CN116904417A - Method for producing ferulic acid by using caffeic acid-O-methyltransferase and application thereof - Google Patents

Method for producing ferulic acid by using caffeic acid-O-methyltransferase and application thereof Download PDF

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CN116904417A
CN116904417A CN202310929672.XA CN202310929672A CN116904417A CN 116904417 A CN116904417 A CN 116904417A CN 202310929672 A CN202310929672 A CN 202310929672A CN 116904417 A CN116904417 A CN 116904417A
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caffeic acid
methyltransferase
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ferulic acid
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张岩峰
梁美祺
毛启红
陈永丽
潘朝智
顾丽红
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Shenzhen Upfo Biotech Co ltd
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Abstract

The invention discloses a method for producing ferulic acid by using caffeic acid-O-methyltransferase and application thereof, belonging to the technical field of bioengineering. The invention overexpresses caffeic acid-O-methyltransferase COMT25468 and S-adenosylmethionine synthetase MetK screened in a North-pole moss metagenome sample in escherichia coli, and knocks out SAM degradation pathway gene speD to construct escherichia coli genetic engineering bacteria. The escherichia coli genetic engineering bacteria are fermented in a 5L fermentation tank for 72 hours, the accumulation amount of ferulic acid reaches 12g/L, and the molar conversion rate of the caffeic acid is 74.2%.

Description

Method for producing ferulic acid by using caffeic acid-O-methyltransferase and application thereof
Technical Field
The invention relates to a method for producing ferulic acid by using caffeic acid-O-methyltransferase and application thereof, belonging to the technical field of bioengineering.
Background
Caffeic acid-O-methyltransferase (COMT), which is an important precursor for ferulic acid biosynthesis, catalyzes the direct production of ferulic acid from caffeic acid. Therefore, caffeic acid-O-methyltransferase is one of key enzymes for ferulic acid biosynthesis, and mainly uses adenosylmethionine (S-adenosyl methionine, SAM) as methyl donor to catalyze methylation of various substrates, which is ascribed to no need of Mg 2+ O-methyltransferase (OMT) of type II plants, is mainly localized in the cytoskeleton and cytoplasm.
Ferulic acid is a natural methylated phenolic acid, the chemical name of which is 4-hydroxy-3-methoxy cinnamic acid, and is widely found in plants in nature. The ferulic acid has strong oxidation resistance, and can well remove hydrogen peroxide, hydroxyl free radicals, superoxide free radicals and nitroso peroxide. Meanwhile, the ferulic acid also has the function of regulating physiological functions, not only inhibits the enzyme generating free radicals, but also can increase the activity of eliminating the free radical enzyme. Ferulic acid is easy to be absorbed by human body, can be discharged from urine metabolism, has low toxicity and safer use, and has more and more important medical value.
Currently, methods for producing ferulic acid mainly include plant extraction, chemical synthesis and biological synthesis. The plant extraction method destroys other high-value chemical components in the plant in an acid-base hydrolysis mode, and meanwhile, byproducts are increased, and products are difficult to separate, so that the energy consumption is high and the environment is polluted. The chemical synthesis method synthesizes ferulic acid through an organic reaction, has long reaction time, causes serious environmental pollution by using toxic or environment-friendly catalysts, requires a large amount of energy for production, and generates toxic intermediate products or byproducts. Thus, the production of ferulic acid by synthetic biological methods starts to be more visible.
Through a synthetic biological strategy, construction of engineering bacteria with ferulic acid synthesis capability is the main research idea at present. The reported ferulic acid synthesis engineering bacteria mainly take caffeic acid as a precursor, and the caffeic acid-O-methyltransferase (COMT for short) with high catalytic activity is heterologously expressed in escherichia coli to generate the ferulic acid. Meanwhile, the production of ferulic acid by using caffeic acid requires S-adenosylmethionine (SAM) as a methyl donor, and hypomethylation is the biggest challenge of ferulic acid biosynthesis at present. Screening for highly active caffeic acid-O-methyltransferase and increasing SAM utilization is therefore necessary for ferulic acid production.
