformation time 24 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 44.2 g / L, the conversion rate was 96.7%, and the production efficiency was 1.84 g / L / h.

[0072] Example 9

[0073] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 7 g / L, p-hydroxybenzaldehyde 43 g / L, acetic acid 23 g / L, PLP 0.05 g / L, pH 8.0, temperature 30℃, shaking speed 200 rpm, and transformation time 24 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 56.2 g / L, the conversion rate was 97.2%, and the production efficiency was 2.34 g / L / h.

[0074] Example 10

[0075] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 9 g / L, p-hydroxybenzaldehyde 53 g / L, acetic acid 30 g / L, PLP 0.05 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, and transformation time 24 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 68.5 g / L, the conversion rate was 96.1%, and the production efficiency was 2.85 g / L / h.

[0076] Example 11

[0077] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 10 g / L, p-hydroxybenzaldehyde 30 g / L, acetic acid 16 g / L, PLP 0.05 g / L, pH 8.0, temperature 30℃, shaking speed 200 rpm, and transformation time 12 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 39.3 g / L, the conversion rate was 97.5%, and the production efficiency was 3.28 g / L / h.

[0078] Example 12

[0079] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 10 g / L, p-hydroxybenzaldehyde 18 g / L, acetic acid 10 g / L, PLP 0.05 g / L, pH 8.0, temperature 30℃, shaking speed 200 rpm, and transformation time 8 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 23.6 g / L, the conversion rate was 97.5%, and the production efficiency was 2.95 g / L / h.

[0080] Example 13

[0081] Following the induction expression method described in Example 2, after induction expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 8 g / L, p-hydroxybenzaldehyde 20 g / L, acetic acid 11 g / L, PLP 0.05 g / L, pH 8.0, temperature 30℃, shaking speed 200 rpm, and transformation time 10 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 25.8 g / L, the conversion rate was 96.0%, and the production efficiency was 2.58 g / L / h.

[0082] Example 14

[0083] Following the induction expression method described in Example 2, after induction expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 7 g / L, p-hydroxybenzaldehyde 30 g / L, acetic acid 17 g / L, PLP 0.04 g / L, pH 8.0, temperature 30℃, shaking speed 200 rpm, and transformation time 17 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 38.8 g / L, the conversion rate was 96.2%, and the production efficiency was 2.28 g / L / h.

[0084] Example 15

[0085] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 6 g / L, p-hydroxybenzaldehyde 18 g / L, acetic acid 10 g / L, PLP 0.04 g / L, pH 6.0, temperature 35℃, shaking speed 200 rpm, and transformation time 12 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 23.4 g / L, the conversion rate was 96.7%, and the production efficiency was 1.95 g / L / h.

[0086] Example 16

[0087] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 5 g / L, p-hydroxybenzaldehyde 15 g / L, acetic acid 8 g / L, PLP 0.03 g / L, pH 7.0, temperature 35℃, shaking speed 200 rpm, and transformation time 12 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 19.4 g / L, the conversion rate was 96.2%, and the production efficiency was 1.62 g / L / h.

[0088] Example 17

[0089] Following the induction expression method described in Example 2, after induction expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 4 g / L, p-hydroxybenzaldehyde 11 g / L, acetic acid 6 g / L, PLP 0.03 g / L, pH 7.5, temperature 35℃, shaking speed 200 rpm, and transformation time 12 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 14.4 g / L, the conversion rate was 97.4%, and the production efficiency was 1.2 g / L / h.

[0090] Example 18

[0091] Following the induction expression method described in Example 2, after induction expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 2 g / L, p-hydroxybenzaldehyde 7 g / L, acetic acid 4 g / L, PLP 0.02 g / L, pH 8.5, temperature 35℃, shaker speed 200 rpm, and transformation time 12 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 9.2 g / L, the conversion rate was 97.8%, and the production efficiency was 0.77 g / L / h.

