Synthesis and Accumulation of Vanillin in Genetically Engineered Escherichia coli

Abstract

The invention discloses the synthesis and accumulation of vanillin in genetically engineered Escherichia coli, belonging to the technical field of microbial fermentation. The invention heterologously expresses the feruloyl-CoA synthase gene (Atfcs) derived from Amycolatopsis and the feruloyl-CoA hydrolase / aldolase gene (Pfech) derived from Pseudomonas fluorescens in Escherichia coli to construct a vanillin synthesis pathway, and uses the CRISPR CAS9 technology to knock out aldehyde-ketone reductase and / or vanillin oxidase, reducing the metabolism of vanillin in Escherichia coli, successfully blocking the degradation pathway of vanillin, improving the utilization rate of the substrate ferulic acid, and the yield can reach 10.8 g / L after 11 h of fermentation in a fermenter, laying a foundation for the further optimization of recombinant Escherichia coli for vanillin production and having good industrial application prospects.

Description

Technical Field

[0001] The present invention relates to the synthesis and accumulation of vanillin in the genetically engineered bacterium Escherichia coli, belonging to the technical field of microbial fermentation. Background Art

[0002] Vanillin (VA) is one of the derivatives of ferulic acid (FA) and is a typical aromatic aldehyde. Also known as methyl protocatechuic aldehyde, vanillin, and vanilla aldehyde, its chemical name is 3-methoxy-4-hydroxybenzaldehyde, and its molecular formula is C 8 H 8 O 3 , with a relative molecular mass of 152.12. Vanillin is a typical aromatic substance with phenolic hydroxyl and aldehyde groups and has a unique and strong aromatic odor. Due to the presence of both phenolic hydroxyl and aldehyde groups, it can undergo both oxidation-reduction reactions of the aldehyde group and reactions of the phenolic hydroxyl and aromatic ring. Vanillin has a very wide range of uses and is one of the most important spices in the world, enjoying the reputation of "the king of food flavors" and being widely used in multiple fields such as cosmetics, food, medicine, agriculture, and plastics. As of 2019, the global annual demand for vanillin reached 17,000 tons and is still increasing, indicating that vanillin plays an important role. Currently, vanillin is mainly prepared by three methods: extraction from natural plants, chemical synthesis, and biotransformation. Vanillin extracted from natural plants is the most natural, green, and safe product, but due to its low yield, long cycle, limited resources, and high price, it cannot meet the market demand alone; chemical synthesis is an alternative method. Although it is economical and fast, it causes environmental pollution, and it lacks substrate selectivity, thus reducing the processing efficiency and increasing the downstream processing cost; according to the regulations of the United States and the European Union (EC no. 1334 / 2008), only vanillin obtained by biotransforming natural raw materials recognized by the FDA can be considered natural vanillin, and vanillin produced by natural and biotechnological methods is considered a food-grade additive by most food control authorities in the world. Therefore, the biopreparation of vanillin has become the most promising method for producing natural vanillin. Based on biotechnological means, producing vanillin by microbial transformation of suitable substrate precursors such as eugenol, isoeugenol, ferulic acid, etc., and biosynthesis using fungi, bacteria, or genetically engineered bacteria has become a very promising development trend.

[0003] Ferulic acid is the most promising substrate for the production of vanillin by biological methods. Compared with eugenol, ferulic acid has a wide source and is one of the most abundant phenolic compounds, and it has less toxic effects on microorganisms. In the project of producing vanillin by engineering Escherichia coli in our laboratory, through literature review and gene sequence alignment in the early stage, it was found that there is no direct vanillin catabolic pathway in Escherichia coli, and feruloyl-CoA synthase from Amycolatopsis sp. ATCC 39116 with high enzyme activity when heterologously expressed in Escherichia coli has been screened. The synthetic pathway of vanillin has been successfully constructed in Escherichia coli, and the vanillin production is also at the highest level among the current production of vanillin by recombinant engineering Escherichia coli. Although the highest yield has been obtained, it was found during the experiment that most of the vanillin will be degraded to vanillyl alcohol and a small amount will be metabolized to vanillic acid. In order to increase the accumulation of the product vanillin and improve the utilization rate of the substrate ferulic acid, ways should be found to reduce the degradation of vanillin.

[0004] After investigation, vanillin is respectively oxidized and reduced to vanillic acid and vanillyl alcohol in Escherichia coli because there are many endogenous broad-substrate-spectrum aldehyde-ketone reductases and oxidases in Escherichia coli. To solve this problem, currently, Francesca Luziatelli et al. from the University of Tuscia in Italy have taken the measure of controlling the pH at pH 10 in the alkaline environment during fermentation to inhibit the activities of endogenous broad-substrate-spectrum aldehyde-ketone reductases and oxidases in Escherichia coli; the research group of Tao Fei from Shanghai Jiao Tong University has taken the measure of whole-cell catalysis at 50 °C during fermentation to inhibit the activities of endogenous broad-substrate-spectrum aldehyde-ketone reductases and oxidases in Escherichia coli. Whether it is controlling the pH or the temperature, the operation is carried out at the fermentation level. The fermentation conditions are not easy to control, with large variable factors. And by controlling the pH and temperature, not only the activities of endogenous broad-substrate-spectrum aldehyde-ketone reductases and oxidases in Escherichia coli are reduced, but it is also possible to affect the expression of key genes for vanillin synthesis and genes related to the metabolic pathway of Escherichia coli itself, reducing the synthesis of vanillin and even affecting cell growth. Therefore, how to find a method to reduce the degradation of vanillin and produce safe and natural vanillin in large quantities and efficiently has become an urgent problem to be solved. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a recombinant bacterium suitable for high-density fermentation production of natural vanillin. Through exploration of fermentation conditions, tank conditions, etc., efficient synthesis is achieved to meet the market demand for vanillin.