Disclosure of Invention
The invention provides caffeic acid-O-methyltransferase (named COMT 25468) screened in a North-pole moss metagenome sample, and the amino acid sequence of the caffeic acid-O-methyltransferase is shown as SEQ ID NO. 1.
The invention also provides a gene comT for encoding the caffeic acid-O-methyltransferase, and the nucleotide sequence of the gene comT is shown as SEQ ID NO.2.
The invention also provides expression vectors carrying the genes, including but not limited to pBAD/HisA.
The invention provides a genetically engineered bacterium of escherichia coli, over-expresses caffeic acid-O-methyltransferase COMT25468 and S-adenosylmethionine synthetase MetK, and knocks out SAM degradation pathway gene speD; the amino acid sequence of the caffeic acid-O-methyltransferase COMT25468 is shown in SEQ ID NO. 1; the amino acid sequence of the S-adenosylmethionine synthetase is shown as SEQ ID NO. 4; the nucleotide sequence of the gene speD is shown as SEQ ID NO. 6.
In one embodiment, caffeic acid-O-methyltransferase is expressed in free form.
In one embodiment, the promoter of the S-adenosylmethionine synthetase gene metK on the genome is replaced by the promoter shown in SEQ ID NO. 7.
The invention also provides a method for producing ferulic acid, which comprises the steps of fermenting the escherichia coli genetic engineering bacteria in a culture medium containing glucose, and feeding caffeic acid and methionine.
In one embodiment, the medium comprises: glucose, KH 2 PO 4 、(NH 4 ) 2 HPO 4 、MgSO 4 ·7H 2 O, citric acid, trace elements and defoamer; the microelements comprise: feSO 4 ·7H 2 O、ZnSO 4 ·7H 2 O、CuSO 4 ·5H 2 O、MnSO 4 ·5H 2 O、CaCl 2 ·2H 2 O and Na 2 B 4 O 7 ·5H 2 O。
In one embodiment, the conditions of the fermentation are: 30-37 ℃, the rotation speed of the stirring paddle is 300-700 rpm, the pH value of the fermentation liquor is 6.5-7.4, and the ventilation rate is 0.5-1.5 vvm.
In one embodiment, when the E.coli genetically engineered bacterium is in mid-log phase, 1.5-2g/L L-arabinose is added for induction for at least 6 hours.
In one embodiment, the mid-log phase refers to the OD of the genetically engineered E.coli 600 25-35.
In one embodiment, the caffeic acid-containing solution is fed after induction to maintain a caffeic acid content of 5-7g/L.
The invention also provides application of the escherichia coli genetic engineering bacteria in production of ferulic acid.
The beneficial effects are that:
the invention provides a caffeic acid-O-methyltransferase (named COMT 25468) screened in a North-pole moss metagenome sample, the amino acid sequence of which is shown as SEQ ID NO.1, and the yield and the production efficiency of ferulic acid are improved by enhancing SAM supply and taking caffeic acid as a substrate. And (3) overexpressing caffeic acid-O-methyltransferase COMT25468 and S-adenosylmethionine synthetase metK in a North-pole moss metagenome sample in the escherichia coli, and knocking out SAM degradation pathway gene speD to construct the escherichia coli genetic engineering bacteria. The escherichia coli genetic engineering bacteria are fermented in a 5L fermentation tank for 72 hours, the accumulation amount of ferulic acid reaches 12g/L, and the molar conversion rate of the caffeic acid is 74.2%.
Drawings
Fig. 1: screening caffeic acid-O-methyltransferase and homology comparison;
fig. 2: the BW1 strain (a) and the BW2 strain (b) catalyze caffeic acid to synthesize ferulic acid, and the yield is evaluated;
fig. 3: the conversion efficiency of catalyzing caffeic acid to produce ferulic acid in different chassis bacteria is evaluated by COMT 25468;
fig. 4: and (5) evaluating the yield of the caffeic acid synthesized ferulic acid on the fermentation tank of the FA4 strain.