[0092] Example 19

[0093] Following the induction expression method described in Example 2, after induction expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 2 g / L, p-hydroxybenzaldehyde 6 g / L, acetic acid 4 g / L, PLP 0.02 g / L, pH 9.0, temperature 35℃, shaking speed 200 rpm, and transformation time 12 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 7.81 g / L, the conversion rate was 96.8%, and the production efficiency was 0.65 g / L / h.

[0094] Example 20

[0095] Following the induction expression method described in Example 2, after induction expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 6 g / L, p-hydroxybenzaldehyde 36 g / L, acetic acid 20 g / L, PLP 0.03 g / L, pH 7.5, temperature 15℃, shaking speed 200 rpm, and transformation time 24 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 47.1 g / L, the conversion rate was 97.3%, and the production efficiency was 1.96 g / L / h.

[0096] Example 21

[0097] Following the induction expression method described in Example 2, after induction expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 4 g / L, p-hydroxybenzaldehyde 25 g / L, acetic acid 14 g / L, PLP 0.03 g / L, pH 7.5, temperature 25℃, shaking speed 200 rpm, and transformation time 24 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 32.5 g / L, the conversion rate was 96.7%, and the production efficiency was 1.35 g / L / h.

[0098] Example 22

[0099] Following the induction expression method described in Example 2, after induction expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 5 g / L, p-hydroxybenzaldehyde 31 g / L, acetic acid 18 g / L, PLP 0.03 g / L, pH 7.5, temperature 40℃, shaking speed 200 rpm, and transformation time 24 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 40.7 g / L, the conversion rate was 97.7%, and the production efficiency was 1.7 g / L / h.

[0100] Comparative Example 1

[0101] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with a cell wet weight of 12 g / L, p-hydroxybenzaldehyde 70 g / L, acetic acid 40 g / L, PLP 0.08 g / L, pH 7.5, temperature 35℃, shaking speed 200 rpm, and transformation time 36 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 14.1 g / L, the conversion rate was 15.0%, and the production efficiency was 0.39 g / L / h.

[0102] Comparative Example 2

[0103] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 5 g / L, p-hydroxybenzaldehyde 30 g / L, acetic acid 20 g / L, PLP 0.03 g / L, pH 5.5, temperature 35℃, shaker speed 200 rpm, and transformation time 36 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 5.24 g / L, the conversion rate was 13.0%, and the production efficiency was 0.15 g / L / h.

[0104] Comparative Example 3

[0105] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 3 g / L, p-hydroxybenzaldehyde 20 g / L, acetic acid 12 g / L, PLP 0.03 g / L, pH 9.5, temperature 35℃, shaker speed 200 rpm, and transformation time 36 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 4.84 g / L, the conversion rate was 18.0%, and the production efficiency was 0.13 g / L / h.

[0106] Comparative Example 4

[0107] Following the induction expression method described in Example 2, after induction expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 4 g / L, p-hydroxybenzaldehyde 24 g / L, acetic acid 15 g / L, PLP 0.03 g / L, pH 7.5, temperature 10℃, shaking speed 200 rpm, and transformation time 36 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 5.48 g / L, the conversion rate was 17.0%, and the production efficiency was 0.15 g / L / h.

[0108] Comparative Example 5

[0109] Following the induction expression method described in Example 2, after induction expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following parameters: cell wet weight 6 g / L, p-hydroxybenzaldehyde 34 g / L, acetic acid 20 g / L, PLP 0.04 g / L, pH 8.0, temperature 45℃, shaker speed 200 rpm, and transformation time 36 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 6.85 g / L, the conversion rate was 15.0%, and the production efficiency was 0.19 g / L / h.

[0110] Comparative Example 6

[0111] Following the induction expression method described in Example 2, after inducing expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR, bacterial cells were collected and placed in a 50 mL system with the following concentrations: cell wet weight 0.1 g / L, p-hydroxybenzaldehyde 0.5 g / L, acetic acid 0.3 g / L, PLP 0.005 g / L, pH 8.0, temperature 30℃, shaker speed 200 rpm, and transformation time 36 h. HPLC analysis showed that the yield of p-hydroxycinnamic acid was 0.13 g / L, the conversion rate was 19.3%, and the production efficiency was 0.004 g / L / h.