[0006] In the research on heterologously expressing vanillin synthesis genes in Escherichia coli and completing the conversion of ferulic acid to vanillin through whole-cell catalysis, attempts were also made to knockout genes related to vanillin degradation in Escherichia coli to directly block the metabolic pathway of vanillin in Escherichia coli. After investigation, there are no directly reported genes related to vanillin degradation in Escherichia coli currently. Through literature reading, Aditya M. Kunjapur et al. from the Massachusetts Institute of Technology reported genes related to the reduction of benzaldehyde in Escherichia coli. After knockout, the reduction of benzaldehyde to benzyl alcohol was effectively reduced. For the reduction of vanillin to vanillyl alcohol in this study, the genes reported by Aditya M. Kunjapur et al. for the reduction of benzaldehyde can be referred to; for the oxidation of vanillin to vanillic acid in this study, an attempt was made to compare the vanillin dehydrogenase (vdh) in the original vanillin-producing strain in Escherichia coli using the NCNI database to find related metabolic pathway genes, and the genes related to vanillin degradation in Escherichia coli were knocked out using the CRISPR CAS9 system to allow vanillin to accumulate in Escherichia coli, and an identification of the vanillin synthesis metabolic pathway was constructed in Escherichia coli as Figure 1 , and through exploration of fermentation conditions and tank conditions, a large amount of high-efficiency synthesis of vanillin was achieved to meet the market demand for vanillin.

[0007] The first object of the present invention is to provide a recombinant bacterium for producing natural vanillin, with Escherichia coli as the host, freely expressing feruloyl-CoA synthase fcs and enoyl-CoA hydratase / aldolase ech, and knocking out or inhibiting the aldehyde-ketone reductase gene.

[0008] In one embodiment, the recombinant bacterium also knocks out or inhibits the vanillin oxidase gene.

[0009] In one embodiment, the aldehyde-ketone reductase gene is selected from one or more of yqhC, yqhD, dkgA, ahr, yahK.

[0010] In one embodiment, the vanillin oxidase gene is selected from one or more of paoC, aldA.

[0011] In one embodiment, the feruloyl-CoA synthase fcs is derived from Amycolatopsis sp. ATCC39116, and the enoyl-CoA hydratase / aldolase ech is derived from Pseudomonas fluorescens.

[0012] In one embodiment, the nucleotide sequence of the gene encoding feruloyl-CoA synthase fcs is as shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding enoyl-CoA hydratase / aldolase ech is as shown in SEQ ID NO.2. In one embodiment, the NCBI accession number of yqhC is WP_001350547.1, the NCBI accession number of yqhD is WP_001058802.1, the NCBI accession number of dkgA is WP_000013149.1, the NCBI accession number of ahr is WP_001309160.1, the NCBI accession number of yahK is EFF8973622.1, the NCBI accession number of paoC is EJD5186076.1, and the NCBI accession number of aldA is WP_000115943.1.

[0013] In one embodiment, the Escherichia coli includes Escherichia coli BW25113 or Escherichia coli BL21(DE3).

[0014] In one embodiment, the recombinant bacterium uses pBAD-HisA as a vector.

[0015] In one embodiment, the gene encoding feruloyl-CoA synthase fcs is expressed under the initiation of the promoter araBAD.

[0016] In one embodiment, the nucleotide sequence of the promoter araBAD is as shown in SEQ ID NO.3.

[0017] The second object of the present invention is to provide a whole-cell catalyst containing the above-mentioned recombinant bacterium.

[0018] The third object of the present invention is to provide a method for producing vanillin, which uses ferulic acid as a substrate and the above-mentioned recombinant bacterium or the above-mentioned whole-cell catalyst as a fermentation bacterium to produce vanillin.

[0019] In one embodiment, the seed solution of the above-mentioned recombinant bacterium or the above-mentioned whole-cell catalyst is inoculated into a fermentation medium. When the OD reaches 20-40, an inducer is added and cultured for 10-15 h, and then ferulic acid is added dropwise for 10-15 h. Glucose is added dropwise throughout the fermentation process.

[0020] In one embodiment, ferulic acid at a concentration of 80-120 g / L is added dropwise at a rate of 2-6 g / L / h.

[0021] In one embodiment, glucose at a concentration of 400-600 g / L is added dropwise at a rate of 2-6 mL / L / h.

[0022] In one embodiment, the inducer is L-arabinose or IPTG.

[0023] In one embodiment, the seed liquid is obtained by inoculating the above-mentioned recombinant bacterium or the above-mentioned whole-cell catalyst into a seed culture medium and culturing overnight at 35-38°C and 180-220 rpm to obtain the seed liquid.

[0024] In one embodiment, the fermentation medium contains 8-12 g / L of tryptone, 3-6 g / L of yeast extract, and 8-12 g / L of sodium chloride.

[0025] The present invention also provides the application of the above-mentioned recombinant bacterium, the above-mentioned whole-cell catalyst, or the above-mentioned method in the fields of cosmetics, food, medicine, agriculture, and plastics.

[0026] The present invention also provides the application of the above-mentioned recombinant bacterium, the above-mentioned whole-cell catalyst, or the above-mentioned method in the preparation of vanillin or products containing vanillin.