Detailed Description
The main reagents, instruments, genetic material and media used in the following examples: ferulic acid standard (source leaf organism, shanghai yuanye Bio-Technology co., ltd.) the comT25468 gene was synthesized by the engine organism (Tsingke biotechnology co., ltd.) and the gene sequence was SEQ ID No.2.
The culture medium in the following examples is as follows:
seed medium (LB) (1L): 5g of yeast powder, 10g of tryptone and 10g of sodium chloride.
M9 medium (1L): glucose 10g, NH 4 Cl 1g，Na 2 HPO 4 6g，KH2PO 4 3g，NaCl 0.5g，MgSO 4 240mg，CaCl 2 5.35mg, VB1 2mg, trace element (H) 3 BO 3 1.25mg，Na 2 MoO 4 ·2H 2 O 0.15mg，CoCl 2 ·6H 2 O 0.7mg，CuSO 4 ·5H 2 O 0.25mg，MnCl 2 ·4H 2 O 1.6mg，ZnSO 4 ·7H 2 O 0.3mg)。
Fermentation medium (1L): glucose 5.0g, KH 2 PO 4 6.65g，(NH 4 ) 2 HPO 4 2.0g，MgSO 4 ·7H 2 O0.6 g, citric acid 0.85g, trace elements (FeSO 4 ·7H 2 O 100mg，ZnSO 4 ·7H 2 O 22.5mg，CuSO 4 ·5H 2 O10mg，MnSO 4 ·5H 2 O 5mg，CaCl 2 ·2H 2 O 20mg，Na 2 B 4 O 7 ·5H 2 O 2.3mg)。
The detection method involved in the following examples is as follows:
1. sample preparation: centrifuging the fermentation broth sample at 12000rpm for 1min, collecting supernatant, and mixing with 5% methanol and 85% ddH 2 After 10-fold dilution of O, filtration was performed using a 0.22 μm filter.
2. Liquid phase conditions: chromatographic column: infinityLab Poroshell HPH-C18, 4.6X105 mm,2.7-Micron; mobile phase a: water (0.1% acetic acid), mobile phase B: pure methanol. Elution procedure: 0-4 min,5% B; 4-14 min, 5-85% B; 14-16 min, 85-5% B; 16-20 min,5% B. Flow rate: 1.0mL/min, column temperature: 30 ℃, sample injection volume: 10 mu L. A detector: DAD (Diode array Detector) detector, wavelength detected: 323nm. And (3) determining target substances by comparing the target substances with retention time and a spectrogram of a standard substance, and quantitatively analyzing caffeic acid and ferulic acid according to peak areas.
3. The catalytic efficiency of the enzyme is characterized by molar conversion, calculated as follows:
4. the caffeic acid-O-methyltransferase enzyme activity definition criteria are:
1IU = under the optimal reaction conditions, the amount of enzyme required to catalyze 1 micromolar caffeic acid to ferulic acid for 1 minute is determined to be 1 enzyme activity unit, i.e., 1IU = 1 μmol/min.
Specific enzyme activity = activity IU/protein mg.
Example 1: screening and homology comparison of caffeic acid-O-methyltransferase
Protein sequences of arctic moss metagenomic samples were analyzed for homology using the BLAST function of NCBI with the commonly reported 5 caffeic acid-O-methyltransferases, atCOMT (from arabidopsis thaliana), osccomt (from rice alfalfa), ntCOMT (from tobacco), msCOMT (from alfalfa), ptCOMT (from populus tomentosa), respectively. The amino acid sequences are aligned as shown in FIG. 1. The 25468 enzyme and the 5 enzymes have 5 sequence conservation regions, so the enzyme is considered to be caffeic acid-O-methyltransferase, named COMT25468, and the amino acid sequence is shown as SEQ ID NO. 1.