[0112] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application. Industrial applicability

[0113] The recombinant bacteria provided in this application can be used as a biocatalyst to convert inexpensive substrates p-hydroxybenzaldehyde and acetic acid into p-hydroxycinnamic acid. This method has high yield, high substrate conversion rate, high production efficiency, is green and environmentally friendly, and has low cost, thus it has good prospects for industrial application.

Claims

1. A recombinant bacterium for producing p-hydroxycinnamic acid, characterized in that, The genome of the recombinant bacteria integrates heterologous aldolase and fumarate genes. The aldolase is derived from Sulfurihydrogenibium sp., Lachnellula hyalina, or Geobacter sp.; the fumarate is derived from Halanaerobium praevalens, Oceanithermus profundus, or Acetohalobium arabaticum.

2. The recombinant bacteria according to claim 1, characterized in that, The aldolases include SsALD with the amino acid sequence shown in SEQ ID NO.1, LhALD with the amino acid sequence shown in SEQ ID NO.2, and GsALD with the amino acid sequence shown in SEQ ID NO.

3. Preferably, the nucleotide sequence of SsALD is shown in SEQ ID NO.4, the nucleotide sequence of LhALD is shown in SEQ ID NO.5, and the nucleotide sequence of GsALD is shown in SEQ ID NO.

6.

3. The recombinant bacteria according to claim 1, characterized in that, The fumarate enzyme comprises HpFR with the amino acid sequence shown in SEQ ID NO.7, OpFR with the amino acid sequence shown in SEQ ID NO.8, and AaFR with the amino acid sequence shown in SEQ ID NO.9; Preferably, the nucleotide sequence of HpFR is shown in SEQ ID NO.10, the nucleotide sequence of OpFR is shown in SEQ ID NO.11, and the nucleotide sequence of AaFR is shown in SEQ ID NO.

12.

4. The recombinant bacteria according to claim 1, characterized in that, The starting strain of the recombinant bacteria includes Escherichia coli; Preferably, the *Escherichia coli* species include *Escherichia coli* BL21(DE3), *Escherichia coli* DH5α, and *Escherichia coli* XL-Blue.

5. The method for preparing recombinant bacteria according to any one of claims 1-4, characterized in that, include: The genes for aldolase and fumarate are inserted into an expression vector to obtain a recombinant vector, and then the recombinant vector is introduced into the starting strain to obtain the recombinant strain. Preferably, the expression vector includes pCDFDuet-1 and pACYCDuet-1.

6. The preparation method according to claim 5, characterized in that, The method for preparing the recombinant bacteria includes: obtaining a recombinant vector by inserting the genes of LhALD and OpFR into an expression vector, then introducing the recombinant vector into a starting strain, and culturing and screening to obtain a recombinant bacteria that produce p-hydroxycinnamic acid; Preferably, the recombinant expression vector includes pCDFDuet-LhALD and pACYCDuet-OpFR.

7. A whole-cell catalyst, characterized in that, It contains the recombinant bacteria as described in any one of claims 1-4.

8. The use of the recombinant bacteria according to any one of claims 1-4 in the synthesis of p-hydroxycinnamic acid and its downstream products.

9. A method for producing p-hydroxycinnamic acid, characterized in that, The method includes adding the recombinant bacteria according to any one of claims 1-4 into a reaction system containing p-hydroxybenzaldehyde and acetic acid to perform whole-cell catalysis to obtain the p-hydroxycinnamic acid.

10. The method according to claim 9, characterized in that, The whole-cell catalytic reaction system comprises: 1-10 g / L of recombinant bacteria (wet weight), 1-60 g / L of p-hydroxybenzaldehyde, 1-35 g / L of acetic acid, and 0.01-0.05 g / L of pyridoxal phosphate; Preferably, the reaction conditions for whole-cell catalysis are: pH = 6.0-9.0, temperature = 15-40℃, and reaction time = 8-24h.

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