A method for producing p-hydroxycinnamic acid

Abstract

The application discloses a method for producing p-hydroxycinnamic acid, and constructs a recombinant strain capable of efficiently synthesizing p-hydroxycinnamic acid by taking escherichia coli as a starting strain. The recombinant strain has the genes of heterologous aldehyde condensing enzyme and fumarase integrated on the genome, and can efficiently synthesize p-hydroxycinnamic acid by taking p-hydroxybenzaldehyde and acetic acid as substrates. The p-hydroxybenzaldehyde and the acetic acid are widely sourced, have simple preparation processes and low prices, and are ideal substrates. The selected enzyme has the advantages of high activity and strong optical specificity. Therefore, the recombinant strain of the application has the advantages of high yield, high conversion rate, high production efficiency, green environmental protection and low cost in the conversion production of p-hydroxycinnamic acid, and has a good industrial application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a method for producing p-hydroxycinnamic acid. BACKGROUND

[0002] P-hydroxycinnamic acid, also known as p-coumaric acid, is a natural phenolic compound widely present in plants, has the effects of anti-mutation, anti-genotoxicity, anti-microbial activity, anti-oxidation, whitening, etc., and is widely used in food, cosmetics and medicine fields.

[0003] The preparation methods of p-hydroxycinnamic acid mainly include extraction method, chemical synthesis method and biological transformation method. The extraction method mainly uses natural plants as raw materials, which are treated by acid or alkali hydrolysis, and then extracted by organic solvent to obtain p-hydroxycinnamic acid. The extraction steps are complex and the cost is high, which limits the industrial application of this method. The chemical synthesis method mainly prepares p-hydroxycinnamic acid through condensation reaction. Toxic by-products are easily produced in the synthesis process, the yield is low, and the separation and purification cost is high, which hinders its large-scale application. The biological transformation method has the advantages of high specificity, green environmental protection, mild reaction conditions and no need for multi-step separation and purification, and has attracted widespread attention.

[0004] At present, some domestic and foreign scholars have reported some biological preparation routes of p-hydroxycinnamic acid. For example, Yuqi Liu expresses phenylalanine ammonia lyase (RgPAL) in Escherichia coli, and prepares p-hydroxycinnamic acid by catalyzing L-tyrosine through whole cell. Under the optimal conditions, the yield of p-hydroxycinnamic acid is only 525 mg / L, and the conversion rate is 57.9%. For example, Chinese patent CN117965514A molecularly modifies phenylalanine ammonia lyase (ZmPAL2) from Zea mays, obtains the optimal ZmPAL2 mutant, and prepares p-hydroxycinnamic acid by transforming L-tyrosine. The highest yield of p-hydroxycinnamic acid reaches 3.12 g / L.

[0005] The biological synthesis method in the prior art has the disadvantages of high substrate cost, low yield, low conversion rate and low production efficiency for preparing p-hydroxycinnamic acid. Therefore, how to develop a p-hydroxycinnamic acid preparation method with low cost, wide substrate source, high yield, high conversion rate and high production efficiency is a problem that researchers in the field need to solve urgently.

[0006] In view of this, the present application is proposed. SUMMARY

[0007] The purpose of the present application is to provide a method for producing p-hydroxycinnamic acid. The method is to construct a recombinant bacterium expressing heterologous aldehyde condensing enzyme and fumarase, and then use it as a biological catalyst to convert cheap substrates p-hydroxybenzaldehyde and acetic acid to obtain p-hydroxycinnamic acid.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] like Figure 1 As shown, the synthesis pathway of p-hydroxycinnamic acid adopted by the present invention is: p-hydroxybenzaldehyde and acetic acid are converted into 3-hydroxy-3-(4-hydroxyphenyl)propionic acid by aldolase (ALD), and 3-hydroxy-3-(4-hydroxyphenyl)propionic acid is converted into p-hydroxycinnamic acid by fumarase (FR). Figure 1 The inventors designed a new recombinant bacterium for the p-hydroxycinnamic acid synthesis pathway shown in Figure 1, which is capable of expressing the key enzymes of the synthesis pathway, namely ALD and FR. The inventors screened three enzymes with high p-hydroxycinnamic acid production efficiency from ALD and FR from various sources.