Example 2: construction of pBAD-COMT25468 and pBAD-AtCOMT plasmids
In order to verify the catalytic activity of COMT25468 obtained by screening in the arctic moss metagenome, an engineering strain is constructed by taking caffeic acid-O-methyltransferase AtCOMT which is most commonly used in the current literature and has the best effect as a control, and a ferulic acid production capacity comparison experiment is carried out.
The pBAD/HisA vector is amplified and linearized by using the primers pBAD-ZT-F and pBAD-ZT-R by taking pBAD/HisA as a template and referring to a method of constructing homologous recombinant plasmids; the artificially synthesized COMT25468 gene (shown as SEQ ID NO. 2) and the AtCOMT gene (shown as SEQ ID NO. 3) are respectively used as templates, the primers COMT25468-F and COMT25468-R are used for amplification to obtain a COMT25468 gene fragment, and the primers AtCOMT-F and AtCOMT-R are used for amplification to obtain an AtCOMT gene fragment; the vector was then ligated with the fragment using homologous recombinase, and plasmid construction was performed using E.coli DH 5. Alpha (commercial) to construct plasmids pBAD-COMT25468 and pBAD-AtCOMT.
TABLE 1 construction of primer sequences for caffeic acid-O-methyltransferase plasmids
Example 3: purification of enzyme and conversion efficiency of catalyzing caffeic acid to produce ferulic acid
The pBAD-COMT25468 and pBAD-AtCOMT plasmids constructed in example 2 were transformed into BW25113 wild-type E.coli, respectively designated BW1 (experimental group) and BW2 (control group), and the obtained BW1 and BW2 strains were transferred into LB liquid medium containing 100. Mu.g/mL ampicillin, respectively, and cultured at 37℃and 200rpm for 8 hours. Then 1% (v/v) of the inoculated matter was transferred to a test tube containing 200mL of LB liquid medium, and ampicillin was added thereto at a final concentration of 100. Mu.g/mL, and the culture was carried out at 37℃and 200rpm for 3 hours to OD 600nm About 0.6-0.8, and then L-arabinose with a final concentration of 2g/L was added as inducer, and the culture was continued at 30℃and 200rpm for 12-14 hours toOD 600nm And (3) centrifuging at 5000rpm and 4 ℃ for 5min to collect the bacterial cells. The cells were resuspended in 50mM Tris-HCl buffer pH 7.4 to a final OD 600nm 25-30, recombinant E.coli cell suspension was obtained, and after sonication (20% power, 5 min), centrifugation (12000 rpm,30 min), the supernatant was obtained. The supernatant was filtered through a 0.22 μm filter, and the filtrates were COMT25468 crude enzyme and AtCOMT crude enzyme. By using Ni previously equilibrated by Tris-HCl of 100mL 50mM pH 7.4 (buffer 1) 2+ Crude enzyme supernatant (15 mL) was loaded onto an IMAC column (0.8 cm) 2 X10 cm,1.5 mL/min). After a washing step with buffer 2 containing 0.3M NaCl and 20mM imidazole, the proteins were eluted with buffer 3 (50 mM Tris-HCl pH 7.4,0.3M NaCl and 300mM imidazole) and the eluates collected were pure enzyme COMT25468 and pure enzyme AtCOMT, respectively. The concentrations of the two enzymes were determined using BCA protein quantification kit (available from bioengineering limited). Wherein, the concentration of pure enzyme COMT25468 is 1.2g/L, and the concentration of pure enzyme AtCOMT is 0.8g/L.
