Construction and application of escherichia coli engineering strain with high yield of vanillin

Abstract

The invention discloses construction and application of an escherichia coli engineering strain for high yield of vanillin, and belongs to the field of genetic engineering technology and biochemical engineering. According to the invention, a series of genes for generating vanillic acid and vanillyl alcohol by oxidizing and reducing vanillin are knocked out, and feruloyl coenzyme A synthetase FCS15369 and p-hydroxycinnamyl coenzyme A hydratase ECH25635 which are obtained by screening from a deep sea extreme environment metagene library are overexpressed. According to the engineering strain EcVAN1 with high yield of vanillin, the accumulation of vanillin reaches 15.2 g / L in the industrial application of a 100t fermentation tank, and the molar conversion rate of catalyzing ferulic acid to synthesize vanillin reaches 90.9%.

Description

Technical Field

[0001] The invention relates to the construction and application of an Escherichia coli engineering strain with high vanillin production, and belongs to the field of genetic engineering technology and biochemical engineering. Background Art

[0002] Vanillin, also known as vanillin, has a chemical name of 4-hydroxy-3-methoxybenzaldehyde. It is the first flavor synthesized by humans and one of the most important aromatic compounds. Vanillin is an aromatic compound with functional groups such as aldehyde, phenol and ether. It is water-soluble and appears as a white crystalline powder with a strong sweet and creamy vanilla smell. Natural vanillin is mainly found in vanilla beans, clove oil and beetroot, in the form of free state or glucoside. Vanillin has a strong milky aroma and has a huge application market as a flavoring agent for cakes, beverages and other foods and medicines.

[0003] At present, the vanillin used in the market is mainly divided into two types: chemically synthesized vanillin and natural vanillin. More than 90% of the vanillin on the market is obtained by chemical methods. The market price of this type of vanillin is relatively cheap and it is easy to cause environmental pollution and other problems. The cost of natural vanillin extracted from plants is expensive, about 300 times that of chemically synthesized vanillin. However, due to the limitations of plant growth environment and climate, it is difficult to increase the output of plant extraction. The annual output is only about 40 tons, accounting for about 0.25% of the market share. The price of vanillin produced by microbial fermentation is about US$100 per kilogram. The market demand for vanillin exceeds 15,000 tons per year, while natural vanillin can only supply 2,000 tons per year. Therefore, the production of vanillin by microbial fermentation conversion has become the most promising method.

[0004] Microbial transformation is based on the in vivo metabolic activity mechanism of microorganisms to convert different substrates into target products. This method has the advantages of short cycle, natural raw materials, mild reaction conditions, high production efficiency, easy product extraction and environmental friendliness. Therefore, further improving the production of vanillin by combining genetically engineered strains with microbial fermentation will become a promising research field. According to literature reports, the strains that produce vanillin mainly include fungi, bacteria, molds and genetically engineered bacteria, and the precursor substances mainly include eugenol, ferulic acid, isoeugenol, glucose, vanillic acid and lignin. Escherichia coli has become a common strain for genetic engineering modification due to its clear genetic background, simple genetic manipulation, simple culture medium components, fast growth rate and suitability for large-scale fermentation. However, Escherichia coli is not a natural vanillin production strain. It needs to be transformed into an engineered strain using genetic engineering techniques and means before it can be used to produce vanillin.

[0005] According to existing reports, Jiangnan University used Escherichia coli to heterologously express the vanillin synthesis gene and completed the conversion of ferulic acid to vanillin through whole-cell catalysis. The highest yield was about 4.27g / L, which is still far from meeting the demand. Therefore, how to mass-produce safe and natural vanillin has become an urgent problem to be solved. Summary of the invention

[0006] The purpose of the present invention is to provide a method for biosynthesizing vanillin using ferulic acid as a substrate. The method has high yield and production efficiency of vanillin and is suitable for large-scale industrial production.

[0007] The invention provides a recombinant Escherichia coli, in which feruloyl-CoA synthetase FCS15369 and p-hydroxycinnamoyl-CoA hydratase ECH25635 are overexpressed in a chassis strain; the amino acid sequence of the feruloyl-CoA synthetase FCS15369 is shown in SEQ ID NO.1; the amino acid sequence of the hydroxycinnamoyl-CoA hydratase ECH25635 is shown in SEQ ID NO.2.