[0010] Among them, ALD includes SsALD derived from Sulfurihydrogenibium sp., LhALD derived from Lachnellulahyalina, and GsALD derived from Geobacter sp.

[0011] FRs include HpFR from Halanaerobium praevalens, OpFR from Oceanithermus profundus, and AaFR from Acetohalobium arabaticum.

[0012] The Genbank number of SsALD is ACD65676.1, its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.4; the Genbank number of LhALD is TVY29750.1, its amino acid sequence is shown in SEQ ID NO.2, and its nucleotide sequence is shown in SEQ ID NO.5; the Genbank number of GsALD is GFE61120.1, its amino acid sequence is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO.6.

[0013] The Genbank number of HpFR is ADO76634.1, its amino acid sequence is shown in SEQ ID NO.7, and its nucleotide sequence is shown in SEQ ID NO.10; the Genbank number of OpFR is ADR36409.1, its amino acid sequence is shown in SEQ ID NO.8, and its nucleotide sequence is shown in SEQ ID NO.11; the Genbank number of AaFR is ADL12064.1, its amino acid sequence is shown in SEQ ID NO.9, and its nucleotide sequence is shown in SEQ ID NO.12.

[0014] The method for constructing the recombinant bacteria comprises the following steps: connecting the genes of ALD and FR to an expression vector, and then introducing the obtained recombinant expression vector into a starting strain to obtain the recombinant bacteria.

[0015] 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, which are not specifically limited in the present application.

[0016] In some embodiments, the Escherichia coli is selected from any one of Escherichia coli BL21 (DE3), Escherichia coli DH5α and Escherichia coli XL-Blue.

[0017] In some embodiments, the expression vector comprises pCDFDuet-1 plasmid and pACYCDuet-1 plasmid.

[0018] The above expression vector can be used to construct the following recombinant vectors: pCDFDuet-SsALD, pCDFDuet-LhALD, pCDFDuet-GsALD, pACYCDuet-HpFR, pACYCDuet-OpFR and pACYCDuet-AaFR.

[0019] More preferably, in the construction of the recombinant Escherichia coli, the combination of pCDFDuet-LhALD and pACYCDuet-OpFR can make the production of p-hydroxycinnamic acid higher.

[0020] Further, the present application also provides a whole-cell catalyst containing the above-mentioned recombinant bacteria.

[0021] The above-mentioned recombinant bacteria as a whole-cell catalyst can efficiently convert p-hydroxybenzaldehyde and acetic acid into p-hydroxycinnamic acid by expressing ALD and FR.

[0022] The present application also provides the use of the above-mentioned recombinant bacteria in the synthesis of p-hydroxycinnamic acid and its downstream products. Based on this, the present application can use the recombinant bacteria to produce p-hydroxycinnamic acid, and the method is as follows:

[0023] The above-mentioned recombinant bacteria is added to a reaction system containing p-hydroxybenzaldehyde and acetic acid for whole-cell catalysis to obtain p-hydroxycinnamic acid.

[0024] In some embodiments, the recombinant bacteria is induced and cultured before whole cell transformation, and the induction and culture process is as follows: the recombinant bacteria is inoculated into LB medium containing 30-60 mg / L chloramphenicol and 30-60 mg / L streptomycin, and a seed liquid is obtained after culture; then the seed liquid is inoculated into fresh LB medium until the OD600nm of the bacteria concentration reaches 0.6-0.8, an inducer is added, and the wet bacteria are separated and washed after induction.

[0025] In some embodiments, the culture conditions of the above-mentioned recombinant bacteria are as follows: the temperature is 32-38℃, the rotation speed is 150-250 rpm, and the culture time is 10-16 h.