The enzyme reaction system comprises (in final concentration): 10mM caffeic acid, 10mM SAM, 50mM PBS, in a total volume of 5mL. 200mg/L of pure enzyme COMT25468 and 200mg/L of pure enzyme AtCOMT were added respectively, and the reaction was carried out on a constant temperature shaking table at 30℃and 200 rpm. Sampling time is 0h, 2h, 4h, 6h, 8h, 12h and 24h, and the generation condition of ferulic acid is measured. As can be seen from FIG. 2a, the accumulation of ferulic acid was highest after 12h in the experimental group and was not increased any more, and the molar conversion rate of caffeic acid to ferulic acid was about 85%. As can be seen from fig. 2b, the molar conversion of caffeic acid to ferulic acid in the control group was 45%. From this, it can be concluded that COMT25468 has higher catalytic efficiency than the control AtCOMT.
The enzyme activity units of the two enzymes were calculated, with COMT25468 activity IU of 0.23 and atcot activity IU of 0.12. The addition amount of the two enzymes in the reaction system is consistent, and the specific enzyme activity of COMT25468 is 0.058IU/mg, which is 1.93 times of the specific enzyme activity of AtCOMT (0.03 IU/mg).
Example 4: overexpression of S-adenosylmethionine synthetase MetK and knockout of speD
(1) Construction of speD knockout DonorDNA
E.coli BW25113 (or genome thereof) is used as a template, primers speD-UF/speD-UR and speD-DF/speD-DR are respectively used for amplifying homologous arms at the upstream and downstream of the speD, the primers speD-UF/speD-DR are used for carrying out overlapping extension PCR (overlap PCR) after purification and recovery, whether a band with correct size and base is obtained is judged according to a sequencing result, and the Donor DNA with the speD knocked-out is recovered.
(2) Construction of pTargetF-speD plasmid
And (3) taking the pTargetF plasmid as a template, performing inverse PCR by using a primer pTF-speD-F/pTF-speD-R, purifying and recovering the transformed competent cell escherichia coli DH5 alpha (commercial), culturing at 37 ℃ for 16 hours (or overnight culture), and selecting a single colony for sequencing, wherein the plasmid with correct sequencing is the pTargetF-speD plasmid.
(3) Construction of Chassis cells harboring pCas9 plasmid
The pCas9 plasmid was introduced into BW25113 competent cells by electrotransformation or chemical transformation, plated with kanamycin resistance plate, and cultured at 30℃to obtain the strain BW25113 pCas9.
(4) BW25113 genome knockout speD
200ng of pTargetF-speD plasmid and 800ng of Donor DNA were added to co-transform BW25113 pCas9 competent cells, and after resuscitating at 30℃and 200rpm for 45-60min, the cells were spread on LB solid medium containing spectinomycin and kanamycin resistance and cultured overnight at 30 ℃. And (3) picking single bacterial colonies for colony PCR, and selecting positive results for measurement to determine the strain from which the speD gene has been knocked out.
(5) Loss of pTargetF-speD plasmid
The correct knockouts obtained above were inoculated into LB medium containing kanamycin, induced by addition of 0.5mM IPTG, and cultured overnight at 30 ℃. The kanamycin-resistant plates are diluted and coated, single colonies are selected for photocopying screening (respectively coating the kanamycin plate and the kanamycin and spectinomycin double-resistant plates, and culturing at 30 ℃), and strains which grow on the kanamycin plate and do not grow on the double-resistant plates are selected, namely the strains which have lost pTargetF-speD and can be used for subsequent continuous gene editing and bacteria preservation, and are named BW25113 delta speD pCas.
(6) Loss of pCas plasmid
BW25113 delta speD pCas glycerol bacteria are inoculated into an antibiotic-free LB medium and cultured overnight at 42 ℃. The non-resistant plates (cultured at 37 ℃) are diluted and coated, single colonies are selected for photocopying screening (coated with non-resistant LB solid medium and kanamycin LB solid medium respectively, cultured at 37 ℃) and strains which grow on the non-resistant LB solid medium and do not grow on the kanamycin LB solid medium are selected, namely the strains which have lost the pCas9 plasmid, and at the moment, the chassis strains which have successfully knocked out target genes and have no resistance are obtained and are named as BW25113 delta speD.