[0008] In one embodiment, the chassis strain is based on Escherichia coli BW25113, but the aldehyde reductase genes yqhK, yqhD and the aldehyde oxidase gene paoC are knocked out.

[0009] In one embodiment, the nucleotide sequence of the aldehyde reductase gene yahK is shown in Gene ID: 944975; the nucleotide sequence of the aldehyde reductase gene yqhD is shown in Gene ID: 947493; and the nucleotide sequence of the aldehyde oxidase gene paoC is shown in Gene ID: 944961.

[0010] In one embodiment, the nucleotide sequence of the feruloyl-CoA synthetase gene Fcs15369 is shown as SEQ ID NO.3, and the nucleotide sequence of the hydroxycinnamoyl-CoA hydratase gene Ech25635 is shown as SEQ ID NO.4.

[0011] In one embodiment, plasmid pBAD is used as an expression vector to express the feruloyl-CoA synthetase gene Fcs15369 and the hydroxycinnamoyl-CoA hydratase gene Ech25635.

[0012] In one embodiment of the present invention, the chassis cell is based on E. coli. BW25113 strain as the starting strain, and the yqhK, yqhD and paoC genes are first knocked out to obtain the chassis strain EcVAN, and then the recombinant plasmid pBAD-Fcs15369-Ech25635 expressing the feruloyl-CoA synthetase gene Fcs15369 and the hydroxycinnamoyl-CoA hydratase gene Ech25635 is introduced to obtain the EcVAN1 strain.

[0013] The invention also provides a method for preparing vanillin by using the recombinant Escherichia coli.

[0014] In one embodiment, the method is to use the recombinant Escherichia coli to catalyze ferulic acid to produce vanillin in M9 medium.

[0015] In one embodiment, after the recombinant Escherichia coli reacts for 3 hours, ferulic acid is added to continue the reaction.

[0016] In one embodiment, the method is to culture the recombinant Escherichia coli in LB medium to obtain seed liquid, and then transfer the seed liquid to a fermentation medium for fermentation.

[0017] In one embodiment, the fermentation medium contains: glucose, yeast powder, KH2PO4, (NH4)2HPO4, MgSO4, citric acid and trace elements.

[0018] In one embodiment, the trace elements include but are not limited to: FeSO4, ZnSO4·, CuSO4, MnSO4, CaCl2, Na2B4O7, (NH4)6Mo7O 24 .

[0019] In one embodiment, the added amount of glucose is: 10-20 g / L.

[0020] In one embodiment, the added amount of the engineered bacteria EcVAN1 is 5% to 10%.

[0021] In one embodiment, the reaction conditions are: 30-37° C., stirring blade speed 300-700 rpm, fermentation liquid pH 6.5-7.4, and ventilation volume 0.5-1.5 vvm.

[0022] In one embodiment, feeding is also performed during the fermentation process.

[0023] In one embodiment, glucose and / or ferulic acid are fed.

[0024] The present invention also provides the use of the above-mentioned recombinant Escherichia coli engineered strain in the preparation of vanillin or products containing vanillin.

[0025] Beneficial Effects

[0026] (1) The present invention obtains the chassis strain EcVAN that can maintain the stability of vanillin by knocking out the genes encoding aldehyde-keto reductase and the genes yqhK, yqhD and paoC encoding alcohol dehydrogenase as chassis cells;

[0027] (2) The present invention heterologously expresses feruloyl-CoA synthetase FCS15369 and p-hydroxycinnamoyl-CoA hydratase ECH25635 from the deep-sea extreme environment metagenomic library in the engineered strain EcVAN to achieve the synthesis of vanillin with a higher yield;

[0028] (3) The high-vanillin-yielding engineered strain EcVAN obtained by the present invention can produce 3.8 g / L of vanillin in a shake flask; in a 5L fermenter, fed-batch fermentation was carried out for 45 h, and the accumulation of vanillin reached 15.6 g / L; in a 100t fermenter, fed-batch fermentation was carried out, and the accumulation of vanillin reached 15.2 g / L, which is the highest level of vanillin synthesis by Escherichia coli engineered strains reported so far. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Metabolic flow chart for the synthesis of vanillin from ferulic acid.

[0030] Figure 2 The figures are liquid chromatograms of vanillin and ferulic acid; wherein A is the liquid chromatogram of the standard products of vanillin and ferulic acid; and B is the liquid chromatogram of the fermentation sample.