[0026] In some embodiments, the culture conditions of the above-mentioned seed liquid are as follows: the temperature is 32-38℃, and the rotation speed is 150-250 rpm.

[0027] In some embodiments, the induction conditions are as follows: the inducer is 0.2-0.6 mM IPTG, the temperature is 25-30℃, and the time is 10-16 h.

[0028] In some embodiments, the separation and washing conditions are as follows: the rotation speed is 7500-8500 rpm, and the centrifugation time is 5-15 min; the wet bacteria are washed with saline, and the concentration of the saline is 0.9%.

[0029] In some embodiments, the reaction system for whole cell catalysis comprises: 1-10 g / L wet weight of the recombinant bacteria, 1-60 g / L p-hydroxybenzaldehyde, 1-35 g / L acetic acid, and 0.01-0.05 g / L pyridoxal phosphate (PLP).

[0030] In some embodiments, the reaction conditions for whole cell catalysis are as follows: the pH is 6.0-9.0, the temperature is 15-40℃, the rotation speed is 150-250 rpm, and the reaction time is 8-24 h.

[0031] The present application has the following advantages:

[0032] The present application constructs a recombinant strain capable of efficiently synthesizing p-hydroxycinnamic acid by using Escherichia coli as a starting strain, and the genome of the recombinant strain is integrated with the genes of heterologous aldehyde aldolase and fumarase, which can efficiently synthesize p-hydroxycinnamic acid by using p-hydroxybenzaldehyde and acetic acid as substrates. Among them, p-hydroxybenzaldehyde and acetic acid are widely available, simple to prepare, and low in price, and are ideal substrates. The selected enzymes have the advantages of high activity and strong optical specificity, etc. Therefore, the recombinant bacteria of the present application has high yield, high conversion rate, high production efficiency, green environmental protection, and low cost in the production of p-hydroxycinnamic acid by transformation, and has good industrial application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some of the embodiments of the present application, and therefore should not be regarded as a limitation on the scope, and for those of ordinary skill in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0034] Figure 1 The synthesis route of p-hydroxycinnamic acid of the present application. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments not mentioned by the manufacturers are all conventional products that can be purchased in the market.

[0036] The features and performances of the present application will be further described in detail below in combination with the embodiments.

[0037] 1. Selection of bacteria and plasmids

[0038] The pCDFDuet-1 plasmid, pACYCDuet-1 plasmid, Escherichia coli BL21(DE3), Escherichia coli DH5α and Escherichia coli XL-Blue purchased from Novagen company.

[0039] 2. Selection of enzymes

[0040] (1) Selection of aldolase

[0041] The amino acid sequences of aldolases SsALD, LhALD and GsALD were obtained from the NCBI database, and the codons were optimized according to the preference of Escherichia coli. Two nucleotide sequences were synthesized by conventional genetic engineering operations in a total synthesis method, as shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6. The amino acid sequences of the enzymes are shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3. Enzymatic cleavage sites EcoRI and HindIII were added at both ends of the nucleotide sequences.

[0042] (2) Selection of fumarase

[0043] The amino acid sequences of fumarase HpFR, OpFR and AaFR were obtained from the NCBI database, and the codons were optimized according to the preference of E. coli. Two nucleotide sequences were synthesized by conventional genetic engineering methods, as shown in SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12. The amino acid sequences of the enzymes are shown in SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9. Enzyme cleavage sites EcoRI and HindIII were added at both ends of the nucleotide sequence.