(7) Overexpression of S-adenosylmethionine synthetase MetK with a strong promoter
Based on BW25113 ΔspeD, the metK promoter P on the genome was obtained by the methods of (1) to (6) above metK Substitution with strong promoter P J23119 A strain with enhanced metK expression, designated BW25113 ΔspeD ΔP, was obtained metK ::P J23119 This strain was designated FA1.
TABLE 2 primers used in this example
Example 5: obtaining recombinant strains FA2, FA3 and FA4
(1) The constructed pBAD-COMT25468 plasmid is transformed into BW25113 wild type escherichia coli to obtain a FA2 strain: BW25113/pBAD-COMT25468.
(2) The constructed pBAD-COMT25468 plasmid was transformed into BW25113 ΔspeD to obtain the FA3 strain: BW25113 ΔspeD/pBAD-COMT25468.
(3) The constructed pBAD-COMT25468 plasmid was transformed into FA1 to obtain the FA4 strain: FA1/pBAD-COMT25468.
Example 6: evaluation of conversion efficiency of COMT25468 for catalyzing caffeic acid to produce ferulic acid
(1) The recombinant strains FA2, FA3 and FA4 prepared in example 5 were transferred to LB liquid medium containing 100. Mu.g/mL ampicillin, respectively, and cultured at 37℃and 200rpm for 8 hours to prepare seed solutions.
(2) The seed solution thus prepared was transferred to a test tube containing 100mL of LB liquid medium at an inoculum size of 1% (v/v), and ampicillin was added thereto at a final concentration of 100. Mu.g/mL, and incubated at 37℃for about 2 hours to OD at 200rpm 600nm About 0.6-0.8, then L-arabinose with a final concentration of 2g/L was added as inducer, the flask was transferred to 30℃and cultured at 200rpm for 24 hours to obtain an OD 600nm The cells were collected by centrifugation at 5000rpm for 5min at 4 ℃.
Preparing a whole cell catalytic system: suspending the cell obtained in step (2) in 20mL of M9 medium (50 mM sodium phosphate buffer pH 7.0), weighing 0.036g of caffeic acid, and adding into the bacterial liquid to obtain whole cell catalytic reaction system, wherein the final cell OD 600nm About 20, caffeic acid was present at a final concentration of 10mM. The reaction was carried out at 30℃and 200rpm on a constant temperature shaking table. Sampling every 24 hours, and measuring the generation condition of caffeic acid, wherein the result is shown in figure 3, the generation amount of ferulic acid of the FA4 recombinant strain is highest, the generation amount of ferulic acid of the FA2 recombinant strain is lowest, and the molar conversion rate of ferulic acid is 78% when the FA4 recombinant strain is used for 48 hours. This result demonstrates that enhancing SAM supply, weakening SAM degradation, can effectively increase caffeic acid to ferulic acid conversion.
Example 7: transformation verification of 5L fermentors
The FA4 recombinant strain of example 6 was tested for its ability to produce ferulic acid on a 5L fermenter, the upper tank fermentation medium formulation was as set forth above, and the specific procedure was as follows:
(1) Inoculating recombinant strain FA4 in LB culture medium, culturing at 37deg.C and 200rpm for 8 hr to obtain primary seed solution; inoculating the primary seed liquid into 250mL of LB culture medium according to the volume ratio of 1%, and culturing for 8 hours at 37 ℃ and 200rpm to obtain a secondary seed liquid;
(2) Inoculating the prepared secondary seed liquid into a 5L fermentation tank containing a fermentation medium according to an inoculation amount of 10% by volume, controlling the liquid loading amount to be 3L, controlling the fermentation temperature to 37 ℃ before the fermentation starts, setting the fermentation starting rotating speed to 400rpm, adjusting the starting pH to 6.8, and setting the dissolved oxygen to 100%, so that the dissolved oxygen in the fermentation process fluctuates at about 30%.