[0031] Figure 3 The figure shows the comparison of enzyme activities after different combinations of FCS and ECH enzymes.

[0032] Figure 4 It is the SDS-PAGE diagram of EcVAN1 strain; among them, lane 1 is protein standard, lane 2 is total bacterial protein, and lane 3 is soluble protein.

[0033] Figure 5 To evaluate the stable production of vanillin in the EcVAN strain.

[0034] Figure 6 To evaluate the production of vanillin from ferulic acid catalyzed by strain EcVAN1 in shake flasks.

[0035] Figure 7 To evaluate the production of vanillin from ferulic acid catalyzed by strain EcVAN1 in a 5L fermenter.

[0036] Figure 8 The EcVAN1 strain catalyzes ferulic acid to synthesize vanillin in a 100t fermentation tank. DETAILED DESCRIPTION

[0037] The main reagents, instruments, genetic materials and culture media used in the following examples: ferulic acid standard (Aladdin reagent), vanillin standard (Aladdin reagent), overexpressed Fcs15369 and Ech25635 were synthesized by Qingke Biotechnology after gene codon optimization for E. coli-K12 strain.

[0038] The pTargetF and pBAD plasmids involved in the following examples were purchased from Youbao Biotechnology, and Escherichia coli BW25113 was retained in the laboratory.

[0039] The culture medium in the following examples is as follows:

[0040] Seed medium (LB) (1 L): 5 g yeast powder, 10 g tryptone, 10 g sodium chloride.

[0041] M9 medium (1L): glucose 10g, NH4Cl 1g, Na2HPO4 6g, KH2PO4 3g, NaCl 0.5g, MgSO4 240mg, CaCl2 5.35mg, V B1 2mg, trace elements (H3BO3 1.25mg, Na2MoO4·2H2O 0.15mg, CoCl2·6H2O 0.7mg, CuSO4·5H2O 0.25mg, MnCl2·4H2O 1.6mg, ZnSO4·7H2O 0.3mg).

[0042] Shake flask fermentation medium (ZYM) (1L): 10g tryptone, 2g yeast powder, 0.5g glucose, 5mL glycerol, 3.55g Na2HPO4, 3.4g KH2PO4, 2.68g NH4Cl, 0.71g Na2SO4; 240mg MgSO4, trace elements (1mMFeCl3, 0.4mM CaCl2, 0.2mM MnCl2, 0.2mM ZnSO4, 0.04mM each of CoCl2, NiCl2, Na2MoO4, Na2SeO3 and H3BO3).

[0043] Fermentation medium (1L): glucose 5.0g, yeast powder 2.5g, KH2PO4 6.65g, (NH4)2HPO4 2.0g, MgSO4·7H2O 0.6g, citric acid 0.85g, trace elements (FeSO4·7H2O 100mg, ZnSO4·7H2O 22.5mg, CuSO4·5H2O 10mg, MnSO4·5H2O 5mg, CaCl2·2H2O 20mg, Na2B4O7·5H2O 2.3mg, (NH4)6Mo7O 241 mg), antifoam, feed (0.5 g / mL glucose solution).

[0044] The strain information involved in the following examples is shown in Table 1 below:

[0045] Table 1: Main plasmid and strain information involved in the present invention

[0046]

[0047] The primer sequences involved in the following examples are shown in Tables 2 and 3:

[0048] Table 2: Primers used in implementation case 2

[0049]

[0050]

[0051] Table 3: Primers used in implementation case 4

[0052]

[0053] The detection methods of vanillin and ferulic acid involved in the following embodiments are as follows:

[0054] 1. Sample preparation: Centrifuge the fermentation broth sample at 12000 rpm for 1 min, take the supernatant, dilute it with 20% methanol aqueous solution to a suitable multiple, and filter it using a 0.22 μm filter membrane.

[0055] 2. Liquid phase conditions: Chromatographic column: InfinityLab Poroshell HPH-C18, 4.6×150mm, 2.7-Micron; Mobile phase A: ddH2O (0.1% acetic acid), Mobile phase B: methanol. Elution program: 0-6min, 20% B; 6-10min, 20-40% B; 10-15min, 40% B; 15-16min, 40-20% B; 16-20min, 20% B. Flow rate: 0.7mL / min, column temperature: 30℃, injection volume: 10μL. Detector: diode array detector, detection wavelength: 280nm (vanillin), 323nm (ferulic acid). The target substance was determined by comparing the retention time and spectrum with the standard, and the measured substance was quantitatively analyzed according to the peak area.