[0027] Beneficial effects:

[0028] In the present invention, the feruloyl-CoA synthetase gene (Feruloyl-CoA synthetase, Atfcs) derived from Amycolatopsis sp. ATCC 39116 and the feruloyl-CoA hydratase / aldolase gene (Feruloyl-CoA hydratase / aldolase, Pfech) derived from Pseudomonas fluorescen BF13 are heterologously co-expressed in Escherichia coli, a vanillin synthesis pathway is constructed in Escherichia coli, and the conversion of ferulic acid to vanillin is completed through whole-cell catalysis. In the present invention, a series of aldehyde-ketone reductases and / or oxidases are knocked out in Escherichia coli using the CRISPR CAS9 knockout technology, reducing the metabolism of vanillin in Escherichia coli. When the finally knocked-out strain BW-Δ7-pBAD-Pfech-Atfcs is fermented in a fermenter, no vanillyl alcohol and vanillic acid are detected at all, and vanillin is not degraded. That is, the knocked-out yqhC, yqhD, dkgA, ahr, yahK, paoC, aldA7 genes successfully block the degradation pathway of vanillin, improving the utilization rate of the substrate ferulic acid, and the yield can reach 10.8 g / L after 11 h of fermentation in a fermenter, laying a foundation for the further optimization of recombinant Escherichia coli for vanillin production. The recombinant bacterium provided by the present invention improves the utilization rate of the substrate ferulic acid, constructs an efficient vanillin synthesis pathway in Escherichia coli, establishes a cell factory for efficient vanillin synthesis, and has good industrial application prospects. Description of the drawings

[0029] Figure 1 Synthetic metabolic pathway of vanillin in Escherichia coli;

[0030] Figure 2Expression of feruloyl-CoA synthase gene and feruloyl-CoA hydrolase / aldolase gene in the co-expressing recombinant bacterium BW-pBAD-Pfech-Atfcs; Lane M--marker; Lanes 1, 2: Supernatant of whole bacteria of BW-pBAD-empty vector; Lanes 3, 4: Supernatant of whole bacteria of BW-pBAD-Pfech-Atfcs;

[0031] Figure 3 Vanillin standard curve;

[0032] Figure 4 Peak positions of 500 mg / L vanillin, 50 mg / L ferulic acid, 50 mg / L vanillyl alcohol and 50 mg / L vanillic acid;

[0033] Figure 5 Comparison of the contents of vanillin, vanillyl alcohol and vanillic acid in the fermentation broth of the knockout bacterium and the original bacterium; a: Bacterium BW-pBAD-Pfech-Atfcs; b: Bacterium BW-Δ5-pBAD-Pfech-Atfcs; VA: Vanillin; FA: Ferulic acid; VAL: Vanillyl alcohol; VAA: Vanillic acid;

[0034] Figure 6 Comparison of the contents of ferulic acid, vanillin, vanillyl alcohol and vanillic acid in the fermentation broth of each knockout bacterium;

[0035] Figure 7 Conversion of the bacterium BW-Δ7-pBAD-Pfech-Atfcs in fermentor culture. Specific implementation manners

[0036] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0037] Example 1 Construction of vanillin synthesis pathway in Escherichia coli

[0038] 1.1 Construction of pBAD-Pfech-Atfcs vector

[0039] The feruloyl-CoA synthase gene fcs of Amycolatopsis sp. ATCC 39116 was obtained through NCBI (NCBI Reference Sequence: WP_020422604.1). The sequence of the feruloyl-CoA hydrolase / aldolase gene ech (Pfech) from Pseudomonas fluorescen BF13 was obtained through NCBI (NCBI Reference Sequence: WP_123394413.1), and the sequence was sent to Tsingke Biotechnology Co., Ltd. for synthesis. The sequence was directly inserted into the pET-21a(+) vector without fragment ligation, and plasmids pET-21a-Atfcs and pET-21a-Pfech were obtained respectively. The promoter of the target gene is the T7 promoter, and the screening marker is ampicillin (Amp).

[0040] The fcs and ech were heterologously tandemly expressed in Escherichia coli to construct the plasmid pBAD-Pfech-Atfcs. When amplifying the linearized pBAD vector, the pBAD-HisA plasmid (purchased from Tsingke) was used as the template, and PCR amplification was carried out using primers P1 (pBAD-F, pBAD-R) to obtain the linearized pBAD vector (3955 bp); using the pET-21a-Pfech plasmid as the template, PCR amplification was carried out using primers P2 (Pfech-F, Pfech-R) to obtain the fragment Pfech; using the pET-21a-Atfcs plasmid as the template, PCR amplification was carried out using primers P3 (Atfcs-F, Atfcs-R) to obtain the fragment Atfcs (1476 bp); the primer sequences used are as follows:

[0041] pBAD-F: 5’aagcttggctgttttggcggatg-3’

[0042] pBAD-R: 5’-ggttaattcctcctgttagcccaaaaaacgg-3’

[0043] Pfech-F: 5’-gctaacaggaggaattaaccATGAGCAACTATGAAGGTCGCTGGA-3’

[0044] Pfech-R: 5’-TCTTAAAGTTAAATTAACGTTTATATGCCTGCAGACCCG-3’

[0045] Atfcs-F: 5’-ACGTTAATTTAACTTTAAGAAGGAGATATACATGCGTAACCAAGGTCTGGGC-3’

[0046] Atfcs-R: 5’-ccgccaaaacagccaagcttTTAGCCGAAACGGCGACGTAC-3’

[0047] Using a recombinant cloning kit (Clone MultiS One Step Cloning Kit, Novizan (C113-02)), the linearized pBAD vector, fragment Pfech, and fragment Atfcs were ligated in three fragments. The ligation product was transformed into competent cells Trans1-T1 (Phage Chemically Comptent Cell, TransGen Biotech (CD501-03)) by calcium chloride chemical transformation method. Positive clones were picked, plasmids were extracted and sequenced. The recombinant plasmid with the correct sequence was designated as pBAD-Pfech-Atfcs. In the plasmid, the fcs gene was linked by the T7 ribosome binding site RBS, the promoter was araBAD, the selection marker was ampicillin Amp, and the replication origin was Pbr322 ori.