(3) In the strain growth stage, the density of the bacterial cells gradually increases, and the demand for oxygen gradually increases, so that the stirring rotation speed needs to be gradually increased. After fermentation for 6 hours, the glucose in the fermentation tank is exhausted, when the dissolved oxygen in the tank rebounds to more than 60-70%, 0.5g/mL of glucose solution starts to flow, and the pH in the tank is controlled to be about 7.0 by ammonia water. The initial sugar supplementing speed is controlled at 1g/L/h, so that dissolved oxygen fluctuates at about 30%, along with the growth of thalli, the sugar supplementing speed and the stirring paddle rotating speed are gradually increased according to the frequency of every 0.5-1 h until the flow acceleration of glucose reaches up to 4g/L/h, and the stirring rotating speed is not higher than 750rpm.
(4) Sampling to determine thallus density of fermentation liquid, and determining OD 600 When 30 (about 20 hours) was reached, the fermentation temperature was lowered to 30℃and L-arabinose was added to the fermenter at a final concentration of 2g/L to induce protein expression. After induction for 6h, a premix containing 10g/L methionine and 10g/L caffeic acid was added, the biomass and the content of caffeic acid and ferulic acid were measured by HPLC every 12h, and when the caffeic acid content was reduced to 1-2g/L, the premix was added to a final concentration of 5g/L. The total input of caffeic acid was 45g when fermented for 72 hours.
(5) The experimental results are shown in FIG. 4, and the accumulation amount of ferulic acid is increased with the extension of fermentation time. When fermentation is carried out for 72 hours, the accumulation of ferulic acid reaches 12g/L, the residual level of caffeic acid is low, and the molar conversion rate is 74.2%.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. A caffeic acid-O-methyltransferase is disclosed, which has the amino acid sequence shown in SEQ ID NO. 1.
2. A gene comT encoding a caffeic acid-O-methyltransferase according to claim 1, wherein the nucleotide sequence of said gene comT is shown in SEQ ID No.2.
3. An expression vector carrying the gene of claim 2, wherein the expression vector includes, but is not limited to, pBAD/HisA.
4. The escherichia coli genetic engineering bacterium containing the expression vector of claim 3, wherein the escherichia coli genetic engineering bacterium also overexpresses S-adenosylmethionine synthetase metK and knocks out SAM degradation pathway gene speD.
5. The genetically engineered E.coli strain of claim 4, wherein the promoter of the S-adenosylmethionine synthetase gene metK on the genome is replaced with the promoter shown in SEQ ID NO. 7.
6. A method for producing ferulic acid, characterized in that the E.coli genetically engineered bacterium of claim 4 or 5 is fermented in a glucose-containing medium and fed with caffeic acid and methionine.
7. The method of claim 6, wherein the medium comprises: glucose, KH 2 PO 4 、(NH 4 ) 2 HPO 4 、MgSO 4 ·7H 2 O, citric acid and trace elements; the microelements comprise: feSO 4 ·7H 2 O、ZnSO 4 ·7H 2 O、CuSO 4 ·5H 2 O、MnSO 4 ·5H 2 O、CaCl 2 ·2H 2 O and Na 2 B 4 O 7 ·5H 2 O。
8. The method according to claim 7, wherein when the genetically engineered bacterium of E.coli is in mid-log growth, the induction is performed by adding arabinose at a final concentration of 1.5-2g/L L-for at least 6 hours.
9. The method according to claim 8, wherein the solution containing caffeic acid and methionine is fed after induction, maintaining the caffeic acid content at 5-7g/L.
10. Use of the caffeic acid-O-methyltransferase of claim 1, or the gene of claim 2, or the expression vector of claim 3, or the genetically engineered escherichia coli of claim 4 or 5 in the production of ferulic acid.
CN202310929672.XA 2023-07-26 2023-07-26 Method for producing ferulic acid by using caffeic acid-O-methyltransferase and application thereof Pending CN116904417A (en)

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