[0056] Example 1: Screening of FCS and ECH enzymes that efficiently catalyze the synthesis of vanillin from ferulic acid

[0057] The present invention screened five FCS sequences (FCS24981, FCS15369, FCS02569, FCS89652 and FCS46345) and three ECH sequences (ECH25635, ECH26431 and ECH05259) from a deep-sea extreme environment metagene library, wherein the nucleotide sequence of FCS24981 is shown in SEQ ID NO.5; the nucleotide sequence of FCS15369 is shown in SEQ ID NO.3; the nucleotide sequence of FCS02569 is shown in SEQ ID NO.6; the nucleotide sequence of FCS89652 is shown in SEQ ID NO.7; the nucleotide sequence of FCS46345 is shown in SEQ ID NO.8; the nucleotide sequence of ECH25635 is shown in SEQ ID NO.4; the nucleotide sequence of ECH26431 is shown in SEQ ID NO.9; and the nucleotide sequence of ECH05259 is shown in SEQ ID NO.10.

[0058] The above genes were combined to obtain 15 different pBAD-fcs-ech recombinant plasmids. The 15 plasmids were transformed into E. coli BW25113 strain separately. In M9 medium, 1g / L ferulic acid was added as substrate, and the catalytic effects of different FCS and ECH combinations were compared. The results showed that the catalytic effect of the enzyme combination of FCS15369 and ECH25635 was the best, with a vanillin yield of 720mg / L and a conversion rate of 92.89% ( Figure 3 ).

[0059] Example 2: Construction of chassis cells, namely vanillin stable strain EcVAN

[0060] Vanillin itself has poor stability and is easily reduced to vanillyl alcohol and easily oxidized to vanillic acid. Referring to the method in the literature (Artificial Biosynthetic Pathway for Efficient Synthesis of Vanillin, a Feruloyl-CoA-Derived Natural Product from Eugenol. J. Agric. Food Chem. 2024, 72, 6463-6470), CRISPR-Cas9 gene editing was performed on the Escherichia coli BW25113 strain to knock out the aldehyde reductase genes yahK and yqhD, and the aldehyde oxidase gene paoC. Taking the knockout of the yahK gene as an example, the specific steps are as follows:

[0061] (1) The genome of Escherichia coli strain BW25113 was used as a template, and primers yahK-up-F and yahK-up-R as well as yahK-down-F and yahK-down-F were used to amplify 500 bp DNA fragments upstream and downstream of the yahK gene, respectively. The two fragments were then connected using overlapping PCR to serve as donor DNA for gene editing.

[0062] (2) Using the original pTargetF plasmid as a template, the primers pTargetF-yahK-N20-F and pTargetF-yahK-N20-R were used to amplify the pTargetF-yahK plasmid by reverse PCR. The obtained Donor DNA and pTargetF-yahK plasmid were co-transformed into the competent strain of Escherichia coli BW25113 expressing the Cas9 protein by electroporation. The single colony obtained was identified by PCR using primers yahK-YZ-F and yahK-YZ-R. Based on the PCR results and combined with sequencing comparison analysis, the strain with the yahK gene knocked out was obtained: BW25113ΔyahK.

[0063] Following the same strategy as above, the genes yqhD and paoC were knocked out, and the resulting chassis strain was named EcVAN.

[0064] Example 3: Chassis EcVAN Vanillin Stability Verification

[0065] The EcVAN strain was inoculated into LB medium at a 1% inoculum, cultured overnight at 30°C, 200 rpm, and then 10 g / L vanillin solution was added. The culture was shaken for 8 hours, and samples were taken every 2 hours. The residual vanillin and the synthesis of vanillic acid and vanillyl alcohol were determined by HPLC.

[0066] The results showed that during the 8-hour cultivation process with the addition of vanillin, the residual vanillin in the control strain (E. coli BW25113 wild strain) gradually decreased, and the accumulation of vanillic acid and vanillyl alcohol continued to increase. After 8 hours of cultivation, the vanillic acid reached 0.92 g / L and the vanillyl alcohol reached 3.48 g / L. In the EcVAN engineered strain, the vanillin content was relatively stable, with only a small amount of vanillic acid and vanillyl alcohol accumulated ( Figure 5 ).