[0048] The obtained correct plasmid pBAD-Pfech-Atfcs was transformed into E. coli BW25113 (Thermo Cat# OEC5042) by calcium chloride chemical transformation method. The resulting recombinant bacteria were designated as BW-pBAD-Pfech-Atfcs respectively.

[0049] 1.2 Culture Medium

[0050] LB medium (1L): Tryptone 10 g, Yeast extract 5 g, Sodium chloride 10 g. Made up to 1L with pure water, natural pH, autoclaved at 121 °C for 20 min.

[0051] 1.3 Preliminary Induction, Expression and Detection of Proteins

[0052] Using the above obtained recombinant bacteria as the test strains, the induction, expression and detection of proteins were carried out according to the following steps:

[0053] The test strains were inoculated into LB medium and cultured overnight at 37 °C with 200 rpm. The bacterial solution was transferred to 5 mL of LB medium containing ampicillin at a final concentration of 100 μg / mL at an inoculation amount of 1% (v / v), and cultured at 37 °C with 200 rpm for about 1.5 h (OD600 reached about 0.6), then the inducer L-arabinose at a final concentration of 2 g / L was added, and induced fermentation was carried out at 30 °C with 200 rpm for 12 h to obtain the fermentation broth.

[0054] Collect 10 OD of fermentation broth into a centrifuge tube, centrifuge at 13,000 rpm for 2 min, discard the supernatant, add 1 mL of 10 mM PBS buffer (pH 7.4) to the cell pellet, and break it using an ultrasonic crusher to obtain a cell lysate. Centrifuge the cell lysate at 13,000 rpm for 2 min, take the supernatant to prepare a protein sample (supernatant), and detect the protein expression using SDS-PAEG.

[0055] The expression of the recombinant enzyme BW-pBAD-Pfech-Atfcs is as Figure 2 shown. According to the protein gel, it can be seen that the size of Atfcs from Amycolatopsis sp. ATCC 39116 is 51.9 KDa, and the size of Pfech from Pseudomonas fluorescen BF13 is 31.0 KDa. Both genes can achieve soluble expression when co-expressed in Escherichia coli.

[0056] 1.4 Establishment of vanillin detection method

[0057] Dissolve vanillin, ferulic acid, vanillyl alcohol, and vanillic acid standard samples in 40% methanol solution, and detect the contents of ferulic acid and vanillin by high-performance liquid chromatography (HPLC). The HPLC detector is Agilent 1260 Infinity LC, the detection column is Agilent Eclipse plus C18 column, the ultraviolet detection wavelengths are 320 nm for ferulic acid, 280 nm for vanillin, 280 nm for vanillyl alcohol, and 280 nm for vanillic acid, the mobile phase is methanol and ddH 2 O (plus one ten-thousandth glacial acetic acid) solution, the flow rate is 1.0 mL / min, and the injection volume is 10 μL. The secondary mass spectrometry uses an LC / MS liquid chromatography / triple quadrupole tandem mass spectrometer (Agilent 1260 / 6460 LC / Triple Quadrupole MS), and the mobile phase uses acetonitrile and ddH 2 O, and the detection column is Agilent Eclipseplus C18 column.

[0058] The HPLC detection method for vanillin was successfully established. The vanillin standard curve is as Figure 3 shown. The peak positions of 50 mg / L vanillin, 50 mg / L ferulic acid, 50 mg / L vanillyl alcohol, and 50 mg / L vanillic acid are 5.909 min, 9.937 min, 2.532 min, and 4.349 min respectively, all of which are single strong absorption peaks (see Figure 4 ), the peak positions of the detected samples and the standard samples are the same, and then the vanillin content of the samples is calculated according to the standard curve.

[0059] Example 2: Optimization of the vanillin synthesis pathway in Escherichia coli

[0060] 2.1 Knocking out aldehyde-ketone reductase genes (yqhC, yqhD, dkgA, ahr, yahK) in Escherichia coli is beneficial to the accumulation of vanillin (I) Knocking out of yqhC, yqhD, dkgA, ahr, yahK genes

[0061] The CRISPR CAS9 knockout system was used to knock out yqhC, yqhD, dkgA, ahr, yahK genes in E. coli BW25113 strain. According to the principle of CRISPR CAS9 knockout, first, the CAS9 plasmid (pCAS9) was transferred into the expression host E. coli BW25113 strain by calcium chloride chemical transformation method to perform the cleavage function. Positive transformants were screened using a kana-resistant plate and denoted as strain BW-pCAS9. A corresponding pTarget plasmid was designed to recognize the knockout gene for each gene. The 20bps gRNA was designed using the website CRISPOR (tefor.net). Then, the upstream and downstream 500bp of the knockout gene were amplified and spliced into a 1000bp homologous arm (HR). The corresponding pTarget plasmid and the 1000bp homologous arm were co-transformed into strain BW-pCAS9, and positive transformants were screened using kana and spe-resistant plates for knockout identification. The specific design is as follows:

[0062] Since the yqhC, yqhD, dkgA genes are on a continuous gene cluster and can be knocked out simultaneously, when amplifying the pTarget vector of these three genes, plasmid pTarget-F was used as a template, and PCR amplification was performed using primers P4 (yqhC N20-F, dkgAN20-R). The correctly sequenced PCR product was denoted as Δ3-pTarget (2118bp); using Escherichia coli as a template, PCR amplification was performed using primers P5 (yqhC-up-F, yqhC-up-R) and primers P6 (dkgA-down-F, dkgA-down-R) respectively to obtain the upstream and downstream homologous arm fragments yqhC-up (500bp) and dkgA-down (500bp) of the yqhC, yqhD, dkgA genes.