[0044] 3. Construction of double-enzyme co-expression system and cultivation of bacterial cells

[0045] Each type of the selected aldehyde dehydrogenase and fumarase was co-expressed with one enzyme selected from each type. The coding genes of two enzymes were co-expressed by using pCDFDuet-1 and pACYCDuet-1 double plasmids; pCDFDuet-1 loaded aldehyde dehydrogenase, and pACYCDuet-1 loaded fumarase. After obtaining the co-expression recombinant plasmid, the two recombinant plasmids were simultaneously transformed into Escherichia coli BL21 (DE3) competent cells, and positive transformants were screened by using a plate containing streptomycin and chloramphenicol, thereby obtaining recombinant E. coli. The obtained recombinant bacteria were inoculated into fresh liquid medium, induced and cultured, centrifuged, and wet bacterial cells were obtained.

[0046] 4. Preparation of p-hydroxycinnamic acid from p-hydroxybenzaldehyde and acetic acid by whole-cell transformation

[0047] Transformation system: the concentration of p-hydroxybenzaldehyde was 1-60 g / L, the concentration of acetic acid was 1-35 g / L, the concentration of pyridoxal phosphate (PLP) was 0.01-0.05 g / L, the pH was adjusted to 6.0-9.0, the amount of fresh bacterial cells was 1-10 g / L, then the transformation was carried out at 15-40℃, 200 rpm, for 8-24 h. After the transformation was completed, the yield of p-hydroxycinnamic acid was determined by liquid chromatography.

[0048] 5. Detection and analysis of samples

[0049] The transformation liquid was analyzed by Shimadzu 2030C high performance liquid chromatograph (HPLC). The chromatographic conditions were as follows: the mobile phase was methanol: water (v / v = 1:1), an Inertsustain C18 chromatographic column (4.6 x 250 mm, 5 μm) was used, the flow rate was 1 mL / min, the column temperature was 30℃, the injection volume was 20 μL, and the detection wavelength was 309 nm.

[0050] Example 1

[0051] Construction of recombinant E. coli

[0052] The recombinant plasmid 1 was obtained by connecting the ALD from different sources to the pCDFDuet-1 vector through T4 DNA ligase after double enzyme digestion of the synthetized ALD recombinant plasmid and the pCDFDuet-1 vector by restriction enzymes EcoRI and HindIII; the recombinant plasmid 2 was obtained by connecting the FR from different sources to the pACYCDuet-1 vector through T4 DNA ligase after double enzyme digestion of the synthetized FR recombinant plasmid and the pACYCDuet-1 vector by restriction enzymes EcoRI and HindIII; and the recombinant E. coli was obtained by transforming the different recombinant plasmids 1 and 2 into the E. coli BL21 (DE3) competent cells.

[0053] Example 2

[0054] Induced culture of the recombinant E. coli

[0055] The recombinant E. coli was inoculated into the 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 the seed liquid. The seed liquid was inoculated into fresh LB medium at an inoculation amount of 2%, and cultured at 37°C and 200 rpm until the bacterial concentration reached OD600 of 0.7. Then, 0.5 mM IPTG was added, and the culture was induced at 28°C for 15 h. After centrifugation at 8000 rpm for 10 min, the supernatant was discarded, and the wet bacterial cells were washed twice with 0.9% physiological saline, and then centrifuged for standby. 600nm

[0056] Example 3

[0057] Comparison of transformation abilities of various recombinant E. coli

[0058] The collected recombinant E. coli was resuspended in 50 mL system, and the final concentration of the cells was 10 g / L, the concentration of p-hydroxybenzaldehyde was 60 g / L, the concentration of acetic acid was 35 g / L, the concentration of PLP was 0.05 g / L, and the pH was 7.0. The reaction was carried out at 30°C, and the rotation speed of the shaking table was 200 rpm. The transformation time was 24 h. After the transformation was completed, the yield of p-hydroxycinnamic acid was determined by HPLC.

[0059] Table 1 Comparison of the yields of p-hydroxycinnamic acid of various recombinant bacteria

[0060]

[0061] Example 4

[0062] ​According to the induction expression method described in Embodiment 2, the E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was collected after induction expression was completed, and was placed in a 50 mL system with a cell wet weight of 1 g / L, p-hydroxybenzaldehyde of 1 g / L, acetic acid of 1 g / L, PLP of 0.01 g / L, pH 8.0, a temperature of 30°C, a shaking bed rotation speed of 200 rpm, and a conversion time of 24 h. The HPLC determination results showed that the p-hydroxycinnamic acid yield was 1.31 g / L, the conversion rate was 97.5%, and the production efficiency was 0.05 g / L / h.