[0067] This indicates that the EcVAN engineered strain, as a chassis cell, can maintain the stability of vanillin in the fermentation broth due to the knockout of intracellular oxidoreductases. This chassis strain has great advantages as a production strain.

[0068] Example 4: Construction of recombinant plasmid pBAD-fcs15369-ech25635 and acquisition of engineered strain EcVAN1

[0069] (1) Using primers pBAD-ZT-F and pBAD-ZT-R and pBAD plasmid as a template, PCR amplification was performed to obtain a linearized pBAD vector, and primers fcs15369-F and fcs15369-R, ech25635-F and ech25635-R were used to amplify the fcs15369 and ech25635 sequence fragments (the nucleotide sequence encoding the feruloyl-CoA synthase FCS15369 is shown in SEQ ID NO.3, and the nucleotide sequence encoding the p-hydroxycinnamoyl-CoA hydratase ECH25635 is shown in SEQ ID NO.4). The PCR reaction system and PCR amplification conditions are shown in Tables 4 and 5.

[0070] Table 4: PCR reaction system

[0071]

[0072] Table 5: PCR amplification conditions

[0073]

[0074] (2) The obtained fragment was connected to the pBAD linearized vector obtained above using homologous recombinase. The connection system is shown in Table 6.

[0075] Table 6: Linearized connection system

[0076]

[0077] The amount of linearized vector x1 or inserted fragment x2 is determined by its length. The most suitable amount is usually: x = [0.02 × base pair length] ng; after transformation of the cloning host, the recombinant plasmid pBAD-fcs15369-ech25635 is obtained by colony PCR identification and sequencing.

[0078] (3) The constructed pBAD-fcs15369-ech25635 plasmid was transformed into the engineered strain EcVAN constructed in Example 2 to obtain the strain: EcVAN / pBAD-fcs15369-ech25635, named EcVAN1.

[0079] Example 5: Evaluation of the conversion efficiency of ferulic acid to vanillin catalyzed by whole cells of EcVAN1 strain

[0080] (1) The engineered strain EcVAN1 prepared in Example 4 was transferred to LB liquid culture medium containing 100 μg / mL ampicillin and cultured at 37° C. and 200 rpm for 8 h to prepare a seed solution.

[0081] (2) Transfer the prepared seed solution to a test tube containing 100 mL of LB liquid medium at a 1% (v / v) inoculum volume, add ampicillin at a final concentration of 100 μg / mL, and culture at 37°C and 200 rpm for about 2 h until the OD reaches 600 The fermentation liquid was centrifuged at 5000 rpm and 4°C for 5 min, and the cells were resuspended in M9 medium. The final cell OD 600 is 10 to obtain a whole-cell catalyst.

[0082] (3) Add a final concentration of 3 g / L ferulic acid to the whole-cell catalytic reaction system and react on a constant temperature shaker at 30°C and 200 rpm. After 3 hours, when it is detected that the ferulic acid is almost exhausted, continue to add 3 g / L ferulic acid solution. Samples are taken every 1 to 2 hours to determine the formation of vanillin and the residual ferulic acid. The results are as follows: Figure 6 shown.

[0083] The results showed that the synthesis amount of vanillin in M9 culture medium continued to increase. After 3 hours of reaction, only 0.2 g / L of the 3 g / L ferulic acid added for the first time remained, and 2.0 g / L of vanillin was generated, with a molar conversion rate of 91%. After 3 hours of reaction, 3 g / L of ferulic acid was added again. After 9 hours of reaction, 0.8 g / L of ferulic acid remained, and finally 3.8 g / L of vanillin was synthesized, and the final molar conversion rate of vanillin reached 93%.

[0084] Example 6: Preparation of vanillin by fed-batch fermentation of EcVAN1 strain in a 5 L fermenter

[0085] In view of the good experimental results obtained in the shake flask experiment of Example 5 for synthesizing vanillin from ferulic acid as a substrate, the EcVAN1 strain was used in a 5L fermenter for fed-batch fermentation to evaluate the production scale-up potential of vanillin. The specific steps are as follows:

[0086] (1) The engineered bacteria EcVAN1 strain prepared in Example 4 was inoculated into LB medium and cultured at 37°C and 200 rpm for 8 h to prepare a primary seed solution; the primary seed solution was then inoculated into 250 mL of LB medium at an inoculum volume of 1% (v / v) and cultured at 37°C and 200 rpm for 8 h to obtain an OD 600 It is the secondary seed liquid of 4.0.