[0063] Similarly, when knocking out the ahr gene, the correctly sequenced PCR product obtained using primers P7 (ahr N20-F, ahr N20-R) was denoted as ahr-pTarget (2118bp), and the correctly sequenced PCR products obtained using primers P8 (ahr-up-F, ahr-up-R) and P9 (ahr-down-F, ahr-down-R) were denoted as ahr-up (500bp) and ahr-down (500bp) respectively.

[0064] When knocking out the yahK gene, the correctly sequenced PCR product obtained using primers P10 (yahK N20-F, yahK N20-R) was denoted as yahK-pTarget (2118 bp). The correctly sequenced PCR products obtained using primers P11 (yahK-up-F, yahK-up-R) and P12 (yahK-down-F, yahK-down-R) were denoted as yahK-up (500 bp) and yahK-down (500 bp), respectively.

[0065] The primer sequences used when knocking out the yqhC, yqhD, dkgA, ahr, and yahK genes are as follows:

[0066] yqhC N20-F: 5’-gtgatctcccgtaaaaccacgttttagagctagaaatagcaagttaaaataaggctagt-3’

[0067] dkgA N20-R: 5’-gtggttttacgggagatcacactagtattatacctaggactgagctagctg-3’

[0068] yqhC-up-F: 5’-attttctgcctacgattgcgattctgac-3’

[0069] yqhC-up-R: 5’-attttctgcctacgattgcgattctgac-3’

[0070] dkgA-down-F: 5’-gtgatctcccgtaaaaccacgttttagagctagaaatagcaagttaaaataaggctagt-3’

[0071] dkgA-down-R: 5’-gtggttttacgggagatcacactagtattatacctaggactgagctagctg-3’

[0072] ahr N20-F: 5’-GTGATATGGTGCATCAACAGgttttagagctagaaatagcaagttaaaataaggctagt-3’

[0073] ahr N20-R: 5’-CTGTTGATGCACCATATCACactagtattatacctaggactgagctagctg-3’

[0074] ahr-up-F: 5’-gttgccgcgttattatctgctaaat-3’

[0075] ahr-up-R: 5’-aaatcattcgcagcgctgatc-3’

[0076] ahr-down-F: 5’-atcagcgctgcgaatgattttttttggtccttctctggtgttgt-3’

[0077] ahr-down-R: 5’-ttggctgctctgactttgatatctg-3’

[0078] yahKN20-F: 5’-GCCGGGCCGGGTAAAAAAGTgttttagagctagaaatagcaagttaaaataaggctagt-3’

[0079] yahKN20-R: 5’-ACTTTTTTACCCGGCCCGGCactagtattatacctaggactgagctagctg-3’

[0080] yahK-up-F: 5’-gcaggatgacgccagctttg-3’

[0081] yahK-up-R: 5’-cagggtatttattaattttttgtgtttactcctgattagctatgtgtatttgg-3’

[0082] yahK-down-F: 5’-aaaaattaataaataccctgtggtttaacatattaacttcgct-3’

[0083] yahK-down-R: 5’-ggtgagtgcgttatcgtctgacc-3’

[0084] Using a recombinant cloning kit (Clone The MultiS One Step Cloning Kit (Vazyme, C113-02)) was used to perform single-fragment ligation on Δ3-pTarget, ahr-pTarget, and yahK-pTarget respectively, and the ligated products were transformed into competent cells Trans1-T1 (Phage Chemically Comptent Cell, TransGen Biotech, CD501-03)) using the calcium chloride chemical transformation method. The selection marker was spectinomycin (spe). Positive clones were picked, plasmids were extracted for sequencing, and the corresponding pTarget plasmids pTarget-Δ3, pTarget-ahr, and pTarget-yahK were constructed.

[0085] The upstream and downstream homologous arms of the yqhC, yqhD, and dkgA genes (yqhC-up and dkgA-down), the upstream and downstream homologous arms of the ahr gene (ahr-up and ahr-down), and the upstream and downstream homologous arms of the yahK gene (yahK-up and yahK-down) were respectively subjected to two-fragment splicing. Then, using the spliced products as templates, PCR amplifications were carried out with primers P13 (yqhC-up-F, dkgA-down-R), P14 (ahr-up-F, ahr-down-R), and P15 (yahK-up-F, yahK-down-R) respectively to obtain the corresponding 1000bp homologous arm sequences. The PCR products with correct sequences were respectively designated as Δ3-HR, ahr-HR, and yahK-HR as homologous arm targeting fragments.