[0063] Embodiment 5

[0064] According to the induction expression method described in Embodiment 2, the E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was collected after induction expression was completed, and was placed in a 50 mL system with a cell wet weight of 1 g / L, p-hydroxybenzaldehyde of 1 g / L, acetic acid of 1 g / L, PLP of 0.01 g / L, pH 8.0, a temperature of 30°C, a shaking bed rotation speed of 200 rpm, and a conversion time of 24 h. The HPLC determination results showed that the p-hydroxycinnamic acid yield was 1.31 g / L, the conversion rate was 97.5%, and the production efficiency was 0.05 g / L / h.

[0065] Embodiment 6

[0066] According to the induction expression method described in Embodiment 2, the E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was collected after induction expression was completed, and was placed in a 50 mL system with a cell wet weight of 1 g / L, p-hydroxybenzaldehyde of 1 g / L, acetic acid of 1 g / L, PLP of 0.01 g / L, pH 8.0, a temperature of 30°C, a shaking bed rotation speed of 200 rpm, and a conversion time of 24 h. The HPLC determination results showed that the p-hydroxycinnamic acid yield was 1.31 g / L, the conversion rate was 97.5%, and the production efficiency was 0.05 g / L / h.

[0067] Embodiment 7

[0068] According to the induction expression method described in Embodiment 2, the E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was collected after induction expression was completed, and was placed in a 50 mL system with a cell wet weight of 1 g / L, p-hydroxybenzaldehyde of 1 g / L, acetic acid of 1 g / L, PLP of 0.01 g / L, pH 8.0, a temperature of 30°C, a shaking bed rotation speed of 200 rpm, and a conversion time of 24 h. The HPLC determination results showed that the p-hydroxycinnamic acid yield was 1.31 g / L, the conversion rate was 97.5%, and the production efficiency was 0.05 g / L / h.

[0069] Example 8

[0070] According to the induction expression method described in Example 2, after E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was induced to complete expression, the bacterial cells were collected in a 50 mL system, the wet weight of the cells was 6 g / L, p-hydroxybenzaldehyde was 34 g / L, acetic acid was 19 g / L, PLP was 0.03 g / L, pH was 8.0, the temperature was 30°C, the shaking speed was 200 rpm, and the conversion time was 24 h. The HPLC determination results 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.

[0071] Example 9

[0072] According to the induction expression method described in Example 2, after E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was induced to complete expression, the bacterial cells were collected in a 50 mL system, the wet weight of the cells was 7 g / L, p-hydroxybenzaldehyde was 43 g / L, acetic acid was 23 g / L, PLP was 0.05 g / L, pH was 8.0, the temperature was 30°C, the shaking speed was 200 rpm, and the conversion time was 24 h. The HPLC determination results 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.

[0073] Example 10

[0074] According to the induction expression method described in Example 2, after E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was induced to complete expression, the bacterial cells were collected in a 50 mL system, the wet weight of the cells was 9 g / L, p-hydroxybenzaldehyde was 53 g / L, acetic acid was 30 g / L, PLP was 0.05 g / L, pH was 8.0, the temperature was 30°C, the shaking speed was 200 rpm, and the conversion time was 24 h. The HPLC determination results 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.

[0075] Example 11

[0076] According to the induction expression method described in Embodiment 2, the E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was collected after induction expression was completed, and was placed in a 50 mL system with a cell wet weight of 10 g / L, p-hydroxybenzaldehyde of 30 g / L, acetic acid of 16 g / L, PLP of 0.05 g / L, pH 8.0, a temperature of 30°C, a shaking bed rotation speed of 200 rpm, and a conversion time of 12 h. The HPLC determination results showed that the p-hydroxycinnamic acid yield was 39.3 g / L, the conversion rate was 97.5%, and the production efficiency was 3.28 g / L / h.