[0087] (2) The prepared secondary seed liquid was inoculated into a 5L fermentation tank containing 2.25L fermentation medium at an inoculation rate of 10% (v / v). Before the start of fermentation, the fermentation temperature was controlled at 37°C, the fermentation starting speed was set to 400rpm, the initial pH was adjusted to 6.8, and the dissolved oxygen was set to 100%, so that the dissolved oxygen during the fermentation process was controlled between 20 and 40%. During the growth stage of the strain, the bacterial density gradually increased and the demand for oxygen gradually increased, so it was necessary to gradually increase the stirring speed. After 6 hours of fermentation, the glucose in the fermentation tank was exhausted. When the dissolved oxygen in the tank rose sharply, glucose was started to be added, and ammonia was used to control the pH in the tank to maintain at about 7.0. The residual sugar in the fermentation tank was controlled at 0.2-0.5 g / L, and the dissolved oxygen fluctuated within the range of 20-40%. As the bacteria grew, the sugar addition rate and the stirring speed were gradually increased at a frequency of 0.5-1 h until the glucose flow acceleration reached a maximum of 4 g / L / h. The residual sugar in the fermentation tank was controlled at 0.2-0.5 g / L, and the stirring speed was 700 rpm. Samples were taken to measure the bacterial density of the fermentation liquid. When the OD 600 When the temperature reached 10 (about 6 hours), the fermentation temperature was lowered to 30°C. Since the activity of the strain decreased at this time, the sugar addition rate was reduced to 3 g / L / h and the stirring speed was reduced to 600 rpm. L-arabinose with a final concentration of 2 g / L was added to the fermenter to induce protein expression. When the fermentation was continued for about 24 hours, ferulic acid was started to be added, and the flow acceleration was maintained at 2.5-4 g / L / h. The residual ferulic acid and the formation of vanillin were detected by liquid phase, and the synthesis of by-products vanillic acid and vanillyl alcohol was monitored.

[0088] The results showed that at the beginning of the reaction, the flow acceleration of ferulic acid was maintained at 4g / L / h, and there was no residual ferulic acid, which was completely converted into vanillin, and the accumulation of vanillin gradually increased; when there was residual ferulic acid, the flow acceleration of ferulic acid was reduced to 2.5g / L, and the flow addition of ferulic acid was stopped after 33h of fermentation. At this time, the cell density was significantly reduced compared with that at the beginning of the conversion reaction. This may be because ferulic acid and vanillin have certain toxicity to the strain, resulting in a decrease in the synthesis rate of vanillin, accompanied by the accumulation of a small amount of vanillic acid and vanillyl alcohol. At 45h of fermentation, the accumulation of vanillin reached 15.6g / L, and there was still 1.2g / L of residual ferulic acid ( Figure 7 ), the cumulative consumption of ferulic acid was about 65 g, and the molar conversion rate of ferulic acid to vanillin reached 91.9%.

[0089] Example 7: Industrial application of 100t fed-batch fermentation of EcVAN1 strain

[0090] In view of the good experimental results of the EcVAN1 strain in the 5L fermenter fed-batch fermentation in Example 5, the industrialization capability of the EcVAN1 strain was evaluated in a 100t fermenter in this implementation case, and the specific implementation is as follows:

[0091] (1) The engineered bacteria EcVAN1 strain prepared in Example 3 was inoculated into LB medium and cultured at 37°C, 200 rpm for 8 h to prepare a first-level seed solution; the first-level seed solution was then inoculated into 2.5 L of LB medium at an inoculum amount of 1% (v / v), and cultured at 37°C, 200 rpm for 8 h to obtain a second-level seed solution; the second-level seed solution was then inoculated into a 10 t seed tank containing 6 t of fermentation medium, and cultured until OD600 was 10 to obtain a third-level seed solution.