[0086] Then, the corresponding pTarget plasmid and the homologous arm targeting fragment were co-transformed into competent cells of strain BW-pCAS9 by electroporation. Positive transformants were screened using LB plates with kana and spe resistance. Identification primers P16 (Δ3-JD-F, Δ3-JD-R), P17 (ahr-JD-F, ahr-JD-R), and P18 (yahK-JD-F, yahK-JD-R) were designed respectively to perform PCR identification to determine whether the target gene was successfully knocked out. The screened knockout strains were inoculated into LB medium and 1 mM IPTG was added for overnight induction at 30 °C to eliminate the pTarget plasmid. pTarget-eliminated bacteria were screened using spe resistance sensitivity. They were inoculated into 5 mL of antibiotic-free LB medium at an inoculation amount of 1% (v / v) and cultured overnight at 42 °C. At the same time, anti-sensitivity screening was performed using spe, kana, and antibiotic-free LB plates. Only when no colonies grew on the LB plates with spe and kana resistance and only grew on the antibiotic-free LB plate could it be ensured that the pCAS9 plasmid and the pTarget plasmid had been eliminated. Thus, a gene knockout strain was obtained. During the knockout process, the knockout of each gene was carried out based on the strain in which gene knockout had been achieved. First, competent cells of the single-gene knockout strain were prepared, and then the knockout plasmid and the homologous fragment were transferred into the single-gene knockout strain and screened, so as to achieve double-gene knockout or multi-gene knockout. The sequences of the identification primers used are as follows:

[0087] Δ3-JD-F: 5’-ccacgctgtttttagaaaacggcc-3’

[0088] Δ3-JD-R: 5’-aacgaccattttccgccacc-3’

[0089] ahr-JD-F: 5’-atggcgggtgaaagctttattttga-3’

[0090] ahr-JD-R: 5’-acaagcagatgcataccgcc-3’

[0091] yahK-JD-F: 5’-ctcgctgtttttagccactggc-3’

[0092] yahK-JD-R: 5’-caaacttaaccgggtgatcagggt-3’

[0093] In this experiment, the strain with the yqhC, yqhD, and dkgA genes knocked out in the E. coli BW25113 strain was designated as BW-Δ3, the strain with the yqhC, yqhD, dkgA, and ahr genes knocked out was designated as BW-Δ4, and the strain with the yqhC, yqhD, dkgA, ahr, and yahK genes knocked out was designated as BW-Δ5.

[0094] The correct plasmid pBAD-Pfech-Atfcs obtained in Example 1 was transformed into BW-Δ3, BW-Δ4, and BW-Δ5 by the calcium chloride chemical transformation method, and the resulting recombinant strains were designated as BW-Δ3-pBAD-Pfech-Atfcs, BW-Δ4-pBAD-Pfech-Atfcs, and BW-Δ5-pBAD-Pfech-Atfcs, respectively.

[0095] (II) Culture Medium

[0096] Transformation medium (1L): 10 g of tryptone, 8 g of yeast extract, 10 g of glucose, 10 g of NaCl, 0.8 g of MgSO 4 0.8 g, made up to 1 L with distilled water, pH 7.0, dispensed after complete dissolution, autoclaved at 115 °C for 25 min.

[0097] (III) Comparison of the Degradation of Vanillin Catalyzed by the Whole Cells of the Strains BW-pBAD-Pfech-Atfcs and BW-Δ5-pBAD-Pfech-Atfcs

[0098] Using the recombinant strains BW-pBAD-Pfech-Atfcs and BW-Δ5-pBAD-Pfech-Atfcs as the test strains, the shake-flask culture and transformation for the synthesis of vanillin were carried out according to the following steps:

[0099] The test strains were inoculated into LB medium and cultured overnight at 37 °C and 200 rpm. The bacterial solution was transferred to 50 mL of transformation medium containing 100 μg / mL ampicillin at a final concentration at an inoculation amount of 1% (v / v), cultured at 37 °C and 200 rpm until the OD600 reached about 0.6, then L-arabinose at a final concentration of 2 g / L was added, and then induced to ferment at 30 °C and 200 rpm for 20 h, and the cells were harvested by centrifugation at a cell concentration of 10 OD / ml.

[0100] The cells were resuspended with fresh transformation medium to prepare a 20 mL reaction system, and 1 g / L ferulic acid as the substrate was added to the reaction system. After catalysis for a certain time, samples were taken to detect the contents of vanillin, vanillyl alcohol, and vanillic acid in the fermentation broth. The HPLC detection method for vanillin was the same as that in Example 1.

[0101] The shake flask transformation results of the recombinant bacteria BW-pBAD-Pfech-Atfcs and BW-Δ5-pBAD-Pfech-Atfcs are as follows Figure 5 , as can be seen from the figure, after knocking out 5 genes of yqhC, yqhD, dkgA, ahr, and yahK, the effect is obvious, the production of vanillyl alcohol decreases, and compared with the control bacterium BW-pBAD-Pfech-Atfcs, the degradation of vanillin by the bacterium BW-Δ5-pBAD-Pfech-Atfcs is inhibited and accumulates.

[0102] 2.2 Knocking out the vanillin oxidase genes (paoC, aldA) in Escherichia coli is beneficial to the accumulation of vanillin

[0103] (1) Knocking out of paoC and aldA genes

[0104] Based on the bacterium BW-Δ5 knocked out in 2.1, the CRISPR CAS9 knockout system was used to continue knocking out the vanillin oxidase genes paoC and aldA in Escherichia coli.

[0105] The knockout principle is the same as that in 2.1, and the specific design is as follows:

[0106] When knocking out the paoC gene, using the plasmid pTarget-tnaA as a template, and using primers P19 (paoC N20-F, paoC N20-R) to obtain a PCR product with correct sequence, denoted as paoC-pTarget (2118bp); using primers P20 (paoC-up-F, paoC-up-R), P21 (paoC-down-F, paoC-down-R) to obtain PCR products with correct sequences, denoted as paoC-up (500bp) and paoC-down (500bp) respectively.