[0077] Embodiment 12

[0078] According to the induction expression method described in Embodiment 2, the E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was collected after induction expression was completed, and was placed in a 50 mL system with a cell wet weight of 10 g / L, p-hydroxybenzaldehyde of 30 g / L, acetic acid of 16 g / L, PLP of 0.05 g / L, pH 8.0, a temperature of 30°C, a shaking bed rotation speed of 200 rpm, and a conversion time of 12 h. The HPLC determination results showed that the p-hydroxycinnamic acid yield was 39.3 g / L, the conversion rate was 97.5%, and the production efficiency was 3.28 g / L / h.

[0079] Embodiment 13

[0080] According to the induction expression method described in Embodiment 2, the E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was collected after induction expression was completed, and was placed in a 50 mL system with a cell wet weight of 10 g / L, p-hydroxybenzaldehyde of 30 g / L, acetic acid of 16 g / L, PLP of 0.05 g / L, pH 8.0, a temperature of 30°C, a shaking bed rotation speed of 200 rpm, and a conversion time of 12 h. The HPLC determination results showed that the p-hydroxycinnamic acid yield was 39.3 g / L, the conversion rate was 97.5%, and the production efficiency was 3.28 g / L / h.

[0081] Embodiment 14

[0082] According to the induction expression method described in Embodiment 2, the E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was collected after induction expression was completed, and was placed in a 50 mL system with a cell wet weight of 10 g / L, p-hydroxybenzaldehyde of 30 g / L, acetic acid of 16 g / L, PLP of 0.05 g / L, pH 8.0, a temperature of 30°C, a shaking bed rotation speed of 200 rpm, and a conversion time of 12 h. The HPLC determination results showed that the p-hydroxycinnamic acid yield was 39.3 g / L, the conversion rate was 97.5%, and the production efficiency was 3.28 g / L / h.

[0083] Example 15

[0084] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of the cells was 6 g / L, p-hydroxybenzaldehyde was 18 g / L, acetic acid was 10 g / L, PLP was 0.04 g / L, pH was 6.0, the temperature was 35°C, the shaking speed was 200 rpm, and the conversion time was 12 h. The HPLC determination results 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.

[0085] Example 16

[0086] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of the cells was 5 g / L, p-hydroxybenzaldehyde was 15 g / L, acetic acid was 8 g / L, PLP was 0.03 g / L, pH was 7.0, the temperature was 35°C, the shaking speed was 200 rpm, and the conversion time was 12 h. The HPLC determination results 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.

[0087] Example 17

[0088] According to the induction expression method described in Example 2, after the induction expression of E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was completed, the bacterial cells were collected, and in a 50 mL system, the wet weight of the cells was 4 g / L, p-hydroxybenzaldehyde was 11 g / L, acetic acid was 6 g / L, PLP was 0.03 g / L, pH was 7.5, the temperature was 35°C, the shaking speed was 200 rpm, and the conversion time was 12 h. The HPLC determination results 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.

[0089] Example 18

[0090] According to the induction expression method described in Embodiment 2, the E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was collected after induction expression was completed, and was placed in a 50 mL system with a cell wet weight of 2 g / L, p-hydroxybenzaldehyde of 7 g / L, acetic acid of 4 g / L, PLP of 0.02 g / L, pH 8.5, a temperature of 35 °C, a shaking bed rotation speed of 200 rpm, and a conversion time of 12 h. The HPLC determination results showed that the production 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.

[0091] Embodiment 19

[0092] According to the induction expression method described in Embodiment 2, the E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was collected after induction expression was completed, and was placed in a 50 mL system with a cell wet weight of 2 g / L, p-hydroxybenzaldehyde of 7 g / L, acetic acid of 4 g / L, PLP of 0.02 g / L, pH 8.5, a temperature of 35 °C, a shaking bed rotation speed of 200 rpm, and a conversion time of 12 h. The HPLC determination results showed that the production 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.