[0092] (2) The prepared 6t of third-level seed liquid was pumped into a 100t fermentation tank containing 54t of fermentation medium through an inoculation tube, so that the initial bacterial density OD600 was 1. Before the start of fermentation, the fermentation temperature was controlled at 37°C, the fermentation starting speed was set to 80rpm, the tank pressure was controlled at 0.03MPa, the initial pH was adjusted to 7.0, the ventilation volume was set to 0.05vvm, and the initial dissolved oxygen was set to 100%. During the growth stage of the strain, the bacterial density gradually increased, and the demand for oxygen gradually increased. Therefore, it is necessary to gradually increase the stirring speed to 120rpm so that the dissolved oxygen in the fermentation process fluctuates within the range of 20-40%. After 6h of fermentation, the glucose in the fermentation tank was exhausted. When the dissolved oxygen in the tank rose sharply, glucose was added and ammonia water was used to control the pH in the tank to maintain at 7.0. Samples were taken to measure the bacterial density of the fermentation liquid. When the OD 600 When the temperature is 10 (about 5h), the fermentation temperature is lowered to 30°C, and L-arabinose with a final concentration of 2g / L is added to the fermenter to induce protein expression. When the fermentation is about 24h and the cell density reaches about 70 OD, ferulic acid is started to be added, and the flow acceleration is maintained at 2.5-4g / L / h. During the conversion stage, the stirring speed is appropriately adjusted between 80 and 120rpm. The residual ferulic acid and the formation of vanillin are detected by liquid phase, and the synthesis of by-products vanillic acid and vanillyl alcohol is monitored.

[0093] The results showed that at the beginning of the reaction, the flow rate of ferulic acid was maintained at 4g / L / h, and there was no residual ferulic acid, which was completely converted into vanillin, and the accumulation of vanillin gradually increased; when there was residual ferulic acid, the flow rate of ferulic acid was reduced to 2.5g / L, and the flow addition of ferulic acid was stopped after 33h of fermentation. HPLC was used to detect the consumption of ferulic acid and the synthesis of vanillin. Finally, at 45h of fermentation, the accumulation of vanillin reached 15.2g / L, and the residual ferulic acid was 0.6g / L ( Figure 8 ), at this time, the volume of the fermentation tank is about 75t, the cumulative synthesis of vanillin is 1.14t, the cumulative consumption of ferulic acid is 1.6t, and the molar conversion rate of ferulic acid to vanillin reaches 90.9%.

[0094] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant Escherichia coli, characterized in that Feruloyl-CoA synthetase FCS15369 and p-hydroxycinnamoyl-CoA hydratase ECH25635 were overexpressed in the chassis strain; the amino acid sequence of the feruloyl-CoA synthetase FCS15369 is shown in SEQ ID NO.1; the amino acid sequence of the hydroxycinnamoyl-CoA hydratase ECH25635 is shown in SEQ ID NO.

2.

2. The recombinant Escherichia coli according to claim 1, characterized in that The chassis strain is based on Escherichia coli BW25113, in which the aldehyde reductase genes yqhK, yqhD and the aldehyde oxidase gene paoC are knocked out.

3. The recombinant Escherichia coli according to claim 1 or 2, characterized in that Plasmid pBAD is used as an expression vector to express the feruloyl-CoA synthetase gene Fcs15369 and the hydroxycinnamoyl-CoA hydratase gene Ech25635.

4. A cell catalyst comprising the recombinant Escherichia coli according to any one of claims 1 to 3.

5. A method for constructing the recombinant Escherichia coli according to claim 3, characterized in that: Using E. coli.BW25113 strain as the starting strain, the yqhK, yqhD and paoC genes were first knocked out to obtain the chassis strain EcVAN, and then the recombinant plasmid pBAD-Fcs15369-Ech25635 expressing the feruloyl-CoA synthetase gene Fcs15369 and the hydroxycinnamoyl-CoA hydratase gene Ech25635 was introduced into the chassis strain EcVAN.

6. A method for preparing vanillin, characterized in that: The recombinant Escherichia coli according to any one of claims 1 to 3 or the cell catalyst according to claim 4 is contacted with ferulic acid to prepare vanillin.

7. The method according to claim 6, characterized in that The method comprises the following steps: the recombinant Escherichia coli is used to catalyze ferulic acid to produce vanillin in an M9 medium containing ferulic acid.

8. The method according to claim 6, characterized in that The recombinant Escherichia coli is fermented in a fermentation medium, and ferulic acid is added during the fermentation process.

9. The method according to claim 8, characterized in that Glucose was also fed during the fermentation process.

10. Use of the recombinant Escherichia coli according to any one of claims 1 to 3 or the cell catalyst according to claim 4 in the preparation of vanillin or a product containing vanillin.

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