[0107] When knocking out the aldA gene, using primers P22 (aldAN20-F, aldAN20-R) to obtain a PCR product with correct sequence, denoted as aldA-pTarget (2118bp); using primers P23 (aldA-up-F, aldA-up-R), P24 (aldA-down-F, aldA-down-R) to obtain PCR products with correct sequences, denoted as aldA-up (500bp) and aldA-down (500bp) respectively.

[0108] The primer sequences used when knocking out paoC and aldA genes are as follows:

[0109] paoC N20-F: 5’-ATTCAGGATGCGAAACTCGAgttttagagctagaaatagcaagttaaaataaggctagt-3’

[0110] paoC N20-R: 5’-TCGAGTTTCGCATCCTGAATactagtattatacctaggactgagctagctg-3’

[0111] paoC-up-F: 5’-agccattgagattccagcccg-3’

[0112] paoC-up-R: 5’-ggaggaacaatgtcatacccgct-3’

[0113] paoC-down-F: 5’-gggtatgacattgttcctcctcatgcctgtgccctcgc-3’

[0114] paoC-down-R: 5’-taagcgaagcctgcattgccac-3’

[0115] aldAN20-F: 5’-TGAGAAGATCATGGCGACTGgttttagagctagaaatagcaagttaaaataaggctagt-3’

[0116] aldAN20-R: 5’-CAGTCGCCATGATCTTCTCAactagtattatacctaggactgagctagctg-3’

[0117] aldA-up-F: 5’-gactggggacaatcccgatg-3’

[0118] aldA-up-R: 5’-gggcgactcctgtgatttatatgtttg-3’

[0119] aldA-down-F: 5’-ataaatcacaggagtcgccccagacccaggtggtttatttacagtcttaa-3’

[0120] aldA-down-R: 5’-taaagatttacgtgcttcacgcgc-3’

[0121] Using a recombinant cloning kit (Clone For the MultiS One Step Cloning Kit (Vazyme, C113-02)), the single-fragment ligation of paoC-pTarget and aldA-pTarget was performed separately, and the products were transformed into the competent cell Trans1-T1 (Phage Chemically Comptent Cell, TransGen Biotech, CD501-03)) using the calcium chloride chemical transformation method. The screening marker was spectinomycin (spe). Positive clones were picked, and plasmids were extracted for sequencing to construct the corresponding pTarget plasmids pTarget-paoC and pTarget-aldA.

[0122] The upstream and downstream homologous arms of the paoC gene, paoC-up and paoC-down, and the upstream and downstream homologous arms of the aldA gene, aldA-up and aldA-down, were spliced in two fragments. Then, using the spliced products as templates, PCR amplifications were performed with primers P25 (paoC-up-F, paoC-down-R) and P26 (aldA-up-F, aldA-down-R) to obtain the corresponding 1000-bp homologous arm sequences. The correctly sequenced PCR products were denoted as paoC-HR and aldA-HR as the targeting fragments.

[0123] The pTarget-paoC plasmid and the paoC-HR targeting fragment were co-transformed into the competent cells of BW-Δ5 bacteria transfected with the pCAS9 plasmid by electroporation. Positive transformants were screened using LB plates with kana and spe resistances. Identification primers P27 (paoC-JD-F, paoC-JD-R) were designed for PCR identification to determine whether the target gene was successfully knocked out. The screened knockout strains were inoculated into LB medium supplemented with 1 mM IPTG and induced overnight at 30 °C to eliminate the pTarget plasmid. The pTarget-eliminated bacteria were screened using a spe resistance-sensitive method and inoculated at an inoculation amount of 1% (v / v) into 5 mL of antibiotic-free LB medium and cultured overnight at 42 °C. At the same time, anti-sensitivity screening was performed using spe, kana, and antibiotic-free LB plates. Only when no growth was observed on the LB plates with spe and kana resistances and growth was only observed on the antibiotic-free LB plate could it be ensured that the pCAS9 plasmid and the pTarget plasmid had been eliminated, and the paoC gene knockout bacteria were obtained. On this basis, the knockout of the aldA gene was superimposed, and identification primers P28 (aldA-JD-F, aldA-JD-R) were designed for PCR identification to determine whether the aldA gene was successfully knocked out. The sequences of the identification primers used are as follows:

[0124] paoC-JD-F: 5’-tagcctgtggctgctgca-3’

[0125] paoC-JD-R: 5'-cgacaccaatcagccctgcaataa-3'

[0126] aldA-JD-F: 5'-cggctatcgttgttcaggaccc-3'

[0127] aldA-JD-R: 5'-ttgcatgacagtttcgggatagaagt-3'

[0128] In this experiment, the gene knockout strains of yqhC, yqhD, dkgA, ahr, yahK, paoC, and aldA7 successfully knocked out were denoted as BW-Δ7.

[0129] The correct plasmid pBAD-Pfech-Atfcs obtained in Example 1 was transformed into BW-Δ7 by the calcium chloride chemical transformation method, and the resulting recombinant strain was denoted as BW-Δ7-pBAD-Pfech-Atfcs.