[0093] Embodiment 20

[0094] According to the induction expression method described in Embodiment 2, the E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was collected after induction expression was completed, and was placed in a 50 mL system with a cell wet weight of 2 g / L, p-hydroxybenzaldehyde of 7 g / L, acetic acid of 4 g / L, PLP of 0.02 g / L, pH 8.5, a temperature of 35 °C, a shaking bed rotation speed of 200 rpm, and a conversion time of 12 h. The HPLC determination results showed that the production 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.

[0095] Embodiment 21

[0096] According to the induction expression method described in Embodiment 2, the E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was collected after induction expression was completed, and was placed in a 50 mL system with a cell wet weight of 2 g / L, p-hydroxybenzaldehyde of 7 g / L, acetic acid of 4 g / L, PLP of 0.02 g / L, pH 8.5, a temperature of 35 °C, a shaking bed rotation speed of 200 rpm, and a conversion time of 12 h. The HPLC determination results showed that the production 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.

[0097] Example 22

[0098] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was induced and cells were collected. In a 50 mL system, the following conditions were added: 5 g / L wet cell weight, 31 g / L p-hydroxybenzaldehyde, 18 g / L acetic acid, 0.03 g / L PLP, pH 7.5, 40°C, a shaker speed of 200 rpm, and a conversion time of 24 h. HPLC analysis revealed a p-hydroxycinnamic acid yield of 40.7 g / L, a conversion rate of 97.7%, and a production efficiency of 1.7 g / L / h.

[0099] Comparative Example 1

[0100] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was induced and cells were collected. In a 50 mL system, the following conditions were added: 12 g / L cell wet weight, 70 g / L p-hydroxybenzaldehyde, 40 g / L acetic acid, 0.08 g / L PLP, pH 7.5, 35°C, a shaker speed of 200 rpm, and a conversion time of 36 h. HPLC analysis revealed a p-hydroxycinnamic acid yield of 14.1 g / L, a conversion rate of 15.0%, and a production efficiency of 0.39 g / L / h.

[0101] Comparative Example 2

[0102] Following the induction expression method described in Example 2, E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was induced and cells were collected. In a 50 mL system, the following conditions were added: 5 g / L wet cell weight, 30 g / L p-hydroxybenzaldehyde, 20 g / L acetic acid, 0.03 g / L PLP, pH 5.5, temperature 35°C, shaker speed 200 rpm, and conversion time 36 h. HPLC analysis revealed a p-hydroxycinnamic acid yield of 5.24 g / L, a conversion rate of 13.0%, and a production efficiency of 0.15 g / L / h.

[0103] Comparative Example 3

[0104] According to the induction expression method described in Embodiment 2, the E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was collected after induction expression was completed, and was placed in a 50 mL system with a cell wet weight of 3 g / L, p-hydroxybenzaldehyde of 20 g / L, acetic acid of 12 g / L, PLP of 0.03 g / L, pH of 9.5, temperature of 35°C, shaking speed of 200 rpm, and conversion time of 36 h. The HPLC determination results showed that the p-coumaric acid yield was 4.84 g / L, the conversion rate was 18.0%, and the production efficiency was 0.13 g / L / h.

[0105] Comparative Example 4

[0106] According to the induction expression method described in Embodiment 2, the E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was collected after induction expression was completed, and was placed in a 50 mL system with a cell wet weight of 3 g / L, p-hydroxybenzaldehyde of 20 g / L, acetic acid of 12 g / L, PLP of 0.03 g / L, pH of 9.5, temperature of 35°C, shaking speed of 200 rpm, and conversion time of 36 h. The HPLC determination results showed that the p-coumaric acid yield was 4.84 g / L, the conversion rate was 18.0%, and the production efficiency was 0.13 g / L / h.