[0130] (2) Fermentation with transformation medium to compare the degradation of vanillin by whole cells of each knockout strain

[0131] Using the recombinant strains BW-pBAD-Pfech-Atfcs, BW-Δ3-pBAD-Pfech-Atfcs, BW-Δ4-pBAD-Pfech-Atfcs, BW-Δ5-pBAD-Pfech-Atfcs, and BW-Δ7-pBAD-Pfech-Atfcs as the test strains, shake flask culture transformation for synthesizing vanillin was carried out according to the following steps:

[0132] The test strains were inoculated into LB medium and cultured overnight at 37°C and 200 rpm. The bacterial solution was transferred to 50 mL of transformation medium containing ampicillin with a final concentration of 100 μg / mL at an inoculation amount of 1% (v / v). When the OD600 reached about 0.6 at 37°C and 200 rpm, L-arabinose with a final concentration of 2 g / L was added, and then induced fermentation was carried out at 30°C and 200 rpm for 20 h. The cells were harvested by centrifugation at a cell concentration of 10 OD / ml.

[0133] The cell pellet was resuspended with fresh transformation medium to prepare a 20 mL reaction system, and 1 g / L ferulic acid as the substrate was added to the reaction system. After catalysis for a certain time, samples were taken to detect the contents of vanillin, vanillyl alcohol, and vanillic acid in the fermentation broth. The HPLC detection method for vanillin was the same as 1.4.

[0134] The shake flask transformation results of the recombinant strains BW-pBAD-Pfech-Atfcs, BW-Δ3-pBAD-Pfech-Atfcs, BW-Δ4-pBAD-Pfech-Atfcs, BW-Δ5-pBAD-Pfech-Atfcs, and BW-Δ7-pBAD-Pfech-Atfcs as the test strains are as follows Figure 6 As can be seen from the figure, after knocking out the 5 genes of yqhC, yqhD, dkgA, ahr, and yahK, the effect is obvious, and the production of vanillyl alcohol decreases. The production of vanillin by the control strain BW-pBAD-Pfech-Atfcs is 125.86 mg / L, and the molar conversion rate of the substrate is 16.15%; compared with the control strain, the degradation of vanillin by the strain BW-Δ5-pBAD-Pfech-Atfcs is inhibited and accumulates. The production of vanillin is 510.12 mg / L, the content of vanillyl alcohol is 9.79 mg / L, and the molar conversion rate of the substrate is increased to 65.18%; after knocking out the two genes of paoC and aldA, the pathway of vanillin degradation to vanillic acid is completely blocked, the degradation of vanillin is inhibited and accumulates, and the content of vanillin in the strain BW-Δ7-pBAD-Pfech-Atfcs is 504.38 mg / L, without an obvious decrease. The content of vanillyl alcohol has decreased by 32.89%, and vanillic acid is no longer produced.

[0135] (III) Fermenter transformation of the strain BW-Δ7pBAD-Pfech-Atfcs

[0136] Select BW-Δ7-pBAD-Pfech-Atfcs for fermenter transformation, and proceed as follows:

[0137] Inoculate the test strain into LB medium and culture overnight at 37 °C and 200 rpm. Inoculate it into 100 mL of seed medium (LB medium) at an inoculation amount of 1% (v / v) and culture overnight at 37 °C and 200 rpm to obtain a seed solution; inoculate the seed solution into 900 mL of fermentation medium containing ampicillin (100 μg / ml) (2 L fermenter, LB medium), stir and culture at 37 °C until the cell concentration reaches 30 OD (λ = 600 nm, about 12 h), add L-arabinose with a final concentration of 2 g / L, and induce and culture at 30 °C. After about 12 h of fermentation, add the substrate ferulic acid (ferulic acid mother liquor: 100 g / L) at a rate of 4 g / Lh. During the whole fermentation process, add a 50 g / 100 mL glucose aqueous solution at a flow rate of 4 mL / L / h, and maintain the pH at about 7.0 with 2.7 M ammonia water. Record the time when the substrate is added as 0 h, and take samples regularly to measure the vanillin content. The results are as follows Figure 7As shown in the figure, the vanillin production continued to increase between 0 and 11 h, and no vanillyl alcohol and vanillic acid were detected. Therefore, vanillin was not degraded. The knockout of the seven genes yqhC, yqhD, dkgA, ahr, yahK, paoC, and aldA successfully blocked the degradation pathway of vanillin, and the highest yield was 10.8 g / L, which is the highest yield of vanillin produced by the whole-cell catalysis of Escherichia coli to transform ferulic acid at present.

[0138] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A recombinant bacterium for producing natural vanillin, characterized in that using Escherichia coli BW25113 as the host, freely expressing feruloyl-CoA synthase fcs and enoyl-CoA hydratase / aldolase ech, knocking out or inhibiting the yqhC, yqhD, dkgA, ahr and yahK genes; the nucleotide sequence of the gene encoding the feruloyl-CoA synthase fcs is as shown in SEQ ID NO.1, the nucleotide sequence of the gene encoding the enoyl-CoA hydratase / aldolase ech is as shown in SEQ ID NO.2, and the recombinant bacterium also knocks out or inhibits the paoC and aldA genes.

2. A whole-cell catalyst, characterized in that it contains the recombinant bacterium described in claim 1.

3. A method for producing vanillin, characterized in that the method uses ferulic acid as the substrate and the recombinant bacterium described in claim 1 or the whole-cell catalyst described in claim 2 as the fermentation bacterium to produce vanillin.

4. According to the method described in claim 3, characterized in that inoculating the seed liquid of the recombinant bacterium described in claim 1 or the whole-cell catalyst described in claim 2 into the fermentation medium, waiting until the OD reaches 20 - 40, adding an inducer and culturing for 10 - 15 h, then feeding ferulic acid and fermenting for 10 - 15 h, and continuously feeding glucose throughout the fermentation process.

5. Use of the recombinant bacterium described in claim 1 or the whole-cell catalyst described in claim 2 or the method described in claim 3 or 4 in the preparation of vanillin or a product containing vanillin.

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