[0107] Comparative Example 5

[0108] According to the induction expression method described in Embodiment 2, the E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was collected after induction expression was completed, and was placed in a 50 mL system with a cell wet weight of 3 g / L, p-hydroxybenzaldehyde of 20 g / L, acetic acid of 12 g / L, PLP of 0.03 g / L, pH of 9.5, temperature of 35°C, shaking speed of 200 rpm, and conversion time of 36 h. The HPLC determination results showed that the p-coumaric acid yield was 4.84 g / L, the conversion rate was 18.0%, and the production efficiency was 0.13 g / L / h.

[0109] Comparative Example 6

[0110] According to the induction expression method described in Embodiment 2, the E. coli BL21(DE3) / pCDFDuet-LhALD+pACYCDuet-OpFR was collected after induction expression was completed, and was placed in a 50 mL system with a cell wet weight of 3 g / L, p-hydroxybenzaldehyde of 20 g / L, acetic acid of 12 g / L, PLP of 0.03 g / L, pH of 9.5, temperature of 35°C, shaking speed of 200 rpm, and conversion time of 36 h. The HPLC determination results showed that the p-coumaric acid yield was 4.84 g / L, the conversion rate was 18.0%, and the production efficiency was 0.13 g / L / h.

[0111] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. The present application can be variously changed and modified by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the present application.

Claims

1. A recombinant bacterium for producing p-hydroxycinnamic acid, characterized in that: The genome of the recombinant bacterium is integrated with heterologous aldolase and fumarase genes; The aldolase source Lachnellula hyalina ; The fumarase is derived from Oceanithermus profundus ; The aldolase is LhALD with an amino acid sequence as shown in SEQ ID NO.2; the fumarase is OpFR with an amino acid sequence as shown in SEQ ID NO.8; The starting strain of the recombinant bacteria is Escherichia coli.

2. The recombinant bacterium according to claim 1, characterized in that The nucleotide sequence of LhALD is shown in SEQ ID NO.

5.

3. The recombinant bacterium according to claim 1, characterized in that The nucleotide sequence of the OpFR is shown in SEQ ID NO.

11.

4. The recombinant bacterium according to claim 1, characterized in that The Escherichia coli includes Escherichia coli BL21(DE3), Escherichia coli DH5α and Escherichia coli XL-Blue.

5. The method for preparing the recombinant bacteria according to any one of claims 1 to 4, characterized in that: include: The genes of aldolase and fumarase 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 bacteria.

6. The preparation method according to claim 5, characterized in that The expression vectors include pCDFDuet-1 and pACYCDuet-1.

7. The preparation method according to claim 5, characterized in that The preparation method of the recombinant bacteria comprises: inserting the genes of LhALD and OpFR into an expression vector to obtain a recombinant vector, then introducing the recombinant vector into a starting strain, and cultivating and screening to obtain the recombinant bacteria producing p-hydroxycinnamic acid.

8. The preparation method according to claim 7, characterized in that The recombinant vector includes: pCDFDuet-LhALD and pACYCDuet-OpFR.

9. A whole-cell catalyst, characterized in that Contains the recombinant bacteria according to any one of claims 1 to 4.

10. Use of the recombinant bacterium according to any one of claims 1 to 4 in the synthesis of p-hydroxycinnamic acid, characterized in that: The substrates for synthesizing p-hydroxycinnamic acid using the recombinant bacteria are p-hydroxybenzaldehyde and acetic acid; The reaction system for converting the substrate into p-hydroxycinnamic acid by the recombinant bacteria 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; The reaction conditions are: pH=6.0-9.0, temperature 15-40°C, and reaction time 8-24h.

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

12. The method according to claim 11, 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.

13. The method according to claim 11, characterized in that The reaction conditions of the whole-cell catalysis are: pH=6.0-9.0, temperature 15-40° C., and reaction time 8-24 h.

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